Mycological Research News features : Second fungal genome sequenced; Gliocladium roseum belongs in Clonostachys; and fungi from coral reefs.

This issue contains 13 papers. The molecular phylogeny of the keratinophilic members of some Onygenales has been examined, with an emphasis on Auxarthron, a genus that proved to be monophyletic with nine species, while Amauroascus was polyphyeltic (pp. 388–396). Concern over oak decline in Europe has led to in-depth studies on Phytophthora species in oak forest soils which have been found to yield not only eight already recognized species, but three species new to science have also been discovered in the rhizosphere, one (P. uliginosa) being particularly aggressive in inoculation experiments (pp. 397–411); these have been compared anatomically, in culture, and by ITS sequence data and are illustrated in colour. The two macrolichens Usnea florida and U. subfloridana have long been regarded as related but differing in their reproductive methods, distributions, and air pollution sensitivity; analyses of rDNA and β-tubulin sequences, however, show that a single species is involved as they formed one monophyletic group with the specimens intermixed in the trees (pp. 412–418). In Phoma exigua, however, rDNA ITS sequences failed amplified fragment length polymorphism (AFLP) fingerprint patterns showed clear clustering of most (pp. 419–427). AFLP's also prove to be a reliable tool for the identification of Alternaria brassicicola and indicate that there this apparently asexual species has a means of generating and maintaining genetic variation (pp. 428–434).

Phylogenetic analyses of nucleotide data from the ITS and 5.8S rDNA gene region were performed on 37 strains of Onygenales representing all the accepted species of Auxarthron (9 strains), several species of Amauroascus (16 strains), and morphologically similar genera (3 strains of Amaurascopsis, 1 of Apinisia, 1 of Shanorella, 1 of Mallochia, 2 of Nannizziopsis, 1 of Pectinotrichum, and 3 of Uncinocarpus). The inferred phylogeny provided strong support for the monophyly of Auxarthron. We propose the new combinations Auxarthron kuehnii and A. pseudoreticulatum combs nov. and provide a key to the accepted species of Auxarthron. Although this study demonstrated that Amauroascus is polyphyletic, relationships among the members of that genus could not be resolved in phylogenies inferred from the analyses of ITS sequences; the new combination A. queenslandicus comb. nov. is also proposed. Molecular data suggested that Amaurascopsis reticulatus, characterised by the absence of discrete ascomata and prominently echinulate ascospores, can be accomodated in Mallochia.

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.

The lichens Usnea florida and U. subfloridana have since long been recognised as distinct species. They show many similarities in morphology, but have different reproductive strategies. Usnea florida is always provided with many apothecia and produces no specialised asexual propagules. Usnea subfloridana has soralia, isidiomorphs and occasionally apothecia. Phylogenetic analyses based on continuous sequences of the ITS and LSU regions of the nuclear ribosomal DNA and the gene coding for β-tubulin, show that specimens of the two species form one monophyletic group of intermixed specimens, and not two groups corresponding to morphology, which would have been expected if two species were at hand. The ‘species pair’ concept in lichenology is discussed. Other Usnea species included in the study are: U. articulata, U. barbata, U. ceratina, U. filipendula, U. hirta, U. rigida and U. wasmuthii.

Several varieties have been distinguished within the phytopathogenic pycnidial fungus Phoma exigua on the basis of cultural characteristics. These varieties have been stated to correlate with differences in ability to attack different host plants. Molecular discrimination of the varieties was investigated using amplified fragment length polymorphism (AFLP) and sequence analysis of the ITS region of the rDNA cluster on a set of 43 strains and 7 outgroup strains. The ITS sequences of the 43 different P. exigua strains were highly similar and revealed no subgroups within P. exigua while the AFLP fingerprint patterns of two primer combinations showed clear clustering of most varieties.

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

The diversity of Tricholoma matsutake basidiomata and their distribution across Japan was investigated, for the first time, by using PCR-RFLP and sequencing. Eight distinct IGS1 rDNA types were identified by using the restriction endonuclease Cfr13I. Four (A, B, G, H) were characterized by one IGS1 dominant sequence, which could then be recorded by direct sequencing of the PCR products. In contrast, the four other types (C, D, E, F) were characterized by up to more than four codominant IGS1 copies that needed to be cloned before sequencing. Among the copies, one was always exhibiting the RFLP pattern of type A basidiomata. Pairwise nucleotide variation among IGS1 spacer copies, recorded in RFLP types A, B, C, D, F, and H, was very low and never exceeded 1·9%. In contrast, type G emerged and its IGS1 spacer sequence exhibited at least 10·6% nucleotide variation compared with all other types. While the data recorded suggests a higher genetic diversity in the southern than in the northern part of Japan, no host/matsutake ribotype specificity could be evidenced. In Japan, the T. matsutake basidiomata belonging to RFLP type A were by far the most frequent. They were found at most sites and were usually the most abundant at each site. Furthermore, this predominance persisted over time during ca 50 years. The dominant RFLP type A may represent the original Japanese population while the other genotypes may have been more recently introduced.

Monilinia fructigena isolates from Japan were compared with isolates from Europe. General colony characteristics, stroma formation, growth rate and conidial dimensions were determined for six isolates each from both groups, as well as sporulation intensity on potato dextrose agar (PDA) and germ tube features. Potential differences in pathogenicity were tested on the pear cultivars ‘Conference’ and ‘Doyenné du Comice’, and on the apple cultivar ‘Cox's Orange Pippin’. A marked difference in stroma formation occurred, the area of stromatal plates ranged from 4·1 to 5·2 cm2 in the Japanese group, and from 0 to 0·9 cm2 in the European. The mean growth rate was significantly higher for Japanese isolates (t-test, P = 0·01). Length and width of conidia were significantly greater in European isolates (t-test, P = 0·01). Conidia measured on average 19×11·5 μm in European isolates, and 16×10 μm in Japanese ones when grown on cherry agar. On fruits, the difference in conidium size was even more pronounced. Sporulation intensity on PDA and germ tube features did not differ between both groups. No differences were found in latency period, lesion growth rate or sporulation intensity on apple and pear fruits between both groups. Together with previously published differences in the ITS region of ribosomal DNA, our results show that the Japanese isolates belong to a distinct species, Monilia polystroma sp. nov. A description of the anamorph is given, as well as a table summarising key features for all four brown rot associated Monilia species.

The root systems of Carex flacca and C. pilulifera, growing in the Burren (western Ireland), were shown to be colonized by Cortinarius (Dermocybe) cinnamomeus. This basidiomycete formed ectomycorrhiza-like structures, which possessed a distinct fungal mantle (85–100 μm thick), hyphal infection in epidermal cells, rhizomorphs and extramatrical hyphae, but lacked a Hartig net. These ectomycorrhiza-like structures were formed on first-order lateral roots but were distinct, morphologically and anatomically, from dauciform roots. Structures typical of arbuscular mycorrhizas were never observed. The colonization of root systems of C. flacca by C. cinnamomeus was confirmed by PCR/RFLPs and DNA sequencing of the ITS region of rDNA.

A set of 23 non-sporulating, unidentifiable endophytic fungi associated with wild Vaccinium macrocarpon were examined for their polyketide metabolite producing potential. Using two degenerate polymerase chain reaction primers (FPKSKSU-2 and FPKSKSD-1), we cloned and sequenced 12 ketosynthase domains from 11 of the 23 cranberry endophytic fungi. Phylogenetic analyses segregated the 12 ketosynthase domains into three groups. One group of four sequences was clustered with polyketide synthase genes involved in melanin formation. The second group of two ketosynthase sequences clustered with aflatoxin encoding fungal polyketide synthases. The remaining six ketosynthase domains were not clustered with any of the known fungal polyketide synthase groups. Of the 12 ketosynthase fragments, five contained one or more introns in the ∼800 bp DNA region. In order to locate the phylogenetic origin of the polyketide synthase genes, phylogenetic relationships of the strains were inferred from small subunit ribosomal DNA sequences. Analyses of small subunit ribosomal DNA sequences showed that all but one of the strains grouped among the major clades of the ascomycetes. The exceptional strain, CR70, was probably an oomycete. Thirteen of the 22 ascomycetous fungi appeared within a clade that included Oidiodendron tenuissimum of the Myxotrichaceae.

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.

Four day/night temperature regimes (11/6, 14/9, 17/12, 20/15 °C), three photoperiods (0, 12, 24 h d−1), and four continuous temperature regimes (10, 13, 16, 19 °) were evaluated for their effects on growth, sporulation, and conidia discharge of two isolates of Valdensinia heterodoxa, a foliar pathogen of salal (Gaultheria shallon). All temperature regimes supported mycelial growth; however, growth rates were faster at higher temperatures. Sporulation and conidia discharge in both isolates was generally highest at 17/12 ° (day/night) or 16 ° constant compared with other temperature regimes. Photoperiods of 12 and 24 h d−1 resulted in faster growth and significantly greater sporulation and discharge compared with a continuous dark treatment. The study shows that temperature and light conditions have a moderate effect on mycelial growth but are crucial for sporulation of V. heterodoxa. Possible implications for the use of V. heterodoxa as a potential biological control agent for salal are discussed.

We report the cloning and sequencing of a DNA fragment encoding a putative C2H2 zinc finger protein from Aspergillus nidulans. The gene was isolated by complementation cloning of a salt sensitive phenotype of the A. nidulans sltA1 mutant. A 3·8 kb PstI fragment that restored wild type salt tolerance contained one large open reading frame of 2202 bp. The predicted protein (StzA) from this reading frame comprises 698 amino acids and has three Zinc fingers along with a putative transcriptional activation domain rich in acidic amino acids. The corresponding sequence from a sltA1 mutant contains a premature STOP codon resulting in loss of the putative transcriptional activator in the C-terminal region. The Zinc fingers show conserved motifs with a number of transcription factors including CreA from A. nidulans and the human Wilm's tumour susceptibility protein WT-1.

The present investigation deals with the main factors involved in early infection of nematode eggs by fungal parasites. We studied the effect of hydrophobicity on appressorium formation by germlings of Pochonia rubescens (syn. Verticillium suchlasporium), P. chlamydosporia (syn. V. chlamydosporium) and Lecanicillium lecanii (syn. V. lecanii). Appressoria were frequently formed on hydrophobic surfaces such as polyvinyl chloride or polystyrene and were infrequently formed on hydrophilic materials such as glass or aluminium. Infected eggs probed with the FITC-labelled lectin Concanavalin-A showed intense labelling corresponding to appressoria formed by fungal parasites on the eggshell surface. Proteolytic activity was found in extracts from conidia and germlings of fungal parasites (especially P. chlamydosporia) in the absence of nematode eggs. Addition of the serine proteinase inhibitors phenylmetylsulphonyl fluoride (PMSF) or diisopropyl fluorophosphonate (DFP) to the extracts reduced their proteolytic activity. PMSF was the most effective inhibitor. Zymography also revealed proteolytic activity in extracts from the three fungi tested. This activity mostly corresponded to bands of Rf's of substrate degradation similar to that of purified main protease (P32) from P. rubescens. Other bands with molecular weight higher than P32 (low Rf) were found especially for P. chlamydosporia extracts. For L. lecanii only bands of low Rf were found. Serum anti-P32 partially inhibited proteolytic activity of extracts from conidia and germlings. Application of PMSF and DFP to the inoculum, reduced egg penetration for the three species studied. PMSF caused the highest reduction in eggs infected by L. lecanii, while DFP significantly reduced egg infection by both P. chlamydosporia and L. lecanii. Our results therefore show hydrophobicity, appresorium formation and protease production as factors involved in early parasitism of nematode eggs.