The genera Hobsonia, Illosporium, and Marchandiomyces include several common lichenicolous (lichen-inhabiting) species that have been taxonomically linked in the past. These mitosporic genera are frequently misidentified because of a paucity of morphological characters, which has led to some confusion about their relationships. We examined the phylogenetic positions of species in these genera using complete sequences of the small subunit (SSU) and internal transcribed spacer (ITS) ribosomal DNA (rDNA), and portions of the mitochondrial (mt) SSU rDNA obtained from isolated cultures of freshly collected specimens. Phylogenetic analysis of the sequence data indicated that Marchandiomyces corallinus and M. aurantiacus are closely related members of a basidiomycete clade. In addition, the clade includes an undescribed non-lichenicolous species that is morphologically similar to M. corallinus. Another undescribed species with marchandiomyces-like sclerotia, appears to be in a separate basidiomycete clade. The three recognized species of Hobsonia are unrelated, with H. christiansenii appearing to be a hypocrealean ascomycete closely related to a sample of Illosporium carneum, H. santessonii an ascomycete of unknown affinity, and H. mirabilis a basidiomycete. The latter, a plant pathogen, is the type species of Hobsonia. The new generic names Illosporiopsis and Hobsoniopsis gens. nov. are described for the lichenicolous species H. christiansenii and H. santessonii, respectively, and the necessary new combinations made.

Chytridiomycota were isolated into pure culture from cool temperate and warm semi-arid soils of eastern Australia. In pure culture these fungi responded variably to the range of temperatures commonly recorded in their environment. All members of the Blastocladiales, Spizellomycetales and Chytridiales grew in culture at temperatures up to 30 °C. Some isolates from the Blastocladiales and Spizellomycetales continued to grow at or above 37 °. Some isolates of the Chytridiales grew up to but not beyond 35 °. All isolates in the Chytridiales were able to resume growth at 20 ° after brief exposure to temperatures higher than the maximum growth temperature, but were killed by exposure to higher temperatures for 7 d. Because in the natural soil habitat temperature may exceed the maximum for growth it may be a limiting factor that determines the distribution of chytrids in the soil.

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.

An in vitro conifer ectomycorrhizal model system was developed to study changes in gene expression during the establishment of symbiotic relationship. Using the model system and the mRNA differential display technique, we have identified cDNA clones from Laccaria bicolor that appear to be involved in establishing and maintaining the symbiotic relationship between L. bicolor and red pine. These differentially expressed cDNA clones were obtained from mRNA extracted from the fungus 6–24 h after initiation of interaction. BLAST analysis showed that sequences of these clones did not match with any of the previously characterized genes. The cDNAs had regions with varying degrees of similarity to genes involved in signal transduction and transcriptional activation.

Stable isotopes in fruit bodies from field studies have been used to infer ectomycorrhizal or saprotrophic status and to understand carbon and nitrogen use, but few controlled culture studies have correlated source and fungal isotopic patterns. Here, we measured natural abundances of 15N and 13C in ten strains of ectomycorrhizal fungi and seven strains of saprotrophic fungi grown on agar with three different primary carbon sources: glucose, glucose plus malt extract, and potato dextrose agar. Eight fungal strains were also grown using position-specific, 13C-labelled glucose (C-1 through C-6 labelled). Most fungi resembled nitrogen sources in δ15N, suggesting that growth on agar media minimizes isotopic fractionation on uptake compared to growth on liquid media, and that in general saprotrophic and mycorrhizal fungi process nitrogen similarly. Saprotrophic fungi were more depleted in 13C than ectomycorrhizal fungi on all media, presumably because of assimilation of 13C-depleted, agar-derived carbon. Results on 13C-enriched glucose indicated that saprotrophic fungi obtained up to 45% of their carbon from the agar substrate. Fungi generally incorporated the individual carbon atoms of glucose in the order, C-4<C-1<C-2, C-3, C-5<C-6, ranging from a mean of 9% for the C-4 atom to 21% for the C-6 atom. Based on these incorporation patterns and intramolecular 13C patterns within glucose, differential incorporation of carbon atoms within glucose among fungal taxa contributed less than 1‰ to isotopic differences among taxa, whereas isotopic fractionation among taxa during metabolism varied up to 4‰. Parallel studies of 13C-enriched and natural abundance substrates were crucial to interpreting our results.

In some filamentous ascomycetes, karyotypic data by conventional light microscopy are not consistent with the electrophoretic karyotypes by pulsed field gel electrophoresis (PFGE). In this study, the three methods, conventional light microscopy on asci, PFGE, and the germ tube burst method (GTBM), were compared for their concordance in karyotyping by using Nectria haematococca (anamorph: Fusarium solani). GTBM was included in the study for cytological karyotyping on mitotic metaphase in germlings of conidia. Conventional light microscopy on asci was concluded to lead to the underestimation of chromosome number in this fungus. PFGE was effective for analysing chromosomes smaller than ca 6 Mb, but did not yield a complete karyotype due to the poor resolution of larger chromosomes. GTBM was useful for determining chromosome numbers as well as chromosome morphology. Analyses by these methods revealed that each of five strains used, two homothallic and the other three heterothallic, has a unique karyotype different from others in number and size of chromosomes. The present results indicate that combination of PFGE and GTBM constitutes a powerful tool to determine karyotypes of filamentous ascomycetes including N. haematococca.

Colletotrichum lagenarium is a plant pathogenic fungus, and produces melanin that is an essential factor for appressorial penetration into host tissues. The melanin biosynthesis pathway of C. lagenarium starts with pentaketide synthesis catalyzed by polyketide synthase Pks1p. We previously confirmed that the direct product of Pks1p is 1,3,6,8-tetrahydroxynaphthalene. Thus, melanin biosynthesis in this fungus requires the reduction of 1,3,6,8-tetrahydroxynaphthalene to scytalone. We made a double mutant 9141-144 from the thr1 mutant 9141 that lacks the ability to metabolize 1,3,8-trihydroxynaphthalene. The double mutant 9141-144 could metabolize neither 1,3,6,8-tetrahydroxynaphthalene nor 1,3,8-trihydroxynaphthalene. However melanin production by the double mutant was restored by THR1, indicating that Thr1p can metabolize both compounds in vivo. These results demonstrate that two enzymes, Thr1p and a deduced 1,3,6,8-tetrahydroxynaphthalene-specific reductase, are involved in the first reduction step of the melanin biosynthesis pathway of C. lagenarium.

Three Cryptosporiopsis species have thus far been isolated from roots of woody plants. A fourth species, which was recently isolated from roots of Calluna vulgaris, Erica tetralix, Vaccinium vitis-idaea, and V. myrtillus in The Netherlands, is described here as new. Sporulation on the natural substratum has not been observed and the morphological description of this fungus is therefore based on characters expressed on oatmeal and malt extract agars. The phenotypic characters indicated a close relationship with the other root-inhabiting species of Cryptosporiopsis and species of the associated teleomorph genus Pezicula. This relationship was confirmed by phylogenetic analyses using sequence data of the 5.8S nuclear rDNA and flanking internal transcribed spacers. In order to facilitate recognition of this possibly under-recognized category of root inhabitants, a key to the root-inhabiting Cryptosporiopsis species based on characters in vitro is given.

Field surveys were carried out in coffee plantations in Chiapas, Mexico, to collect and identify fungi associated with the cuticle, gut, faeces and galleries of the coffee berry borer, Hypothenemus hampei. Insects and coffee berries containing galleries were collected in three coffee farms at different altitudes: Rosario Izapa (425 m), La Alianza (700 m) and Monteperla (950 m). An additional sample consisting of coffee berry borers reared in the laboratory on meridic diets was also included. Results show that there is a great diversity of fungi associated with this insect. 212 cultures, including 40 species distributed in 22 genera, were isolated. The recovery of fungi from the galleries was markedly less than from the borer's body. Three of the isolated species were undescribed; two belonging to the Penicillium and one to Hanseniaspora. Most of the species were collected from the cuticle of the insect, and the presence of fungi was not correlated with altitude. Fusarium, Penicillium, Candida and Aspergillus were the dominant genera with percentage abundance of 26.4, 18.7, 13.4 and 12.5%, respectively. The present study provides a detailed description of the mycobiota associated with H. hampei and represents a significant advance in the understanding of the relationship among this insect and the fungi associated with it.

The ITS of the nuclear ribosomal DNA of Serpula lacrymans and S. himantioides was amplified by PCR to evaluate this DNA region as a diagnostic tool. Sequencing of the amplified ITS region revealed a size of the complete ITS of 654 bp for S. lacrymans and 650 bp for S. himantioides. The base sequence difference between the two fungi was used to design species-specific oligonucleotide PCR primers for both species. With laboratory and field samples, each primer selectively detected its target organism. No cross reaction occurred with other fungi causing rot of indoor wood.

Genetic and physiological diversity was assessed in isolates of the lichen-forming fungus Xanthoria elegans originating from a range of geographic localities and climatic regimes including polar and temperate regions. In vitro cultures were established and isolates examined by production of multi-locus RAPD markers and by sequence comparison of the ITS region of the rDNA tandem repeat unit. Both molecular techniques revealed significant variability. Phylogenetic analysis of RAPD profiles (226 markers) clustered isolates from particular locations together and Mantel's test showed a correlation between genetic divergence and extent of geographic separation. Phylogenies based on ITS sequence divergence were not well supported and did not correlate with the geographic origin of samples, suggesting a single locus approach to be less informative than RAPD data in a study of this nature. Physiological investigations also revealed significant variability between isolates with different geographic origins. Temperature had a significant effect on relative growth rate (RGR) such that X. elegans originating from sites with lower mean annual temperatures had significantly higher RGRs at all test temperatures between 2 ° and 18 °C. Enhanced metabolic activity might be an adaptation for growth in colder climates. The results demonstrate high genetic diversity within this morphologically variable species in response to geographic and environmental factors, and are discussed in relation to data obtained for non-lichenised fungal species and the possibility of cryptic speciation.

A Bayesian analysis of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences was used to infer phylogenetic relationships of 14 Physconia species. The analysis supports the monophyly of the genus. Three well supported clades can be distinguished within Physconia: the series griseae, venustae and pulverulentae. The relationships of these clades, however, is not resolved with confidence. Cortical characters are re-evaluated on the basis of the phylogenetic hypothesis. Anatomical features of the upper cortex are only diagnostic above the species level for two special forms of two-layered cortices, while morphological characters, such as lower surface and rhizine-type are characteristic for distinct clades. P. venusta and P. perisidiosa are not separated in this analysis, but populations of P. muscigena, and European and North American samples of P. americana are clearly distinct and the monophyly of both P. americana and P. muscigena s. lat. is rejected on the basis of a Bayesian hypothesis testing.

Extracellular matrix (ECM) surrounding conidia and germlings of B. sorokiniana was studied using light microscopy (LM), scanning (SEM) and transmission electron microscopy (TEM). Conidial ECM surrounding dry-inoculated, ungerminated conidia was fluid-like and observed only using a cryo-preparation technique, suggesting that the material was water soluble. ECM enveloping germlings appeared fibrillar in LM, TEM and SEM but amorphous in cryo-SEM, indicating that the structure of the ECM is dependent on the water content of the matrix. Fibrillar ECM formed thread-like structures that extended over long distances on the substrate or towards neighbouring conidia and hyphae. TEM of germlings negatively stained with uranyl acetate revealed the presence of fungal fimbriae. The strong resemblance between the extending organization of fibrillar thread-like ECM structures and fimbriae suggested that fimbriae constitute a basic structural component of the ECM and serve as the aggregation centre for the other ECM components. Histochemical labelling revealed significant differences between ECM surrounding the fungus at different morphological stages. The germ tube ECM was labelled for both proteins and polysaccharides whereas germling ECM consisted of two layers: an inner rich in proteins and an outer composed mainly of polysaccharides. Furthermore, the newly released ECM localized on germ tubes and hyphal tips showed affinity for microspheres carrying any type of surface properties while hyphal ECM had affinity only for negatively charged microspheres. This together suggests that ECM after its release is subjected to structural changes.

A method for the early detection and diagnosis of common bunt, caused by Tilletia tritici, has been developed. The technique is based on a nested PCR amplification of DNA from T. tritici in infected plant inflorescence tissue at the stage of internode elongation. Primers amplifying a 212 bp fragment in T. tritici were designed on the basis of sequences in the ITS2 region in the ribosomal RNA genes. Disease diagnosis obtained with nested PCR amplification correlated well with the conventional disease diagnosis, which involves manual observation of disease symptoms at maturity. With the present method, wheat varieties can be screened for resistance towards common bunt at the first node stage. There was no variation between the ITS2 region from T. tritici, T. laevis, T. controversa, and T. bromi and the primers are therefore not species specific. The PCR diagnosis method has only been validated for common bunt caused by T. tritici, but it is likely also to be applicable to T. laevis and T. controversa.

Hyphal growth of the white button mushroom Agaricus bisporus on spawn grains and compost is typically vegetative. Hyphae are loosely arranged, in contrast to the organized texture in tissues, and needle-like calcium oxalate crystals are frequently present on the surface of vegetative hyphae. The mycelial cord is the first well-organized tissue of the fruiting mycelium; it is surrounded by fluffy white hyphae that grow vegetatively. The hyphae of the cord are held together through a semi-fluid medium, the extracellular matrix, which aids in creating a three-dimensional pseudoparenchymatous structure. The matrix material seems to be secreted into the extracellular environment by specifically differentiated cells, but the vegetatively growing hyphae of A. bisporus initially exploit a different mechanism in the production of matrix which involves a type of cell death different from cell necrosis. This primary matrix production leads to the formation of minute cord tissues in which oxalate crystals are no longer present. Once the hyphal cells of A. bisporus pass the threshold from a vegetative form into organized structures, they become differentiated and self-maintaining in the production of the extracellular matrix material. Morphogenetic cell death has been observed before in A. bisporus development and here we show that it occurs in various species of macrofungi: a mucoid zone of the pileipellis typically found in developing fruit bodies of Psilocybe and Panaeolus spp. contains numerous, dying or dead hyphal cells which show ultrastructural features comparable to those observed during the mycelial cord formation of A. bisporus. Studies performed using specimens of Stropharia rugoso-annulata, Coprinus domesticus, Psathyrella candolleana, Tremella mesenterica, Otidea onotica and Peziza ostracoderma in representative growth stages revealed supporting evidence for the view that morphogenetic cell death plays a key role at different stages during the development of fungal fruit bodies. This phenomenon may be related to the programmed cell death occurring in developing plants and animals.

Conidia of the entomopathogenic Metarhizium flavoviride were harvested 8, 10, 12 or 15 d after inoculation at 25 °C and then (as conidiated rice) dried rapidly (10–12% r.h. and 17–20° for 17 h to about 15–22% moisture content) or slowly (50–60% r.h. and 27° for 5 d to about 27–32% moisture content initially). The subsequent survival of these conidia in air-dry storage at 50° with 8·1% moisture content was then assessed. Conidia longevity (assessed by the duration of storage until conidia viability was reduced to 50%, p50) was maximal when conidia were harvested 10 d after inoculation, and was much greater following slow rather than rapid drying. The substantial beneficial effect of slow desiccation to subsequent conidia survival is consistent with that detected in other propagules in anhydrous biology, and is also of considerable practical utility for the biological control of insects by entomopathogenic fungi.

Mycena galopus is among the most important leaf litter decomposers in UK coniferous and angiosperm woodlands, having the potential to utilise all the major constituents of plant litter. Even so, the enzyme or combination of enzymes produced by M. galopus responsible for lignin depolymerisation was previously unknown. A range of media from liquid and semi-solid cultures to more natural substrata was tested to determine whether laccase was produced by an isolate of M. galopus, M9053. Malt extract liquid medium (MEL) with 2,5-xylidine favoured laccase production as compared with the same medium containing the inducers veratryl alcohol, veratryl aldehyde, veratric acid, homoveratric acid, vanillic acid or p-anisic acid. A semi-solid medium of cereal bran in phosphate buffer and a solid medium of Picea sitchensis F1 horizon needle litter were also not as effective as MEL with 2,5-xylidine as an inducer. Compared with six other isolates of the same species grown in MEL without inducers, M9053 exhibited rates of laccase activity fairly typical for M. galopus. An isolate from a dark coloured basidiome of M. galopus, but not var. nigra, exhibited the greatest activity while var. candida showed relatively low laccase activity. Marasmius androsaceus exhibited peak laccase production several days later than M. galopus. In addition, a manganese-dependent peroxidase that was responsible for 15% (in MEL culture fluid) and 39% (in needle litter extract III) of ligninolytic activity was produced by M9053. A further peroxidase was found to be the major ligninolytic constituent in MEL extracts (53%), and had a similar contribution to total activity (29%) as laccase (32%) in needle litter fraction III. Mycena galopus produced water- and buffer-extractable mannases and xylanases when grown on needle litter.

Fifty-five Phytophthora sojae isolates were collected from soil samples and diseased soybean plants from Illinois, Indiana, Iowa, and Minnesota in 1994 and 1995. Races for the isolates were determined. DNA of P. sojae isolates was amplified with 16 Operon decanucleotide primers. Twenty-three of 75 amplified fragments were polymorphic. Based on the 23 RAPD markers, a dendrogram depicting the relatedness of the isolates was constructed using UPGMA. The P. sojae isolates clustered into four distinct groups. The isolates of races 3, 4 and 25 clustered into group I. The isolates of races 1, 8 and 13 clustered into group II. The isolates of race 5 clustered into group III, and the isolates of race 7 clustered into group IV. Genetic diversity was detected among isolates of races 1, 3, 4, 5, 7 and 25 but not among isolates of races 8 and 13.

The ability of Botrytis cinerea to produce cutinolytic, pectolytic and cellulolytic enzymes was re-examined in vivo and in vitro. In liquid culture, B. cinerea produced both constitutive and inductive forms of hydrolytic enzymes. The constitutive levels were 44 mU, 720 mU, 1820 mU, 12·0 U and 22·4 mU for cutin esterase, endopolygalacturonase (endoPG), exopolygalacturonase (exoPG), pectin methyl esterase (PME) and pectate lyase (PL), respectively. Unlike the other enzymes, however, carboxymethyl cellulase (CMCase) production was found to be entirely inducible. The enzymes secreted at the germination and germ-tube growth stages, and during subsequent disease development, were quantified simultaneously. Complete germination was observed within 9 h of incubation of B. cinerea on the surface of bean leaves and disease symptoms gradually increased during days 1–6 of the incubation. Significant amounts of all hydrolytic enzymes studied in the early hours and monitored during 6 d of incubation were detected, supporting the possibility of a major role for the enzymes produced by B. cinerea in the development of disease. The amount of cutinase was found to be uniform from the beginning, indicating that this enzyme may have an important role in penetrating the impervious cutin layer. The exoPG level increased from the beginning of incubation whereas that of endoPG started to rise later. PL and PME were also detected on the leaf surfaces. The patterns of in vitro production of exoPG, endoPG and PL correlated with the in vivo production of these enzymes, but there was only partial correlation in the production of PME and no correlation in the patterns of cutinase and CMCase production.