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.

This paper describes, as a part of the resistance risk assessment, the genetic analyses which were performed to evaluate the resistance risk of the phenylpyrrole fungicide fludioxonil and the implications for cross resistance with the dicarboximide vinclozolin. Cross resistance between dicarboximides and phenylpyrroles, often found in the laboratory, has been extremely rare in the field. Over seven years of monitoring in Switzerland no fludioxonil field resistant isolates were found. Sexual crosses between a fludioxonil field resistant/vinclozolin field resistant strain isolated in France and a sensitive one showed an independent segregation of the resistance to the two fungicides, suggesting that different genes regulate field resistance to fludioxonil and vinclozolin. Sexual crosses between a fludioxonil field resistant B. fuckeliana strain and a fludioxonil laboratory resistant strain indicated that the two fludioxonil resistance phenotypes segregate independently. We concluded that fludioxonil resistance is influenced by at least two different genes. Genetic studies with laboratory strains resistant to both fungicides demonstrated a low segregation rate between fludioxonil and vinclozolin resistant phenotypes. These results let us hypothesize that fludioxonil and vinclozolin resistance induced in laboratory could be due to two genes closely linked. Finally, pathogenicity tests performed with progenies of sexual crosses showed a positive correlation between virulence and phenotypes.

The effect of single or mixtures of antioxidants on the lag phase prior to growth, growth rate, hydrolytic enzyme production (N-acetyl-β-glucosaminidase, β-D-glucosidase and α-D-galactosidase) and fumonisin production by Fusarium verticillioides and F. proliferatum was evaluated on maize-based media at 25 °C, and under different water activity (aW) conditions. An increase in the lag phase (h) was observed for both F. verticillioides and F. proliferatum especially with propyl paraben (PP)+butylated hydroxyanisole (BHA) treatments at all aW levels tested. For both species PP alone or in combination with BHA, at concentrations of 0.5 and 1 mM reduced the growth rates by > 85% at the three aW levels tested (0.995; 0.98 and 0.95). PP+butylated hydroxytoluene (BHT) or trihydroxybutyrophenone (THBP) were less effective in controlling growth, regardless of aW level. Combinations of PP+BHA reduced the fumonisin concentrations produced by F. verticillioides and F. proliferatum at 0.995 and 0.98 aW significantly. However, at low concentrations of antioxidants (0.5 mM) some stimulation in fumonisin production was observed with some treatments. The efficacy of the treatments was reflected in the impact on enzyme production. In the untreated control the highest total enzyme activity of three hydrolytic enzymes was observed at 0.995 aW after 96 h. All the antioxidant treatments alone or combined resulted in a significant reduction (P < 0.001) of the total enzyme activity at the aW levels tested. Only 10 mM THBP produced an increase in the total amount of N-acetyl-β-D-glucosidase by both F. verticillioides and F. proliferatum. For the three enzymes single factors: time, aW and antioxidant treatments, most two and all three way interactions were significant (P < 0.001). The use of combined mixtures of antioxidants could be a good strategy to diminish the entry of fumonisin into the animal feed and human food chains.

Interactions between the mycoparasite Cladosporium tenuissimum and the bean rust Uromyces appendiculatus were studied through light and electron microscopy in vitro at the host–parasite interface. Urediniospore germination decreased on contact with ungerminated C. tenuissimum conidia, possibly due to antibiosis mechanisms. C. tenuissimum grew towards the bean rust spores and coiled around their germ tubes. Penetration of the urediniospores occurred either enzymatically and/or mechanically, through appressorium or infection cushion structures, from which a thin penetrating hypha was generated. Enzyme production by the mycoparasite was suggested by the loosening of the matricial components of the spore wall, which sometimes left chitin fibrils visible. Mycoparasite hyphae grew within the host spore, emptied its content, and emerged profusely forming conidiophores and conidia. C. tenuissimum was able to grow on media containing laminarin, suggesting the ability of producing glucanases, but not when chitin was used as the sole carbon source. Conidia that had been grown on a sugar-rich medium, filtered, and extracted with organic solvents, were found to contain cladosporol and related compounds. Complete control of the bean rust disease was achieved by application of C. tenuissimum culture filtrates but not by conidial suspensions. This is the first report of parasitism by C. tenuissimum on U. appendiculatus. These investigations provide additional observations on a genus besides Melampsora and Cronartium from which this fungus has been isolated and tested to date. The possible role of environmental factors for the exploitation of this organism as a biocontrol agent is also mentioned.

To understand the phylogenetic relationships and evolution of the powdery mildew genus Sawadaea (Ascomycota: Erysiphaceae), obligate parasitic fungi of maples, we performed molecular phylogenetic analyses based on 47 ITS and ten 28S rDNA sequences. Seven major clades of Sawadaea, each represented by powdery mildew specimens collected from a single or a small number of closely related sections of Acer (maples), were identified in this study, suggesting that a close evolutionary relationship exists between Acer (host) and Sawadaea (parasite). A 6–11-base insertion/deletion was found in the ITS1 region of Sawadaea, and the presence or absence of the indel was consistent within the respective clades. Because the outgroup genera Podosphaera and Cystotheca have no deletions in these sites, deletion of the sequences may have occurred during the divergence of the respective clades of Sawadaea. The seven clades of Sawadaea were divided into two geographical groups, viz. an East Asian and a global group, based on the countries of collection. Calculation of the evolutionary timing of Sawadaea using molecular clocks showed that the divergence of different species of Acer occurred many millions of years before the radiation of Sawadaea. Thus, the close evolutionary relationship between Sawadaea and Acer found in this study might not be due to a true coevolutionary process. Powdery mildew fungi belonging to Sawadaea may have jumped onto Acer spp. long after the radiation of the major sections of these trees, and then expanded their host ranges according to the phylogeny and geographical distribution of Acer.

Internally transcribed spacer (ITS) regions of the rRNA gene cluster in isolates of Ascochyta spp. were amplified and digested with restriction endonucleases. Addition of fragment sizes from conventional gel electrophoresis gave anomalous sizes which suggested the presence of more than one PCR product. Separation in HA-Yellow containing agarose gel allowed the detection of fragments of the same size but with different sequences. Isolates of Ascochyta spp. were shown to possess multiple PCR-amplification products of the same sizes, but with significant sequence differences.

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.

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.

Double-stranded RNA (dsRNA) has been isolated from Agaricus bisporus fruit bodies exhibiting a wide range of disease symptoms. The symptoms which occurred singularly or in combination included; bare cropping areas on commercial beds (primordia disruption), crop delay, premature veil opening, off- or brown-coloured mushrooms, sporophore malformations and loss of crop yield. All symptoms were associated with loss of yield and/or product quality. Collectively, these symptoms are described as mushroom virus X (MVX) disease. The dsRNA titre was much lower than that previously encountered with the La France viral disease of mushrooms and a modified cellulose CF11 protocol was used for their detection. A broad survey of cultivated mushrooms from the British industry identified dsRNA elements ranging between 640 bp and 20.2 kbp; the majority have not previously been described in A. bisporus. 26 dsRNA elements were identified with a maximum of 17, apparently non-encapsidated dsRNA elements, in any one sample. Three dsRNAs (16.2, 9.4 and 2.4 kbp) were routinely found in mushrooms asymptomatic for MVX. Previously, La France disease was effectively contained and controlled by minimising the on-farm production and spread of basidiospores. Our on-farm observations suggest that MVX could be spread by infected spores and/or mycelial fragments.

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.

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.

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.

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.

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.

Fungal species richness and abundance were assessed in leaf litter of the Australian rainforest tree Neolitsea dealbata (Lauraceae) using particle filtration. Results were comparable to the species richness and abundance reported in previous studies of tropical leaf litter microfungi. Eight leaf samples yielded 1365 strains. In an assessment of the effect of surface treatments, 736 strains in 112 morphotaxa were isolated, while 639 strains in 141 morphotaxa were recovered to assess the effect of surface treatment. Isolation rates in airdried leaves stored at room temperature for four weeks declined linearly, while the number of morphotaxa remained essentially constant for the first three weeks. Isolates of common morphotaxa were lost preferentially over those of rarer taxa. Such losses of isolates may be acceptable if only presence/absence data are collected. If frequency data are vital for community analysis, only fresh material should be utilised. Two surface sterilisation treatments were applied to N. dealbata leaves. An ethanol/sodium hypochlorite treatment was considered unsuitable because it significantly reduced the number of morphotaxa derived from the leaf lamina. A sodium hypochlorite treatment reduced the number of detectable propagules in the wash water without changing the number of morphotaxa derived from leaf particles in comparison with those of the control group.

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.

The mitosporic fungus Pochonia chlamydosporia is a potential biocontrol agent for cyst (Heterodera spp. and Globodera spp.) and root knot (Meloidogyne spp.) nematodes, which are important agricultural plant pests. 54 isolates from diverse geographical regions and several nematode hosts were used in this study. Genetic variation was examined using enterobacterial repetitive intergenic consensus (ERIC) primed PCR and sequences from the internal transcribed spacer (ITS) rRNA region. ERIC PCR yielded 35 scorable binary characters from all the fungi tested and cluster analysis of the data showed that isolates from cyst nematodes were more genetically variable than those from root knot nematodes. The ITS regions were highly conserved, the only significant difference being an extra thymidine in isolates from Meloidogyne spp. Assays with nematode eggs indicated that isolates differ in their ability to infect different nematode genera. The results indicate host related variation in P. chlamydosporia. This finding has significant implications for the application of P. chlamydosporia as a biocontrol agent.

Sequential effects of colonisation by the wood decaying Fistulina hepatica were examined microscopically in naturally infected and artificially inoculated heartwood of pedunculate oak (Quercus robur). For 6 mo, the deposition of brown materials within parenchyma cells containing hyphae was the only visible effect of colonisation. After 12 mo degradation occurred but was initially confined to parenchyma of xylem rays, in which the secondary cell walls showed helically orientated internal cavities containing hyphae, as in a soft rot. The hyphae were covered with a resinous material and persisted in a herringbone pattern after the secondary walls became heavily degraded. The adjacent libriform wood fibres also showed cavity formation. By contrast fibre-tracheids were mainly affected by a brown rot. uv-microscopy indicated that cell types in which the soft rot mode occurred were rich in syringyl lignin, whereas the brown rot was associated with cells rich in guaiacyl lignin. Thus, the present study shows that F. hepatica possesses dual modes of degradation which appear to correlate with the different lignin composition within cell types of oak.

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.

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.