Effect of temperature and water potential on extension rate, extra-resource biomass and fractal dimension of Stropharia caerulea and Phanerochaete velutina growing in trays of soil was determined non-destructively with time, by image analysis. S. caerulea responded differently from P. velutina, mycelial extension and biomass production rates of the former being considerably affected by changes in temperature and water potential, with greater aggregation of mycelia into cords at 25 °C and at and below −0·02 MPa. These morphological changes were reflected in the fractal dimension values. Mycelial extension of S. caerulea on agar was sensitive to temperatures above 25 ° and water potentials below −1·3 MPa. This is the first reported work on abiotic effects on fractal dimension of cord-forming fungi on soil, and these results are discussed in terms of the function of mycelial cords and foraging strategies.

A search for double-stranded RNA (dsRNA) was conducted among 106 isolates of the pathogenic basidiomycete Heterobasidion annosum. Of these isolates, 47 were tissue isolates from fruit bodies and 59 were isolated from decayed wood. Nucleic acids were extracted from freeze-dried mycelia and dsRNA was separated by cellulose CF-11 chromatography and confirmed by digestions with specific nucleases. dsRNA was present in 19 and 14% of the tissue and wood isolates, respectively. From five of the fruit bodies containing dsRNA basidiospores were investigated and 10–84% of the germinated basidiospores contained dsRNA. On high nutrient media, the germination frequency of basidiospores was reduced by presence of dsRNA in the fruit body (P<0.05); germination frequencies were 34 and 78% for spores from fruit bodies with and without dsRNA, respectively. The same trend was present also on low nutrient media, although not statistically significant; germination was 3 and 10% for spores from infected and dsRNA free fruit bodies, respectively. Transmission of dsRNA in H. annosum from mycelia into basidiospores together with the lowered germination frequency are likely to play a significant role in the life cycle of the fungus. The relative importance of different transmission routes for dsRNA in H. annosum is discussed.

Genetic variation in the pistachio late blight fungus, Alternaria alternata, was investigated by restriction fragment length polymorphism (RFLP) in the rDNA region. Southern hybridization of EcoRI, HindIII, and XbaI digested fungal DNA with a RNA probe derived from Alt1, an rDNA clone isolated from a genomic library of the Japanese pear pathotype of A. alternata, revealed 34 different rDNA haplotypes among 56 isolates collected from four central valley locations in California. Analysis of molecular variation revealed a significant amount of genetic diversity within populations (85.8%), with only marginal variation accounting for differentiation among populations (14.2%, ϕST =0.142). All isolates examined were highly pathogenic. The identity of the four geographic populations sampled was not evident in both cluster and principal component analyses, probably indicating either the selectively neutral nature of rDNA variation or prevalence of widespread gene flow among populations combined with uniform host-selection.

During a study on the occurrence and pathogenicity of oomycetes in the reed-belt (Phragmites australis) of Lake Constance (Germany), a new Pythium resembling the important cereal pathogen species complex P. arrhenomanes/P. graminicola was consistently isolated from necrotic mature reed leaves and reed rhizosphere samples. The new species proved to be significantly more aggressive towards reed leaves and seedlings in vitro than related species. It is characterised by filamentous, inflated sporangia and plerotic oospores with usually more than one antheridium. ITS and cox II sequence data indicate this new species shares a common ancestor with P. arrhenomanes, but the sequence differences are clearly consistent with a divergence of the two taxa and with P. phragmitis being a distinct species. ITS 1 and 2 of 15 isolates of the taxon consistently differed from P. arrhenomanes by 13 positions. Sequence analyses of the cox II gene confirmed the new species' phylogenetic position. This paper gives a formal description of the taxon as P. phragmitis sp. nov., providing information on morphology, ecology and pathogenicity in comparison to related species. As indicated by the close association to Phragmites australis, the high aggressiveness towards reed leaves and seedlings, and the abundance in the investigated stands, Pythium phragmitis might act as a reed pathogen of considerable importance, in particular under flooding situations.

The causal agent of tobacco anthracnose was identified as Colletotrichum destructivum based on the morphology of the fungus and a comparison of the sequence of the rDNA ITS with those of other Colletotrichum species. The infection process on tobacco (Nicotiana tabacum and N. benthamiana) was examined by light microscopy, which revealed that the pathogen acted as an intracellular hemibiotroph. Penetration occurred preferentially at the anticlinal walls of epidermal cells by an appressorium and penetration peg. An infection vesicle formed in the penetrated host cell by 48 h after inoculation, and out of this, a multi-lobed infection vesicle grew which remained limited to the initially infected cell. The interaction at this point was biotrophic, which was confirmed by plasmolysis and accumulation of a vital stain by the infected host cells. Thin secondary hyphae arose from multi-lobed infection vesicles at 60 h after inoculation, which then penetrated the host cell wall and began the necrotrophic phase of the infection. Acervuli formed on the plant surface by 96 h after inoculation, typically with a single melanized seta. In addition to tobacco, the fungus could infect alfalfa, cowpea, and Medicago truncatula, but not soybean. The process of infection of C. destructivum in tobacco was very similar to that previously reported in alfalfa and cowpea.

The production of laccase, an enzyme of industrial interest, was screened among species of the genus Pycnoporus, in particular P. sanguineus. Strains were isolated from various tropical Chinese environments and phylogenetically compared to ones deposited in international collections. Molecular clustering, based on ribosomal ITS1-5.8S-ITS2 genomic sequence analysis, showed that the Chinese strains of P. sanguineus formed an homogeneous phylogenetic group distinguished by its laccase-overproducing character. The dikaryotic strain P. sanguineus G05 was selected for its ability to produce up to 40 000 U l−1 laccase in the presence of 2,5-xylidine, Tween 80 and maize bran. Since fruit bodies of P. sanguineus could be formed in the laboratory, monokaryotic laccase-hyperproducing strains were isolated using classic genetical methods. Among these isolates, strain G05.10 synthesized up to 71 000 U l−1 laccase, with a productivity of 5069 U l−1 d−1. The laccase was purified and identified as a 70 kDa protein with an acidic pI, and was very stable at high temperatures.

Thirty South American species of Cortinarius belonging to the subgenera Telamonia, Dermocybe, Myxacium, Phlegmacium, and Cystogenes were studied using an integrated approach that included morphological, anatomical, and ultrastructural data, and also molecular phylogenetic analysis of nuclear rDNA sequences. The micromorphology of the basidiomes was studied by light microscopy, and the principal structures were illustrated by line drawings. Basidiospore ornamentation was studied by scanning electron microscopy (SEM). Nuclear internal transcribed spacers (ITS, including the 5.8S gene) and the DNA coding for the D1/D2 domains of the large ribosomal subunit (LSU) were sequenced and analysed using a Bayesian Markov chain Monte Carlo method to estimate phylogenetic relationships between the studied Cortinarius species. Morphology and anatomy of the pileus surface and basidiome pigmentation appeared to be the most useful characters to delimit some natural groups, whereas microcharacters related to the structure of pileus context, hymenophoral and stipe trama were of little taxonomic value. Basidiospore morphology and cheilocystidia seem to be taxonomically relevant at the species level. The following five infrageneric groups were supported by the morphological, chemical and molecular data: (1) Telamonia characterized by wide hyaline hyphae of the veil and by small basidiomes; (2) Dermocybe spp. with an epicutis as the most external layer of the pileus, and skyrin and hypericin pigments; (3) Dermocybe spp. with a thin viscid layer on the pileus, and endocrocin and dermolutein pigments; (4) Phlegmacium spp. characterized by a long and radicating stipe; and (5) Phlegmacium spp. that overlap in some macrocharacters with Telamonia species. Our analyses suggest that classification concepts based mainly on macromorphological characters are likely to lead to artificial grouping, whereas certain microscopical and chemical characters seem to be useful in constructing a more natural classification system for Cortinarius.

The influence of Cd(II), Cu(II) and Zn(II) on growth and thiol production in an aquatic hyphomycete (Articulospora tetracladia) and a zygomycete (Mucor racemosus) were studied. Growth of a strain of M. racemosus, isolated from effluents contaminated by heavy metals, was not inhibited by Cd concentrations of up to 100 μM. M. racemosus was more sensitive to Cu and Zn. The production of SH-containing compounds by M. racemosus was positively correlated with Cd concentration in the nutrient medium. By contrast, glutathione concentration in the mycelium was negatively correlated with Cd concentration. Addition of Zn or Cu to the medium had no significant effect on mycelial levels of thiol compounds or glutathione. One strain of A. tetracladia, isolated from a clean stream (At-BB), significantly increased the production of thiol compounds and of glutathione in the presence of Cd; the other strain from a Cu enriched stream (At-CS) showed no significant response. Addition of Zn or Cu did not significantly change levels of thiols or glutathione, with one exception: glutathione levels in At-CS were negatively correlated with external Cu levels. Declining glutathione levels in M. racemosus exposed to Cd was coupled with the appearance of several phytochelatins. No such compounds were found in A. tetracladia.

A method to effect rapid, near-synchronous colonization of tomato root systems by a vesicular arbuscular mycorrhizal fungus was developed. This procedure was used to compare the time course of colonization in roots of tomato that were grown under varying phosphate conditions. It was found that the primary symbiotic interaction developed through a series of regularly timed stages. Furthermore, the formation of intercellular hyphae, arbuscules and vesicles was significantly reduced in plants grown with high soil phosphate. These results show that this method is ideal for the production of material for molecular biological studies of VAM symbiosis, including investigation of the regulatory role of phosphate in the symbiosis and the processes that control the fungal life cycle.

There are approximately 70000 species of described fungi, representing about 5% of the estimated 1·5 M species world-wide. Proportionally there are numerous undescribed species and most habitats and hosts should provide a bounty of novel fungi that can be exploited in a wide variety of ways. Where, however, are these missing fungi? Data indicates that they may occur in poorly studied countries, hosts, habitats, niches or tissues, and are mostly microfungi. Host specificity and/or tissue recurrence are important considerations in biodiversity estimates. We are a long way from establishing where we can find the missing 1·43 M fungi, but evidence presented here resulting from data from Hong Kong indicates many places where we could look.

Persistence of Aschersonia aleyrodis, a fungal pathogen of whitefly, was studied on cucumber, gerbera and poinsettia. Germination capacity and infectivity of conidia, which stayed on the plants for up to 1 month, were assessed. Average germination of conidia on the leaves was low (< 14%), whereas most of the conidia transferred from the leaf to water agar were viable, even after having been on the leaf surface for 1 month. Germination capacity was highest on cucumber, followed by poinsettia and lowest on gerbera. On cucumber leaves, conidia stayed viable and were able to infect 90% of whitefly nymphs 31 d after spore application. On gerbera, germination capacity decreased considerably from 80% (day 0) to 40% (day 31). This was reflected in nymphal mortality, which declined from 75% to 40%. Despite the high germination capacity (60%) of conidia on poinsettia after an exposure of one month, nymphal mortality decreased from 70% at the day of spore application to 10% after 3 d at leaf surface, and remained low throughout the monitoring period. Relations between germination capacity, infectivity and the host plant environment such as phyllosphere microorganisms, secondary plant metabolites and microclimate are discussed.

Two new genera, Neophaeosphaeria and Phaeosphaeriopsis, are described to accommodate species of Paraphaeosphaeria that are not congeneric based on morphological characters and results of 18S rDNA sequence analyses. Paraphaeosphaeria s. str. is restricted to species with two-septate ascospores and anamorphs that produce non-septate, smooth, pale brown conidia enteroblastically from phialides which have some periclinal thickening. Species in Neophaeosphaeria have 3–4-septate ascospores and anamorphs that produce ovoid to ellipsoid, non-septate, brown, verrucose or punctate conidia from percurrently proliferating conidiogenous cells. Paraphaeosphaeria barrii, P. conglomerata, P. filamentosa and P. quadriseptata are transferred to Neophaeosphaeria. At present all species in Neophaeosphaeria occur on Yucca (Agavaceae). Phaeosphaeriopsis is described for species that produce 4–5-septate ascospores. Known anamorphs produce cylindrical, 0–3-septate, brown, punctate conidia from percurrently proliferating conidiogenous cells or bacillar conidia from simple phialides. P. agavensis, P. glauco-punctata, P. nolinae and P. obtusispora are transferred to Phaeosphaeriopsis. P. amblyspora is described as a new species.

The ability of Hymenoscyphus ericae to produce 12 carbohydrolytic activities has been investigated during growth under solid-state fermentation conditions. The fungus produced a complete cellulase enzyme complex (cellulase, cellobiohydrolase and β-D-glucosidase) along with components of the hemicellulolytic (endoxylanase, β-D-xylosidase, α-L- and β-L-arabinosidase and glucoronidase) and mannanolytic (mannanase, β-D-mannosidase, α-D- and β-D-galactosidase) complexes. Specific activities were greatest for endo-acting enzymes and lower for exo-acting and side-branch removing activities in each case. The data indicate that H. ericae possesses a considerable ability to degrade plant polymeric material and are discussed in relation to establishment of the symbiosis and interaction of mycelium with moribund plant organic matter in soil.

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.

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.

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.