Protostelids, dictyostelids and myxomycetes are well known from terrestrial ecosystems in temperate regions but poorly known from the tropics. This study examined the distribution and ecology of these three groups of ‘slime moulds’ in the forest floor litter microhabitat of five different forest types (tabonuco forest, secondary tabonuco forest, palo colorado forest, palm forest, and elfin forest) within the Luquillo Experimental Forest in Puerto Rico. Protostelids associated with aerial litter (dead but still attached plant parts) also were studied. At least 13 species of protostelids, 13 species of dictyostelids and 24 species of myxomycetes were recovered. Both species richness and abundance of dictyostelids and myxomycetes were higher in the secondary tabonuco forest than in any of the other forest types. The tabonuco forest yielded the most species of protostelids, but the next highest total was recorded from the secondary tabonuco forest. Both of these forest types are located at the low end of the elevation gradient. Lowest levels of species richness and abundance occurred in the elfin forest, located at the high end of the elevation gradient. The general pattern observed was that of decreasing diversity with increasing elevation.

The ketosynthase domains of putative polyketide synthase (PKS) genes from 15 species in the lichenized genus Lecanora as well as three representatives of other genera were amplified and sequenced using conserved primers. A phylogenetic analysis was carried out with the corresponding amino acid sequences, including those of non-lichenized fungi. The phylogenetic hypothesis places all PKS sequences from Lecanora and the other genera in a clade of PKSs that produce complex aromatic compounds. The PKSs from Lecanora are found in two distinct clades. One of these forms a monophyletic group with PKSs producing precursors of dihydroxy naphthalenes from non-lichenized species. This includes PKSs from the L. rupicola group and several other species which are not closely related. The other clade represents at least one functionally different protein and has no close relationships with known PKSs from other fungi. A comparison between an ITS phylogeny of the species with their DNA sequences of ketosynthase domains reveals similar PKS genes in certain closely related species. Coevolution was unapparent in other clades, suggesting the presence of paralogous genes.

Species of Melanospora are parasitic on or closely associated with other fungi. The morphological characters traditionally used to place Melanospora and the Ceratostomataceae in Sordariales do not agree with their placement in our phylogenetic study. The nuclear encoded small subunit ribosomal DNA (SSU nrDNA) grouped four species of Melanospora and several allied genera and species in Hypocreales, an order rich in mycoparasites. An analysis based on characters derived from deduced amino acids from the second largest subunit of the RNA polymerase II (RPB2) gene further supports the close relationship of Melanospora and Hypocreales. The nuclear encoded large subunit ribosomal DNA (LSU nrDNA) phylogeny inferred six groups for 40 species in Hypocreales and allied genera (Bionectria, Claviceps, Hypocrea, Nectria, and Niesslia). Four species of Melanospora, including the type, and two Sphaerodes species formed a basal clade. Scopinella was placed in a distinct clade. Two other allies of Melanospora, Syspastospora and Persiciospora, were closely related to or within the Hypocrea and Nectria groups, respectively. The study argues for the reclassification of taxa that previously were placed in the Sordariales.

Two methods are currently being used to describe ECM fungal communities associated with root tips: molecular techniques and morphological classification. Previous studies have found that these two approaches give conflicting results, with several fungal genotypes being identified from different ectomycorrhizas within the same morphotype. This has led researchers to question the usefulness of the morphological approach. The objective of this study was to compare the two approaches on ectomycorrhizas collected from three plant species growing in two different environments. Specifically, mycorrhizas were classified using a detailed morphological approach and then were subjected to PCR-RFLP analysis of the ITS region of the rRNA gene repeat. Ectomycorrhizas of Douglas-fir (Pseudotsuga menziesii) and paper birch (Betula papyrifera) were sampled from three widely dispersed sites with different soil types in the southern interior of British Columbia. Ectomycorrhizas of Douglas-fir and arbutoid mycorrhizas from Arctostaphylos uva-ursi were sampled from a fourth site in a different biogeoclimatic zone. For eight of eleven dominant morphotypes, one main RFLP banding pattern was observed. Ninety-three % of the mycorrhizas analyzed in these eight morphotypes would have been classified in the same way by either method. Five of the eight morphotypes were positively identified as Russula nigricans, Lactarius sp., Leccinum scabrum, Rhizopogon sect. Villosuli, and Thelephora terrestris by matching the RFLPs to those of fungal fruit bodies in our database or by sequencing the ITS region. The other morphotypes producing one dominant RFLP pattern were designated as Cenococcum, E-strain and Mycelium radicis atrovirens (MRA) based on their morphology. Morphotyping did not distinguish amongst major RFLP types for mycorrhizas classified as Amphinema-like, Piloderma-like and Rhizopogon-like A. We conclude that detailed morphological classification can be very useful as the primary method of ectomycorrhizal classification, when used in conjunction with molecular techniques. This approach will allow for an efficient use of research funds.

Twenty-nine field isolates of the broad host range pathogen Claviceps purpurea from various host plants and different geographical origin were compared by RAPD analysis. The RAPD patterns obtained showed considerable diversity; it was even possible to discriminate all strains tested with single primers, indicating an unusual high degree of genetic diversity within this species. A parsimony analysis using the PAUP software grouped most strains from specific host plants together, indicating some degree of host specificity in this fungus. Those strains derived from rye seem to be genetically less diverse than those from other host plants. However, the geographic origin also seems to be important.

Mycosphaerella ambiphylla sp. nov. (anamorph: Phaeophleospora) and Mycosphaerella aurantia sp. nov., are described from diseased Eucalyptus globulus leaves. In addition, a new fungal record in Australia, M. mexicana, and two new records for Western Australia, M. gregaria and M. parva, are discussed. A key is provided to Mycosphaerella species on E. globulus in Western Australia.

Dark septate root endophytes (DSE) are an artificial assemblage of fungi that have darkly pigmented, septate hyphae and that are frequent or distinctive intracellular associates of roots of apparently healthy plants. Based on isolates obtained from the roots of Salix spp., the distribution of a common DSE fungus, Phialocephala fortinii, was examined along a latitudinal transect in Canada running from the high arctic to the 49° N parallel. Non-sporulating isolates were provisionally identified as P. fortinii through analysis of DNA sequence data of the ITS2 region of rDNA. P. fortinii was isolated frequently from boreal and arctic habitats, but rarely from grassland habitats. Patterns of genetic variation were examined through analysis of amplified fragment length polymorphisms (AFLP). All AFLP profiles were unique with the majority of genetic variation occurring among individuals within the collecting sites at each latitude. Neighbour-joining analysis of genetic distances yielded eight well-supported clusters, three of which included individuals from more than one latitude. Some linkage disequilibrium, possibly due to partial clonality, was detected.

Vegetative growth rate, and size of sporangia, chlamydospores and oospores from 94 P. ramorum isolates were measured and the isolates were paired in vitro with four different heterothallic Phytophthora species isolated from infected nursery plants in Germany. P. ramorum isolates originated from different European countries and from Canada and the USA. 66 of the 67 European isolates were determined as mating type A1; only one isolate was of mating type A2. Of the 27 North American isolates tested, seven (all from nurseries) were determined to be the A1 mating type and 17 to be the A2 mating type. Three isolates did not produce gametangia during the incubation period. Discriminant analysis of all data allowed a grouping based on the vegetative growth rate. The two groups corresponded with the mating type no matter whether the isolates originated from Europe or North America. The A1 isolates were much more homogeneous in their morphology than the A2 isolates. They grew faster, had larger chlamydospores and did not produce gametangia with P. cambivora. Within the A2 group, the single European isolate of mating type A2 (BBA 16/02) and three US isolates showed intermediate characters and were classified with the discriminant function into that of the opposite mating type. The morphological characters and the mating behaviour of the isolates will be discussed.

Three new Cortinarius species, Cortinarius conopileus, C. keralensis, and C. phlegmophorus spp. nov., are described from Kerala State in southern India. This is the first record of ectomycorrhizal Cortinarius spp. in the tropical part of India. In addition to distinct morphological characters, the comparative analysis of rDNA ITS sequences of the collections from India and morphologically similar species support the recognition of these taxa as new species. Phylogenetic analyses demonstrate that the three Indian Cortinarius spp. belong to both larger subclades of the genus Cortinarius, clade/cortinarius and clade/telamonia. As supported by morphological and molecular data, C. phlegmophorus belongs to Cortinarius subgen. Myxacium sect. Defibulati. Based on classical morphological characters, both C. keralensis and C. conopileus are representatives of subgen. Telamonia. However, C. conopileus belongs to clade/obtusi, which is a well-supported subclade of clade/cortinarius. Thus, in contrast to classical taxonomy, the clade/obtusi represents an independent evolutionary origin of telamonioid taxa. This result is also reflected by the distinct morphological characters of taxa of clade/obtusi, namely the lamellar trama with ellipsoid inflated hyphae and the presence of cystidia. In contrast, C. keralensis is a typical member of clade/telamonia. Within/telamonia, only relationships of closely related taxa are resolved due to the low genetic divergence found in ITS sequences. Based on morphological and molecular criteria, C. keralensis is a distinct taxon of sect. Saturnini.

The ITS II and the first part of the LSU rDNA were amplified from 26 isolates within the genus Entomophthora. The specificity of the primers allowed the use of both in vivo and in vitro material. Size polymorphism and long amplification of the ITS II regions, ranging from 1200 to 2000 bp, were observed. The PCR-products were cut with eight different restriction endonucleases and analysed by UPGMA, one analysis from each of the two regions. Conidial morphology was of predictive value for the overall taxonomy of the genus Entomophthora, as the genus clustered together in the analysis of the LSU rDNA. In both analyses the E. muscae complex clustered into three different clades, which support the validity of E. schizophorae and E. syrphi as separate species. Considerable variation was detected in the E. muscae clade, but it could not be grouped by host, geographic origin or conidial morphology, though the E. muscae s. str. isolates in both analyses grouped together. One isolate with E. muscae-like conidia found on Hymenoptera clustered out within the E. muscae clade, widening the host range for E. muscae significantly.

DNA sequence alignment of the nuclear 5·8S ribosomal RNA (rRNA) gene and internal transcribed spacers (ITS) from Beauveria bassiana demonstrated that 6·62% sequence variation existed between nine isolates. A higher level of mutation was observed within the ITS regions, where 8·39% divergence occurred. Polymerase chain reaction restriction fragment length polymorphism, PCR–RFLP, and DNA sequence alignment of the ITS1 and ITS2 regions identified seven polymorphic restriction endonuclease sites, Alu I, Hha I, Hinf I, Sin I, Tru 9AI and two Tha I restriction sites. The allelic frequency of each genetic marker was determined from 96 isolates. PCR–RFLP defined 24 B. bassiana genotypes within the sample set, from which eight phylogenetic clusters were predicted to exist. AMOVA and Fst (θ) indicated that no significant correlation existed between B. bassiana haplotype and insect host range as defined by insect order from which each isolate was derived.

Growth and ochratoxin A accumulation by two isolates of Penicillium verrucosum, IBT5010 and IBT12803, were examined in co-cultures with the antagonistic yeast Pichia anomala or baker's yeast Saccharomyces cerevisiae. Each yeast was co-cultured with Penicillium verrucosum in malt extract agar supplemented with 1% yeast extract and incubated at 25°C for 14 d. Ochratoxin A was found to be stable at 25°, whether or not yeast was present. Pichia anomala and Saccharomyces cerevisiae reduced mould growth to about the same extent in vitro. Both yeasts significantly reduced the growth of Penicillium verrucosum isolates at 3×103 yeast c.f.u./plate. In all co-cultures in vitro accumulated ochratoxin A was reduced to below the detection limit (100 ng/plate) by yeast amounts as low as 3×103 c.f.u./plate. In wheat, the growth of Penicillium verrucosum IBT5010, measured as c.f.u., was not reduced in the presence of Pichia anomala, whereas the growth of Penicillium verrucosum IBT12803 was clearly inhibited by this yeast. A similar pattern was observed for ochratoxin A accumulation in wheat when the two mould strains were co-cultured with Pichia anomala. Mycotoxin production was more sensitive to the presence of yeasts than was mould growth. Co-cultivation with yeasts gave no detectable stimulation of ochratoxin A accumulation in agar or wheat.

Basidiomes of 43 samples of eight Amanita species were gathered from different habitats of Hungary. The mineral composition (22 elements) was analysed by the ICP method in three independent replications, and mineral compositions found as discussed and compared. The Amanita species analysed were very different in As-, Cd-, Cr-, Mo-, Mn-, Se- and mainly in V-content. Other elements (Al, B, Ba, Ca, Co, Cu, Fe, Mg, Na, Ni, Sr, Ti, Zn) occur in the basidiomes in balanced concentrations. The K and P contents have the lowest differences. Summarizing all the data (n=43), the average mineral status of species of Amanita can be deduced. The lowest variability measured was for K and P, and the highest for chromium, nickel and vanadium.

Specific, significant accumulation was found only for vanadium, due to the previously demonstrated occurrence of a binding molecule ‘amavadine’ in the basidiomes of A. muscaria. Remarkable Cd-levels were estimated in A. pantherina and A. muscaria (11.4 and 12.3 mg kg−1D.M., respectively). The higher contents of other elements (e.g. K, practically in all species; Se in A. strobiliformis) are analytical facts but, not accumulations.

The mineral compositions of the ectomycorrhizal genus Amanita, of litter decomposing Agaricus and of wood decaying Trametes were compared. Some significant differences were found (AsAmanita<AsAgaricus; CdAmanita<CdAgaricus; CuAmanita<CuAgaricus; PAmanita<PAgaricus; KAmanita>KTrametes; PAmanita>PTrametes) but it seems that the mineral composition of the basidiomes is practically independent of the ectomycorrhizal habit. The specificities of the fungi-tree symbiotic interactions are known, and well documented (higher uptake and transport of certain elements first of all of P), however, the differences found in the mineral components are due to other factors (e.g. substrates, accumulating ability) and not to the mycorrhizal status.

Thirty-three strains of Fusarium species from clinical sources including F. solani, F. oxysporum, F. moniliforme, F. proliferatum, F. subglutinans and F. chlamydosporum were examined for their physiological characteristics and mycotoxin production. Of this collection 29 exhibited growth at 37 °C and 21 were resistant to cycloheximide. Two strains of F. moniliforme and two strains of F. proliferatum produced fumonisins. No strains in this collection produced detectable amounts of trichothecenes. All 18 strains of F. solani tested produced the immunosuppressive agent cyclosporin A. The cyclosporin A-producing ability of clinical isolates of F. solani may influence their pathogenic potential.

Epicoccum purpurascens was evaluated as a biocontrol agent of Sclerotinia head rot of sunflower. The potential of antifungal compounds produced by E. purpurascens as fungicides was also assayed. Treatments consisted of application of E. purpurascens conidia and of partially-purified antifungal compounds produced in broth culture before inoculation with ascospores of S. sclerotiorum. Application of conidia resulted in reduced head rot incidence on greenhouse grown plants, but the application of antifungal compounds had no effect on head rot development. Capitula colonization by E. purpurascens had no negative effects on capitula development. After application of E. purpurascens to field grown plants, capitula were much less colonized than under greenhouse conditions. As a consequence, this treatment had no effect on head rot development in the field when plants were artificially inoculated with S. sclerotiorum. The search for new isolates of E. purpurascens well adapted to the fluctuating conditions typical of natural environments could contribute to achieving an acceptable level of efficacy of this organism as a biological control agent of head rot.

The morphology of two soil-borne Verticillium species, V. dahliae and V. tricorpus, was studied on two semi-selective agar media, in the absence and presence of soil. Morphology of the fungi differed considerably between the media, with respect to presence and shape of microsclerotia, dark hyphae (i.e. short melanised hyphae attached to the microsclerotia) and dark mycelium (i.e. melanised mycelium throughout the colony). On modified soil extract agar (MSEA), a pectate based agar, V. dahliae always had globose to elongate microsclerotia, without dark hyphae or dark mycelium, whereas V. tricorpus always had dark hyphae or dark mycelium, and microsclerotia, whenever present, were globose to irregular in shape. On ethanol agar (EA), V. dahliae had large microsclerotia and abundant dark hyphae, whereas V. tricorpus did not form microsclerotia, but always abundant dark mycelium. For the first time we observed the formation of dark hyphae by V. dahliae to a great extent. In the presence of soil, most characteristics were less pronounced, and V. dahliae microsclerotia were smaller, but V. tricorpus produced large microsclerotia, even when they were absent in pure culture. Morphological characteristics suitable for discrimination between the two species on MSEA plates in the presence of soil were selected and tested with fresh isolates from agricultural fields. The two fungi could be distinguished using qualitative characteristics and microsclerotial size. Molecular analysis and morphology on potato dextrose agar confirmed all identifications made on soil dilution plates.

Mycoparasites collected from aerial parts of the cocoa plant (Theobroma cacao) have shown great promise in the control of black pod, caused by Phytophthora palmivora, and moniliasis, caused by Moniliophthora roreri. However, the ecology of epiphytic mycoparasites is still poorly understood although it has a direct bearing on applied biocontrol practices, ranging from the identification and isolation of promising biocontrol candidates to formulation needs and required application frequency. One objective of this study was to determine the natural abundance of mycoparasites on cocoa flowers and pods in relation to crop development stage and cultivar. For this purpose, native mycoparasites were detected on cocoa flowers and pods using the precolonised plate baiting technique. Furthermore, the survival of an applied Clonostachys rosea isolate on cocoa pods on shaded and non-shaded trees was compared as well as the recolonisation patterns of surface-sterilised pods by native mycoparasites under these conditions. Clonostachys spp. were the most commonly isolated native mycoparasites, followed by Fusarium spp. No differences in the occurrence of native, epiphytic mycoparasites were observed between the three main cocoa cultivars, ‘Criollo’, ‘Forastero’ and ‘Trinitario’, nor between clones within these groups. Thus, a single biocontrol inoculum can be suitable for application to cultivar mixtures of cocoa commonly grown together in a field. Different susceptibility classes of segregating F1 populations of hybrids with resistance against M. roreri and P. palmivora supported similar population levels and taxonomic assemblages of mycoparasites. Therefore, we reject the hypothesis that these antagonists mediate resistance. Mycoparasite abundance and genetic disease resistance to black pod and moniliasis are independent phenomena and should lead to additive effects if employed simultaneously in an integrated disease management programme. The survival of applied C. rosea was not affected by the shading regime or any other meteorological parameter measured. On the other hand, recolonisation of surface-sterilised cocoa pods by most native mycoparasites was faster in the shade. Only Trichoderma spp. colonised pods exposed to direct sunlight faster than shaded ones. The implications for the design of biocontrol inocula and formulation technology are discussed.

Genetic variability of 95 isolates of Beauveria bassiana from different geographical regions and hosts was assessed using RAPDs and ITS-RFLP molecular markers in order to characterize its genetic variability. Clusterings were obtained using UPGMA and Principal Coordinates Analysis. Four groups were identified with RAPDs. A 930 bp fragment was amplified with primers ITS1 and PN16, and polymorphisms were observed with AluI and MspI, generating two main clusters. Results indicate low genetic variability present in the Colombian B. bassiana population, no association to host type or geographic locality, and a population structure that is not clonal. This characterization sets criteria for determination of bio-insecticide potentiality, introduction of foreign strains and improvement of entomopathogens within an IPM program.

Pycnidiospores of Phyllosticta ampelicida germinate only after they become attached to a hydrophobic substratum. They do not readily attach or germinate on hydrophilic surfaces or on nutrient-rich media. The properties of the spore surface that function in attachment and signal mediation for germination were investigated in this study. Pycnidiospores were surrounded by a sheath that bound Concanavalin A (Con A)-TRITC and only weakly bound wheat germ agglutinin (WGA)-FITC. Galanthus nivalis lectin-FITC selectively bound primarily to the apical region of the sheath associated with the appendage. Con A, but not WGA or GNL, disrupted spore attachment. The Con A effect on spore attachment was significantly reduced when the lectin was pre-mixed with methyl-D-α-mannoside. The sheath as well as the associated appendage was removed with either Novozym 234 or crude β-glucuronidase. Such sheathless spores were initially adhesion incompetent but, by 40 min, the spores regained their ability to adhere to hydrophobic surfaces through the secretion of material that had an affinity toward colloidal gold. Non-treated pycnidiospores surrounded by sheath remained spatially separate; however, sheathless spores readily agglutinated. Addition of cations enhanced attachment to both hydrophobic and hydrophilic surfaces presumably through the masking of electronegative groups on the sheath or by reduction of the ordered water-shell surrounding the sheath. In particular, Ca2+ induced both attachment and germination on normally non-inductive hydrophilic surfaces, suggesting an additional role for Ca2+ in signal mediation for germination.

A Melampsora lini β-tubulin gene and flanking sequence has been isolated and characterised. The gene contains 8 introns, is expressed in germinating urediospores and vegetative mycelia and encodes a 448 amino acid protein that is similar to other fungal β-tubulin genes. Evidence for a second β-tubulin gene within the M. lini genome is also presented. The predicted M. lini β-tubulin protein was compared with 34 other fungal β-tubulin proteins in a phylogenetic analysis and, in general, good agreement was observed between the derived β-tubulin based phylogeny and the current taxonomic status of these species. However, a second β-tubulin protein found in five fungal species is atypical when compared to other fungal β-tubulin proteins. Analysis of intron positions within 33 fungal β-tubulin genes showed that intron location was generally characteristic of the fungal class, with euascomycete fungi showing particularly striking conservation of intron position, although intron number was variable and intron sequences showed little conservation.