A monoclonal antibody (MAb), UB31, is described that binds to the extracellular matrix (ECM) surrounding germ-tubes and appressoria, but not conidia, of the bean anthracnose fungus, Colletotrichum lindemuthianum. Comparative localisation studies with MAb UB26, which has the same cell type specificity, suggest that the ECM is heterogeneous in composition. Immunofluorescence showed that UB31 labelled appressoria more intensely than germ-tubes, whereas UB26 labelled these structures to a similar extent. Immunofluorescence and TEM-immunogold labelling showed that UB31 antigens were located close to the appressorial wall, while UB26 antigens extended further away from the wall. MAb UB31 bound to the ECMs of all six Colletotrichum species tested. Western blotting and ELISA indicated that the antibody recognises a high Mr glycoprotein (>200000) that may be linked to melanin. The glycoprotein recognised by UB31 was not removed from substrata by ultrasonication, suggesting it may contribute to germling adhesion.

Pseudocercospora mori produced three kinds of hyphae – primary infection hyphae, internal hyphae and secondary infection hyphae. The primary infection hyphae were developed from conidia and they penetrated the leaf through stomata directly or by lateral penetration branches – the stomatopodia. Inside the stomatal chambers the stomatopodia swelled to become vesicle-like structures. The internal hyphae produced compact stromata in the sub-stomatal chambers. Each stromata carried 1–5 conidia at a time on short unbranched conidiophores. The secondary infection hyphae were developed from internal hyphae directly or from stromata. The secondary infection hyphae emerged through stomata, branched profusely on the leaf surface and produced conidia singly on short conidiophores. The secondary infection hyphae re-entered the leaf directly or by stomatopodia. Conidia were developed also from stomatopodia and from their substomatal vesicles. The conidia developed from substomatal stromata, and those produced from the secondary infection hyphae on leaf surface were similar in their morphology and size of conidiophores. The conidia were obclavate to cylindrical, 3–17 celled and measured 4–35×1.5–2 μm. Germ tubes were often found to emerge from the conidial cell nearer to stomata. The primary infection hyphae and secondary infection hyphae produced stomatopodia above stomata.

In the present study we investigated the abundance and spatial distribution of dark septate root endophytes (DSE) in a 3×3 m plot in a spruce stand (Picea abies). A total of 144 DSE isolates were obtained by means of a hierarchical sampling design. Most roots were colonised, as DSE were isolated from 81.7% of root segments. ISSR–PCR fingerprinting was used to identify 21 unique ISSR types. Dominant types were isolated from adjacent points that covered an area of up to 6.8 m2 of the study plot, and ISSR types were intermingled extensively. Frequency of isolation of the different ISSR types was uneven with two dominant types that accounted for 38% and 28% of all DSE isolates, respectively. Seven DSE strains representing six different ISSR types were identified as Phialocephala fortinii based on the morphology of fertile conidiophores and/or ITS 1 and 2 sequence comparisons.

The fungal pathogen responsible for Colletotrichum leaf disease of Hevea, develops three different disease symptoms: raised spots, anthracnose and papery lesions. These diseases have been attributed to Colletotrichum gloeosporioides (teleomorph Glomerella cingulata) and the fungi involved are morphologically indistinguishable. Twenty-five Colletotrichum isolates, which originated from three different disease symptoms, were characterized initially using RAPD markers. Two major RAPD profiles were detected which were related to the type of disease symptom developed. Colletotrichum isolates causing raised spot symptom were easily distinguished from isolates originating from either anthracnose or papery lesions. Restriction analysis of the PCR amplified 5·8S ribosomal DNA (rDNA), including both the flanking internal transcribed spacers (ITS) of representative isolates from the three different disease symptoms, also revealed two distinct RFLP patterns reflecting the same groupings as detected through RAPDs. Both molecular approaches suggested that there were two species of Colletotrichum associated with Hevea inciting the development of three different symptoms: Colletotrichum acutatum causing raised spot symptom, and C. gloeosporioides causing both anthracnose and papery lesions. This is the first record of Colletotrichum acutatum on Hevea in India.

Motile zoospores of species of oomycetes encyst rapidly to form walled cysts that germinate and infect host plants. Differences in the protein composition of the plasma membrane and endomembranes of zoospores and cysts of the oomycete Phytophthora nicotianae have been explored by comparing patterns of polypeptides in one- and two-dimensional gels of microsomal fractions. Against a backdrop of common components, this comparison revealed that at least 53 proteins were specific to, or occurred preferentially in, the microsomal fraction of one spore type or the other. In addition, proteins common to zoospore and cyst plasma membranes were further investigated by immunocytochemical labelling with a panel of 10 monoclonal antibodies raised against P. nicotianae spore components. The results of immunolocalisation studies and immunoblotting showed that the antibodies reacted with four different sets of membrane proteins, and were grouped accordingly. Group 1 antibodies bound preferentially to the bladder of the water expulsion vacuole or a region of the cell surface associated with it. Group 2 antibodies reacted with a protein of high relative molecular weight (>200 kDa) found in zoospores and cyst plasma membranes and in the cleavage membranes of sporangia. Group 3 antibodies reacted with a set of proteins occurring in the plasma membrane of zoospores and cysts, in the peripheral cisternae, in the spongiome membranes of the water expulsion vacuole, in the cleavage membranes of sporangia, and in the plasma membrane and apical vesicle membranes in hyphae and germinating cysts. Group 4 antibodies bound to a set of proteins present in the zoospore and cyst plasma membrane, in sporangial cleavage membranes and in the spongiome but not present in the peripheral cisternae, hyphae or germinating cysts. The results document the presence of proteins that are common to the plasma membrane of all stages of the asexual life cycle of P. nicotianae, of proteins that occur in the plasma membrane of the asexual spores but not of hyphae, and of proteins that occur in the membranes of either zoospores or cysts. The results also indicate that zoospore and cyst plasma membrane proteins may be present in specific subsets of other membranes within the zoospores.

The genus Tectimyces gen. nov. (Harpellales: Legeriomycetaceae) is described with two species, T. leptophlebiidarum and T. robustus spp. nov., collected on the hindgut of the mayfly nymph Habroleptoides confusa (Ephemeroptera: Leptophlebiidae) in northern Spain. This is the second report of a trichomycete inhabiting a member of this family of ephemerids. Diagnostic for the new genus are type II zygospores and unappendaged trichospores, borne on long generative cells and carrying a very short collar after release. The position and morphological traits of the newly described taxa are discussed and compared with other genera and species, such as Bojamyces repens and Orphella spp.

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.

Oidiodendron maius is accepted widely as an ericoid mycorrhizal endophyte because it is isolated primarily from the roots of ericaceous plants. Reports of the species from other materials are much fewer, suggesting a limited role as a free-living saprobe. We show that assumptions concerning the prevalence of O. maius in a common substrate (i.e. peat) are affected by isolation protocols. Oidiodendron maius was observed on 99.6% of peat samples incubated in moist chambers but only 0–9% of the peat fragments plated on different isolation media. These results, and previous studies showing ability to degrade Sphagnum, indicate that O. maius could inhabit a much broader niche than previously suspected. Sterile ascocarps with peridia morphologically similar to Myxotrichum arcticum were observed on moist incubated peat and in a series of controlled crosses. These observations provide evidence that the teleomorph of O. maius is a species of Myxotrichum.

Rimularia insularis forms an photosynthetic thallus on lichens of the Lecanora rupicola group. In the thalli of R. insularis, some hyphae of the host are detectable, and they are situated mainly in the basal part of the Rimularia's medulla. No Lecanora hyphae were present in the Rimularia's algal layer and they were indistinct in the upper parts of its medulla, from where we obtained clean ITS-sequences of Rimularia. Direct PCR from various sections of the algal layer detected only one photobiont genotype, although the algal cells could be assigned to two morphologically different types. Calcium deposits were found in the upper parts of the medulla, while abundant rock substrate fragments with different mineral compositions were present in the lower parts. Cavities and fissures in the rock below the infected thalli were filled by fungal cells, PCR analyses of such parts indicated that the Rimularia hyphae extend below the host, and into the rock.

Production of lignin-modifying enzymes by 10 white-rot fungi, as measured by decolorization of Poly R 478 dye, varied in response to different carbon and nitrogen regimes. Fastest decolorization rates were achieved with wood polysaccharide monomers (glucose, xylose) as carbon source, although cellulose was the only carbon source to facilitate decolorization by all test fungi. Enzyme production by most isolates was strongly dependant on nitrogen levels, with high nitrogen conditions generally suppressing enzyme production. One unidentified isolate (HKUCC 4062) displayed apparent nitrogen deregulation of enzyme production. A cellulose-low nitrogen growth medium is recommended for agar-based screening procedures of lignin-modifying enzyme production by white-rot fungi.

A method for the observation of the three-dimensional structure of intact ectomycorrhizal rhizomorphs is described. The method is based on a combination of clearing the material with KOH followed by staining with congo red and subsequent imaging under a laser scanning confocal microscope (LSCM). The images obtained are of a much higher three-dimensional resolution than those obtained previously by use of conventional light microscopical techniques. The structure of highly differentiated and undifferentiated rhizomorphs is described. Applications of the method are briefly discussed.

The presence of the 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway was demonstrated in several sapstain fungi including Ceratocystis and Ophiostoma, using both chemical inhibitors and molecular techniques. The inhibitor compounds tricyclazole and carpropamid effectively reduced pigmentation at low concentrations in all tested fungal species, but also lead to growth inhibition at higher concentrations. The inhibitor cerulenin prevented fungal growth in all tested fungi at all tested concentrations, likely due to its inhibitory effect on another enzyme, the metabolically critical fatty acid synthase. Partial DNA sequences for the gene encoding scytalone dehydratase (SD) were obtained from species of Ceratocystis and Ophiostoma and found to have homology with known respective DHN-SD gene sequences. Sequence analysis of the partial SD amino acid sequences showed greater than 80% similarity among the sapstain species, and corresponded well with known phylogenies of sapstain fungi based on rDNA sequences. Aside from the work carried out on the isolate O. floccosum 387N, this is the first known documentation of the melanin pigmentation pathway used by species of the sapstain fungi Ceratocystis, Leptographium and Ophiostoma. Furthermore, since no fungus has ever been found, to our knowledge, to have more than one melanin synthesis pathway, we can state that these species are likely only to use the DHN pathway for melanin production.

The occurrence of disease outbreaks associated with the genus Sorosporella on grasshoppers and locusts (Orthoptera: Acridoidea) in Africa is reported. Infected hosts, representing ten genera within five acridoid subfamilies, are characterized by red, thick-walled chlamydospores which completely fill the cadaver. On selective media, the chlamydospores, up to seven-years-old, germinated to produce a Syngliocladium anamorph which is considered to be undescribed. The new species Syngliocladium acridiorum is described and two varieties are delimited: var. acridiorum, on various grasshopper and locust genera from the Sahelian region of West Africa; and, var. madagascariensis, on the Madagascan migratory locust. The ecology of these insect-fungal associations is discussed. Sorosporella is treated as a synonym of Syngliocladium.

The secondary metabolites, helvolic acid and cerulenin were detected in liquid culture of Sarocladiumoryzae, causal agent of sheath rot of rice, and also in diseased sheath tissues. These metabolites were examined by thin-layer chromatography (TLC), column chromatography, and by high-performance liquid chromatography (HPLC). Helvolic acid and cerulenin showed the Rf value of 0.49 and 0.61, respectively using TLC. Cerulenin produced two spots in the TLC chromatogram and produced two major peaks in the HPLC chromatogram due to its instability. External application of helvolic acid and cerulenin produced lesions similar to those caused by the fungus. When infiltrated both of these metabolites caused electrolyte leakage in susceptible cultivar, IR 36 and induced lower levels of leakges in White Ponni, the moderately resistant cultivar to S. oryzae. The concomitant production of helvolic acid and cerulenin by S. oryzae in liquid culture and in planta was confirmed and the role of helvolic acid and cerulenin on the virulence of S. oryzae and sheath rot disease of rice is discussed.

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

The influences of temperature on the fruit body induction of Flammulina velutipes were investigated. The fruit body could be induced after reducing of the ambient temperature under complete darkness, but could not be induced after light irradiation without temperature reduction. Fruit bodies formed under complete darkness elongated without pileus formation (pinhead fruit body). Protein expression patterns were investigated by two-dimensional electrophoresis, and 22 protein spots were found to be newly expressed in the mycelia and fruit bodies after reducing of the ambient temperature, but few protein spots were newly expressed in the mycelia after light irradiation without temperature reduction. Four of the spots had identical N-terminal sequences to polypeptides, Pf1, Pf3, Pfd3, and Pf6 which are previously sequenced as polypeptides expressed specifically during fruit body formation of F. velutipes.

A fungus was discovered causing a progressive twig dieback on stems of Vaccinium vitis-idaea (lingonberry) in Oregon. Both morphological and molecular data suggest that the fungus belongs in Diaporthe/Phomopsis but is distinct from P. vaccinii, cause of a dieback and fruit rot of blueberry and cranberry (Vaccinium spp.). This fungus is described and illustrated as a new species, Phomopsis columnaris. It is distinguished from other species of Phomopsis by the distinctive conidiophores that consist of vertically aligned cells lining the base and sides of the conidiomata. Another species of Phomopsis described on Vaccinium, Phomopsis myrtilli, known from V. myrtillus, is redescribed and illustrated based on authentic herbarium material.

Conifer needles, like many grasses, are infected by systemic fungal endophytes. Following suggestions made in the early 1980s that (1) conifer needle endophytes may produce anti-insectan compounds, and (2) population dynamics of the eastern spruce budworm in New Brunswick could not be completely explained based on existing knowledge, we discovered that a low percentage of needle endophytes made a range of known and new metabolites toxic to this insect. Here, we report that wound inoculations of toxigenic endophytes of seedlings from a breeding population of white spruce were successful across a range of genotypes. The needles colonized by a rugulosin-producing endophyte were found to contain rugulosin in concentrations that are effective in vitro at retarding the growth of spruce budworm larvae. Larvae presented with endophyte infected needles containing rugulosin do not gain as much weight as those eating uncolonized needles. This represents the first positive evidence that the kind of mutualism between toxigenic endophytes and grasses affecting insect herbivory also may occur in white spruce.

A Phytophthora species was found on blighted foliage of Ipomoea longipedunculata, a morning glory native to the highlands of central Mexico. Based on host range, morphology, allozymes, mitochondrial DNA haplotype and rDNA sequences it is concluded that a new Phytophthora species, P. ipomoeae sp. nov., is the causal agent of leaf blight disease on I. longipedunculata.

The genus Dioicomyces is reviewed on the basis of the examination of types, authentic material, or personal collections. Three new species from Spanish anthicids are described: D. denticulatus, D. ladoi, and D. leptalei spp. nov. Eleven taxa (including eight species, two forms, and one variety) are synonymized and included under D. anthici, which is considered a very variable species. With additions and changes, Dioicomyces currently includes 23 species. In this synopsis no taxa at infraspecific rank are recognised. For each accepted species synonyms are listed, mostly based on the study of type material. Types for each taxon are included and lectotypes designated when necessary. This monograph also includes descriptions, photographs and/or drawings for all species studied and a key for their determination.