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.

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.

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.

Economic mass production is a crucial stage in the development of biocontrol agents. This paper compares different solid substrates with and without preceding liquid culture for efficacy and economics in the scaled-up production of the mycoparasitic biocontrol agent Clonostachys rosea. In a basic liquid medium (BLM) containing (in g l−1): molasses (80), neopeptone (10) and yeast extract (2), Clonostachys rosea produced 107 conidia ml−1 after 4 d in agitated culture. Spore viability was 99% and did not differ from that of conidia derived from potato dextrose agar plates. Guata, an inert polyester fibre, coated with BLM and cassava starch (30 g l−1), was the best solid matrix yielding 109 conidia g−1 after 20 d. On a volume basis after extraction and on a production unit basis, Guata was in a similar range to Biodac®, a granular cellulose complex, which had also been enriched with BLM, producing up to 107 conidia ml−1 or over 1010 conidia per 23×28 cm polypropylene bag, the production unit in which all solid matrices were incubated. Rice was the lowest-yielding solid matrix regardless of quality. Chlamydospores were not observed in liquid or on solid media. All conidia produced on solid matrices had a viability >90%. Conidia produced on Biodac had a higher viability (95.6%) than spores produced on Guata (93.1%) after 24 h on potato-dextrose plates. However, production on Biodac was more variable and negatively affected by prefermentation in liquid culture, whereas production on Guata and rice was unaffected by the origin of the starting inoculum. Guata of medium thickness (1.27 cm) was superior to thicker (2.54 cm) or thinner (0.64 cm) sheets with respect to both yield per gram and per production unit. Although prefermentation neither improved yield nor reduced cost, it increased throughput by a factor five and may be advantageous where fermentor and/or incubator space is limited. Biodac and Guata were the most economical solid matrices; inoculum to treat one hectare (3×1012 conidia) cost US $ 27, whereas rice-based production cost US $ 613 under Costa Rican conditions. Liquid culture alone would be fast but too costly: US $ 1078 ha−1. Experiments to further reduce costs in the Guata system are suggested.

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.

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.

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.

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 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.

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°.

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.

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.

Corky root of tomato caused by Pyrenochaeta lycopersici is a disease of concern in Italy and for many tomato growing areas in the world. Isolates of the fungus were characterized at both the physiological and molecular level. The optimal in vitro growth temperature for all isolates was 23 °C. All Italian isolates of P. lycopersici showed similar RAPD and esterase banding patterns. No relevant polymorphisms were detected after enzymatic digestion of PCR-amplified ITS and IGS regions. The overall results indicate a low degree of genetic variability within a collection of 43 Italian isolates. These data are of interest in breeding programs for resistance against corky root of tomato and they provide useful information for the development of molecular diagnostic tools for the rapid identification and detection of P. lycopersici.

The ability of an unidentified mycobiont from Pisonia grandis mycorrhizas to utilize organic nitrogen and phosphorus sources was determined in axenic liquid cultures. The fungus utilized inorganic phosphate and both freely- and poorly-soluble salts of inositol hexaphosphate as sole phosphorus sources. With the exception of uric acid which did not support growth, the yield of the mycobiont on all organic nitrogen sources tested was significantly greater than the yield on either NH4+ or NO3−. It is suggested that the major ecological role of the Pisonia mycobiont in stands of P. grandis on coral cays may be rapid absorption of allantoin and urea produced during degradation of uric acid by the soil microflora. This may be important in retaining nitrogen in the soil[ratio ]plant system prior to loss from the coral cay by further degradation, volatilization and/or leaching.

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.

Isolates of Verticillium lecanii, from insects and other substrates, were tested for vegetative compatibility by observing heterokaryon formation among complementary nitrate-nonutilizing (nit) mutants, Among 33 V. lecanii isolates, 17 were self-incompatible. The 16 self-compatible isolates were divided into 13 vegetative compatibility groups (VCGs). Eleven VCGs were single-member VCGs, and the remaining two included two or three isolates each. Self-incompatibility occurred more frequently among isolates with abundant aerial mycelium, whereas most of the isolates with short or reduced aerial mycelium were self-compatible. Virulence to larvae of Bemisia tabaci ranged from 0 (four isolates) to 83% mortality 4 d after treatment with conidial suspensions. There was no correlation between the capacity of isolates to anastomose and their virulence to larvae of Bemisia tabaci: highly virulent isolates were found at similar frequencies among self-compatible and self-incompatible isolates. Those with abundant aerial mycelium were collectively more virulent (28·5% mortality) than those with reduced mycelium (10·7% mortality). Three isolates belong to VCG VL-11 were all highly virulent.

All isolates of Rhizoctonia solani (teleomorph Thanatephorus cucumeris) anastomosis group (AG) 3 from potato tubers in commercial fields in Scotland were found to contain more than one double-stranded RNA (dsRNA) element ranging in size from 0·3 to 15 kb. Strains of other AGs (2-1, 3, 4, 5) from culture collections also contained dsRNA elements. Isolates of AG 3 from different black scurf lesions on single tubers showed similar dsRNA profiles and, when opposed on agar plates, underwent compatible hyphal fusion where the colonies met. Isolates from different tubers in the same commercial field often showed somatic incompatibility (mean 44%) leading to cytoplasmic death when colonies fused. Isolates from different fields always showed somatic incompatibility after hyphal fusion, suggesting that the complex population structure of AG 3 could significantly limit the transmission of dsRNA between strains. The incidence of different dsRNA elements in AG 3 isolates could not be related to pathogenicity in seedling tests on tomato, carrot, lettuce, radish, cress or onion. Double-stranded RNA elements could not be eliminated from AG 3 isolates by repeated hyphal-tip subculture or extended (8 week) incubation on agar containing cycloheximide. Some isolates lost the smaller dsRNA elements (1·0 to 1·6 kb) during these procedures, and one isolate (of AG 4) lost a 6·5 kb dsRNA element, but the larger elements (up to 9·5 or 12 kb) were always retained. When assessed by low stringency hybridisation, the dsRNA elements of R. solani AG 3 and of strains from AGs 2-1, 4 and 5 showed no homology with a plasmid containing cDNA of the complete hypovirus dsRNA genome of Cryphonectria parasitica.

The white rotting Armillaria mellea induced a spatially very distinctive pattern of degradation in sycamore wood, under both natural and artificial conditions. Degradation began preferentially within groups of fibres containing intercellular spaces, whereas fibre regions lacking such spaces were undegraded and remained largely intact even when decay had become advanced elsewhere. The two types of fibre region differed not only in the presence of intercellular spaces, and hence in the potential for gas exchange, but also in their degree of lignification. This was higher in the more resistant type, as shown by staining of undecayed wood with toluidine blue-O, by microspectrometry after staining for the Mäule colour reaction, and by uv-microscopy. A spatially similar pattern of cellulose degradation was induced by the brown rotting Laetiporus sulphureus, which is known to cause preferential degradation of less strongly lignified cell walls. By contrast, the white-rotting Ganoderma pfeifferi showed a tendency to degrade the stronger lignified cell walls. Thus, in combination with the application of conventional histological methods, the wood degradation modes observed give additional evidence for stronger and weaker lignified fibre regions within the wood of sycamore.

The infection process of Alternaria cirsinoxia was studied on Canada thistle (Cirsium arvense) in the controlled environment and in the field. In the controlled environment, germination of conidia began at 2 h and appressoria formation at 4 h after inoculation. Approximately 75% of appressoria formed at the anticlinal wall junctions of the epidermis. Leaf penetration occurred between 6 and 24 h, most commonly in between adjoining anticlinal walls. Penetration through stomata was rare. After penetration, large, intracellular infection hyphae formed and branched within epidermal cells, ramifying throughout the leaf tissues inter- and intracellularly by 24 h. In field conditions, infection coincided with prolonged rainfall and conidia remained viable on the leaf surface for 8–9 days (d) before causing infection on leaves. A host response occurred after penetration, involving deposition of lignin and callose in the infected epidermal and mesophyll cell walls. High levels of silicon were detected in epidermal cells directly below appressoria, often appearing to form entirely silicified infected cells which were resistant to collapse after air-drying. This study shows that A. cirsinoxia has the potential for rapid invasion of the leaf tissues of Canada thistle under good moisture conditions. The implications of the host responses, in terms of defence against infection, are discussed.