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.

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.

The fluG gene proved to be essential in the initialisation of autolysis in Aspergillus nidulans (teleomorph Emericella nidulans) cultures, while a loss-of-function mutation in only one out of the flbB-E genes had only minor effects on autolysis. In contrast to its important role in sporulation, brlA regulated only some, but not all, elements of the autolytic process. The tightly coupled autolytic events (chitinase and proteinase production, hyphal fragmentation, disorganisation of pellets, autolytic loss of biomass) observable in ageing cultures of A. nidulans were disconnected by loss-of-function mutations in some genes of the FluG-BrlA regulatory network. The tight correlation between pellet morphology and size and hydrolase production was also erased by these mutations. On the other hand, the mutations studied did not affect the glutathione metabolism of the fungus.

Uptake of 75[Se]-selenite by Saccharomyces cerevisiae has been characterized. At a 0.5 mM selenite, ~0.14 nmol Se (106 cells)−1 was rapidly accumulated by the cells at a rate of ~56 pmol.min−1 (106 cells)−1 which was independent of temperature and glucose. This rapid phase was followed by a slower uptake phase which was sensitive to glucose, temperature and metabolic inhibitors [2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), potassium cyanide (KCN) and sodium azide (NaN3)] and therefore presumed to be metabolism-dependent. Two transport systems appeared to be involved in selenite uptake. At the low range of selenite concentrations used (0.025–0.1 mM), a high affinity transport system occurred with apparent Km and Vmax values of 54.0 μM and 3.14 pmol.min−1 (106 cells)−1 respectively. A low affinity system was present at higher concentrations (0.1–1.0 mM) with apparent kinetic parameters of Km=435 μM and Vmax=11.6 pmol.min−1 (106 cells)−1. Elevated sulphate concentrations (up to 2.5 mM) did not affect the accumulation of selenite. However, the transport rate from 0.5 mM selenite was stimulated by sulphite, with the maximum effect occurring at 0.5 mM sulphite. Methionine had a detectable inhibitory action on selenite uptake whereas cystine and cysteine completely inhibited active transport of selenite. Transmission electron microscopy of 5 mM selenite-grown cells revealed the presence of abundant small cytoplasmic vesicles containing electron-dense granules which could represent an intracellular selenium-detoxification mechanism.

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.

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.

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.

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.

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.

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.

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.

Studies were performed to assess the genetic variation amongst isolates of the aphid-pathogenic fungus Pandora neoaphidis (syn. Erynia neoaphidis). 37 isolates were examined, from a range of pest and non-pest aphid species, as well as 21 from eight other entomophthoralean species. Universal primers were used to amplify the ITS rDNA regions and all of the species tested produced discrete ITS groups, with the exception of Conidiobolus spp. Neighbour-joining analysis of the ITS2 regions from P. neoaphidis, P. kondoiensis and Zoophthora radicans demonstrated that these three species formed distinct groups with sequence identities of 58–82% between the groups. An ITS size of ca 1100 bp was diagnostic for P. neoaphidis, while ca 1450 bp was characteristic of P. kondoiensis. ITS-RFLP analysis failed to yield intraspecific polymorphisms in any of the P. neoaphidis isolates screened, although it was useful in distinguishing between different entomophthoralean species. Some intraspecific variation in the ITS region was detected in a number of isolates of Z. radicans and Conidiobolus spp. We propose that two isolates previously identified as P. neoaphidis based on conidia morphology, are actually P. kondoiensis based on molecular studies. Sequencing analysis of the complete ITS region from P. neoaphidis and P. kondoiensis allowed species-specific primers to be developed for P. neoaphidis and P. kondoiensis. These were used to screen aphids infected in laboratory bioassays and from field-collected samples, without prior isolation of the fungus. The primers are useful tools for quantifying the epizootiology of P. neoaphidis in aphid populations, as well as assessing competitive interactions between these two species.

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.

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.

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 genus Paraphaeosphaeria (Loculoascomycetes) was described as a segregate of Leptosphaeria for species producing brown, usually punctate ascospores with rounded ends, a submedian primary septum, an inflated cell above the primary septum, and a Coniothyrium sensu lato anamorph. This study evaluated the taxonomic value of the morphological characters, host range, ecology, and anamorphs in relation to DNA sequence data in order to determine the relationship between species described in Paraphaeosphaeria. Nine species were characterized morphologically and the ITS and 18S regions of the rDNA were sequenced and a phylogeny was inferred. Morphological studies and results of the sequence analyses provide strong support for polyphyly of this genus. Based on these analyses, the concept of Paraphaeosphaeria should be narrowed to reflect a monophyletic generic concept. Characters such as spore septation and method of conidiogenesis were found to predict relatedness.

Various fungi and myxomycetes have been tested for their synthesis and hydrolytic degradation of β-poly(L-malate) (PMLA), a highly anionic polyester of L-malate-malate. The PMLA content of the culture medium, and also of some cell extracts, has been assayed by L-malate-malate dehydrogenase (EC 1.1.1.83)-catalysed reduction of NAD+ after the alkaline hydrolysis of the polymer. All eight myxomycete isolates were producers. Eight producers were Mitosporic fungi but only one Ascomyete was productive. Aureobasidium pullulans contained PMLA in yeast-like cells but not in hyphae. The polymer was bound to membrane fractions from where it was slowly released during preparation for analysis. Only 12 of the 237 fungi and myxomycetes showed PMLA-hydrolase activity; and two of these were producers of PMLA. Fungi with hydrolase activity were mostly different from those releasing L-malate. The Physarum polycephalum-type unbranched polymer of high molecular mass was not recognized in fungi. Fungi, typically A. pullulans, produced polymer of low molecular mass which seemed to be covalently bound to polysaccharide.

Many species formerly listed in Entyloma have been removed and are now placed in several orders. This study aims to clarify the framework of the genus Entyloma and the phylogeny of these plant parasitic smut fungi with molecular data. Analyses of LSU and ITS sequences are presented and support the monophyly of the order Entylomatales. The sequences show higher similarities within the examined species of Entyloma than within other smut families and genera, suggesting a recent radiation. Within the Entylomatales a cluster of anamorphic Tilletiopsis washingtonensis, T. lilacina and T. cremea collections is the sister group to Entyloma. The phylogenetic relationships in the genus Entyloma are a result of joint evolution with their hosts. The analyses of the sequence data show unresolved groups on Ranunculales and a well-supported group on Asteridae.

Indoor air contamination with mould spores currently experiences an increasing interest with respect to their relevance to health. To assess adverse health effects, epidemiological studies combine the health outcome of individuals with their concomitant exposure to airborne spores, which is observed, for example, during the current month. While the latter is representative for the studied period, health effects might also be the result of long term-exposure or emerge in consequence of a peak of pollution throughout the year. To consider such questions, additional information about the spatiotemporal distribution of airborne spores is necessary.

This paper aims at elucidating the spatial and temporal variation of spore concentrations in Leipzig, Germany. The analysis is based on 1165 matched pairs of indoor and outdoor measurements taken in the period 1998–2002. All data were collected in the frame of previous epidemiological studies and refer to apartments. The analysis comprised spore concentrations (as CFU m−3 in air) of the most important genera, such as Penicillium, Aspergillus, Alternaria, Mucorales, Cladosporium, and also for yeasts.

We found two groups of fungi differing in their spatiotemporal distribution. As this behaviour can be explained by the predominant origin and growing conditions, we call them indoor-relevant and outdoor-relevant genera. Penicillium species are a representative of the former group, while the latter is well represented by Cladosporium. In the studied period we did not observe a clear trend in the spore concentration. Outdoors there is a year-to-year variation of Cladosporium spore concentrations, which follow the prevalent climatic conditions.

For the spore concentration of the outdoor-relevant group a significant annual cycle was observed. Highest concentrations occurred during the summer months and were about 100× the winter burden. That means, for a direct comparison of measurements of spore concentrations taken during different months the season has to be considered. We summarise the findings in a seasonal model, which is fitted to our measurements. Based on the model we developed a procedure for seasonal adjustment, which enabled us to estimate the annual peak spore concentration utilising one monthly observation.