This paper reports the first biochemical characterisation of Tuber borchii mycelial strain ATCC 96540, grown in liquid media containing either glucose, fructose or sucrose. A new biochemical method, based on the estimation of total protein content, was developed for determining the amount of mycelium growth. This method is more sensitive than other methods, allowing growth to be monitored in the lag phase and when small amounts of mycelium are grown on a solid medium. Mycelium of T. borchii utilizes glucose and fructose as carbohydrate sources but grows poorly if at all on sucrose. In these experiments the functional state of the mycelium was evaluated by determining the activity of enzymes of the glycolytic and pentose phosphate pathways which are involved in producing energy and in supplying reducing power through the formation of reduced pyridine coenzymes. The biochemical data on mycelium growth were supported by observations on ultrastructural morphology which revealed the different steps in hyphal depletion during ageing. In addition, the monitoring of alanine content of the liquid media during mycelial growth indicated that there was an increasing loss of alanine from their cytoplasm as hyphae aged. The development of this procedure makes it possible to identify the conditions under which T. borchii is able to synthesize a mycorrhiza with a compatible host.

Twenty-four strains of six Cylindrocladiella spp. were compared with respect to their morphology, nuclear ribosomal DNA (nuclear rDNA) polymorphisms, and restriction fragment length polymorphisms of A+T-rich DNA (AT-DNA). Two distinct DNA profiles were observed for the ten strains of C. camelliae/peruviana-complex studied. One profile corresponded with the type strain of C. peruviana, which was earlier regarded as a synonym of C. camelliae. Based on Southern analyses and AT-DNA, C. parva, C. elegans and C. lageniformis were confirmed to be distinct species with little intraspecific variation. Although C. infestans was shown to have some variation among the four strains studied, they could not clearly be distinguished as separate taxa. Based on general morphology, nuclear rDNA and AT-DNA polymorphisms, seven species of Cylindrocladiella are recognized.

Anthostomella petrinensis and A. theobromina are described from endemic Pandanus species in Mauritius. They are compared with other Anthostomella species reported on Pandanaceae and Palmae. A table is included, facilitating comparison between the new species and previously published taxa with similarly sized ascospores.

The effects of varying carbon (glucose), nitrogen ((NH4)2SO4, KNO3) and phosphate (KH2PO4) source on solubilization of insoluble Co3(PO4)2.8H2O, Zn3(PO4)2.2H2O and ZnO by the soil fungus Aspergillus niger were assessed. Solubilization activity was quantified by measuring the clear zones produced around colonies of A. niger growing on solidified mineral salts medium amended with the insoluble metal compounds. Effects of nutrient variation on solubilizing properties were compared using ratios of colony growth rate on the metal compounds (Rm) to control growth rate (Rc) and the rate of extension of the zone of solubilization (Rs) compared to the colony growth rate on the metal compound (Rm), i.e. Rm[ratio ]Rc and Rs[ratio ]Rm. Ratios of solubilization rate to growth rate (Rs[ratio ]Rm) on all the compounds decreased with decreasing glucose concentration; there was no solubilization of ZnO below 60 mm glucose and no solubilization of the metal phosphates below 6 mm glucose. Reducing the concentration of ammonium sulphate in the growth medium decreased Rs[ratio ]Rm but these values were increased when the nitrogen source was nitrate. Reducing the phosphate concentration increased solubilization of Co3(PO4)2 but reduced solubilization of Zn3(PO4)2. These findings demonstrate that manipulation of carbon, nitrogen and phosphate sources in the growth medium, and variation of the form of the nutrient source, can be used to alter the solubilizing ability of A. niger. Whilst, in the natural environment, this response to different nutrient sources allows optimal exploitation of resources, the potential to manipulate nutrients for maximum solubilizing ability may prove beneficial for the optimization of the solubilization of metal compounds with respect to the bioremediation of metal-contaminated wastes and polluted ecosystems. It could also prove useful in other biotechnological applications such as metal recycling and extraction of metals from low-grade ores.

A new species of Penicillium, Subgenus Biverticillium, was isolated from the root of a wheat seedling during a survey of the ability of rhizosphere fungi to solubilize phosphate. The species, described here as Penicillium radicum, has affinities with P. variabile, P. allahabadense and Talaromyces wortmannii, but can be distinguished by differences in morphology, secondary metabolite profiles and unique DNA banding patterns obtained using RAPD PCR.

Rotiferophthora ellipsospora and R. japonica spp. nov., are described as endoparasites of bdelloid rotifers, forming large dictyochlamydospores and small infection conidia. Verticillium bactrosporum was found in association with a new host species of bdelloid rotifer and was cultured for the first time: resting spores were formed in culture.

Two rapid, independent molecular assays have been developed for discriminating between the closely related rusts of hard pines, Cronartium flaccidum and Peridermium pini. A portion of the ITS region and a portion of the IGS region from the ribosomal RNA operon of the two organisms were amplified with the PCR. Amplifications were made using DNA extracted from aeciospores collected from unopened aecia, taking a small number of spores as a source of template DNA. Amplified fragments were subjected to RFLP or to SSCP analysis. Digests of the amplified products from the IGS1 region were electrophoresed on polyacrylamide gel and stained with ethidium bromide. Hinf I digestion of these fragments created polymorphic restriction profiles which allowed differentiation of the autoecious P. pini from the heteroecious C. flaccidum. Similar-sized DNA fragments representing the ITS2 region of the two rusts were denatured, subjected to electrophoresis as single strands on polyacrylamide gel under non-denaturing conditions and ‘silver stained’. The different mobility displayed by these short fragments revealed sequence polymorphism in the examined portion of the ITS region. This technique therefore represents an accurate and sensitive method for detection of base changes in given sequences of genomic DNA. A high level of homology was found between the two biotroph organisms in the loci screened. Results obtained in this trial indicate that PCR-RFLP and PCR-SSCP can be used as simple, speedy taxonomic tools for elucidating relationships among related organisms.

Norway spruce needles were sampled from two series of stand areas located in southern Finland. Both series consisted of five sampling areas in mature managed stands and one in a mature virgin stand. The proportion of spruce varied from 8 to 100% of the basal tree area and the major other species were pubescent birch and Scots pine. From each sampling area (some of which consisted of several sites) 40 mature spruces were randomly chosen and healthy looking needles of the third age class were sampled from heights of 5–8 m and incubated on water agar for isolation of endophytic fungi.

The majority of isolates were identified by their combined fatty acid and sterol profiles (FAST-profiles) as Lophodermium piceae. Tiarasporella parca was less common and Rhizosphaera kalkhoffii, Sclerophoma pythiophila, Lirula macrospora and Thysanophora penicillioides occurred occasionally. For calculations of fungal diversity all isolates were classified by their FAST-profiles into 81 groups (=operational chemotaxonomic units) according to a defined upper variation limit, i.e. an upper FAST-profile mismatch limit.

The highest percentage of endophytically infected needles was found in pure spruce stands and dense virgin stands. Location of the stand, its proportion of spruce and total basal area of trees (i.e. tree density) explained 82% of the variation of the overall infection rate. The effect of location was probably a consequence of differences in air quality between the various sampling areas.

The endophyte diversity, expressed as the number of FAST-groups per 40 spruces of each sampling area, correlated positively and statistically significantly with the percentage of needles infected or with the proportion of spruce in the stand. The highest endophytic diversity, expressed as FAST-groups per tree, was found in pure spruce and mixed virgin stands.

An in vitro screen of 83 isolates of coprophilous fungi revealed that many of them were capable of stimulating growth of Sphaeronaemella fimicola at a distance. When in direct contact with S. fimicola, many (excluding the Zygomycotina) also stimulated the production of perithecia. Species of Coniochaeta, Ryparobius, Thelebolus and Trichobolus were the strongest inducers. These and related genera were also associated with perithecia of S. fimicola on dung collected from the field. Hyphae of many of the strong perithecium inducers and a few non-inducing species were parasitized by necrotrophic intrahyphal growth of S. fimicola. Perithecium formation was stimulated even when parasitism was prevented by separation of growing mycelia of Ryparobius pachyascus and S. fimicola by Cellophane. In contrast, prior growth and removal of R. pachyascus followed by inoculation with S. fimicola caused enhanced growth and asexual reproduction without concomitant perithecium formation.

Dicellomyces calami sp. nov. was collected on rotang palm in southern India. It is the first Dicellomyces species on palms. A key to the known Dicellomyces species is presented and possible relationships of the genus are discussed.

Candelabrum desmidiaceum sp. nov. and C. clathrosphaeroides sp. nov. are illustrated and described. Morphologically, they represent a new type of conidium development previously unknown in Candelabrum: the formation of a basal plate by dicho- or trichotomous branching of the conidial body in combination with branches growing upwards at the outer margin of the basal plate which are distinctly verrucose outside, forming a hollow sphere enclosing an air bubble in the centre. In Austria, both species were found mainly on submerged beech leaves, but in the U.S.A., C. clathrosphaeroides was isolated from submerged leaves of many different deciduous species, indicating a wider host range. Both species are aero-aquatic fungi, and C. clathrosphaeroides, at least, is widely distributed in temperate climates. A key to the species of Candelabrum is provided.

Astrocystis fimbriata, A. rarissima and A. cepiformis spp. nov. are described and illustrated from dead leaves of Pandanus. Their affinities with related taxa are discussed, especially with Astrocystis species on Palmae and with Rosellinia as interpreted in recent studies. One species has stromata which develop from a mat of superficial hyphae, which has not previously been reported for Astrocystis.

Eleven cultures representing eight genera of five families of Loculoascomycetes were obtained from ATCC and IMI. Partial 18S and 28S nuclear ribosomal DNA (rDNA) were amplified using PCR and sequenced. About 1100 bps of 18S rDNA (NS1/NS4 region) and 459 bps of 28S rDNA (F63/R635 region) for each taxon were subject to phylogenetic analysis using parsimony. The reconstructed phylogenetic trees supported the separation of two monophyletic groups, containing the type genera of the Leptosphaeriaceae and the Pleosporaceae, respectively. The members of the Leptosphaeriaceae (sensu Barr) consisted of the paraphyletic clade in the phylogenetic trees. The members of the Pleosporaceae (sensu Barr) plus Pyrenophora trichostoma formed a monophyletic clade. The results generally supported the separation of the Leptosphaeriaceae from Pleosporaceae and the placement of P. trichostoma, and Comoclathris baccata in the Pleosporaceae. The taxonomic position of Pleospora betae, whose coelomycetous anamorph is more closely related to Leptosphaeriaceae, was not well resolved because of low bootstrap values and decay indices. Leptosphaeria bicolor, whose ascospore morphology is not congeneric with the type of the genus, L. doliolum, does not belong to Leptosphaeria based on the 18S sequence data. The taxonomic disposition of Lewia, Pyrenophora, and Comoclathris based on the phylogenetic analyses are discussed.

Cladobotryum dendroides, causal agent of cobweb disease of Agaricus bisporus, has become increasingly resistant to methylbenzimidazole carbamate (MBC) fungicides following the extensive use of MBC in cultivated mushroom production in Ireland. Of 38 isolates of C. dendroides obtained from Irish mushroom units, 34 were resistant to carbendazim. Primers based on conserved regions of the β-tubulin gene were used to amplify and sequence a portion of the β-tubulin gene in C. dendroides. A point mutation was detected at codon 50 in isolates resistant to benzimidazole fungicides, causing an amino acid substitution from tyrosine to cysteine. Species-specific PCR primers were designed to amplify the region of the β-tubulin gene containing this substitution. The point mutation removed an Acc I restriction site in the β-tubulin gene sequence of resistant isolates. Digestion of the PCR product with Acc I thus provides a rapid diagnostic test to differentiate sensitive and resistant isolates of this fungus. EMBL accession number: Y12256.

Hemibeltrania saikawae sp. nov., found on decaying leaves in Cuba and characterized by cylindrical to clavate, mammillate, one-celled conidia, is described and illustrated. A key to Hemibeltrania species is provided.

Assessment of diversity (including both species richness and intraspecific variation) within a habitat may often be difficult when morphological criteria are used for identification and classification of isolates. Here we describe how combined fatty acid and sterol profiles (FAST-profiles) can be used for classification of fungal isolates into FAST-groups (i.e. operational chemotaxonomic units) according to a defined upper variation limit. Data analysis includes a two stage statistical approach. First, the isolates are grouped into taxonomic species using a discriminant model based on well identified ‘model’ isolates. These species groups together with one group that includes isolates which could not be identified with the discriminant model are further divided into operational chemotaxonomic units using a FAST-profile mismatch threshold. This method is demonstrated with Norway spruce needle endophytes.

The model isolates were obtained from fallen spruce needles and identified by morphological criteria. Five species fruited on the needles: Lophodermium piceae, Rhizosphaera kalkhoffii, Lirula macrospora, Tiarosporella parca, and Thysanophora penicillioides. Additionally, mycelia of Sclerophoma pythiophila were frequently noted. These model isolates were separated by their FAST-profiles by applying a multivariate discriminant model into six distinct non-overlapping clusters corresponding to their species. This model was used for identification of 1740 isolates obtained from surface sterilized Norway spruce needles. As an example, isolates identified as T. parca were further divided into FAST-groups by applying a FAST-profile mismatch threshold value. This threshold value was the average within-species mismatch-value obtained for isolates identified by morphological criteria. This method allows a division of any batch of fungi cultivable in vitro into operational chemotaxonomic units according to a defined upper variation limit.

Colletotrichum xanthorrhoeae sp. nov. causes leaf spots on Xanthorrhoea preissii and X. platyphylla in south-western Western Australia.

Zoospore cysts of four isolates of Aphanomyces euteiches with different host-specific pathogenicity germinated in response to peptone, pea root extract, CaCl2 or gum arabic but, in tests on one isolate, did not respond to several individual sugars, amino acids or polysaccharides. A. euteiches showed differential responses to substances applied at different times in the encystment process. Gum arabic triggered germination only when added to motile zoospores, which it caused to encyst. CaCl2 triggered germination when added to motile zoospores or during vortex-encystment of zoospores, but seldom immediately (<2 min) after encystment. Peptone and pea root extract triggered germination when added to motile zoospores, during vortex-encystment or immediately after encystment, but not ca 20 min after encystment. In comparative tests, Phytophthora parasitica was less stage-specific than A. euteiches, because it germinated in response to gum arabic applied to motile zoospores, during encystment and immediately after encystment.

Zoospore taxis, encystment and germination of A. euteiches and P. parasitica cysts were recorded on pea roots by video microscopy. The speed of germination and pattern of sporeling development on roots were most closely matched by treatment of motile spores with gum arabic in pure culture. The behaviour of A. euteiches conformed to a previous model for Pythium and Phytophthora spp., in which zoospores encyst by recognition of root surface components (simulated by gum arabic), germinate autonomously by a calcium-mediated process (simulated by calcium treatment during vortexing), and the germ-tube produces an assimilative hypha (simulated by peptone or pea root extract).

Genetic variation in Alternaria brassicae, A. brassicicola, and A. raphani collected from geographically diverse regions of the world was studied with RAPD and RFLP markers. Twenty 10-mer primers of arbitrary nucleotide sequences were tested for amplification of genomic DNA of A. brassicae using PCR. Of these, five primers amplified the genomic DNA from 20 A. brassicae isolates and produced reproducible RAPD profiles. UPGMA analysis of RAPD data showed that isolates collected from geographically distinct regions could be broadly classified into four groups. Intra-regional variation between isolates was less apparent. Variation was, however, higher in A. brassicicola, as based on RAPD analysis. Two isolates (from Canada and France) of A. raphani also showed variability with different RAPD profiles generated by all five primers tested. Five polymorphic, distinct RAPD products were used as hybridization probes for RFLP analysis to detect inter- and intra-specific variation. Variation among A. brassicae, A. brassicicola, and A. raphani was evident. Non-radioactive probes were also used to hybridize with Southern blots of A. brassicae, A. brassicicola, Leptosphaeria maculans, Rhynchosporium secalis and Brassica juncea for the selection of A. brassicae-specific probe(s). Probe AbP3 specifically hybridized with restriction digests of A. brassicae but not with those of A. brassicicola or other tested species. This probe should, therefore, be useful for distinguishing between two important pathogens of crucifers, i.e. A. brassicae and A. brassicicola both in culture and infected tissues.

Two new taxa from rain forest in Cuba are described and illustrated. They are Endophragmiella sphaeroproliferata anam. sp. nov., characterized by cymbiform, 3-septate, pale brown, smooth conidia, and Sporidesmiella intermedia anam. sp. nov., distinguished by obovoid, 2–5 distoseptate, pale brown to subhyaline conidia. A comparative tabulation of the described species of Sporidesmiella is provided.