The polygalacturonase (PG)-encoding gene (rpg1) of Rhizopus oryzae, the causal pathogen of rhizopus rot of mulberry, was cloned and sequenced. PGs were partially purified from incubation mixture of 2% pectin medium and their N-terminal amino acid sequences were determined by a gas-phase protein sequencer. RT-PCR was performed using degenerate primers designed from the amino acid sequences, which resulted in part of a PG-encoding gene being obtained. By 3′-RACE and TAIL-PCR analyses, the entire region of the PG-encoding gene was cloned and sequenced. The structural gene comprised 1199 bp coding for 383 amino acids with a putative signal peptide of 26 amino acids, and the open reading frame was interrupted by single intron of 47 bp. Phylogenetic analysis using the deduced amino acid sequence revealed that R. oryzae RPG1 belonged to a clade consisting of exo-PGs of ascomycete fungi.

In 1993 a destructive new Phytophthora pathogen of riparian Alnus trees was discovered in the UK and subsequently shown to be present in other parts of Europe. The new Phytophthora comprised a group of emergent heteroploid hybrids, probably between P. cambivora and a species related to P. fragariae. These included a common, near tetraploid standard hybrid, the presumptive allopolyploid; and four scarcer major variant types with chromosome numbers intermediate between diploid and tetraploid, named the Swedish, Dutch, German and UK variants. The standard hybrid type is formally designated here as Phytophthora alni subsp. alni. The Swedish variant is designated as P. alni subsp. uniformis; and the Dutch, German and UK variants collectively as P. alni subsp. multiformis. The properties of the Dutch, German and UK variants within subsp. multiformis are informally described. The problems of designating emergent species hybrids under the International Code of Botanical Nomenclature and the reasons for the taxonomic choices made are discussed.

The current circumscription of Lasiosphaeria includes taxa with a wide variety of ascomatal walls, ascomatal wall vestitures, and ascospore morphologies and a broad range of putative anamorphs. Despite the complexity of morphological characters in the genus, species within Lasiosphaeria can be arranged into four groups based on ascospore morphology. Taxa which possessed ascospores in each of the four groups were used in phylogenetic analyses of partial nuclear large subunit (LSU) rDNA sequences to test the monophyly of the genus and determine relationships among its species. Lasiosphaeria was found to be highly polyphyletic in that species segregated into seven well-supported monophyletic clades dispersed among several orders. Three new genera, Echinosphaeria, Hilberina, and Immersiella, are erected for three of these clades while the genus Lasiosphaeris is reintroduced for a fourth clade. These data support Ruzenia as a previously established genus and the transfer of Lasiosphaeria raciborskii to Chaetosphaeria. The circumscription of Lasiosphaeria has been considerably narrowed to better reflect a natural classification. These taxonomic changes are additionally supported by a combination of morphological characters which are discussed in relation to the phylogenetic trees.

Lactarius angiocarpus sp. nov. is described from miombo woodlands in Zambia. It is the third sequestrate representative of the Russulaceae described from tropical Africa. Morphological characters and DNA sequence data support its placement in Lactarius subgen. Plinthogali. Molecular kinship analyses confirm a multiple origin of sequestrate Russulaceae spp. As none of the previously sequenced sequestrate Russulaceae spp. has been assigned to the Plinthogali clade, the placement of L. angiocarpus indicates an additional point of origin of derivative sporocarp types within the Russulaceae. Within the same subgenus, another tropical African sequestrate species, L. dolichocaulis comb. nov. (syn. Arcangeliella dolichocaulis) is recognized.

36 fungal β-tubulin sequences were analysed to study the evolution of this gene and the phylogeny of basidiomycetes. The analysis comprises a representative selection of all major lineages of basidiomycetous fungi and some selected ascomycetes for comparison. Intron positions vary between the different lineages, but seem to be conserved in the Hymenomycetes and Ustilaginomycetes. The most conserved regions seem to be highly susceptible for introns. Splicing and branching sites of the introns are more variable in basidiomycetes than reported from other fungal groups so far.

Basidiomycete monophyly was confirmed with our data in respect to the ascomycetes studied. By analysing amino acid sequences, the Hymenomycetes and the Ustilaginomycetes were resolved as monophyletic groups. The phylogeny within these two groups is similar to that obtained with other genes. Based on β-tubulin data Naohidea sebacea, Chionosphaera apobasidialis, Jaculispora submersa, Platygloea pustulata, Platygloea disciformis and Melampsora lini, representing the Urediniomycetes, are not resolved in most analyses. The early radiation of major basidiomycetous lineages seems to be reflected in the highly conserved β-tubulin gene.

Sowerby described Agaricus cossus in 1799. The fungus possessed a smell, resembling that of a wounded larva of Cossus cossus (Lepidoptera). The species belongs in Hygrophorus, and since more than one white Hygrophorus species has this distinctive smell the epithet cossus has been variously interpreted. The complete internal transcribed spacer (ITS) region of the original type collection made in 1794, preserved in the Royal Botanic Gardens Kew herbarium, was successfully sequenced. Comparison with the ITS sequences from four other white aromatic-acidulous smelling Hygrophorus species, including the type specimen of H. quercetorum, showed that H. cossus is a species associated with Quercus and an older name for H. quercetorum. The differences in basidiome colouration developing with age and host-tree association appear to be the most useful characters to discriminate between the four species with a Cossus cossus smell. A table of morphological and ecological characters is provided.

Leptographium wingfieldii is a well-known fungal associate of the pine shoot beetle, Tomicus piniperda, in Europe. This fungus is pathogenic to pines and is an important cause of blue-stain in the sapwood of infested trees. Tomicus piniperda was first found in a Christmas tree plantation in Ohio, USA, 1992, but isolation of the fungi associated with these intercepted insects was not attempted. Fungal strains resembling L. wingfieldii were recently isolated from pines attacked by T. piniperda, Dendroctonus valens and Ips pini in the northeastern United States. These strains were morphologically similar to the ex-type and other reference strains of L. wingfieldii. Strains were also compared based on sequences of the partial ITS ribosomal DNA operon, β-tubulin and elongation factor 1-alpha (EF-1α) genes. Based on these DNA sequence comparisons, reference strains of European L. wingfieldii were conspecific with North American strains from pines attacked by T. piniperda, D. valens and I. pini. A single strain from Canada, collected in 1993 near the Ontario border with the USA, shortly after the discovery of T. piniperda in that area and tentatively identified as L. wingfieldii, was also included in this study. Its identification was confirmed, suggesting that L. wingfieldii has been present in this region and probably over the whole range of the insect's distribution for at least a decade. This represents the first record of L. wingfieldii associated with the introduced and damaging pine shoot beetle T. piniperda in North America. It shows that the fungus is well established and can become associated with other native bark beetles that attack stressed and/or dying trees. The occurrence and spread of this highly pathogenic fungus associated with North American bark beetles should be monitored.

This study aimed at testing the hypothesis that the genetically distinct Tuber aestivum population on the island of Gotland, Sweden, is adapted to habitats different from French T. aestivum populations. The soil structure, soil chemistry, bedrock, climate, vegetation and host tree continuity of 18 T. aestivum sites on Gotland were analysed and compared with data from France. We conclude that T. aestivum can grow in soils with a broad soil structure range and that no striking differences in soil chemistry were found. No T. aestivum indicator plants other than the host trees were found, but the host tree continuity on the T. aestivum sites on Gotland was more than 300 yr. If the T. aestivum population on Gotland constitutes an ecotype it is rather an adaptation to the colder and drier climate on Gotland. Selecting local T. aestivum inoculum for truffle orchards in northern Europe could be important for successfull truffle production.

Studies were performed to assess the genetic variation amongst isolates of the aphid-pathogenic fungus Pandora neoaphidis (syn. Erynia neoaphidis). 37 isolates were examined, from a range of pest and non-pest aphid species, as well as 21 from eight other entomophthoralean species. Universal primers were used to amplify the ITS rDNA regions and all of the species tested produced discrete ITS groups, with the exception of Conidiobolus spp. Neighbour-joining analysis of the ITS2 regions from P. neoaphidis, P. kondoiensis and Zoophthora radicans demonstrated that these three species formed distinct groups with sequence identities of 58–82% between the groups. An ITS size of ca 1100 bp was diagnostic for P. neoaphidis, while ca 1450 bp was characteristic of P. kondoiensis. ITS-RFLP analysis failed to yield intraspecific polymorphisms in any of the P. neoaphidis isolates screened, although it was useful in distinguishing between different entomophthoralean species. Some intraspecific variation in the ITS region was detected in a number of isolates of Z. radicans and Conidiobolus spp. We propose that two isolates previously identified as P. neoaphidis based on conidia morphology, are actually P. kondoiensis based on molecular studies. Sequencing analysis of the complete ITS region from P. neoaphidis and P. kondoiensis allowed species-specific primers to be developed for P. neoaphidis and P. kondoiensis. These were used to screen aphids infected in laboratory bioassays and from field-collected samples, without prior isolation of the fungus. The primers are useful tools for quantifying the epizootiology of P. neoaphidis in aphid populations, as well as assessing competitive interactions between these two 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 toxic naphthoquinone juglone (5-hydroxy-1,4-naphthoquinone) is efficiently degraded by the ligninolytic fungus Pleurotus sajor-caju, as demonstrated by the total bleaching within 9 d of a conventional liquid culture medium supplemented with 0.6 mM juglone. The oxidative degradation involves the production of hydrogen peroxide arising from both enzymic and non-enzymic oxidation reactions, promoted by the fungus. Juglone is not directly attacked by the oxidative enzymes of the ligninolytic machinery of P. sajor-caju, such as laccase, manganese peroxidase and arylalcohol oxidase. On the other hand, this naphthoquinone is a good substrate for a reductase, which triggers an auto-oxidative process producing reactive oxygen species and leading to juglone degradation. The degradation process continues to completion by means of a direct, presumably non-catalysed reaction with hydrogen peroxide.

The devastating dry rot fungus, Serpula lacrymans, has a worldwide occurrence in buildings. We investigated the genetic variation in European isolates belonging to five vegetative compatibility groups (VCGs) by AFLP analysis. Our results indicate that S. lacrymans in Europe is genetically extremely homogenous; only five out of 308 scored AFLP fragments (1.6%) were polymorphic. In contrast, S. himantioides, the closest relative of S. lacrymans, possessed 31.3% polymorphic fragments (84 out of 268). AFLP polymorphisms observed in S. lacrymans were distributed independently of the VCG boundaries, indicating that the VCGs do not represent clones but that different genets of S. lacrymans frequently share similar vic alleles due to low genetic variation. Thus, although the European S. lacrymans is genetically extremely homogeneous, and our results suggest that the species reproduces and spreads mainly sexually and not by clones.

Geosmithia putterillii is an anamorphic fungus with connections to bark beetles. Genetic variability of 89 isolates traditionally grouped in G. putterillii and G. lavendula isolated from different geographical regions from subcorticolous insects and from other unspecific substrata was assessed using RAPD, sequencing of the ITS region (ITS1-5.8SrDNA-ITS2) and morphological characters. RAPD analysis revealed eight distinct groups. One group was represented by G. lavendula type strain and showed no relations to any other isolate. Five RAPD-types with similar ITS sequences and phenotype were related to the ex-type strain of Penicillium pallidum (generally given as a synonym of G. putterillii). Because of unique phylogenetic position and a phenotype markedly different from G. putterillii, the new combination G. pallida is made here. For another group of isolates formerly identified as G. putterillii the new species G. flava is described based on a characteristic RAPD-type, a unique ITS sequences and a different phenotype. These newly recognized species are stable in culture and with worldwide distribution.

A new powdery mildew infecting papaya (Carica papaya) in Brazil, Streptopodium caricae sp. nov., is described. The species is compared with other anamorphic Erysiphales known to infect papaya: Oidiopsis sicula, Ovulariopsis papayae, Oidium caricae, O. papayae, O. caricicola, O. indicum, O. caricae-papayae, Podosphaera (syn. Sphaerotheca) spp., and Erysiphe spp. An emended description Streptopodium and a key to the anamorphs of powdery mildews on papaya are also presented. A re-examination of the type material of Phyllactinia caricaefolia showed that conidia in this material are dimorphic, indicating that its anamorph does not belong to Ovulariopsis and that the teleomorph is not conspecific with Phyllactinia guttata. Oidium caricae, the common powdery mildew of papaya, was re-examined, recognized as a member of subgenus Pseudoidium, an emended description was prepared, and a new type was indicated. O. papayae was recognized as a synonym of O. caricae, and many of the records of this fungus are considered to be doubtful or incorrect, either omitting a description of the fungus or including a description or illustration of an euodium conidiophore morphology.

The oxidizing enzyme laccase produced by many fungi is generally considered to be active in the biodegradation of lignin, a major plant cell wall component highly resistant to microbial attack. The enzyme is secreted at high levels by the P-type of the highly aggressive pathogen Heterobasidion annosum, but at much lower levels by the S-type which correlated with their varying wood decay capability. To investigate the evolutionary relationship between laccase genes of the different H. annosum types from several geographical regions we have compared the nucleotide sequence of the laccase gene from 32 different isolates of the fungus together with two other Asian isolates (H. araucariae, H. insulare). In addition to nucleotide sequence assessment, we have also cloned, characterized and analysed the partial sequences of the laccase gene from the homokaryotic (FSE-7: S-type; Sä16-4: P-type) and heterokaryotic (Faf-8: F-type) strains of H. annosum. Using degenerate primers, two distinct laccase gene fragments of 1.64 and 2 kb were amplified from the genomic DNA of this fungus. DNA sequence analyses showed that the 1.64 kb laccase fragment in all three H. annosum types shared significant homology (86–96%). But comparative analyses of the 1.64 and 2 kb laccase gene fragment revealed only 48% nucleotide sequence similarity. Using the cDNA sequence information, exon regions were predicted and this revealed that about nine small introns interrupted the genomic DNA. Southern hybridization analysis indicated a single copy of the gene in the homokaryotic S-type (FSE-7) examined but presence of double bands in the homokaryotic and heterokaryotic P-type strains of the fungus suggest the existence of two laccase genes. Northern analyses revealed that the gene is constitutively expressed but appear to be enhanced several fold with the addition of ferulic acid or oxalic acid. Alignments of the nucleotide sequences and phylogenetic analyses are presented allowing estimations of evolutionary relationships to be made. These comparisons indicate that laccase gene of P-type is distinct from other Heterobasidion sequences, including the outgroups H. araucariae and H. insulare, while the relationship between the S- and F-groups could not be resolved. Comparative phylogenetic analyses using predicted amino acid sequence also showed strong similarity to the laccases from other basidiomycetes (Pleurotus ostreatus, Phlebia radiata and Trametes versicolor) but least similar to laccases from ascomycete fungi. In addition, the results of McDonald-Kreitman test for possible evidence of selection based on analyses of two exon regions of the H. annosum laccase gene are presented and discussed.

Two pathogenic special forms (f. sp.) of the Fusarium oxysporum species complex f. sp. lycopersici (Fol) and f. sp. radicis-lycopersici (Forl) are morphologically indistinguishable. Although they are pathogenic to the same host genus Lycopersicon (tomato), and infect the same tomato cultivar, they form distinct diseases; Fol causes wilt and Forl causes crown rot and root rot. These two special forms apparently exist as genetically isolated populations, based on vegetative compatibility and molecular variation at the DNA level. In seeking efficient diagnostic tools for differentiating Fol and Forl isolates, we examined three techniques: isozyme analysis, mitochondrial DNA (mtDNA) RFLP by HaeIII-digestion of total genomic DNA, and an osmotic method using high performance liquid chromatography (HPLC) to detect fungal pigments. The isolates were collected from geographically widespread locations. Distinct HPLC-profile differences were found between an endophytic non-pathogenic isolate and the other pathogenic isolates. However, the direct mtDNA RFLP technique proved to be an efficient diagnostic tool for routine differentiation of Fol and Forl isolates.

The taxonomy of the marine species of Collemopsidium in northwest Europe was investigated using morphological and molecular evidence. 210 specimens were collected from the west coasts of Norway and Ireland, and morphological and ecological variables recorded. ITS1 rDNA sequences were obtained from 24 specimens. A phylogenetic analysis, resulting in a single optimal tree, was performed under the unweighted least squares optimality criterion based on maximum likelihood distances obtained from unaligned sequences. Principal components analysis (PCA) was performed on morphological variables of the sequenced specimens, and classification was carried out by maximizing agreement between the phylogenetic tree and the PCA. Thallus immersion, and perithecial immersion and size, were the most important characters for discriminating between taxa. Apart from substratum, niche separation between taxa was small but statistically significant as shown by a redundancy analysis (RDA). Variance partitioning indicated that genetic variation is vastly more important than ecology for explaining phenotypic variation. Five species of marine Collemopsidium are recognized, including two new combinations: C. foveolatum (syn. Arthopyrenia foveolata) and C. ostrearum (syn. Lecanactis ostrearum) combs. nov.

Uptake of 75[Se]-selenite by Saccharomyces cerevisiae has been characterized. At a 0.5 mM selenite, ~0.14 nmol Se (106 cells)−1 was rapidly accumulated by the cells at a rate of ~56 pmol.min−1 (106 cells)−1 which was independent of temperature and glucose. This rapid phase was followed by a slower uptake phase which was sensitive to glucose, temperature and metabolic inhibitors [2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), potassium cyanide (KCN) and sodium azide (NaN3)] and therefore presumed to be metabolism-dependent. Two transport systems appeared to be involved in selenite uptake. At the low range of selenite concentrations used (0.025–0.1 mM), a high affinity transport system occurred with apparent Km and Vmax values of 54.0 μM and 3.14 pmol.min−1 (106 cells)−1 respectively. A low affinity system was present at higher concentrations (0.1–1.0 mM) with apparent kinetic parameters of Km=435 μM and Vmax=11.6 pmol.min−1 (106 cells)−1. Elevated sulphate concentrations (up to 2.5 mM) did not affect the accumulation of selenite. However, the transport rate from 0.5 mM selenite was stimulated by sulphite, with the maximum effect occurring at 0.5 mM sulphite. Methionine had a detectable inhibitory action on selenite uptake whereas cystine and cysteine completely inhibited active transport of selenite. Transmission electron microscopy of 5 mM selenite-grown cells revealed the presence of abundant small cytoplasmic vesicles containing electron-dense granules which could represent an intracellular selenium-detoxification mechanism.

The sequence variation within the genus Letharia in the Caucasus and Morocco was investigated. Twelve thalli from each area were sequenced at eight different loci. Phylogenetic analysis of the multiple loci data revealed the cryptic species Letharia ‘lupina’ in Morocco, previously known only from western North America. The two cryptic species L. vulpina and L. ‘lupina’ locally share the same ecology but are genetically isolated from each other. In the Caucasus, five different haplotypes of L. vulpina were detected, and in Morocco four L. vulpina haplotypes and six L. ‘lupina’ haplotypes were found. For L. vulpina, allelic differences were detected at five of the eight loci in the Caucasus and Morocco. The populations of L. vulpina in both the Caucasus and Morocco contain more genetic variation than those previously investigated in Europe, which indicates that the Caucasus and Morocco acted as refugia during quaternary glaciations, and that central and northern Europe may have been recolonised from one or both of these areas.

The diversity and vertical transmission of double-stranded (ds) RNA in Helicobasidium mompa and Rosellinia necatrix was examined by electrophoresis and Northern hybridization. These two fungi share the similar niche as root rot pathogens of trees in forests and orchards, and had diverse dsRNAs. The detection frequency of dsRNA in both fungi was different; in H. mompa, 68.4% (132 out of 193 MCGs; mycelial compatibility groups) had dsRNA, whereas 20.9% (53 out of 254 MCGs) in R. necatrix. dsRNA banding patterns and Northern blot analyses revealed the presence of various dsRNA elements in both fungi. Hyphal tip isolation was mostly unsuccessful to remove dsRNA with some exceptions. Sexual reproduction functioned to remove dsRNA in both fungi since dsRNA was not detected from single sexual spore cultures. Possible explanations for the difference in the detection frequency of dsRNA are discussed in terms of the differences in their sexual reproduction and other factors.