Penicillium nodositatum induces the formation of nodules on the roots of Alnus. The analysis of RFLPs in the PCR-amplified ITS1-5·8S-ITS2 ribosomal sequence demonstrates that all the nodule-inducing strains isolated belong to a homogeneous taxon, P. nodositatum, distinct from other morphologically close species of Penicillium. A gene tree inferred from the ITS1-5·8S-ITS2 PCR/RFLP data shows that the P. nodositatum sequence clusters with those of Penicillium species belonging to subgenus Biverticillium. RAPDs generated using four 10-mer primers, allowed us to identify four distinct groups of strains in a sample of 28 strains of P. nodositatum collected in 10 different geographic sites. Only one group was found in each sampled site. No link could be established between RAPD-based groups and the geographic origin of the strains. However, our results suggest a possible link between these groups and Alnus species and/or habitats associated with each of the four French Alnus species.

Fusarium isolates were extracted from stems and rhizosphere soils of 79 populations of four Gossypium species native to two regions of inland Australia in 2001. Six Fusarium species were isolated from 31 (3%) of the 919 stem samples. F. semitectum was predominant, occurring mostly in G. bickii populations and accounting for 81% of the isolates recovered. Twenty Fusarium species were isolated from 68 (87%) of the 78 composite soil samples, with F. solani (71%), F. compactum (8%), F. oxysporum (5%), F. graminearum (3%), and F. crookwellense (3%) being the most prevalent. Significant differences in the relative densities indicated that F. solani was more common in the soil from G. sturtianum populations than from G. bickii populations, and that F. crookwellense was more common in South Australian soils than in Queensland and Northern Territory soils. Fusarium oxysporum had a relatively greater relative density in the rhizosphere soils of G. australe plants than in that of sympatrically growing G. sturtianum plants. Fifteen (17%) of the 89 F. oxysporum isolates produced typical Fusarium wilt symptoms on cultivated cotton (G. hirsutum cv. ‘Siokra 1–4’), therefore they were classified as wild Fov. Soil samples collected from G. sturtianum populations in the Arkaroola-Leigh Creek region in South Australia had the highest incidence of wild Fov (24%). Two wild Fov isolates were similar in virulence to a cotton field Fov isolate in the glasshouse experiments, indicating that they could incite Fusarium wilt disease in cotton fields. The presence of wild Fov in native Gossypium populations suggests that the Fov occurring in Australian cotton fields may be of indigenous origins.

The efficacies of three nematophagous fungi, Paecilomyces lilacinus, Plectosphaerella cucumerina and Pochonia chlamydosporia, for controlling potato cyst nematodes (PCN) as part of an Integrated Pest Management (IPM) regime were studied. The compatibility of the nematophagous fungi with commonly used chemical pesticides and their ability to compete with the soil fungi Rhizoctonia solani, Chaetomium globosum, Fusarium oxysporum, Penicillium bilaii and Trichoderma harzianum were tested in vitro. Paecilomyces lilacinus was the most successful competitor when the ability to grow and inhibit growth of an opposing colony at both 10 and 20 °C was considered. P. lilacinus also showed potential for control of the soil-borne fungal pathogen R. solani, releasing a diffusable substance in vitro which inhibited its growth and caused morphological abnormalities in its hyphae. Pochonia chlamydosporia was least susceptible to growth inhibition by other fungi at 20° in vitro, but the isolate tested did not grow at 10°. Plectosphaerella cucumerina was a poor saprophytic competitor. Radial growth of Paecilomyces lilacinus and Plectosphaerella cucumerina was slowed, but not prevented, when grown on potato dextrose agar incorporating the fungicides fenpiclonil and tolclofos-methyl, and was not inhibited by the addition of pencycuron or the nematicide oxamyl. Radial growth of Pochonia chlamydosporia was partially inhibited by all the chemical pesticides tested. The efficacy of Paecilomyces lilacinus as a control agent for R. solani was further investigated in situ. Treatment with P. lilacinus significantly reduced the symptoms of Rhizoctonia disease on potato stems in a pot trial. The effectiveness of P. lilacinus and P. cucumerina against PCN was also tested in situ. Three application methods were compared; incorporating the fungi into alginate pellets, Terra-Green® inoculated with the fungi and applying conidia directly to the tubers. Both formulations containing P. lilacinus and formulation mixtures alone, particularly alginate pellets, significantly reduced multiplication of PCN in soil. We conclude that P. lilacinus showed the greatest potential for use in combination with selected fungicides and nematicides as part of an IPM programme for the control of PCN, but further work is required to confirm whether it is effective against PCN in soil.

The taxonomy of lichenized fungi (lichens) is relatively well-known compared to that of other fungi because lichens are long-lived and possess a thallus that requires exposure to light. Therefore they are easily observed on their natural substratum and sampling can be carried out much more thoroughly than in other fungal groups. Comprehensive catalogues of lichen taxa known worldwide are available and currently about 13500 species are accepted.

An analysis of selected, recently revised groups suggests that there has been a mean increase in species number of about 25% since 1931. One third of the species recognized before 1931 were withdrawn, and almost half of the currently recognized species have been described after 1931. Some 3000 names seem to be ‘orphaned’, not included in currently accepted genera and not appearing in recent species counts. New species are being described at an increasing rate, so that large numbers of ‘missing lichens’ seem to be present. However, there is no clear indication how numerous they are, only an estimation that they amount to some 25% of all species.

A geographical analysis of checklists suggests that most missing species will be found in the tropics and the southern hemisphere. A taxonomic analysis of recently published species suggests that there is no particular group yielding more novelties than others. Our own fieldwork impressions suggest that tropical primary forests are the most productive in revealing undescribed species. These are found in particular on leaves and on bark high up on the tree trunks. In well-studied areas like western Europe the new discoveries tend to be cryptic species or species rarely producing ascocarps.

In conclusion, the ‘missing’ lichens are estimated at about 4000, and are to be found everywhere and in all taxonomic groups, but predominantly in primary tropical forests. This would result in a total of about 18000 lichen species.

Volatile organic compounds (VOCs) emitted by two wood-rotting basidiomycete fungi, Serpula lacrymans (dry rot fungus) and Coniophora puteana (cellar fungus), and the timber of Pinus sylvestris (Scots pine), were identified. Several volatile collection techniques were employed including dichloromethane solvent extraction, solid-phase microextraction (SPME) and thermal desorption of VOCs entrained on Tenax GR. In addition, a new method of solid sample injection (SSI) is described which utilises a low injector temperature and an all-glass deactivated injector liner designed to minimise both the formation of pyrolysis products and analyte degradation. All the volatile compounds collected were analysed using electron impact capillary gas chromatography-mass spectrometry (GC-MS) on HP-5, HP-Innowax and β-cyclodextrin columns. SSI and Tenax thermal desorption were found to be the most effective extraction methods. A total of 19 VOCs were observed from S. lacrymans grown on glass slides and pine, 15 from C. puteana grown on glass slides and 12 from P. sylvestris timber. S. lacrymans was found to emit, in low abundance, six unique VOCs, of which 2-methylbutanal was the greatest. The major volatile compound emitted by S. lacrymans was 1-octen-3-ol, which was also found in lower abundance from C. puteana. Six VOCs, including diethylene glycol and 4-methyl methylbenzoate, were found to be unique to C. puteana, all in medium abundance. From P. sylvestris, the major volatiles identified were S-α-pinene and 3-carene.

We have designed primers for identification of the economically important plant pathogenic Rhizoctonia solani, for AG 2 and for each subgroup and an ecological type of AG 2. These specific primers have been designed based on specific sequences of the ITS regions in the R. solani species complex. The PCR procedure involves amplification of the 5.8S ribosomal DNA and part of the ITS regions, using the designed primers in combination with the general fungal primers ITS1F and ITS4B. Two of the primers amplify under optimal PCR conditions R. solani AG 1, AG 2, AG 3, AG 4, AG 5 and binucleate Rhizoctonia (BNR), and six more primers amplify specifically R. solani AG 2, the subgroups, AG 2-1, AG 2-2 and AG 2-3, and the ecological type AG 2-t. In this study DNAs from R. solani AG 2 and AG 4 growing on infected radish were amplified similarly to DNAs from axenic cultures. PCR detection has time saving advantages over traditional isolation methods for detection of Rhizoctonia on infected plant tissue and provides a powerful tool of identification.

Analysis of Phytophthora cactorum using Random Amplified Microsatellites (RAMS) revealed considerable variation among isolates, most of which correlated with the original host plants. The lack of variation among isolates originating from strawberry suggests that crown rot of strawberry is caused by a single clone within the geographical area studied. It was also shown that P. cactorum isolates form a unique group different from other Phytophthora spp. from Group I.

P. cactorum isolates from necrotic stem lesions on Betula pendula seedlings or Fragaria × ananassa plants suffering from crown rot were highly pathogenic to their original host plants. P. cactorum isolates from strawberry inoculated via wounds also caused necrotic lesions on B. pendula. On the other hand isolates from B. pendula did not cause disease symptoms on strawberry plants. On Alnus glutinosa the percentage of successful inoculations with a birch isolate was 40.

Suitability and reproducibility of ISSR–PCR to find strain-specific and taxon specific markers for strains of the tree-root endophytes Phialocephala fortinii and ‘Type 1’, a non-sporulating dematiaceous mycelium, were examined. The results were compared with data previously generated by isozyme analyses. P. fortinii and ‘Type 1’ are two DSE taxa and are abundant colonisers of coniferous forest-tree roots in the North Temperate zone. ‘Type 1’ was never observed to sporulate in pure culture but is well defined by its cultural characteristics. DNA of 14 strains per taxon was amplified with three short, 17–18-nucleotide-long, tandemly-repeated primers (CCA, CGA, ACA) with two (CCA) or three degenerated bases at their 5′-ends. The resulting DNA products were separated by agarose gel electrophoresis. The bands were scored for absence/presence, respectively, and the binary matrix subjected to multiple correspondence analysis (MCA). ISSR–PCR was found to be highly reproducible since amplification of DNA from several single-hyphal-tip cultures of the same strain resulted in identical banding patterns. Eighty-five (92.4%) of the 92 DNA fragments were polymorphic. The fragments ranged from 320 to 4100 bp. ISSR–PCR was found to be a powerful tool to find strain-specific and taxon-specific markers. Each strain showed a unique banding pattern and diagnostic bands for the two taxa could be identified. ISSR–PCR data correlated neither with the geographical nor the host origin of the strains. The strains grouped into similar clusters independently of whether MCA was performed with ISSR–PCR or isozyme data. However, ISSR–PCR allowed the differentiation of strains with the same allozyme phenotype.

This study shows for the first time that Tolypocladium species produce efrapeptins, a group of toxic peptides, in vivo but the quantities are too small to account for insect death, suggesting that these insecticidal compounds work in concert with other pathogenicity determinants. There is inter- and intraspecific variation in efrapeptin production in vitro by Tolypocladium species. T. parasiticum produced only efrapeptin E, in small quantities. Efrapeptins were detectable 48 h after inoculation and increased with biomass. The relative amounts of individual efrapeptins (C, D, E, F, G) produced by T. niveum in vitro were D > E > F > C > G but in vivo they were D > F > C > E > G. Efrapeptins were toxic to a wide range of insects when injected into the haemocoel. Mortality was dose-related. Efrapeptins also exhibited limited antifungal and antibacterial activity. Micrococcus luteus was considered an excellent indicator of efrapeptin presence in culture filtrate extracts because of its extreme sensitivity to these compounds.

The relationship of Venturia nashicola, the scab fungus of Japanese and Chinese pears, to the European pear scab fungus, V. pirina, was reexamined. Ascospores of V. pirina were longer and wider than those of V. nashicola and the conidia of V. nashicola significantly shorter than those of V. pirina. Results from mating experiments in culture clearly showed sexual isolation between the species. V. nashicola was only pathogenic on Japanese and Chinese pears and V. pirina was only pathogenic on European pear. It is concluded that V. nashicola is distinct from V. pirina.

This study reports novel terricolous mitosporic fungal morphs nested in the genus Tuber according to molecular phylogenetic analysis. Fungal DNA was amplified directly from field-collected anamorph samples. Nuclear ribosomal DNA (nrDNA) sequences including the ITS regions and the D1 and D2 domains of the LSU identify the anamorphs as mitosporic Tuber borchii and Tuber oligospermum. The link of the novel anamorphs to the genus Tuber is confirmed by the comparative analysis of five collections from four sampling sites.

Ectomycorrhizas with characteristic features of Tuber borchii ectomycorrhizas were found in the soil volume collected with one of the mitosporic T. borchii collections. A nrDNA sequence amplified from these ectomycorrhizae is identical with the corresponding anamorph sequence.

The possible role of the newly discovered anamorphs in the Tuber life-cycle and the potential significance of anamorphs for the propagation of ectomycorrhizal fungi are discussed.

The phylogeny of the family Micareaceae and the genus Micarea was studied using mitochondrial small subunit ribosomal DNA sequences. Phylogenetic reconstructions were performed using Bayesian MCMC tree sampling and a maximum likelihood approach. The Micareaceae in its current sense is highly heterogeneous, and Helocarpon, Psilolechia, and Scutula, all thought to be close relatives of Micarea, are shown to be only distantly related. The genus Micarea is paraphyletic unless the entire Pilocarpaceae and Ectolechiaceae are included, as also indicated by an expected likelihood weights test. It is suggested that the Micareaceae is reduced to synonymy with the Pilocarpaceae, which also includes the Ectolechiaceae, and that Micarea may have to be divided into a series of smaller genera in the future. Micarea species with a ‘non-micareoid’ photobiont group with Psora and the Ramalinaceae, whereas Micarea intrusa appears to belong in Scoliciosporum. Three species fall inside the paraphyletic Micarea: Szczawinskia tsugae, Catillaria contristans, and Fellhaneropsis vezdae. Tropical foliicolous taxa are nested within groups of mainly temperate and arctic-alpine distribution. A ‘micareoid’ photobiont appears to be plesiomorphic in the Pilocarpaceae but has been lost a few times.

Marker genes were introduced in the biocontrol strain Clonostachys rosea IK726 (IBT 9371) as a tool for monitoring the strain in ecological studies. The β-glucuronidase (GUS) reporter gene and a gene encoding the green fluorescent protein (GFP) were, in separate experiments, integrated into the genome of IK726 using the Hygromycin B (HygB) resistance gene as selective marker. In order to select GUS and GFP transformants that resembled the wildtype strain, growth rate, production of enzymes and of metabolites of four GUS positive and six GFP positive transformants were tested in vitro. In addition, the biocontrol efficacy against disease caused by seed-borne Fusarium culmorum was evaluated on barley grown in sand. Compared to the wildtype, two selected GUS and GFP transformants, IK726c5 and IK726d11, did not vary in physiological properties. Both maintained the ability to colonize barley roots, and to reduce efficiently the severity of F. culmorum without affecting plant emergence. Quantification of GUS activity of IK726c5 in peat and vermiculite and on seeds was carried out. The GFP transformant, IK726d11, was visualized by epifluorescence and confocal scanning laser microscopy directly in soil, vermiculite, on carrot seed and roots, and on barley leaves. It was shown that C. rosea can thrive in very different niches. Conidia germination, colonization and conidiogenesis were demonstrated in vivo in all four environments. This is the first report on transformation of Clonostachys rosea with marker genes.

Inter simple or short sequence repeat (ISSR) fingerprinting was used to differentiate Botryosphaeria species and related anamorphic fungi. Fifty-one isolates representing 10 different Botryosphaeria species and related taxa, including two RAPD marker groups and one special form of Sphaeropsis sapinea were investigated using five ISSR primers. A total of 171 fingerprint fragments were generated from these isolates, and sizes of fragments were mostly between 200 bp and 1650 bp. Both cluster and principal coordinate analyses were used to examine separately the fingerprint data from the two previously proposed sections Hyala and Brunnea of Botryosphaeria. The results of each analysis indicated that B. dothidea and B. ribis are distinct; B. ribis and B. parva are closely related, but separable; B. mamane is distinct from other Botryosphaeria species with similar morphology or ITS sequences; the A and B RAPD marker groups of S. sapinea and S. sapinea f. sp. cupressi are probably three distinct species; B. obtusa and S. sapinea B group are closely related but distinct; and the name B. stevensii may have been applied to more than one species. This study has shown that ISSR fingerprinting can be a powerful tool for differentiating closely related filamentous fungi with very similar morphologies or ITS sequences, dissecting species complexes, and justifying newly described species.

A field study was conducted to determine the size and spatial distribution of mycelial individuals of Pisolithus tinctorius at a site in NSW, Australia. Following collection and mapping of carpophores and isolation of the fungi into axenic culture, genomic DNA was extracted and combined data from RAPD and microsatellite analyses used to identify and map mycelial individuals. Thirty-three genetically distinct individuals were recognized at the field site and, while one large individual (at least 30 m diam.) was identified, most individuals appeared relatively small (<3 m diam.). The size and distribution of individuals is discussed in relation to growth of P. tinctorius mycelia through soil.

A considerable number of species of lichen-forming fungi have wide geographical distributions, but studies of their genetic variability are minimal. ITS rDNA sequences of 32 populations of Parmelia saxatilis from five continents revealed two monophyletic groups. β-tublin gene sequences from a subset of nine collections supported these conclusions. While the number of collections sequenced is limited, one monophyletic group (the Atlantic Population. AtP) was recognized as occurring in Arctic and Antarctic regions and also included collections from more atlantic sites. Samples from more mesic environments in the Mediterranean region belonged to a second monophyletic group (the Mediterranean Population, MeP). In addition, four subgroups were distinguishable within the Atlantic Population. Norstictic and protocetraric acids are reported from the species for the first time, the norstictic acid only being found in the Atlantic Population. Living thalli from the Atlantic Population were provenance-tested; specimens transported from the UK to central Spain where the Mediterranean Population occurs showed adverse symptoms after six months. These results demonstrate that there can be substantial large-scale genotypic variability within widespread lichen phenospecies, something which has implications for comparative ecological, physiological, and air pollution sensitivity studies as well as for lichen conservation.

RFLP, DNA fingerprinting and VCG methods were used to characterize fourteen Verticillium dahliae isolates collected from southwestern Ontario, Canada. The isolates were typed as not pathogenic to tomato (NP), race 1 (avirulent on cvs carrying the Ve resistance gene) or race 2 (virulent on Ve cvs). On the basis of RFLPs, RAPDs, and DNA fingerprints detected by hybridization to a dispersed, repetitive genomic DNA probe, the isolates were classified into five DNA types. Type I included two NP isolates. Type II included four race 2, and three NP isolates. Types III and V were represented by single race 2 and race 1 isolates, respectively. Type IV included one race 2, and two race 1 isolates. Vegetative compatibility was determined for selected NP, race 1, and race 2 isolates of each race type/DNA type combination. Isolates of the same DNA type were compatible, as were type II and III isolates (VCG 4B), and type IV (VCG 2A) and V isolates (VCG 2B). These data show a level of genetic diversity not previously identified in the V. dahliae tomato pathogen population, and suggest multiple origins for the Ontario race 2 pathotype.

110 isolates of Beauveria (104 B. bassiana, 5 Beauveria spp., 1 B. brongniartii) were obtained from Sunn Pests (Eurygaster and Aelia species), litter, and other insect samples at overwintering sites in seven countries in the Middle East and West Asia. DNA was extracted from these isolates, and four techniques were used to characterize and to investigate genetic diversity at the molecular level: ITS-RFLP, ITS sequencing, ISSR-PCR, and AFLP. The ITS-RFLP and ITS sequences did not detect significant genetic variation among the isolates. However, both ISSR-PCR and AFLP analyses gave indications of intraspecific groupings correlated with geographical origin and relative genetic diversity among some isolates, but no obvious association with Sunn Pest hosts. There was no obvious genotypic grouping of B. bassiana isolates from E. integriceps, perhaps suggesting the overwintering populations were infected by generalist native isolates rather than by host-specific ones that might be more suitable for biocontrol purposes.

Metal-contaminated soils often contain a spatially heterogeneous distribution of metal concentrations, and the ability of fungi to colonize such metal-contaminated domains will be influenced by the nutritional resources available. An experimental system based upon tessellated agar tiles was used to study the influence of nutrients upon the ability of soil fungi Trichoderma virens and Clonostachys rosea to colonize spatially discrete toxic metal (copper and cadmium) containing domains. The growth parameters recorded demonstrated a decrease in apparent metal toxicity with increasing concentration of available carbon source. It was shown that maximum hyphal extension rates and the efficacy of carbon substrate utilization of both species decreased with increasing concentration of toxic metals. It was also observed that in the gap between metal-free and metal-containing tiles, the presence of toxic metals led to negative chemotropic reactions and cessation of growth, swelling and lysis of some hyphal tips. Penetration of the hyphae into the metal-containing domain was often followed by the formation of very dense mycelia or mycelial ‘bushes’ representing an associative (constraining, exploitative or ‘phalanx’) growth strategy of the mycelial system. After the fungi entered the toxic metal-containing domains, they often produced long sparsely-branched or branchless explorative hyphae representing a dissociative (expansive, explorative or ‘guerrilla’) growth strategy. Our data therefore demonstrate that fungi efficiently use both ‘phalanx’ and ‘guerrilla’ states of the mycelial system as well as shifts in these growth strategies as a response to toxic metal stress combined with nutritionally-poor conditions.

A particular group of saprotrophic and ectomycorrhizal fungi produced reproductive structures successively on the forest floor after treatment with some nitrogenous compounds (N compounds) such as urea and aqua ammonia. These fungi were also observed after the decomposition of dead bodies or faeces of animals. Their growth in pure culture was examined in liquid media containing ten different N sources (four inorganic and six organic). The saprotrophic Amblyosporium botrytis, Tephrocybe tesquorum, Coprinus echinosporus and C. phlyctidosporus grew well on ammonium, some amino acids (asparagine, glycine), urea and bovine serum albumin, although A. botrytis did not grow on bovine serum albumin. Coprinus phlyctidosporus grew on nitrate as well as on the above N compounds. Peziza urinophila and Pseudombrophila deerata did not grow on any of the N compounds examined. The ectomycorrhizal Laccaria bicolor, Hebeloma vinosophyllum and Hebeloma sp. showed different patterns of nitrogen utilization: L. bicolor grew well on ammonium, nitrate and urea; Hebeloma spp. grew well on ammonium, nitrate, amino acids, urea and bovine serum albumin. None of the fungi tested grew on ethylenediamine or putrescine. The patterns of growth in relation to the nature of nitrogen supply were discussed in relation to the conditions of soil where these fungi occur.