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 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.

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 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.

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.

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.

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.

Sphaeropsis sapinea is an endophyte and latent pathogen of pines, assumedly introduced to the Southern Hemisphere along with its host. There are at least three recognised forms of S. sapinea that differ from each other morphologically and can also be separated based on molecular characteristics. Pinus radiata is a native to California but has been used extensively for afforestation in the Southern Hemisphere. For this study, populations of S. sapinea were collected from exotic P. radiata plantations in South Africa, South and Western Australia, Tasmania and New Zealand and from native P. radiata in California. The genotypic diversity of the populations was assessed and compared using vegetative compatibility tests. SSR markers were used to determine the morphotype of isolates from each vegetative compatibility group. All Californian isolates of S. sapinea were found to be of the ‘B’ morphotype, while all introduced isolates in the Southern Hemisphere were of the ‘A’ morphotype. The genotypic diversities of S. sapinea populations ranged from extremely low in Australia to very high in South Africa with New Zealand having an intermediate genotypic diversity. S. sapinea is an asexual fungus and, therefore, different genotypes in an exotic population represent separate introductions. Our results suggest there have been very few introductions into Australia and multiple introductions into South Africa. In addition, it appears that S. sapinea isolates from P. radiata in the Southern Hemisphere did not originate from native P. radiata, but rather the widely planted exotic P. radiata has acquired this fungal endophyte from other Pinus within the exotic environment.

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.

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.

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.

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.

Metarhizium anisopliae exhibits two different developmental patterns under nutrient-deprived conditions: appressorium formation in early stages and conidiation in late stages of pathogenesis in its insect hosts. In this study we isolated genes enriched during mature conidial production under nutrient-deprived conditions in M. anisopliae by using the method of suppression subtractive hybridization. We sequence-identified seven conidiation-associated genes (cag) in M. anisopliae. One of the genes, cag7, encoded an extracellular subtilisin-like protease, Pr1, that plays a fundamental role in cuticular protein degradation. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed that cag cDNAs are expressed during the development of mature conidia under nutrient-deprived conditions. RT-PCR analysis was also performed for Pr1 during infection of greater wax moth larvae (Galleria mellonella). Results showed up-regulation of Pr1 in the infected insect as the mycelia emerge and produce conidia on the surface of the cadaver. It is well documented that Pr1 is produced during the initial stages of transcuticular penetration by M. anisopliae. Here we show that Pr1 is also up-regulated during the final stages of pathogenesis as the fungus emerges from the dead host and subsequently conidiates on the cadaver.

Mature basidiomes of the hypogeous ectomycorrhizal fungi Rhizopogon vinicolor and R. truncatus were fed to three species of mycophagous mammals in captivity. Spores which passed through the digestive tract of the mammals were isolated from faecal pellets. The metabolic activity and mycorrhizal effectiveness of these spores was assessed. Flouricene-diacetate (a vital stain) was used to assess spore metabolic activity. Mycorrhizal effectiveness was determined by inoculating Douglas-fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa) seedlings with spore slurries. The same assays were performed on spores isolated from uneaten basidiomes. Spores from R. vinicolor were viable after passage through the gut of each mammal species but the digestive process did not substantially enhance or detract from the ability of spores to form ectomycorrhizas relative to spores from uneaten basidiomes. Spores recovered from faeces of Californian red-backed vole (Clethrionomys californicus) and Townsend's chipmunk (Tamias townsendii) had significantly higher metabolic activity than spores from uneaten basidiomes and those recovered from northern flying squirrel (Glaucomys sabrinus) faeces. Spores from eaten and uneaten basidiomes of R. truncatus failed to show any signs of metabolic activity and largely failed to form ectomycorrhizas on seedlings. The reasons for this are unclear but we suggest that germination of spores from this fungus may depend upon other factors. Our findings add to the growing body of literature demonstrating that spores of at least some species of hypogeous fungi remain viable after passage through the digestive tract of mycophagous animals.

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.