Genetic variation within and between eighteen isolates of Alternaria brassicicola, five isolates of A. alternata, and a single isolate of Rhynchosporium secalis was compared using Amplified Fragment Length Polymorphisms (AFLPs). The AFLPs consistently distinguished between the three species. AFLPs thus provide a reliable tool for identifying A. brassicicola. The analysis of eighteen isolates of A. brassicicola from different sites also indicated at least moderate levels of genetic diversity within A. brassicicola along the New South Wales coast. Of 43–66 markers scored per primer combination, 16·7–27·9% were polymorphic. On the basis of an unweighted paired group method of arithmetic averages analysis using data from four primer combinations, most isolates were identified as separate genotypes. However, multiple isolates from particular locations tended to cluster together, implying the potential for population structure, and there was a significant positive correlation between Nei's measure of genetic distance and physical distance (r = 0·5486, P<0·001). Despite the absence of an identified sexual stage, A. brassicicola would appear to have a means for generating and maintaining significant variation.

Variation within the internal transcribed spacer (ITS) and the intergenic spacer (IGS) of the ribosomal RNA gene of isolates representing seven European species of Armillaria was examined by PCR, coupled with RFLP analysis and partial sequencing of the ITS region.

The amplified-ITS region was about 840 bp long and uniform in length among the European isolates, while the amplified-IGS region showed four different lengths which corresponded to A. mellea, A. tabescens, A. ectypa and a group including the four other species. A. borealis, A. ostoyae, A. cepistipes and A. gallica.

Restriction digestions of the ITS and IGS regions by Alu I and Rsa I, respectively, gave rise to the same four polymorphic groups. However, A. borealis and A. ostoyae were easily separated from A. cepistipes and A. gallica by digestion of the two rDNA spacers with Hinf I and Taq I. Nde II digests of the amplified ITS could distinguish A. borealis from A. ostoyae and each of the seven species were separated by Alu I digestion of the IGS region. A North American A. mellea isolate, partly examined in this work, was found to be different from the European A. mellea isolates, while there was a close similarity between the European A. ostoyae and an isolate of the same species isolated from the U.S.A.

Cluster analysis based on the presence or absence of comigrating restriction fragments indicated more than 80% similarity between A. borealis and A. ostoyae and between A. cepistipes and A. gallica, while A. mellea, A. tabescens and A. ectypa were found in separate clusters exhibiting, respectively, about 40, 38 and 32% average similarity with the other species.

Although little intraspecific variation was observed in many species, A. gallica and A. cepistipes were found to be heterogeneous. In view of recent results suggesting several groups in A. borealis and A. cepistipes, the Alu I restriction patterns obtained in this work would identify the former species as type B, characterized by an Alu I pattern different from that of A. ostoyae, and the latter species as composed of one isolate (C4) of type B and three isolates (C1, C2 and C3) of type A.

Nucleotide sequences of the rDNA internally transcribed spacer 1 (ITS1) of one isolate of each species showed that A. mellea and A. ectypa differed from the other species by several insertions and point mutations giving rise to a level of similarity ranging from 66 to 79%. This DNA region was highly conserved within the other species which revealed a similarity of 97 to 100%.

These results demonstrate that analysis of rDNA spacers provides appropriate data to circumscribe taxonomic entities within the Armillaria complex and that the phylogenic relationships among species deduced from the present study are consistent with previous analyses based on pairing tests as well as morphological and physiological characters.

In experimental microcosms, three Glomus spp. were subjected to heating to over 200°C at the soil surface and 70° at 5 cm to determine the effect of fire on survival of arbuscular mycorrhizal fungi. Heating reduced the quantity of propagules surviving at the soil surface and the effect declined with depth. While all propagules are likely to be affected by heat, we argue that the hyphal network is most severely disturbed and probably responsible for declines in density of fungi observed in the field following fire.

In the present study we investigated the abundance and spatial distribution of dark septate root endophytes (DSE) in a 3×3 m plot in a spruce stand (Picea abies). A total of 144 DSE isolates were obtained by means of a hierarchical sampling design. Most roots were colonised, as DSE were isolated from 81.7% of root segments. ISSR–PCR fingerprinting was used to identify 21 unique ISSR types. Dominant types were isolated from adjacent points that covered an area of up to 6.8 m2 of the study plot, and ISSR types were intermingled extensively. Frequency of isolation of the different ISSR types was uneven with two dominant types that accounted for 38% and 28% of all DSE isolates, respectively. Seven DSE strains representing six different ISSR types were identified as Phialocephala fortinii based on the morphology of fertile conidiophores and/or ITS 1 and 2 sequence comparisons.

PCR-based methods to detect Verticillium chlamydosporium on infected plant roots were developed. Arbitrary ERIC primers and those based on rRNA genes, to identify fungi grown in pure culture, were unsuitable for DNA extracted from nematode-infested roots, because of interference by plant and nematode DNA. A novel method utilizing specific primers designed from an amplified and cloned fragment of the V. chlamydosporium β-tubulin gene was developed. Although it could not discriminate between different isolates of V. chlamydosporium, one primer set could identify the fungus on tomato roots infested with root-knot nematodes. The V. chlamydosporium β-tubulin sequence data showed close homology to sequences from plant endophytic Acremonium and Epichloë species and the saprotrophic Trichoderma viride.

Verticillium fungicola, a severe mycopathogen of the cultivated mushroom Agaricus bisporus, was successfully transformed using both PEG-mediated and Agrobacterium-mediated techniques. PEG-mediated co-transformation was successful with hygromycin B resistance (hph), uidA (β-glucuronidase GUS), and green fluorescent protein (GFP) genes. Agrobacterium-mediated transformation was successful with the hph gene. Transformation frequencies of up to 102 transformants per μg DNA and 4068 transformants per 105 conidia were obtained for PEG-mediated and Agrobacterium-mediated transformation respectively. Expression of integrated genes in co-transformants was stable after 18 months of successive sub-culturing on non-selective medium, and following storage at −80 °C in glycerol. Molecular analysis of PEG-mediated transformants showed integration of the transforming genes into the target genome. Molecular analysis of Agrobacterium-mediated transformants showed integration of transforming DNA as single copies within the target genome. Co-transformants exhibited symptoms of disease in inoculation experiments and were at least as virulent as the wild-type fungus. GFP and GUS expression were observed in-vivo with the GFP-tagged strain showing great potential as a tool in epidemiological and host-pathogen interaction studies. The development of transformation systems for V. fungicola will allow in-depth molecular studies of the interaction of this organism with A. bisporus.

About 15000 species of ascomycete fungi lack a known sexual state. For fungi with asexual states in the anamorph genera Embellisia, Ulocladium, and Alternaria, six species have known sexual states but more than 50 species do not. In sexual filamentous ascomycetes, opposite mating type information at the MAT1 locus regulates mating and the opposite mating type genes each have a clonal, non-recombining phylogenetic history. We used PCR to amplify and sequence fragments of the opposite mating type genes from three supposedly asexual species, A. brassicae, A. brassicicola and A. tenuissima. Each haploid fungal isolate had just one mating type, but both mating types were present in all the three species. We sequenced the ribosomal ITS regions for isolates of opposite mating type, for the three asexual species and four known related sexual species. In a phylogenetic analysis including other ITS sequences from GenBank®, the three asexual species were not closely related to any of the known sexual species. Isolates of opposite mating type but the same species had identical ITS sequences. During any period of asexual evolutionary history, lineages of each mating type would have had a separate evolutionary history and any ITS substitutions shared between isolates of opposite mating type would have had to accumulate by convergence. Allowing for varying substitution rates and assuming a Poisson distribution of substitutions, the probability that isolates of opposite mating type shared an ITS substitution through convergence was low. This suggests that isolates of opposite mating type of A. brassicae, A. brassicicola and A. tenuissima were exchanging substitutions through sexual or parasexual reproduction while the ITS was evolving. If sexuality was lost, it was lost after the period of evolutionary history represented by the shared substitutions.

Genetic variation within Gremmeniella abietina var. abietina was studied using the Random Amplified Microsatellite-technique (RAMS). Ninety-three isolates were investigated, originating mostly from Finland, but also from Canada, U.S.A., Japan, Norway, Italy, Iceland and Sweden. Four banding pattern types were observed corresponding to the present division of this species in Asian, North American and two types of European races. Furthermore, an additional banding pattern was also observed. Intraracial variation was observed within all races, the North American one being the most polymorphic. Isolates of the large tree type (LTT) Gremmeniella from North America, Italy and Iceland contained RAMS alleles not observed in Finland, Sweden or Norway. Therefore, the isolates of LTT Gremmeniella should not be transported even within the area of its natural occurrence.

Two types of genetic markers, vegetative compatibility and RAPD, were evaluated for their ability to assess genetic variation among 38 isolates of the entomopathogenic fungus, Paecilomyces fumosoroseus. Both markers revealed significant polymorphisms. Nitrate non-utilizing mutants were generated from chlorate-containing media and used as forcing markers to observe heterokaryon formation and determine vegetative compatibility. Nineteen separate vegetative compatibility groups (VCGs) were identified, only seven having at least two members and 12 represented by single isolates. No correlation was observed between VCG and either insect host or geographic origin. Nine RAPD primers produced 365 unique fragments and could distinguish three different phenetic groups. RAPD profiles conclusively showed that strains within some VCGs were genetically unrelated, indicating that these strains were not clonal in origin. The large number of VCGs suggests that formation of heterokaryons and possible genetic exchange are uncommon. DNA fingerprinting with RAPD-PCR provides high resolution of genotype distribution and could be useful in tracking the fate of biocontrol agents released into the environment.

Twenty-four isolates of Aspergillus sojae, A. parasiticus, A. oryzae and A. flavus, including a number that have the capacity to produce aflatoxin, have been compared using amplified fragment length polymorphisms (AFLPs). Based on analysis of 12 different primer combinations, 500 potentially polymorphic fragments have been identified. Analysis of the AFLP data consistently and clearly separates the A. sojae/A. parasiticus isolates from the A. oryzae/A. flavus isolates. Furthermore, there are markers that can be used to distinguish the A. sojae isolates from those of A. parasiticus, which form the basis for species-specific markers. However, whilst there were many polymorphisms between isolates within the A. oryzae/A. flavus subgroup, no markers could be identified that distinguish between the two species. Sequencing of the ribosomal DNA ITS (internal transcribed spacers) from selected isolates also separated the A. sojae/A. parasiticus subgroup from the A. oryzae/A. flavus subgroup, but was unable to distinguish between the A. sojae and A. parasiticus isolates. Some ITS variation was found between isolates within the A. oryzae/A. flavus subgroup, but did not correlate with the species classification, indicating that it is difficult to use molecular data to separate the two species. In addition, sequencing of ribosomal ITS regions and AFLP analysis suggested that some species annotations in public culture collections may be inaccurate.

The effect of hydroxyl radical scavengers dimethyl sulphoxide, p-nitrosodimethylaniline, ethanol, benzoate, salicylate and thiourea was studied on sclerotial differentiation and growth of Sclerotinia sclerotiorum and Rhizoctonia solani. There is a correlation between both scavenger concentration and scavenger-hydroxyl radical reaction rate with delay and inhibition of differentiation at growth-noninhibiting scavenger concentrations. Growth-inhibiting scavenger concentrations further increased delay and inhibition of differentiation, and eventually stopped fungal growth, acting as antifungal antioxidant alternatives to traditional fungicides. p-nitrosodimethylaniline (with the highest hydroxyl radical reaction rate) was the most effective inhibitor of sclerotial differentiation and growth. The results strongly support the theory that oxygen free radicals induce differentiation in sclerotium-producing phytopathogenic fungi.

Leaf-cutting ants finely fragment the leaf material that they bring to the nest and place faecal droplets on this substrate before incorporating it in the upper part of the fungus-garden. The faecal droplets contain enzymes of which some have been shown to be of fungal origin. Here we explicitly address the enzymatic activity of faecal droplets in the leaf-cutting ants Acromyrmex echinatior and Atta colombica. We used isoelectric focusing and specific staining to show that faecal droplets of both species contain carboxymethylcellulases, laccases, proteases, and pectinases (both pectin esterases and pectin lysases) and we demonstrate that these enzymes originate from the symbiotic fungus and not from the ants themselves. The level of activity of fungal pectin lyase in faecal droplets indicates that fungal enzymes may be protected and possibly concentrated during their passage through the ant gut. This would imply that enzymes that are transferred by ants from mature parts of the fungus gardens may play an important role for the colonization of new substrate by new mycelium of the same fungal clone.

Rust fungi cause economically important diseases of cereals, and their ability to rapidly evolve new virulent races has hindered attempts to control them by genetic resistance. PCR-based molecular tools may assist in understanding the genetic structure of pathogen populations. The high multiplex DNA fingerprinting techniques, amplified fragment length polymorphisms (AFLP), selectively amplified microsatellites (SAM) and sequence-specific amplification polymorphisms (S-SAP) were assessed for their potential in investigations of the genetic relationships among isolates of the wheat rust pathogens, Puccinia graminis f. sp. tritici (Pgt), Puccinia triticina (Pt), and P. striiformis f. sp. tritici (Pst), the oat stem rust pathogen P. graminis f. sp. avenae (Pga), and a putative new P. striiformis special form tentatively designated Barley grass yellow rust (Bgyr). Marker information content, as indicated by the number of species-specific fragments, polymorphic fragments among pathotypes, percentage of polymorphic loci, and the marker index, was highest for the SAM assay, followed by the AFLP and S-SAP assays. UPGMA analysis revealed that all marker types efficiently discriminated the five different taxa and Mantel tests revealed significant correlations between the marker types. Within pathogen groups, the marker types differed in the amount of variation detected among isolates; however, the major differences were consistent and polymorphism was generally low. This was reflected by the AMOVA analysis that significantly partitioned 90% of the genetic variation between taxa. Of the three marker types, SAMS were the most informative, and have the potential for the development of locus-specific microsatellites.

We investigated the influence of changes in external phosphorus (P) concentration on the proportion of phosphatase-active structures of the arbuscular mycorrhizal fungus Gigaspora margarita associated with Allium cepa. The P treatment was started when mycorrhizal colonisation had been established, and plant systems were harvested three times after the start of the P treatment. Higher shoot dry weights and P contents were observed in the high-P treated plants at the last harvest. We did not find any change in the proportion of phosphatase-active extraradical mycelium following P treatment. However, the proportion of alkaline phosphatase-active mycelium was positively correlated for extraradical and intraradical mycelium. Also, the proportion of alkaline phosphatase-active arbuscules seemed to increase with the shoot fresh weight, whereas the proportion of acid phosphatase-active arbuscules decreased with higher shoot P concentration and dry weight. We have shown experimentally that the intraradical mycelium of G. margarita, but not the extraradical mycelium, responds metabolically to plant P concentration, and possibly also to external P availability.

Scleroderris canker of conifer is caused by Gremmeniella abietina var. abietina, which comprises several taxa, including races, varieties, and biotypes. The European race of G. abietina var. abietina was introduced into North America early in the century, most likely on asymptomatic infected pine seedlings, and is currently widespread in eastern North America. In order to detect latent infections and differentiate the North American and European races of the fungus, we developed oligonucleotide primers to amplify by PCR portions of the ITS of the ribosomal DNA of G. abietina var. abietina. The 417 bp amplified DNA fragment comprises two Msp I restriction sites in the NA race but only one in the EU race. DNA extractions directly from infected asymptomatic needles, or from single fruiting bodies, followed by PCR amplification and Msp I digestion allowed the detection and race identification of both races of G. abietina var. abietina from seedlings and branches of Pinus resinosa and P. banksiana. A nested PCR assay was sensitive enough to detect the equivalent of a single infected seedling in a bulk sample of 1000 healthy seedlings. Validation tests were conducted by comparing PCR and isolation assays with 104 fascicles. All samples for which the fungus was isolated yielded a positive PCR assay and there was no false negative, i.e. samples for which the fungus was isolated but not detected by PCR. Among the samples from which the fungus was not isolated, most yielded a negative PCR assay (71%), but a proportion (29%) yielded positive PCR assays. In several of those cases, aggressive contaminants had apparently overgrown the pathogen. The method described here can lead to the detection and race identification of the NA and EU races of G. abietina var. abietina directly from infected tissues without the need to culture the fungus and should find applications in nursery inspection and quarantine.

The mountain pine beetle, Dendroctonus ponderosae, and its fungal associates are devastating the lodgepole pine forests in British Columbia, Canada. During our fungal survey, an unknown Leptographium species has been consistently isolated from both D. ponderosae and infested lodgepole pine (Pinus contorta var. latifolia). This Leptographium species has similar morphology with the Leptographium anamorph of Ophiostoma clavigerum whose association with the D. ponderosae is well known. However, thorough morphological comparisons showed that this fungus is distinct from all the other Leptographium species described in the literature, and especially from O. clavigerum. Comparison of DNA sequences of multiple loci and the profiles by the PCR-RFLP marker also confirmed that this Leptographium species represents an undescribed taxon. Based on its distinct morphological, physiological characteristics and phylogenetic position, we describe it as L. longiclavatum sp. nov.

Needles of Pinus strobus (white pine) were cleared and stained to survey the occurrence and location of Lophodermium sp., a fungal endophyte. Cytoplasmically dense endophytic hyphae with a pronounced lobed morphology and containing lipid bodies were localized intercellularly between the epidermis and hypodermis. These fungal infections did not appear quiescent, but rather exhibited signs of continual slow growth. A few associated host cells exhibited a hypersensitive response. Material embedded in resin and examined by light microscopy and transmission electron microscopy confirmed the location of hyphae between epidermal and hypodermal cells, and the presence of lipid bodies within the hyphae. In senescing needles, aggressive colonization of needle tissues occurred. Thus, for Lophodermium in white pine, endophytic infection is active rather than quiescent, and displays an alternate hyphal strategy to that seen in the reproductive phase.

The ectomycorrhizal fungus Hebeloma velutipes consists of two biological species (BSP 16 and 17). Within BSP 17 a dikaryon was found with two divergent types of the ribosomal Internal Transcribed Spacer (ITS1 and 2). The two ITS types segregated in monokaryotic progeny of that dikaryon, showing that these different ITS types represent different alleles at homologous rDNA loci in the two nuclei. RFLP analysis of a number of strains of BSP 17 showed that the polymorphism is widespread in Europe. There was no deficiency of the heterokaryotic type, demonstrating that ITS divergence in this species is not correlated with reduced intercompatibility. A strain from North America, not assigned to a biological species, showed the same polymorphism. Cladistic analysis of the two ITS sequences showed that they were not sister groups. One of the ITS types formed a monophyletic group together with the ITS type of BSP 16, the other type formed a clade with the ITS type of H. incarnatulum (BSP 18). BSP 16 and 17 showed partial intercompatibility. However, several lines of evidence suggest that the polymorphism of BSP 17 is not the result of frequent and continuing hybridisation with BSP 16. Instead, we give arguments for the hypothesis that the polymorphism evolved in allopatry and that the two types have come together relatively recently. The results of the polymorphism indicate a potential problem for molecular identification of fungal species based on ITS fingerprinting. The results also show that no generalisations are possible about the relation of speciation (the formation of BSP) and nuclear ITS divergence.

Almost 900 species were encountered during a seven year survey of fungal fruit bodies in 13 forests and grasslands plots measuring about 1 ha. The data were comparable to those from a Swiss forest plot monitored for 21 years. Species richness and abundance were almost linearly correlated on a log/log scale. Abundance and yearly frequency of species were tightly correlated. The geographic range of species may also be related to these two parameters. Rare species scored low on abundance, yearly frequency and geographic range. About half of the species were rare, and occurred in only one out of seven years. The two major functional groups of saprotrophic and mycorrhizal species behaved similarly over the years, as in the Swiss study.

Annual variation was high for species richness alone and for richness combined with abundances. The log transformed species richness of plots correlated well with parameters that take the abundances or yearly frequencies of species into account. If other mushroom assemblages show similar regularities, it may be possible to omit abundance counts in future surveys. The species composition of the plots varied strongly over the years. Many species did not reach their maximum abundance in the richest year. Species showed their maximum abundances in different plots in the same year more often than expected by chance. Thus the presence and abundance of species depended on factor(s) other than the general productivity of a year, and years showed (a) different aspect(s) than productivity alone. Long-term surveys are important for understanding the structure of mushroom assemblages and their biodiversity.