The aim of this study was to compare the ectomycorrhizal community structure in spruce stands treated with different levels of dolomite lime. ITS-typing of randomly sampled mycorrhizas, without prior morphotyping, was used. Sixteen different ITS-RFLP patterns were found. By comparison with the available reference material, nine of these could be identified at least to genus level. Variation within treatments was large and statistically there were no significant differences between treatments with respect to specific taxa. A similarity assessment did, however, show less similarity between control and high dolomite (HD) than between low dolomite (LD) and either control or HD, suggesting a shift in the community structure as a result of the treatment. The fruitbody production at Hasslöv had been recorded in a different study during 1985–92 and 28 ectomycorrhizal species had been found in the treatments examined in the present study. Except for three species, all were present in the reference material used for identification of the ITS-types. Only three species, Russula ochroleuca, Lactarius necator and Boletus chrysenteron were found as both mycorrhizas and fruitbodies. There were five taxa which occurred on over 5% of the screened roots. These were Thelephora terrestris, 21.5%; Tylopilus felleus, 13%; Tylospora fibrillosa, 13%; and two unidentified taxa, 10% and 6%. Together these five taxa colonized over 60% of the mycorrhizal roots investigated, yet none of them was found among the fruitbodies recorded in the above-ground study. Despite the differences in taxa found below and above ground, similarity tests between treatments using the fruitbody data also revealed a shift in community structure.

Beauveria spp. from New Zealand were compared with isolates from other countries using morphological and genetic methods. The New Zealand isolates could be divided into B. bassiana and B. brongniartii based on conidial dimensions; isolates with conidia longer than 3 μM were classified as B. brongniartii, isolates with shorter, spherical conidia were B. bassiana. RAPD analyses using 10 random primers and scoring 330 bands divided isolates into four large groups: a heterogeneous B. bassiana/B. brongniartii group from New Zealand and other countries, a group containing only New Zealand B. bassiana, a group of only B. brongniartii from both New Zealand and overseas, and a group of other Beauveria species (B. velata, B. caledonica, B. amorpha and B. vermiconia). Restriction digestion of the internal transcribed spacer regions of the nuclear rDNA supported the existence of a genetically distinct New Zealand group of B. bassiana isolates, separating these from a heterogeneous group of B. bassiana and B. brongniartii.

The phylogenetic position of members of the entirely asexually reproducing lichen-forming genera Lepraria and Leproloma was investigated using sequence data from the ITS1-5.8S-ITS2 and small subunit (SSU) nuclear ribosomal DNA. Phylogenetic reconstructions were carried out using a likelihood-based Bayesian Markov chain Monte Carlo (MCMC) tree sampling technique and the unweighted least squares optimality criterion, the latter based on maximum likelihood distances obtained via an alignment-free distance estimation technique. The results indicate that most species currently referred to Lepraria and Leproloma form a single monophyletic group. This monophyletic group is the sister group to Stereocaulon and Muhria and belongs in the Stereocaulaceae (Lecanorales, Lecanoromycetes, Ascomycota). This indicates that an ancestor of Lepraria switched from a sexual to an asexual mode of dispersal. Subsequent speciation must have taken place in the absence of sexual processes, which contradicts the view of asexual taxa as ‘evolutionary dead ends’. Leproloma is polyphyletic and nested within Lepraria. Lepraria flavescens is a Lecanora, probably belonging in subgenus Glaucomaria. Lepraria lesdainii and L. obtusatica are unrelated to each other and to other species currently referred to Lepraria or Leproloma. Leprocaulon and Crocynia are distantly related to the core group of Lepraria and Leproloma.

Female flies (Phorbia phrenione) cross fertilize Epichloë typhina via a specific behaviour in which they transfer spores among self-incompatible fungi. Flies ingest spores while visiting fungi for egg laying. Only 11% of stromata lacking P. phrenione eggs produced perithecia and these perithecia covered an average of only 8% of the stromatal surfaces. In contrast, 71% of stromata that possessed fly eggs produced perithecia, covering 54% of the stromatal surfaces. Immediately following oviposition, flies stereotypically drag their abdomen across the fungus in a spiral pattern while excreting faeces. Transfer of fly faeces to unfertilized stromata resulted in cross fertilization. We observed spiralling patterns of perithecial development on fungal surfaces, signifying the behaviour of the fly results in cross fertilization of the fungus. This is the first documentation of active fertilization in an insect-fungus mutualism.

Partial rDNA sequences were obtained from 26 isolates representing species of Cercospora, Passalora, Paracercospora, Pseudocercospora, Ramulispora, Pseudocercosporella and Mycocentrospora. The combined internal transcribed spacers (ITS) including the 5·8S rRNA gene and 5′ end of the 25S gene (primer pairs F63/R635) on rDNA were amplified using PCR and sequenced directly. The ITS regions including the 5·8S varied in length from 502 to 595 bp. The F63/R635 region varied from 508 to 519 bp among isolates sequenced. Reconstructed phylogenies inferred from both regions had highly similar topologies for the taxa examined. Phylogenetic analysis of the sequences resulted in four well-supported clades corresponding to Cercospora, Paracercospora/Pseudocercospora, Passalora and Ramulispora, with bootstrap values greater than 92% for each clade. Based on the results of the analysis, a new combination for Pseudocercosporella aestiva is proposed in Ramulispora, and Paracercospora is reduced to synonymy with Pseudocercospora.

Two distinct groups (A and B) of the conifer shoot blight and canker pathogen Sphaeropsis sapinea have been recognized on pine hosts in the northcentral United States. Their occurrence in a collection of isolates from much more diverse hosts and locations was studied by analysis of RAPD markers and isozymes. Seventy-nine isolates were used for RAPD marker analysis, and 37 for isozyme analysis, with 33 isolates in common. Hosts included 19 species in Pinus and species of Cedrus, Larix, Picea, Pseudotsuga, located in Africa, Australasia, Europe, and North America. Relationships among these isolates were determined using cluster analyses of presence or absence data for each amplification product or isozyme. Analyses of either RAPD marker or isozyme data placed most isolates in a group that contained previously characterized RAPD marker group A isolates. There was no strong indication of either host or geographic clustering within this highly similar, widely distributed group. Analysis of RAPD marker data placed several other isolates in a separate group that contained previously characterized RAPD marker group B isolates. This is the first report of isolates from hosts other than Pinus banksiana or P. resinosa, and from outside Michigan, Minnesota, or Wisconsin, that have been characterized as members of the RAPD marker group B.

Forty-seven isolates of Pythium irregulare from different hosts and geographic origins were compared from molecular, morphological and physiological viewpoints. They were divided into four groups (I–IV) based on ITS-RFLP analysis and RAPD analysis. Groups I and II included 32 and eight isolates, respectively, collected from diverse hosts and geographic origins, and groups III and IV comprised seven isolates derived from sugar beet and sugar beet field soil. Group I had smaller oogonia and oospores than did the other three groups. In groups I and II, a significantly higher percentage of the oogonia produced multiple projections compared to groups III and IV which occasionally produced one projection. The growth rate of the four groups was similar at 5–30 °C. At 33°, many isolates of group I grew rapidly but most of the isolates of other groups grew slowly, and at 35°, the former grew but the latter did not. In phylogenetic analysis based on sequences of the ITS region, four groups of P. irregulare were included in one cluster with P. sylvaticum. Groups I–II and III–IV clustered more tightly in the same branch, respectively. The genetic divergence between I–II and III–IV was higher than between each group (I–II and III–IV) and P. sylvaticum, indicating that groups I–II and III–IV may represent two different species.

A survey of fungicide resistance among isolates of the mushroom pathogens Cladobotryum mycophilum and C. dendroides Types I and II was undertaken, with respect to the active ingredients thiabendazole, carbendazim (benzimidazoles) and prochloraz manganese following an epidemic in Britain and Ireland in 1994/95. The majority of isolates (41/57) were strongly resistant to thiabendazole (ED50 > 200 ppm) and were exclusively C. dendroides Type II. All C. mycophilum and C. dendroides Type I isolates, and four C. dendroides Type II isolates, were weakly resistant to thiabendazole (ED50 1–10 ppm). Thiabendazole-resistant C. dendroides Type II isolates were only weakly resistant to carbendazim (ED50 2–10 ppm) and isolates which were weakly resistant to thiabendazole were carbendazim-sensitive (ED50 < 1 ppm), demonstrating a lack of complete cross resistance between these two benzimidazole fungicides. The ED50 values for all isolates with respect to prochloraz manganese ranged from 0.14 to 7.8 ppm. Benzimidazole resistance was considered to have been an important factor influencing the severity of the 1994/95 cobweb epidemic but 25% of isolates collected were benzimidazole sensitive.

The forest pathogen Dothistroma pini (Scirrhia pini) infects the needles of many pine species, causing needle loss and consequently retarded wood growth. Only one strain of Dothistroma pini is present in New Zealand. Because over 90% of commercial forests in New Zealand are planted with the susceptible species Pinus radiata, a study of the global diversity of D. pini strains was initiated to assess the threat of further unwanted introductions of the pathogen. A collection of D. pini strains from eight countries was studied in the UK. The production of dothistromin toxin by the strains, and DNA sequence analysis of the ribosomal ITS region, confirmed their identification as D. pini, although strains from the central USA contained two nucleotide substitutions in the ITS region. Colony morphologies and growth rates were diverse, but all strains which sporulated showed a similar wide range of spore size. The morphological features examined did not support separation of the strains into the two groups shown by ITS sequences. Most striking was the production, in axenic culture, of extremely high levels of dothistromin toxin by strains from Germany and, to a lesser extent, some from the USA (>500 times and >40 times as much as the New Zealand strain, respectively). The high level of production of dothistromin toxin by some strains is a concern for forest health as well as for forest workers and needs to be evaluated further.

The time course of release of an extracellular matrix (ECM) from conidia of the barley powdery mildew pathogen Blumeria graminis was investigated by scanning electron and light microscopy. Conidia released the ECM material preferentially onto a hydrophobic surface and release was detected within 20 s of contact of a conidium with the substratum. No such rapid release of an ECM could be detected when conidia were deposited onto the hydrophilic surface of clean glass. A limited amount of ECM was, however, released from conidia onto the hydrophilic surface of a hydrated cellulose membrane. A time study on hydrophobic plastic revealed that although the ECM was clearly present within the first hour after contact of the conidium with the substratum, it was much reduced in quantity after 12 h incubation. The ECM was demonstrated by micromanipulation and light microscopy to be present as a liquid at the contact interface of the conidium and the substratum. The ECM could not be detected by SEM beneath conidia on leaves, but light microscopy and micromanipulation demonstrated that the ECM could be detected beneath some conidia on the waxy surface of an epidermal strip taken from the abaxial surface of a barley leaf. The ECM material stained positively for protein and could not be removed from the conidium interface by vacuum desiccation in the scanning electron microscope indicating that the ECM is not simply water.

Puccinia recondita f. sp. tritici pathotype (pt) 64-(6),(7),(10),11 was first detected in Australia in northern New South Wales in 1990. Greenhouse tests of pathogenicity on wheat and triticale genotypes indicated that this pathotype was similar to two closely related pathotypes, but differed from them in at least three pathogenic features indicating that it had not arisen by a simple mutational event. Pathotype 64-(6),(7),(10),11 combined several pathogenic and isozymic features which, prior to its detection, were known only in two different groups of pathotypes, strongly suggesting that it had arisen via somatic hybridisation between isolates from each group. This hypothesis was supported by the RAPD phenotypes of isolates representing pathotypes present in Australia at the time pt 64-(6),(7),(10),11 was first detected. The hybrid pathotype combined virulence for Lr1 with partial virulence for gene Lr13 and was fully virulent in greenhouse adult plant tests on two hybrid wheats heterozygous for these genes. This is the first case where compelling evidence has been obtained for somatic hybridization in Prt and only the second of inter pathotype somatic hybridization in a wheat rust under field conditions.

The level of molecular variability in the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was assessed. We established several monoxenically growing single-spore cultures of the G. intraradices collected from a long-term soil tillage experiment in Tänikon (Switzerland). They were grown in symbiosis with Ri T-DNA transformed carrot roots. The ITS region was amplified from genomic DNA of fungal spores using ITS1 and ITS4 primers. The amplified ITS region of three of the fungal isolates was cloned and sequenced. All sequences obtained from our samples closely resembled those previously reported for G. intraradices, and no evidence for genetic exchange was found on the interspecific or intergeneric level. Phylogenetic analysis showed the presence of at least two different sequence families within a single-spore isolate. The single strand conformation polymorphism (SSCP) method detected different sequence types in a single PCR sample. This technique enabled us to detect several distinct ITS sequences within each of the single-spore isolates of the AMF. Estimates of intra- and intersporal sequence diversity were obtained by comparing diversity within and among the single-spore isolates. A higher level of diversity was found among spores than within a single spore.

Cladobotryum dendroides, causal agent of cobweb disease of Agaricus bisporus, has become increasingly resistant to methylbenzimidazole carbamate (MBC) fungicides following the extensive use of MBC in cultivated mushroom production in Ireland. Of 38 isolates of C. dendroides obtained from Irish mushroom units, 34 were resistant to carbendazim. Primers based on conserved regions of the β-tubulin gene were used to amplify and sequence a portion of the β-tubulin gene in C. dendroides. A point mutation was detected at codon 50 in isolates resistant to benzimidazole fungicides, causing an amino acid substitution from tyrosine to cysteine. Species-specific PCR primers were designed to amplify the region of the β-tubulin gene containing this substitution. The point mutation removed an Acc I restriction site in the β-tubulin gene sequence of resistant isolates. Digestion of the PCR product with Acc I thus provides a rapid diagnostic test to differentiate sensitive and resistant isolates of this fungus. EMBL accession number: Y12256.

On nutrient-poor rock surfaces, yeast-like black fungi (also called microcolonial fungi, MCF) may derive organic carbon either from the atmosphere or from interactions with other rock-inhabiting microorganisms. Interactions between free-living rock inhabiting heterotrophic fungi and phototrophic algae were investigated using axenic cultures. Five typical MCF strains were incubated with pure cultures of four lichen photobionts isolated from lichens growing in similar locations. After 2–12 months of combined cultivation, the fungal and algal colonies developed an overlapping structure involving both partners. Microscopic, histological and ultrastructural methods were used to investigate the changes that occur after prolonged contact. Histological analysis demonstrated changes in the spatial organisation of mixed colonies when in contact with each other. The branching of fungal hyphae was more pronounced in the vicinity of algal cells, thus increasing the potential contact surface. Photobiont cells were not changed in size, but a reduction of sporulation could be observed in some Trebouxia strains. Close cell wall contacts between fungal and algal cells were formed and mucilage accumulated at the contact places. The ability of MCF to form associations with potential photobionts present on rock surfaces was thus demonstrated for pure cultures in vitro.

Of three hundred and sixty Trichoderma strains investigated, fourteen, identified as T. aureoviride, T. harzianum and T. viride, grew well at 5 °C on both minimal and yeast extract agar media. In dual culture tests at 10°, most strains antagonized the phytopathogens Rhizoctonia solani and Fusarium oxysporum f. sp. dianthi. T. aureoviride and T. viride were more effective against Pythium debaryanum than T. harzianum. The activities of extracellular chitinases (EC 3.2.1.30), β-glucosidases (EC 3.2.1.21) and proteases (EC 3.4.21.1; EC 3.4.21.4), which are thought to be involved in the mycoparasitic process, were also examined and results showed that these enzymes were highly active at 5° in the cold-tolerant strains.

We used ITS-RFLP and sequence analysis to determine the identities of the fungal endophytes of six terrestrial orchid species from southeastern Queensland, a region previously unexplored in this context. Pure cultures of orchid – colonising fungi were obtained and fungal identities were assessed by means of ITS-PCR, RFLP analysis, sequence comparison, and protocorm colonisation tests. ITS-PCR and RFLP analysis resulted in five main groupings. Sequencing and GenBank comparison of these five groups showed that the fungal endophytes isolated from the three Pterostylis species were probably Thanatephorus species. There was close sequence identity (90%) of the fungus isolated from Acianthus spp. to Epulorhiza repens, suggesting these may be the same fungal species. However, that only E. repens succeeded in colonising protocorms of Thelymitra pauciflora suggests these may be different species of Epulorhiza. Analysis of the ITS and LSU sequences of the fungus isolated from Caladenia carnea showed high identities with a sequence from a Sebacina vermifera originally isolated from Caladenia dilatata. These results show that there is specificity for fungal partners within the orchid genera Acianthus, Caladenia and Pterostylis.

A technique was developed for transforming the ectomycorrhiza-forming basidiomycetes Suillus bovinus, Hebeloma cylindrosporum, and Paxillus involutus based on Agrobacterium tumefaciens-mediated T-DNA transfer. The selection marker employed was the Shble gene conferring resistance to phleomycin under control of the Schizophyllum commune GPD promoter and terminator. Transformants from all three investigated species were shown by PCR to contain the GPDScP-Shble-GPDScT construct, although the fate of the foreign DNA (integrated vs episomal, single-copy vs multi copy) could not be determined. The mycorrhiza formed between S. bovinus Bler transformants and Pinus sylvestris did not reveal any differences from those formed with untransformed Suillus bovinus.

Colletotrichum lindemuthianum is a pathogen of the common bean plant (Phaseolus vulgaris) causing anthracnose. Large numbers of isolates can rapidly arise with different genetic and chromosomal compositions but their origin is unknown since sexual fruit bodies have only been found in the laboratory. We have recently described the occurrence of special kinds of hyphae that create anastomoses directly between conidia. In this work we show that conidial anastomoses can occur between two different Colletotrichum species. The implications of this observation on the generation of genetic diversity in these species are discussed.

Competition and replacement interactions between species of wood-decomposing basidiomycetes were studied with a culture technique using various sized pieces taken from 6 cm diam. wood discs as a substrate. Based on field studies, 17 species of basidiomycetes, either primary decayers or late successional saprotrophs which show strict dependencies on certain preceding pioneer decayers in the field, were isolated and inoculated on the wood sectors. After precolonization, the sectors were attached to each other pairwise in various species combinations and incubated on water agar. The discs were regularly inspected and photographed for detection of mycelial overgrowth. After 6 months, samples were taken from the wood of the sectors and cultured for identification of prevailing mycelia. Measured by the replacement of opposing fungi, the selective late successional species are more combative than the primary decayers. Species of Antrodiella had the greatest competitive success. The results of our experiment corresponds well with field observations and explain, at least partly, the physiological background of the interactions.

Phoma medicaginis was isolated as the dominant endophyte from surface-sterilized shoots of Medicago sativa and M. lupulina growing outdoors. Plants were either symptomless or showed signs of infection in the shape of limited lesions which sometimes contained melanized pycnidial initials. Rapid colonization of host tissue and sporulation were observed within 9 d on dead plant material upon incubation in a moist chamber. Such colonized material, but not freshly harvested living tissue, contained brefeldin A (1.7 μg g−1D.W.). This toxin was also produced in pure culture (20 mg l−1) and in artificially inoculated autoclaved M. sativa stems (3 mg g−1D.W.=920 μg ml−1). The latter concentration of brefeldin A should be similar to that produced within a fruiting lesion of P. medicaginis and suppressed spore germination and growth of nine of 11 common phylloplane fungi tested. This metabolite may thus have a function in substrate defence after the switch from the endophytic to the saprotrophic period in the life-cycle of P. medicaginis following the death of infected host tissue.