The production of chloromethane (CH3Cl) by wood rotting fungi of the Hymenochaetaceae is discussed with particular emphasis on emissions by species of Phellinus and Inonotus. Recent work on the metabolic role of CH3Cl as a methyl donor in the biosynthesis of secondary metabolites both in the Hymenochaetaceae and other families of white-rot fungi is reviewed. The parameters affecting the fungal emissions of CH3Cl in forest ecosystems are considered and where possible quantified. The annual global input to the atmosphere from this source is provisionally estimated at 160000 t of which 75% is released from tropical and subtropical forests and 86% is attributable to Phellinus. The possible impact of the contribution from fungi and other biological sources on the atmospheric CH3Cl burden and stratospheric ozone depletion is assessed.

The genetic variability within 600 bp of DNA sequence from the ribosomal internal transcribed spacers (ITS 1 and ITS 2) and the 5·8 S rRNA gene was examined in 52 strains belonging to 29 terverticillate taxa in Penicillium subg. Penicillium from diverse sources and locations. The sequenced region is extremely conserved within the terverticillate penicillia, with only 29 positions differing in one or more taxa. The highest degree of ITS variability was seen among the species close to Penicillium roqueforti (P. roqueforti, P. carneum and P. paneum) whereas the ITS variability was very low between closely related taxa, e.g. among the taxa near P. aurantiogriseum. The relationships among the terverticillate penicillia, and related teleomorphs in Eupenicillium, were analysed from bootstrapped ITS sequence data sets using the neighbour-joining method. The terverticillate penicillia form a well supported clade with Eupenicillium crustaceum. Sequence analysis generally confirmed the overall taxonomy in Penicillium subg. Penicillium but relationships between all the terverticillate taxa could not be clearly established due to the low degree of ITS variability. Nevertheless, clades of Penicillium species sharing environmental characteristics did emerge, e.g. species growing on protein and lipid rich substrates (e.g. P. crustosum and P. commune), species prevalent in dry habitats (e.g. P. chrysogenum and P. nalgiovense), and those prevalent on carbohydrate rich substrates (e.g. P. aurantiogriseum and P. freii).

The green fluorescent protein (GFP) has been widely used as an extremely useful vital marker in a large number of organisms, but good expression in filamentous ascomycetes has not been reported. To facilitate the research of fungal development and fungal-plant interaction, we constructed two plasmid vectors for the expression of the synthetic SGFP-TYG gene in ascomycete species, and used these vectors for transformation of the maize pathogen Cochliobolus heterostrophus. High level expression of GFP was obtained, as detected by anti-GFP antibodies and by fluorescence microscopy. The intense fluorescence was used as a highly efficient vital marker to detect cytoplasmic and developmental changes that occur in the fungus, and to follow phytopathogenic development of the fungus on and inside maize leaves. The hyphae within the leaf form a unique parallel growth pattern, closely associated with, and apparently determined by, the anatomy of the leaf. Fluorescence intensity was quantified by digital analysis of the green fluorescence image and was highly correlated with the amount of mycelium and levels of disease. Expression of GFP was obtained in additional ascomycetes that were transformed with the new constructs, indicating that SGFP-TYG can be used as a highly effective vital marker in ascomycetes.

Endophytic fungi were isolated from nine plant species growing on gypsum and saline soils in central Spain. The plants sampled were Arundo donax, Atriplex halimus, Diplotaxis erucoides, Ephedra nebrodensis, Phragmites australis, Rosmarinus officinalis, Scirpus holoschoenus, S. maritimus and Stipa tenacissima. A total of 152 fungal species were recovered from 2880 samples of leaves, stems or twigs, taken from 45 individual plants. Ephedra and Rosmarinus showed the highest diversity of endophytes, whereas both species of Scirpus showed the lowest. The most frequently isolated fungi were Alternaria alternata, Sporormiella intermedia, Rhizoctonia sp., Epicoccum purpurascens, Pleospora herbarum, Cladosporium herbarum, Sporormiella australis and a sterile fungus. A total of 187 strains belonging to 136 species were tested for the production of antimicrobial activities, using a panel of bacteria and yeasts, some of them of clinical relevance. Production of antimicrobial compounds was detected in 45 strains, belonging to 37 species. Large differences were observed among isolates from the same species, with respect to their ability to produce metabolites with antimicrobial activity.

The concept of species and species complexes of lichenized fungi is reviewed in the light of recent molecular approaches. Species concepts based on chemistry, morphology, reproductive mode, photobiont choice and habitat preference provide working hypotheses from which the delimitation of species or relationships within species complexes can be tested with molecular data. In studies in which a single locus such as ITS is used, a phylogenetic species concept can be applied only when the sequence data delimits genetically discrete clades that correlate with phenotypic characters or biogeographical distribution. This will be the case when sufficient time has passed for genetic isolation to result in the coalescence of different character states among the sibling species of a complex. In the case of recently diverged species, a single locus may not accurately separate species, and is not sufficient evidence to reject putative species based on a phenotypic species concept. In such cases, several genetic loci must be used to delimit species by a phylogenetic species concept. These phylogenetic species may corroborate phenotypic characters or biogeography, or they may be cryptic for all observed characters. Molecular analyses at the species level will lead to the re-evaluation of phenotypic characters for species delimitation, and will improve the understanding of speciation in lichenized fungi. The results from new molecular approaches have implications for taxonomic revisions. Formal changes in nomenclature based solely on molecular evidence should be made conservatively, and only after studies based on a thorough sampling with sufficient data of all kinds is performed, and after the taxonomic history of the group has been reviewed. In the meantime, informal names to describe new phylogenetic hypotheses are sufficient. Formally named cryptic species cause problems in distributional studies, where old material and literature references are included, but are useful in high resolution studies such as ecophysiology, especially where phenotypic differences may exist among cryptic species.

The aim of the present work was to determine the identity and molecular relationships between 127 strains of dark septate (DS) fungi isolated from healthy root tips, decayed coarse roots, live healthy-looking stems, coarse (stumps, snags and logs) and fine (tree branches and tops) woody debris in temperate-boreal forests in Sweden and Lithuania. Sequence analysis of ITS rDNA was used to identify the fungi. In a neighbour-joining similarity tree, all sequences were grouped into five distinct clusters. Within each of these, ITS rDNA sequence variation consisted of 2–18 nucleotides, corresponding to 1–3% of their total length. The four least variable clusters were supported with high bootstrap values of 86–100%. Comparisons with the sequences in the GenBank database showed that all our strains had a 95–100% homology with identified Phialocephala species, and they were thus assigned to this genus. The representatives of two clusters were identified, as P. fortinii and P. dimorphospora. The representatives of three remaining clusters were defined as Phialocephala sp. 35, Phialocephala sp. 6 and Phialocephala sp. 18. Within each of these clusters, ITS rDNA sequence uniformity was higher than that observed within P. fortinii and P. dimorphospora. Consequently, their clusters were most discrete, supported with bootstrap values of 100%. Genetic variation in the five distinguished Phialocephala species and their possible ecological roles are discussed. Phialocephala sp. 6 was confined to healthy root tips of conifers. P. dimorphospora was only associated with dead woody tissue of P. abies. P. fortinii, Phialocephala sp. 18 and sp. 35 were isolated from both dead and living conifers and Betula pendula. In conclusion, the present study revealed the ability of fungi from the genus Phialocephala to colonise and persist in live and dead trees under strikingly different ecological conditions.

Based on 43 taxa, the relationships of the European poroid Hymenochaetales were examined by molecular sequence data of a region of the large subunit of the nuclear-encoded ribosomal DNA. The molecular data were discussed referring to morphological and anatomical features of the fruit bodies, nuclear behaviour of the vegetative mycelium, and the pattern of reproduction. As a result, the genera Phellinus s. lat. and Inonotus s. lat. are subdivided into five subunits each. The following genera are accepted: Phellinus s. str. (P. igniarius group), Porodaedalea (P. pini group), Fomitiporia (P. robustus group), Fuscoporia (P. ferruginosus and relatives, including P. torulosus and P. wahlbergii), Phellinidium (P. ferrugineofuscus and relatives), Phylloporia (P. ribis) and Inonotus s. str. (I. hispidus and relatives), Inocutis (I. rheades group), Mensularia (I. radiatus and relatives), Pseudoinonotus gen. nov. (I. dryadeus and relatives), and Inonotopsis (I. subiculosus). Their relationships to non-European members of these groups are discussed. Onnia and Coltricia were confirmed as separate genera. Phaeolus schweinitzii was placed outside of the Hymenochaetales.

In several studies of oak decline in Europe, one semi-papillate (Phytophthora psychrophila sp. nov.) and two non-papillate homothallic Phytophthora species (P. europaea and P. uliginosa spp. nov.) were isolated, together with other Phytophthora species, from rhizosphere soil samples which could not be assigned to existing taxa. P. psychrophila differs from other semi-papillate species of Waterhouse's morphological Group IV, like P. ilicis and P. hibernalis, by its uniform, dome-shaped and cottonwool-like colony growth pattern on V8 juice agar and malt extract agar, the occurrence of sympodially branched primary hyphae, the high variation in size and shape of the sporangia, shorter pedicels, lower optimum temperature for growth, and ITS sequences. P. europaea is distinguished from related non-papillate Group V and VI species, namely P. fragariae, P. cambivora, and the ‘alder phytophthora’, by producing oogonia with tapered bases, irregular walls and exclusively paragynous antheridia, its cardinal temperatures for growth, and ITS sequences. P. uliginosa differs from related Group V and VI species by its large oogonia with exclusively paragynous antheridia, the predominant occurrence of ellipsoid sporangia with markedly wide exit pores, its slow growth, low cardinal temperatures, its colony growth patterns, and ITS sequences. P. uliginosa is separated from P. europaea by its larger oogonia without tapering bases, lower cardinal temperatures and growth rates, different colony growth patterns, and greater aggressiveness on Q. robur.

Downy mildew of sweet basil (Ocimum basilicum) has become a serious disease issue for the producers of sweet basil in Switzerland since it was first recorded in 2001. Reported in Africa in Uganda as early as 1933, major outbreaks of this disease in Europe were first noted in Italy in 1999 and in the USA from 1993. Previous reports have named the pathogen as Peronospora lamii. Its preferential hosts belong to the Lamiaceae family including basils (Ocimum spp.), mints (Menta spp.), sages (Salvia spp.) and other aromatics. This study investigated the taxonomic status of the downy mildew pathogen, using both morphological characters and molecular analysis of the ITS region of the rDNA. The inherent variability of conidial dimensions made species differentiation difficult. Sequence homology and phylogenetic analysis of nine collections of the Peronospora on sweet basil showed unique ITS sequences distinct from those of P. lamii and any other sequenced Peronospora species. This paper describes and illustrates the morphology of this presumably undescribed species of Peronospora. Its taxonomic position and relationships with other related species in the same genus are presented and discussed. In addition to this work, PCR primers for real time PCR analysis have been developed for the specific detection of this downy mildew pathogen from infected tissues or seeds. It is shown that these primers can also be used in classic PCR.

Concentrations of potassium nitrate, potassium phosphate, or calcium chloride were varied in stream-simulating microcosms containing leaf discs colonized by the aquatic hyphomycetes Anguillospora filiformis or Lunulospora curvula. Cumulative conidium production of the fungi determined from rates of sporulation at 2 d intervals was stimulated by increasing concentrations of both potassium nitrate and potassium phosphate but not by calcium chloride. Leaf weight loss was also stimulated by increasing concentrations of potassium nitrate and potassium phosphate. The amount of fungal biomass (determined as ATP concentrations) associated with leaf discs increased significantly only in the treatments in which A. filiformis received increasing concentrations of potassium nitrate. These results indicate that as aquatic hyphomycetes grow on leaf litter, they can obtain at least a portion of their inorganic nutrition from the water flowing over the leaves. They also suggest that sporulation is more sensitive to changes in nutrient concentrations than growth.

Conifer needles, like many grasses, are infected by systemic fungal endophytes. Following suggestions made in the early 1980s that (1) conifer needle endophytes may produce anti-insectan compounds, and (2) population dynamics of the eastern spruce budworm in New Brunswick could not be completely explained based on existing knowledge, we discovered that a low percentage of needle endophytes made a range of known and new metabolites toxic to this insect. Here, we report that wound inoculations of toxigenic endophytes of seedlings from a breeding population of white spruce were successful across a range of genotypes. The needles colonized by a rugulosin-producing endophyte were found to contain rugulosin in concentrations that are effective in vitro at retarding the growth of spruce budworm larvae. Larvae presented with endophyte infected needles containing rugulosin do not gain as much weight as those eating uncolonized needles. This represents the first positive evidence that the kind of mutualism between toxigenic endophytes and grasses affecting insect herbivory also may occur in white spruce.

Sequence analysis of the ribosomal DNA internal transcribed spacers (ITS) was used to establish phylogenetic relationships among 23 isolates of Pythium insidiosum, the etiological agent of pythiosis in mammals. The isolates were divided into three distinct clades that exhibited significant geographic isolation. Clade I consisted of isolates from North, Central, and South America, while clade II contained isolates from Asia and Australia. Also present in clade II was an isolate from a patient in the USA, but the origin of the infection may have been in the Middle East. Clade III was comprised of isolates from Thailand and the USA. All 23 P. insidiosum isolates were more closely related to each other than to any other Pythium species in this study. Additionally, all Pythium isolates formed a clade separate from both outgroup species, Phytophthora megasperma and Lagenidium giganteum. The ITS sequence results tend to support the existence of geographic variants or cryptic speciation within P. insidiosum. The sequence information obtained also provides an abundance of data for applications in the diagnosis of pythiosis and identification of P. insidiosum from clinical samples.

We have screened the genome of ecto- and endo-mycorrhizal fungi by using primers designed on microsatellite sequences: (CT)8, (CA)8, (GACA)4, (TGTC)4, (GTG)5. PCR experiments proved that microsatellites such as (GTG)5 exist as short repeated sequences in 11 species of Tuber (Ascomycetes) and seven species within Glomales (Zygomycetes). Variations in the banding pattern obtained by DNA fingerprinting enabled all these species and some isolates to be distinguished according to the number, size and intensity of the fragments. (GACA)4 and (TGTC)4 also led to successful amplifications in some isolates from Tuber and Glomales. These experiments demonstrate that microsatellite primers are reliable, sensitive and technically simple tools for assaying genetic variability in mycorrhizal fungi, and can be used to discriminate mycorrhizal symbionts with different taxonomic features. (GTG)5 in fact led to species-specific fingerprints in both truffles, which are closely related species, and in Glomales, which are quite separate species in evolutionary terms.

Inter- and intra-specific variation in destruxin production was detected in Metarhizium and may be important in determining virulence and/or specificity against insects. Strains of M. anisopliae var. anisopliae produced different amounts of destruxins A, B and E, but strain V220 did not produce any destruxins. M. anisopliae var. majus, M. flavoviride and M. album which are reported to be specific towards Coleoptera, Orthoptera and Hemiptera, respectively, had different destruxin profiles with destruxin A predominant. In time course studies on M. anisopliae var. anisopliae V245, destruxin E levels declined with time while destruxin A levels increased. The most virulent strains, Ma23 and V245, produced large quantities of destruxins but some low toxin producers were also virulent, suggesting that destruxins are not the only pathogenicity determinants. Some weakly to moderately pathogenic strains were highly pathogenic when injected into Galleria mellonella larvae, demonstrating the importance of the cuticle as a barrier to fungal infection. Only trace amounts of destruxin A or a combination of A and B could be detected in Galleria larvae infected with M. anisopoliae var. anisopliae or M. anisopliae var. majus. No destruxins were detected in larvae infected with M. flavoviride. Destruxin production may be influenced by the nutrients in insects or culture media.

The elemental composition of vacuolar granules in different ectomycorrhizal fungi, Suillus bovinus, Paxillus involutus, Pisolithus tinctorius and Laccaria laccata was analysed by energy dispersive X-ray spectroscopy (EDXS) either after chemical preparation or cryofixation, freeze-drying and pressure infiltration. Vacuolar inclusions were present in living hyphae and were not an artifact of specimen preparation, and they can be referred to as polyphosphate granules. These granules were localized in fungal vacuoles and the main counter-ions were K and Mg. The postulated association of these granules with the divalent cation Ca has to be interpreted as an artifact of the specimen preparation and was caused by the chemical preparation of former EDXS studies. The incorporation of cations, such as K, Na and Zn, depended on the external supply and emphasized the importance of these granules for the intracellular homeostasis of fungal cells. EDXS studies of polyphosphate granules in ectomycorrhizal associations of Pinus sylvestris showed that the elemental composition of granules differed between the sheath and the Hartig net. In polyphosphate granules of the Hartig net a higher content of K was detectable, whereas the incorporation of Mg was reduced. These results indicated a possible role of K in the transfer of short chained, mobile polyphosphates through the hyphae to the Hartig net. The concentration of potassium and phosphate in the fungal vacuole and the cytoplasm was closely correlated, which can be explained by a possible linkage between the phosphate and K uptake, which would maintain the charge balance and the pH of the fungal cell. The estimation of different phosphate pools in axenic cultures and mycorrhizal roots revealed that the fungal metabolism was changed by the association with an ectomycorrhizal host plant. In a mycorrhizal association higher proportions of absorbed phosphate were translocated into the metabolically inactive polyphosphate pool, which demonstrates the importance of this pool for the nutrition of the ectomycorrhizal host plant.

An innovative ex situ fungal baiting method using soil collected from field sites which allows the simultaneous detection of mycorrhizal fungi for multiple terrestrial orchids is presented. This method demonstrated that coarse organic matter (>2 mm) in the litter and topsoil was the most important reservoir of inoculum of these fungi. A new in situ seed baiting method using multi-chambered packets to simultaneously assess germination for different orchid species within soil is also introduced. These in situ and ex situ methods are compared using seed of orchids in the genera Monadenia, Microtis, Caladenia, Pterostylis and Diuris, using urban Banksia woodland sites with high or low weed cover. Both these seed baiting methods detected compatible fungi for these orchids, but common orchids germinated more frequently than those which were uncommon at the field sites. Germination rates were not significantly affected by weed cover even though adult orchids were rare in areas with high weed cover. The two new seed baiting methods vary in efficiency and applicability depending on the situation where they are used. However, the ex situ method allowed the time-course of germination to be observed, resulting in the production of more protocorms and facilitation of the isolation of mycorrhizal fungi. These techniques provide valuable new tools for detection of compatible mycorrhizal fungi to assist orchid research and conservation.

In order to evaluate substrate dependence of the symbiotic fungal associations in leafy liverworts (Jungermanniopsida), 28 species out of 12 families were investigated by transmission electron microscopy and molecular methods. Samples were obtained from the diverse substrates: from naked soil, from the forest floor on needle litter, from between peat moss, from rotten bark of standing trees, and from stumps and rotten wood. Associations with ascomycetes were found in most of the specimens independent from the substrate. Seven species sampled from soil were found to contain basidiomycete hyphae. Ultrastructure consistently showed dolipores with imperforate parenthesomes. Molecular phylogenetic studies revealed that three specimens belonging to the Jungermanniales were associated with members of Sebacinaceae, while Aneura pinguis (Metzgeriales) was associated with a Tulasnella species. These taxa are so far the only basidiomycetes known to be symbiotically associated with leafy liverworts. The probability that the associations with Sebacinaceae are evolutionary old, but the Tulasnella associations more derived is discussed. The sebacinoid mycobionts form a similar interaction type with the jungermannialian leafy liverworts as do the associated ascomycetes. The term ‘jungermannioid mycorrhiza’ is proposed for this distinctive symbiotic interaction type.

Correct identification of fungi to species level is important because a specific epithet embodies a set of characters that enables us to predict, for example, the mycotoxin production of a species. Many small-spored Alternaria isolates have been misidentified due to inappropriate growth conditions and the use of spore size as the only identifying character. In this study 39 Alternaria isolates were grown under standardised conditions and subjected to chemical, morphological and physiological analyses. All isolates were extracted and analysed by HPLC–DAD. Analysis showed that both A. gaisen and A. longipes were able to produce altertoxin I, which has not previously been reported. The resulting metabolite profiles were subjected to cluster analysis and principal component analysis. A subset of the isolates was grown at five different temperatures. Colony colour and diameter were recorded and the diameter measurements were subjected to principal component analysis. Analysis of chemical and physiological data showed that the 39 isolates segregated into the same distinct groups that are morphologically identifiable as A. alternata, A. longipes or A. gaisen. The results showed that A. longipes, A. gaisen and A. alternata are different species that can be distinguished morphologically, physiologically and chemically. Therefore, the continued use of the name Alternaria alternata for A. longipes and A. gaisen is unwarranted and pathotypes should not be used.

Phyllosticta capitalensis (teleomorph Guignardia mangiferae) occurs as a foliar endophyte in woody trees belonging to different families of both temperate and tropical regions. We isolated this endophyte from plants in different habitats, such as mangroves, dry deciduous forest, moist deciduous forest and semi-evergreen forest. This endophyte was found to produce a black pigment that was characterized to be melanin based on UV-visible, IR and ESR spectra and chemical tests. Tricyclazole, a specific inhibitor of pentaketide melanin biosynthesis, inhibited synthesis of the pigment indicating it is a 1-8, dihydroxynaphthalene. This appears to be the first report of such a melanin in Phyllosticta or other foliar endophytes. Melanin in the hyphae of P. capitalensis may be responsible for the success of this fungus as a cosmopolitan endophyte, since melanin is known to enhance the survival capability of fungi in stressful environments.