This contribution of Mycological Research News features Pseudotulostoma: the find of the century?

This issue contains 15 original research papers. Fast-tracked is the description of a new and serious plant pathogen of Rhododendron and Viburnum, Phytophthora ramorum. Pistachio gummosis is found to be caused by two species of Phytophthora, one new to science. New molecular data confirm that the loculoascomycetes are not monophyletic, and show Chaetothyriomycetes to be a sister group of Eurotiomycetes, and that the Laboulbeniales warrant recognition as a separate ascomycete class (the Laboulbeniomycetes). Molecular studies on the hypogeous Arcangeliella borziana reveal a relationship to Lactarius sect. Russularia. A major study of Daldinia using a wide variety of biochemical and molecular methods is presented, and biometric studies on Pythium oospores demonstrate the efficacy of the approach in species separations.

The development of Sphaerotheca fusca on different melon cultivars is compared, Neotyphodium endophytes in grasses have been demonstrated inside vascular bundles for the first time, and the adhesion of conidia and germlings of Bipolaris sorokiana on solid surfaces investigated. In Agaricus bisporus the transcript levels of genes when stored is compared to that at harvest, and the behaviour of carbon-starved Penicillium chrysogenum cultures has been examined. The ability of citric and oxalic acids, and also culture filtrates of Aspergillus niger, to bind cobalt and zinc ions has been examined.

A remarkable new ascomycete discovered in Guyana, Pseudotulostoma volvata, resembles a gasteromycete, and a second species of Merimbla has been found in Mexico.

The following new scientific names are introduced: Pseudotulostoma gen. nov.; Merimbla humicoloides, Phytophthora pistaciae, P. ramorum, and Pseudotulostoma volvata spp. nov.

Since 1993, a hitherto unidentified Phytophthora species has been found associated with twig blight disease in Rhododendron and, sporadically, Viburnum. The morphology and growth characteristics of fourteen isolates from Germany and the Netherlands were investigated, together with their breeding system, the internal transcribed spacer (ITS) regions of the ribosomal DNA, amplified fragment length polymorphism (AFLP) fingerprints, and isozyme profiles, which were compared to those of a number of outgroup species. Morphologically the isolates are characterized by abundant production of chlamydospores and elongate, ellipsoid, deciduous sporangia with a short pedicel, in which they resemble P. palmivora. However, sporangia were semi-papillate, chlamydospores were much larger and cardinal temperatures much lower than those of P. palmivora. Oogonia with amphigynous antheridia and plerotic oospores were produced in dual cultures with an A2 mating type strain of P. cryptogea. ITS1 and ITS2 sequences of the unidentified species were closest to those of P. lateralis, but differed in three and eight nucleotides respectively from the latter species. AFLP fingerprints and isozyme patterns of malate dehydrogenase (MDH-2) and malic enzyme (MDHP) showed that the isolates formed a homogeneous group, distinct from all examined outgroup species, including P. lateralis. It was concluded that they represent a new Phytophthora species, described here as P. ramorum sp. nov. In pathogenicity tests all isolates of P. ramorum were pathogenic to Rhododendron.

Two non-papillate species of Phytophthora are the principal cause of pistachio gummosis in Iran. Their previous description as P. megasperma and P. drechsleri was re-examined in the light of RFLPs and sequence comparisons of internal transcribed spacer (ITS) regions of rDNA. Both taxa were more closely related to a clade comprising P. sojae, P. cajani, P. vignae, P. melonis and P. sinensis than to the unrelated P. megasperma s. str. or P. drechsleri. The P. megasperma-like isolates from pistachio differed from all the above taxa in morphology, ITS sequence and AFLP patterns, and are described as a new species, P. pistaciae. The P. drechsleri-like isolates from pistachio had identical ITS sequences to those of P. melonis, P. sinensis and P. drechsleri-like isolates from cucurbits in Iran and the AFLP profiles of P. melonis, P. sinensis and the P. drechsleri-like isolates from pistachio were virtually identical. This and other published isozyme and mtDNA evidence suggests these taxa should be considered conspecific and all subsumed within P. melonis with a revised host range and geographical distribution. The relationships of P. melonis and P. pistaciae with other members belonging to the same ITS clade are considered.

The loculoascomycetes are defined by the initiation of ascoma development prior to dikaryotisation and functionally bitunicate asci. Previous molecular studies suggested non-monophyly of loculoascomycetes and consequently, Chaetothyriomycetes and Dothideomycetes were distinguished. We have used sequences of nu SSU rDNA, nu LSU rDNA, and mt SSU rDNA to re-evaluate the monophyly of loculoascomycetes. 13 new sequences of these regions from 10 species, including two representatives of Pezizomycetes used as outgroup, were aligned with sequences obtained from GenBank. A combined data set was analysed phylogenetically using maximum parsimony. The Chaetothyriomycetes and Eurotiomycetes form a sister-group, supported by a bootstrap value of 97%, suggesting that loculoascomycetes are not monophyletic. A topology constrained to loculoascomycete monophyly can be rejected as being significantly worse using parametric bootstrapping. Our results also indicate that mt SSU rDNA sequence data are useful as additional characters to elucidate the phylogeny of ascomycetes at the rank of different classes. Additional data from more representatives of these groups and other ascomycetes are required to clarify whether the loculoascomycetes are a paraphyletic or polyphyletic assemblage.

The Laboulbeniales have had a clouded taxonomic history. Initially interpreted as abnormal cuticular outgrowths of arthropods, they have since been considered to be acanthocephalans, basidiomycetes, and zygomycetes as well as ascomycetes. Phylogenetic analysis of partial sequences of SSU rDNA with species of Chytridiomycota as outgroup taxa, and including trichomycete and entomophthoralean Zygomycota, provides evidence of the placement of a Laboulbeniales-Pyxidiophora clade among the Ascomycota. Support for parsimony analyses came from maximum likelihood ratio tests applied to trees with ten different topological constraints. The placement among Ascomycota, excluded from Sordariomycetes, is well supported, and indicative of a distinct lineage, class Laboulbeniomycetes. At this time, however, we are unable to discover close relatives of the Laboulbeniomycetes clade among other filamentous ascomycetes.

In a polyphasic classification approach, stromata and cultures of Daldinia and allied Xylariaceae from around the world were studied for: (1) morphology of teleomorphs and anamorphs; (2) metabolite patterns in stromata and cultures, employing analytical HPLC-UV-visible (diode array) detection and mass spectrometric detection; (3) amplified 18S rDNA restriction patterns (ARDRA); and (4) PCR amplified minisatellite regions. Comparison of type material, herbarium specimens, cultures, and freshly collected material revealed new evidence on their geographic distribution. Several Daldinia spp. were identified in Europe and other locations for the first time. The results point towards the existence of further undescribed species. Stromata of a given species never contained the same major metabolites as corresponding cultures. Most cultures of Daldinia spp. produced naphthalene and chromane derivatives, differing from allied genera by the absence of mellein. Stromata of Daldinia spp. did not produce mitorubrin, but generally contained binaphthyls. Metabolite profiles were correlated with colours of KOH-extractable stromatal pigments. The yellow azaphilones and benzophenones found in D. childiae were lacking in species with purple stromatal pigments. Cytochalasins were found in stromata of D. eschscholzii. Genetic fingerprints helped to distinguish morphologically closely related taxa. ARDRA gave specific results for species whose 18S rDNA contained insertions, while minisatellite PCR provided specific genetic fingerprints. A combination of both PCR based techniques provided a fair resolution of genetic subtypes, reflecting the intrageneric variance in Daldinia as established from morphological data and secondary metabolite profiles.

Evidence that the biometric characters of the oogonial unit of Pythium are of significant taxonomic value, allowing for a good species separation, has been presented by other workers, based on 40 species, each represented by fairly few isolates. Since broad intraspecific variation and interspecific overlapping of biometric parameters was observed in isolates of four Pythium species from Denmark, it was suspected that a such wide variability would weaken the conclusions obtained by previous biometric approaches. Species to be studied should be represented by higher numbers of isolates to reflect intraspecific variation. In the present biometric study 49 isolates were used to test the robustness of the biometric approach and to examine the taxonomic value of biometric characters from a practical perspective.

Good species separation was obtained despite intraspecific variation. The biometric parameter set for the oogonium, consisting of oogonial, oospore and ooplast diameters, supplemented with the thickness of the oospore wall or the protoplast diameter and with derived indices of volume or linear ratios of the measured parameters were all of taxonomic value for the separation of species, as shown from canonical variate plots after linear discriminant analysis. More detailed information on species separation, isolate-species or isolate–isolate associations could be obtained by separation based on the Mahalanobis distance function in the linear discriminant analysis. Both the derived volume and linear indices contributed significantly to species separation and appeared to be equally satisfactory.

The substrates used for oogonium production may influence the biometric parameters to an extent which may seriously affect the results of the biometric approach to taxonomy. These results stress the importance of standardizing substrates in morphological taxonomy and identification work.

Development of the powdery mildew fungus Sphaerotheca fusca on leaves of susceptible, resistant and temperature-sensitive resistant melon cultivars at 21 °C and 26 ° was examined by light and scanning electron microscopy. On the susceptible cv. ‘Rochet’ the time course of the infection process was similar at each temperature. Development of powdery mildew colonies paralleled the progressive branching of mycelia and conidiophore formation. On the resistant cv. PMR-6, however, development was arrested at the primary appressorium stage irrespective of the temperature. When the temperature-sensitive resistant line ANC-57 was infected by a race 1 isolate, incubation temperature had a clear effect on the outcome of the interaction. At 21 °, it took longer for symptoms to appear on cv. ANC-57 than on cv. ‘Rochet’, while colonies were limited and sparse. At 26 ° resistance was complete, there being no development.

We screened a cDNA library generated from harvested and stored sporophores of Agaricus bisporus and identified 19 genes with higher transcript levels than at the time of harvest. Five of these genes had no detectable mRNA levels prior to detachment from the mycelium. Sequence analysis of ten clones revealed significant similarities to known genes, these code for proteins involved in polymer breakdown and metabolism, cell wall synthesis, stress tolerance, cytochrome P450 activity and DNA binding. The diversity of functions of these genes suggests the changes in the sporophore after harvest involve several different physiological processes.

Sporocarps of Arcangeliella borziana, a hypogeous russuloid fungus, were found in a subalpine spruce forest in the Swiss Alps. We identified the ectomycorrhizas formed by this species using the ITS–PCR–RFLP method and described them in terms of morphological, anatomical, and molecular features. Phylogenetic placement of A. borziana was studied by comparing nucleotide sequences of the mitochondrial large subunit rRNA gene and the ITS region of nuclear rRNA, respectively, with existing sequence databases, and by comparing the ITS–RFLP pattern with a reference library of approx. 180 mainly basidiomycetous fungi. Sequence analysis of the mitochondrial large subunit rRNA gene confirmed the affiliation of A. borziana to the Russulaceae. Sequence as well as RFLP pattern comparison of the ITS region revealed a close affinity of this species to Lactarius sect. Russularia. This is in accordance with morphological and anatomical studies of the ectomycorrhizas, which share many features with ectomycorrhizas of Lactarius sect. Russularia.

Neotyphodium endophytes have not been reported within vascular tissue of grass hosts even though hyphae were observed in close proximity to vascular bundles. However, examination by light microscopy and transmission electron microscopy of a range of natural and artificially introduced associations involving Neotyphodium spp. has now shown that hyphae of these endophytes can colonize vascular tissue. In natural associations, hyphae were seldom present within vascular bundles and if present the number was low. Small vascular bundles lacking a bundle sheath were more likely to contain hyphae than large vascular bundles that were surrounded by a bundle sheath. Examination of a range of Lolium×hybridium (hybrid rye-grass) and L. multiflorum (annual rye-grass) plants, as well as F. arundinacea (tall fescue) and F. pratensis (meadow fescue) plants, that were inoculated as seedlings with either a strain of N. lolii or a strain of an interspecific hybrid Neotyphodium sp., revealed extensively colonised vascular bundles in four Lolium plants. Each of these plants was infected with the N. lolii strain and each had strong L. multiflorum characteristics. The most extensively colonised large vascular bundles were those first formed during leaf growth. Growth within these vascular bundles was continuous along their length. Hyphal extension and branching of hyphae within these vascular bundles ceased when leaf growth ceased. Grasses with extensively colonised vascular bundles were not noticeably stunted and formed inflorescences without external stromata.

In submerged carbon-limited autolysing cultures of Penicillium chrysogenum, round-ended yeast-like fragments became the dominant surviving morphological forms and a new equilibrium between cell lysis and growth was observed, termed as a phase of ‘cryptic growth’. The formation of yeast-like fragments and their cryptic growth represented the only way that enabled the fungus to cope with harsh environmental conditions and survive. The surviving morphological forms reverted to vegetative growth after carbon supplementation. The glutathione metabolism and formation of reactive oxygen species (ROS) were influenced by numerous endogenous and exogenous factors depending on the phase of growth. Consequently, no firm correlation between the redox status, ROS levels and the observed morphological and physiological characteristics of the cells was found. The Dioxygen Avoidance Theory of Cell Differentiation failed to explain the complex physiological changes observed prior to and during autolysis, and any possible intrinsic analogy between the autolysis of P. chrysogenum and the oxidative stress dependent apoptosis of higher eucaryotes was also questioned. Nevertheless, ageing yeast-like fragments accumulated ROS as a function of incubation time and seem to be promising objects for future apoptosis research in filamentous fungi.

Soon after coming in contact with its host, the plant pathogenic fungus Bipolaris sorokiniana produces an extracellular material that appears to be important for adhering conidia and germlings to the host surface. To further understand this step of the infection, the adhesion of B. sorokiniana to artificial solid surfaces was examined. On a hydrophobic (polystyrene) surface adhesion occurred in two stages, the first by conidia and the second by germlings. Conidial adhesion occurred shortly (0–1 h) after hydration. The conidia were easily detached by increasing the shear force and including detergents in the washing buffer. As conidia were hydrophobic, these observations indicate that conidial adhesion to polystyrene is due to weak, hydrophobic interaction. The second stage of adhesion was accompanied by conidial germination and occurred 1–2 h after hydration and contact with the surface. Concomitant with the delayed adhesion, the fungus produced an extracellular matrix (ECM). The adhesion of germlings was firm and surface-unspecific since they adhered to both hydrophobic and hydrophilic (glass) surfaces. Except for strong bases, hydrochloric acid and broad-specificity proteases (including Pronase E), none of the hydrolytic enzymes, electrolyte solutions, ionic and hydrophobic detergents and organic solvents removed germlings from the solid surfaces. The adhesion of germlings incubated in the presence of the protein glycosylation inhibitor tunicamycin or the lectins Con A (Concanavalin A) and GNA (from Galanthus nivalis) was significantly reduced, which indicates the involvement of surface glycoproteins in this process. The surface proteins of germlings were labelled with 125I, extracted and analysed by two-dimensional gel electrophoresis. This revealed about 40 surface proteins over a wide pH range (4–10) with molecular masses between 10 and 100 kDa.

The ability of commercially-available citric, oxalic and gluconic acids to bind Co2+ and Zn2+ was investigated and compared with culture filtrates from Aspergillus niger, a fungus capable of citric, gluconic and oxalic acid production, grown in the presence and absence of cobalt or zinc phosphate. This work demonstrated that citric and oxalic acid and the A. niger culture filtrates can bind Co2+ and Zn2+ and in some cases, the culture filtrates were more efficient than commercial organic acids. Gluconic acid did not bind Co2+ or Zn2+ under the conditions used in this study. The presence of insoluble metal phosphates in the growth medium was found to markedly influence the production of organic acids and, while large concentrations of gluconic acid were produced in the presence of Co3(PO4)2, the culture filtrate was unable to bind Zn2+. The production of oxalic acid by A. niger when grown in the presence of Zn3(PO4)2 led to the precipitation of insoluble zinc oxalate, a phenomenon with implications for metal tolerance and toxicity. The significance of these findings in relation to the environmental mobility of metals and phosphate, and the role of fungi in such transformations are discussed.

Pseudotulostoma volvata gen. sp. nov. is described from the south-central Pakaraima Mountains of Guyana. Pseudotulostoma volvata is associated with ectomycorrhizal Dicymbe corymbosa trees (Caesalpiniaceae) and placed in the Ascomycota, Eurotiales, Elaphomycetaceae. Included are a description of the genus and species, illustrations of the macroscopic and microscopic features, and a discussion of the distinctive features and phylogenetic placement of this fungus.

Merimbla humicoloides sp. nov. was isolated after heat treatment of soil collected in a pine forest of Veracruz state, Mexico. The fungus is characterized by pale buff to cinnamon colonies that become dark brown to black in reverse and conidiophores that vary from irregularly asymmetrical penicilli to symmetrical penicilli with inflated metulae, and Humicola-like chlamydospores on the submerged hyphae. Phylogenetic inferences made from the ITS1-5·8S-ITS2 rDNA sequences indicate that M. humicoloides is a member of the Trichocomaceae and that it is related to M. ingelheimensis, Hamigera avellanea, and Penicillium species.