The mechanisms responsible for controlling hyphal extension and branching are still poorly understood. We have investigated these processes by studying their dependence on temperature and nutrient concentration. Tip growth is highly responsive to temperature change, increasing linearly from 4 to 37 °C. Over this range of temperatures the branching pattern shows virtually no response. Likewise, varying nutrient concentration does not affect branch distribution. Colonies subjected to rapid extreme temperature downshifts (for example from 25 to 4°) display a strong and highly predictable branching response. There are three stages to this response. First there is an initial lag phase of growth without branching. After this, the growing tips display a series of tightly spaced, dichotomous branches dubbed starbursting. Following continued growth at 4°, tips enter a recovery phase, returning to normal branching frequencies with lateral branches. The strength of the response to cold is correlated with the ratio of growth rates before and after the downshift. The combined observations point to a homeostatic set point for branch distribution that compensates for temperature and nutrient concentration.

The abilities of isolates of Amanita alboverrucosa, A. conicoverrucosa, A. fuscosquamosa, A. nauseosa, A. ochrophylla, A. pyramidifera, A. roseolamellata, A. xanthocephala and six unidentified Amanita species from eastern Australian temperate sclerophyll forests to utilise a range of inorganic and organic nitrogen sources for growth was examined in axenic liquid cultures. All taxa utilised NH4+ and at least some amino acids readily, while biomass yields on NO3− and histidine were generally low. All taxa were able to utilise bovine serum albumin, but for most taxa biomass yields on this substrate were significantly lower than on NH4+. Significant intraspecific variation in biomass yield was observed on all substrates for taxa for which multiple isolates were screened. As a group, eastern Australian Amanita species thus have the potential to utilise nitrogen from a broad range of organic substrates and this might be important in the nitrogen nutrition of their ectomycorrhizal tree hosts in sclerophyll forests.

Strains of Trichoderma harzianum with either elevated or reduced production of cellulase-complex enzymes β-glucosidase and endoglucanase were constructed using uv-light mutagenesis. In tests for rhizosphere competence two of the cellulase minus strains were found to have significantly enhanced rhizosphere competence, whilst two cellulase overproducers were found not to colonize either the rhizosphere or rhizoplane of bean plants. These results indicate that rhizosphere competence of Trichoderma strains is not always associated with extracellular enzyme production.

Claviceps purpurea specific to grasses in salt marsh habitats (Group G3) has previously been identified on Spartina spp. in two locations: New Jersey, USA and southern England. We have identified this subgroup of C. purpurea (G3) in 11 distinct populations including western Europe, South America, and along the Atlantic and Pacific Coasts of the USA. In addition, G3 C. purpurea was discovered on a new host grass genus, Distichlis. Unweighted pair group mean analyses of AFLP and RAPD data reveal distinct structure in G3 C. purpurea populations. Pacific coast populations show little diversity, suggesting they may have been introduced recently in that region. 43 isolates, representing 11 populations were identified as G3 based on the presence of an EcoRI restriction site in the 5.8S ribosomal DNA, and a clear genetic separation from isolates representing the other two C. purpurea subgroups (G1 and G2). In addition, all isolates originating from Spartina densiflora, S. foliosa, S. alterniflora, and S. anglica were identified as belonging to G3. RAPD and AFLP analyses supported the recognition of three discrete groups within C. purpurea and revealed high genetic variability between groups, with only 1.8% of polymorphic markers shared across all isolates. Similarly, analysis of molecular variation (AMOVA) revealed that genetic variability was mainly due to variations between groups (63.5%) rather than within groups (28.5%) or within populations (7.96%). G3 isolates were 35% similar, Pacific coast isolates 83% similar. Ninety percent similarity among isolates from the San Francisco Bay Area suggests this is a recently introduced population.

Hirsutella versicolor, H. citriformis and H. nivea sp. nov. are described from Homoptera in Thailand. H. versicolor was usually associated with the ascomycete Torrubiella pruinosa and was isolated from ascospores. H. citriformis could not be linked with a teleomorph but was successfully isolated from conidia. H. nivea was not associated with a teleomorph and could not be isolated in pure culture.

Picoa lefebvrei and Tirmania nivea are reported for the second and first time from Spain and Europe, respectively. They are illustrated and compared with material and descriptions of the related P. juniperi, T. pinoyi and P. lefebvrei is compared with material from Israel. The collection of T. nivea was originally recorded as T. pinoyi, which should, therefore, be removed from the European Catalogue of hypogeous fungi.

A total of 34 isolates of Cylindrobasidium evolvens were obtained from wounded Picea abies in five sample plots in Sweden and Lithuania. Population structure and genetic variation within the species were assessed by somatic incompatibility and arbitrary primed DNA (AP-DNA) analysis, using the M13 core sequence as a primer. All somatic incompatibility pairings between different C. evolvens isolates resulted in weak mycelial antagonism. The AP-DNA profiles indicate genetic variation was high in the species with no differentiation among isolates being found either on a large (more than 700 km) (R2=0·108) or a fine scale (200 m) (R2=0·024). In a plot, genetic similarity was significantly higher (P<0·02) among isolates from wounds infected during the previous 2 years than among the rest of the same population.

Brunneosporella aquatica gen. et sp. nov., Aqualignicola hyalina gen. et sp. nov., Jobellisia viridifusca sp. nov. and Porosphaerellopsis bipolaris sp. nov. are described and illustrated from wood submerged in freshwater collected in Hong Kong. Both Brunneosporella and Aqualignicola have characteristic features of the Annulatascaceae and their placement within this family is discussed. Jobellisia viridifusca sp. nov. differs from all described species in having an ascomatal wall with a bright orange middle wall layer and fusiform, greenish-brown ascospores. Porosphaerellopsis bipolaris sp. nov. is unique in producing multiseptate ascospores with bipolar mucilagilous pads.

PCR and DNA sequence analysis have become standard tools for identification, detection and phylogenetic analysis of fungi. A large number of species are incapable of growth in the laboratory, making the preparation of pure DNA problematical. The amplification of DNA samples from impure material is subject to misinterpretation if more than one species is present. To overcome this problem, we designed an in situ PCR technique that links PCR amplification to the light microscopic image. The amplified tissue is stained, thus confirming which morphotype has been amplified. The PCR product can then be sequenced. We tested the technique on fixed Blumeria graminis spores and mycelia using primers derived from the sequence of the gene encoding the catalytic subunit of protein kinase A (bka1). This is the first report of in situ PCR on phytopathogenic fungal material. This technique allows positive confirmation of the origin of genes cloned from obligate pathogenic fungi and could be adapted for use on any samples containing mixed fungal species.

Primary colonization and the early growth of the mycelium of Agaricus bisporus in compost could be improved to increase the rate of substrate utilization. The production of enzyme activities during the vegetative growth of a commercial strain in compost were determined. N-acetyl-glucosaminidase activity peaked during the first week whereas the activities of polysaccharidases decreased with the replacement of autochthonous micro-organisms by A. bisporus. A low level of water soluble sugars was maintained in compost inoculated with A. bisporus in comparison with a non-inoculated compost. Microbial residues and hemicelluloses appeared to be important nutrient sources during the early growth.

An assessment of the inter- and intraspecific resolution of 5′ nrDNA ITS TRFLP was conducted by computer-simulated restriction analysis of 316 ectomycorrhizal GenBank sequences. Generally, sequences with a similarity of <90% could be distinguished with two to three independent enzyme digests, although sequences with similarity >95% were likely to remain unresolved. Choice of restriction enzyme strongly influenced resolution, especially when less than three enzymes were used. Although our results showed that 5′ nrDNA ITS TRFLP is a powerful tool for distinguishing between species of ectomycorrhizal fungi, closely related species, including species of Rhizopogon, Dermocybe, Hebeloma, and Lactarius produced indistinguishable 5′ nrDNA ITS TRFLP – profiles. 5′ nrDNA ITS TRFLP may also split a species, and the probability of this occurring reflected intraspecific sequence variation. For groups of closely related species, the use of 3′ nrDNA ITS in conjunction with 5′ nrDNA ITS produced improved resolution comparable with RFLP analyses. Overall, our results show that nrDNA ITS TRFLP is a valuable addition to the array of molecular tools available to ectomycorrhizal ecologists. However, simple assessments of ectomycorrhizal species diversity based on numbers of unique TRF created with a single restriction enzyme must be viewed with caution, as for each enzyme we examined, identical TRF common to groups of unrelated species were observed. In addition, to maximize the effectiveness of TRFLP in surveys of ectomycorrhizal fungi it will be useful to combine information from at least two independent enzyme digests in order to distinguish and track species in the field.

Relative quantitative RT-PCR and western blotting were used to investigate the expression of three genes with potentially regulatory functions from the arbuscular mycorrhizal fungus Glomus intraradices in symbiosis with tomato and barley. Standardisation of total RNA per sample and determination of different ratios of plant and fungal RNA in roots as colonisation proceeded were achieved by relative quantitative RT-PCR using universal (NS1/NS21) and organism-specific rRNA primers. In addition, generic primers were designed for amplification of plant or fungal β-tubulin genes and for plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes as these have been suggested as useful controls in symbiotic systems. The fungal genes Ginmyc1 and Ginhb1 were expressed only in the external mycelium and not in colonised roots at both mRNA and protein levels, with the proteins detected almost exclusively in the insoluble fractions. In contrast, mRNA of Ginmyc2 was identified in both external and intraradical mycelium. In mycorrhizal roots, Ginmyc2 and fungal β-tubulin mRNAs increased in proportion to fungal rRNA as colonisation proceeded, suggesting that accumulation reflected intraradical fungal growth. Fungal α-tubulin protein and β-tubulin mRNA both appeared to be more abundantly accumulated in AM hyphae within heavily colonised roots than in external hyphae, relative to fungal rRNA. Tomato GAPDH mRNA accumulation was proportional to tomato rRNA, but accumulation of tomato β-tubulin mRNA was reduced in colonised roots compared to non-mycorrhizal roots. These results provide novel evidence of differential spatial and temporal regulation of AM fungal genes, indicate that the expression of tubulin genes of both plant and fungus may be regulated during colonisation and validate the use of multiple ‘control’ genes in analysis of mycorrhizal gene expression.

Optimization of protoplast release (202×104 ml−1) from a strain of Rhizoctonia solani anastomosis group three was achieved by treating 3 d old mycelia from broth cultures with Novozyme 234 for 3 h at 30 °C. The only other treatment, of many tested, that yielded any protoplasts (15·6×104 ml−1) was commercial lysing enzymes from Trichoderma harzianum. Various combinations of commercial enzyme preparations, variations of osmotic stabilisers, and mycelial age, were ineffective in raising the protoplast yield. Regeneration of colonies from protoplasts was maximal (83–87%) when protoplasts were incorporated in cooled agar containing 5% glucose, malt extract and yeast extract, supplemented with 0·8 or 1·0 M mannitol as osmotic stabiliser. Liquid culture broth and other potential osmotic protectants at various concentrations yielded fewer regenerating colonies (5% in broth and 13–75% in agar). The Rhizoctonia strain was highly sensitive to hygromycin B, so transformation of protoplasts was achieved with a polyethylene glycol-based procedure, using plasmid pES200 containing the bacterial hygromycin B phosphotransferase gene driven by a trpC promoter from Aspergillus nidulans. Transformation was achieved using the DNase inhibitor aurintricarboxylic acid and incubation temperatures below 10–12 °. It was confirmed by high stringency hybridization of DNA extracted from the transformants, using pES200 as a radiolabelled probe. The transformants grew slowly (25–35 mm diam after 14 d) then ceased growth, and never produced fast-growing sectors indicative of integrated plasmid DNA. Transformation was also achieved with pAXHY2 from Cryphonectria parasitica, and colonies reached 55–65 mm diam before they stopped growing. Southern blots confirmed the presence of non-integrated pAXHY2, but no fast-growing sectors were produced.

This study examined the occurrence of macrofungi and the decay of roofs thatched with water reed, Phragmites australis. Sampling from 20 north- and 20 south-facing roof sides showed that several ascomycetes usually associated with reed in situ are common on thatch. The only basidiomycetes recorded were Mycena species. There was no significant difference in the representation of macrofungi on north- and south-facing roof sides, but Mycena spp. only produced basidiomes on surfaces facing north, suggesting that the dry environment of the south side prevents fruiting. Eleven species were recorded in total, and the average of 2·4 species per roof did not increase with the age of thatch or degree of decay. The same species were generally present on young and old thatch, and no successional stages of fungal communities could be distinguished with increasing roof age.

Deterioration of thatch occurs at the exposed surface of the roof and progresses inwards. Within the layer of reeds outer, middle, and inner zones develop representing different stages in the decay. The zones move inwards as thatch deteriorates. A comparison of the rate of decay among roofs with pitch 30°, 45° and 60° showed that the innermost zone appeared ca 20 cm from the exposed reed butts in both of the steeper roofs, whereas it was no longer present in the roof with low pitch. This suggests that the depth of the zones depends upon the roof slope, and the outer and middle zones move inwards at a higher rate in roofs with a low pitch, resulting in an increased rate of deterioration.

Phragmites thatch appears to harbour its own characteristic macrofungal community, with certain Mycena species likely to represent the principal decomposers. A common feature of fungi occurring on thatch is that they must endure unfavourable conditions.

Inter-simple sequence repeat PCR (ISSR-PCR) was used to develop markers for simple sequence repeat-rich (SSR) regions for investigation of genetic relatedness of Pisolithus isolates collected from eastern mainland Australia. Primers were designed to amplify ten SSR-rich regions and these were used to screen 14 Pisolithus isolates. Two amplified loci showed size polymorphisms among the isolates (regarded as polymorphic), two were monomorphic for all isolates, while the remainder amplified alleles for only some isolates. UPGMA analysis of the alleles for each isolate at each locus together with ITS-RFLP analysis, separated the isolates into two groups. These two groups appear to correspond to isolates that ITS sequence data have previously separated as P. albus and P. microcarpus.

The relative complexity measure (RCM) is a new approach to evaluate relatedness of DNA sequences which eliminates the requirement to align sequences prior to analysis, a step required with standard reference methods. The value of the RCM approach to generate distance matrices for use in phylogenetic analysis of organisms has not been determined. This study compared RCM with the algorithmic and tree searching reference methods for phylogenetic analysis using fungal sequences. Sequences of the cytochrome b gene and the 18S rDNA gene were obtained from the GenBank database to determine feasibility of this method for phylogenetic relatedness. The RCM approach was also used to construct a phylogenetic tree using internal transcribed spacer (ITS) sequences from 23 medically relevant fungal species. The robustness of the RCM and reference approaches was determined by comparing the topology of seven medically relevant fungi within the phylogenetic trees generated after progressive removal of 10, 20, 30, 40 and 50% of the nucleotide bases from either the 5′ or 3′ end of the three genomic target sequences. The results demonstrated that the RCM method was equivalent to the reference methods for construction of phylogenetic trees from cytochrome b and 18S rDNA gene sequences. The phylogenetic tree constructed using the ITS sequence generated no contradictory topology. The RCM generated trees retained the appropriate topology after removal of up to 50% of the cytochrome b sequence, 40% of the ITS sequence, and 30% of the 18S gene target sequence. Comparatively, the reference methods failed to maintain topology after only a 10% sequence deletion for each genomic target. The results showed the RCM to be a reliable and robust computational approach for use in the construction of fungal phylogenetic trees without the requirement for prior sequence alignment.

Mycelial preparations of Erynia neoaphidis were tested under different drying and storage conditions. For unformulated mycelial discs and formulated alginate granules, sporulation was best maintained if drying at 20–22 °C was performed at 60 or 90% r.h. rather than an ambient humidity of 30%. Storage experiments were performed on preparations dried at 90% r.h. for 72 h. Estimates for a 50% decline in sporulation with storage time (ST50) were computed if there were statistically significant fits using exponential or non-parametric linear regression. For mycelial discs, values of ST50 were between 36–39 d when maintained at 10° with silica gel or 20% r.h., and 14–26 d when kept at 10° and 35% r.h. or with silica gel and 15 or 20°. For alginate granules, ST50 values of 6–9 d were obtained at 10° and 20–55% r.h. A 5- to 9-fold increase in sporulation was produced with granules containing 2% w/v sucrose and 5% w/v starch compared to granules without additives. Elevated humidities are recommended for drying E. neoaphidis mycelia and viability of dried preparations is best maintained if they are stored at 10° compared with 15 or 20°.

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 distribution and behaviour of nuclei in conidia of 11 coelomycete species with tri- and tetraradiate conidia and belonging to five genera has been investigated: Cornutispora (C. ciliata, C. intermedia, C. lichenicola, C. limaciformis, and C. pittii), Eriosporella (E. calami), Furcaspora (F. abieticola, F. pinicola), Suttoniella (S. eriobotryae, S. gaubae), and Tetranacrium (T. gramineum). They have been studied by the HCl-Giemsa technique using dried, preserved material including holotypes and isotypes with ages ranging from 3 to 116 yr. Conidia of Cornutispora species showed different ploidy levels, and C. limaciformis showed a very high (>90%) frequency of stable and viable micronuclei with an unusual type of ploidy level, occurring naturally. Frequency of ploidy levels in nuclei within conidia of Cornutispora species appeared to be associated with changes in gross conidial morphology. This is the first report of micronuclei in coelomycetes. The types of appendages on arms or parts of conidia have been studied using various stains including erythrosin in ammonia and a modified Leifson's flagella staining technique. In Furcaspora species the apical and basal conidial appendages are cellular maintaining protoplasmic continuity with the arms on which they are sited. The results have been compared with those of Crucellisporium species which have tetraradiate conidia. The new species, Cornutispora intermedia, C. pittii and Furcaspora abieticola spp. nov. are described, and illustrated, and a key to all known Cornutispora species is provided.

Recently, a powdery mildew anamorph infecting snowberry (Symphoricarpos albus) has been reported as a new plant pathogen in several European countries. We report here its occurrence in the UK, Germany and, for the first time, Switzerland. Based on morphological and scanning electron microscope (SEM) patterns, this novel pathogen appeared similar to the anamorph of a common North American powdery mildew, Erysiphe symphoricarpi (syn. Microsphaera symphoricarpi). A phylogenetic analysis of the internal transcribed spacer (ITS) sequences of the ribosomal DNA (rDNA) of an English and a North American snowberry powdery mildew fungus showed that they are conspecific. Ascomata of the pathogen were found only in one European collection, that being in Germany in 2002. The similarity in morphology of the ascomata also confirmed the co-identity of the European and American snowberry powdery mildews.