The dynamics of competitive colonisation of necrotic cyclamen tissue by the plant pathogenic fungus Botrytis cinerea and the saprophytic fungal antagonist Ulocladium atrum were studied immuno-histologically, while sporulation was studied macroscopically. The effect of different time intervals between inoculation of both fungi on resource capture by each species was explored. Colonisation and sporulation were used as indicators for competitive resource capture and the effectiveness of biological control of B. cinerea, using U. atrum. Mycelial biomass and sporulation showed logistical time courses in both species, in monocultures as well as in mixed cultures. Final colonisation and sporulation levels were lowered by competition, indicating competitive resource capture. Analysis of the extent to which sporulation of either fungus could be reduced by co-inoculation with the other fungus at different times, showed that B. cinerea can be completely excluded by ‘early’ pre-inoculation with U. atrum, but not vice versa, indicating that U. atrum can exploit resources in the leaf that are not accessible to B. cinerea. A model of competitive substrate colonisation and resource capture was developed on the basis of the experimental results. Model results confirm that competition for resources provides a sufficient biological explanation for the dynamic interactions between the fungi. The model provides a tool to optimise dose and timing of U. atrum applications providing effective biological control of B. cinerea.

Sclerotinia sclerotiorum produced erythroascorbate at levels dependent on oxidative growth conditions, developmental stage and strain differentiating capacity. The transition of S. sclerotiorum from the undifferentiated to differentiated state under high oxidative stress growth conditions, was accompanied by a shift in the ratio of reduced/oxidized erythroascorbate towards its oxidized form. At low oxidative stress, the reduced form of erythroascorbate predominated. In a non-differentiating strain of S. sclerotiorum, reduced erythroascorbate predominated over the oxidized form during growth. The reduced/oxidized erythroascorbate ratio in this strain was 5-fold higher than the corresponding ratio in the differentiating strain. The level of oxidative stress (lipid peroxidation) in colonies of the differentiating strain (one day before they differentiated) was 3-fold higher than that formed in colonies of the non-differentiating strain. Exogenous ascorbate exhibited its antioxidant activity in the differentiating strain by causing a dose-dependent decrease in lipid peroxidation, and proportional inhibition of sclerotial development. It is suggested that during its initial growth stages, S. sclerotiorum cannot decrease lipid peroxidation to levels found in the non-differentiating strain, and then forms sclerotia. We propose that oxidative stress triggers differentiation in sclerotium-producing filamentous phytopathogenic fungi.

Phenotypes of species hybrids created from in vitro fusion of zoospores from Phytophthora nicotianae and P. capsici were characterized and compared. The species hybrids were created as part of a study of sources of genetic variation in populations of the parent species that are pathogenic over a similar range of plants. Four isolates of species hybrids proved to be similar to both P. capsici and P. nicotianae in relation to vegetative and reproductive morphologies. As in a previous study, DNA of P. capsici was detected more readily than that of P. nicotianae in all hybrid isolates. In the present study, DNA of P. nicotianae was detected in three of four hybrids by hybridization of RAPD-PCR products with species-specific DNA from P. nicotianae. By thermal denaturation analyses, DNA melting temperatures and GC contents of parent species and species hybrids were similar. The mean GC content of 47·2% was similar to GC contents reported for other Phytophthora spp. Additionally, the distributions of GC-rich regions of hybrids were more similar to the distribution in P. capsici than in P. nicotianae. By these molecular analyses, the hybrids were shown to be more similar to P. capsici than to P. nicotianae. Even though interspecific somatic fusion is likely to occur rarely under natural conditions, it could contribute to the genetic diversity of heterothallic species of Phytophthora.

The entomopathogenic fungus Metarhizium anisopliae was physiologically manipulated by culturing in liquid culture under a range of water-stress conditions (0.98, 0.97 and 0.96 water activity, aw) imposed by either PEG 200, KCl or NaCl at 25 °C to optimise blastospore production and quality in terms of accumulation of endogenous compatible solutes and germinability. Optimum spore yield occurred at 0.98aw, when PEG 200 was the solute. Blastospores produced under different water-stress levels were subjected to hypo-osmotic and iso-osmotic washing treatments before quantification of endogenous sugar alcohols (polyols) and germinability studies. Endogenous amounts of the low molecular weight polyol erythritol were greatly affected by culture age, water-stress and hypotonic or isotonic washing treatment. Endogenous erythritol in blastospores produced under 0.98 and 0.97aw increased with culture age. Osmoprotection (washing with isotonic PEG 200 solution) of blastospores produced under all water-stress conditions resulted in retention of higher amounts of erythritol. Increased endogenous retention of the high molecular weight polyol mannitol was also observed with an isotonic washing treatment but only in blastospores produced under intermediate water-stress (0.98 and 0.97aw). Germination of all treatments was >90% on a medium with freely available water. Under water-stress conditions (0.96aw) only 10% of unmodified blastospores washed with water germinated. However, those grown under modified water availability conditions (0.98aw) and washed with isotonic solutions had significantly improved germination (38–90%). Blastospores produced at [les ]0.97aw washed with isotonic solutions had decreased germination. These results could have significant implications for conserving ecophysiological quality of fungal biocontrol propagules during harvesting and have an impact on storage and formulation.

Cryopreservation of ectomycorrhizal fungi is often difficult, but needed for patent deposits and for enabling experiments on model strains with constant characteristics. In this study we report a protocol for successful cryopreservation of the ectomycorrhizal mushroom Cantharellus cibarius, together with a brief summary of some unsuccessful attempts. A slow freezing rate (0.3°C min−1) of agar cubes carrying the mycelium, and a gentle addition of the cryoprotectants sorbitol and DMSO were important features. This protocol was successfully repeated by ATCC who used it to deposit a patented strain of C. cibarius, and therefore the protocol should also be tested for other ectomycorrhizal basidiomycetes.

Zoospore cysts of four isolates of Aphanomyces euteiches with different host-specific pathogenicity germinated in response to peptone, pea root extract, CaCl2 or gum arabic but, in tests on one isolate, did not respond to several individual sugars, amino acids or polysaccharides. A. euteiches showed differential responses to substances applied at different times in the encystment process. Gum arabic triggered germination only when added to motile zoospores, which it caused to encyst. CaCl2 triggered germination when added to motile zoospores or during vortex-encystment of zoospores, but seldom immediately (<2 min) after encystment. Peptone and pea root extract triggered germination when added to motile zoospores, during vortex-encystment or immediately after encystment, but not ca 20 min after encystment. In comparative tests, Phytophthora parasitica was less stage-specific than A. euteiches, because it germinated in response to gum arabic applied to motile zoospores, during encystment and immediately after encystment.

Zoospore taxis, encystment and germination of A. euteiches and P. parasitica cysts were recorded on pea roots by video microscopy. The speed of germination and pattern of sporeling development on roots were most closely matched by treatment of motile spores with gum arabic in pure culture. The behaviour of A. euteiches conformed to a previous model for Pythium and Phytophthora spp., in which zoospores encyst by recognition of root surface components (simulated by gum arabic), germinate autonomously by a calcium-mediated process (simulated by calcium treatment during vortexing), and the germ-tube produces an assimilative hypha (simulated by peptone or pea root extract).

Laccaria bicolor and L. laccata are ectomycorrhizal basidiomycetes which fruit abundantly over 1 or 2 months in autumn and sometimes in summer. We investigated the phenology of fruiting of the various Laccaria spp. genets in a Douglas fir plantation in the Nièvre (France), in which all genets had been identified by molecular methods. During the fruiting period of the population, the genets did not fruit synchronously: some fruited early, others later, and some over the whole period. For some genets that survived over 2–3 years (1995–97), the fruiting phenology remained similar from year to year, even when fruiting occurred in summer. We took advantage of the presence of a L. bicolor genet, which had been used to artificially inoculate Douglas firs in this plantation, to assess whether the timing of fruiting was influenced by microsite differences in the plantation (soil, host tree, etc.). In summer 1997, fruit bodies of this genet appeared synchronously everywhere at the beginning of the fruiting period. We conclude that the timing of Laccaria fruiting may be genetically determined, rather than explained by the size and position of the genet or the environmental conditions. The consequences for population studies are discussed.

Mycosphaerella vespa sp nov. is described from diseased leaves of Eucalyptus from south-eastern Australia. It is associated with small, circular to irregular lesions that have red-brown margins and amphigenous pseudothecia. These characters, in combination with ascospore morphology and germination, distinguish M. vespa from previously described species of Mycosphaerella on eucalypt leaves. Lesions associated with this pathogen were often found to be harbouring wasps and this observation is discussed.

Thirty-five isolates of Gaeumannomyces graminis were tested for ability to infect wheat, rye and oats, and for DNA polymorphisms using nuclear ribosomal DNA (rDNA) RFLP patterns and RAPD analysis. In general, the cereal-attacking isolates could be readily assigned to the rye-attacking (R) or non-rye-attacking (N) subgroups of var. tritici, or to var. avenae, on the basis of either of these molecular approaches. A small number of isolates gave anomalous rDNA RFLP patterns, but could nevertheless be assigned to one of the three groups by RAPD analysis. Two related G. graminis var. tritici isolates (T1–1 and T1–2) clearly grouped with the N rather than the R var. tritici subgroup on the basis of molecular analysis but were pathogenic to rye, indicating that the ability to infect this host may have arisen more than once. An earlier phylogenetic study of Gaeumannomyces involving DNA sequence analysis of the internal transcribed spacers of the rDNA indicated that several oat-infecting isolates originally classified as G. graminis var. tritici could be grouped with var. avenae isolates. From the rDNA RFLP and RAPD analysis described here, however, these isolates appear to be intermediate between var. avenae and var. tritici, although it is not clear whether they represent evidence of inter-varietal sexual hybridization.

Fluorescent stains offer an effective means of visualising bacteria and fungi in soil or litter samples. Fluorescent brightener (FB) 28 (also known as Calcofluor White M2R) is commonly used to stain such microorganisms. However, during production of soil thin-sections we observed erratic crystallisation of this stain, particularly in soils colonised by Rhizoctonia solani. We report on the evaluation of alternative stains to FB 28 for their propensity to crystallize in interaction with fungi, staining efficiency, and suitability for application in soil thin-section production. All of the additional candidate stains namely Fluorescent Brightener Agent (FBA) 15/25, FBA 71 and SCRI Renaissance 2200 (SR 2200) were highly effective in staining agar-cultured hyphae, but differed in the degree to which they stained hyphae cultured in soil. All stains tested, except SR 2200, stained hyphae of R. solani insufficiently when growing on or through soil. These stains also showed extensive crystallisation in solutions that had been in contact with R. solani colonised soil. However, SR 2200 stained hyphae of R solani growing over soil as effectively as hyphae growing on agar and showed no evidence of crystallisation; the intensity of staining exceeded that of the ‘benchmark’ FB 28 for hyphae grown in two soil types. These excellent fluorescent properties of FBA 220 persisted in soil thin-sections, resulting in bright hyphae that could be readily visualised in situ in undisturbed soils.

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.

Extracellular enzymes play an important role in the pathogenicity and virulence of phytopathogenic fungi. Several isolates of Colletotrichum coccodes, causal agent of anthracnose on tomato, were screened to determine the relationship between protease activity and virulence. A direct relationship was observed between extracellular protease activity and the induction of disease symptoms of fruit and mortality in plants. Isolate Cc155 exhibited the highest protease activity after five days of growth in protease induction medium and produced an extracellular serine protease (sp78) that was 78 kDa, auto-degradative, glucose repressible, and non-glycosylated. To determine the role of sp78 in pathogenicity, a UV-induced extracellular protease deficient mutant (np155) was generated from the wildtype isolate Cc155. Np155 maintained growth rates comparable to Cc155 and produced wildtype levels of extracellular cellulase but did not produce extracellular protease. Unlike Cc155, np155 caused no disease symptoms on tomato fruit and 0% mortality on tomato seedlings. These results suggest that extracellular protease activity is required for pathogenicity and virulence of C. coccodes, and that the elimination of protease activity transforms a virulent pathogen to a non-pathogenic endophyte.

We report the first successful culturing and confirmation of identity (via sodium dodecyl sulphate polyacrylamide gel electrophoresis, SDS–PAGE) of Serpula lacrymans (the dry rot fungus) derived from basidiomes and mycelia growing in the ‘wild’. The fungus was found growing on well decayed coniferous wood within the Narkanda region of the Western Himalayas at between 2800 and 3100 m above sea level (a.s.l.). The ‘wild’ habitat of the fungus is described, as is the isolation of Serpula himantioides also found in these regions. Temperature, osmotic potential, initial pH and air-current influenced the average colony extension rate of ‘wild’ and a selection of building isolates on 2% malt extract agar. The ‘wild’ and building isolates behaved as two separate cohorts; the ‘wild’ isolates appeared to be less affected by extremes of temperature, whereas building isolates extended more rapidly at the moderate microenvironmental regimes.

Assessment of genetic and phenotypic diversity is necessary to confidently distinguish genotypes of Pleurotus eryngii when seeking traits of interest and to identify strains with high yield potential. We studied 154 strains from Italy for quantitative (shape, size and yield), qualitative (colour, malformations and growing behaviour), and molecular (RAPD and minisatellite) traits. This population consisted of isolates mostly belonging to P. eryngii var. eryngii, var. ferulae or var. nebrodensis, from different regions of Italy. A replicated cultivation trial, with three blocks and three replicates for each strain within the block, was used as an experimental design to calculate trait estimates. Significant differences were observed between strains for basidiome number and weight, while no significant differentiation for quantitative morphological traits was observed between geographical origins and taxonomic groups. Qualitative morphological traits were efficient in differentiating isolates of P. eryngii var. nebrodensis. On average, yield per strain (basidiome weight) was correlated more with basidiome number than with size. The most stable yield traits were basidiome number and weight per strain. An average heritability of 0.31 was estimated for yield related traits. A significant difference between var. ferulae and var. eryngii populations was detected for basidiome production measured as ‘average harvest time’. Molecular markers showed a high level of heterogeneity within populations and a low, but significant, degree of differentiation among populations defined a priori. No population-specific marker was detected and the differential pattern of variation between vars. ferulae and eryngii was due to frequency-dependent alleles. The nebrodensis type was more differentiated from var. eryngii than from var. ferulae using either molecular or qualitative morphological traits.

Spore proteins of different species and isolates of arbuscular mycorrhizal (AM) fungi were compared by PAGE. Reproducibility of protein patterns was assessed by using cultures of the same species either grown on different host plants, or produced during successive propagation cycles and stored up to 5 years. The results consistently showed that host species, different generations and storage, did not affect protein profiles, thus validating the accuracy of the method. Comparison among different geographical isolates of the same species revealed consistent protein patterns. The stability and diversity of spore protein profiles suggested that PAGE could be used to discriminate and identify AM fungal species and isolates. By contrast, the physiological state of spores affected the quality and quantity of bands, with germinating spores showing marked profile changes, as compared to quiescent spores, both in denaturating and native analytical conditions. The disappearance of some polypeptides in germinated spores might be related to the occurrence of storage proteins in AM fungi.

The composition and concentration of airborne fungal propagules are probably determined by many interrelated environmental factors, such as temperature, relative humidity, atmospheric pressure, wind speed, rainfall and gas pollutants. The importance of these variables was assessed in an outdoor sampling survey conducted at regular intervals for 12 months at a single site in Turin. Samples were collected with a single-stage volumetric sieve sampler on potato dextrose agar supplemented with 15 mg l−1 streptomycin and 50 mg l−1 chloramphenicol.

Canonical Correspondence Analysis showed that the four ordination axes accounted for 93·5% of the variance in relationships between fungal entities present in more than 20% of samples and the environmental variables. The Monte Carlo permutation test demonstrated that the ordination was highly significant (P=0·01). The community's qualitative and quantitative composition mainly depended on the factors that have the greatest influence on Turin's climate, i.e., in descending order, temperature, relative humidity and rainfall. The relative importance of these environmental variables on different groups of fungi was assessed. Wind speed positively correlated with the fungi producing conidia of larger sizes.

The identification of plant-associated isolates of Aphanomyces spp. has been mainly based on morphological characters. These types of features, however, can be unreliable due to their high variation and degree of overlap between different taxa. In this work, strains of Aphanomyces, with plant pathogenic and non-pathogenic characteristics, derived mainly from the roots of several leguminous crops in Sweden, as well as a number of reference strains of A. euteiches, A. cladogamus and A. cochlioides were compared by morphology, AT-rich DNA RFLPs and sequence analysis of the total ITS/5.8S rRNA gene region. Parsimony analysis of the sequence data of the total ITS region of 21 strains separated the taxa at the specific level, while two intra-specific groups were also detected within A. euteiches. One comprised all pathogenic isolates of A. euteiches from Sweden, together with two isolates from France and the USA. These isolates formed a homogenous group with identical ITS regions and a single AT-rich DNA RFLP banding pattern. The second ITS subspecific group included reference strains of A. euteiches with preferential pathogenicity to bean and alfalfa, all of which originated from the USA. The bean and alfalfa pathotypes were further distinguished by their different AT-rich DNA RFLPs. The non-pathogenic isolates of Aphanomyces from Sweden associated with the roots of several legumes, formed a genotypically uniform group with an identical ITS sequence and a single AT-rich DNA RFLP pattern. The ITS sequence of this group was identical to that of reference strains of A. cladogamus, suggesting that the non-pathogenic isolates belonged to this species. Intra-specific AT-rich DNA banding profiles were also obtained for isolates of A. cladogamus and these patterns corresponded well to the original hosts.

A piece of Baltic amber (Tertiary, Eocene) contains a sporocarp of a slime mould which is assigned to the recent genus Arcyria and described as A. sulcata sp. nov. Apart from a fossil stemonitoid myxomycete, there are no further unambiguous fossil records of slime moulds and therefore the fossil gives new insights into the evolutionary history of the Myxomycetes.

The anamorphic basidiomycete genus Marchandiomyces presently includes two common lichenicolous (lichen-inhabiting) species, M. corallinus and M. aurantiacus (teleomorph Marchandiobasidium aurantiacum). We describe here a new species, M. lignicola sp. nov., that is similar to M. corallinus in the colour of its sclerotia, but differs in having a wood-inhabiting (lignicolous) habit. The phylogenetic position of this lignicolous fungus was compared with the lichenicolous species of Marchandiomyces and related species currently placed in the basidiomycetous families Corticiaceae and Ceratobasidiaceae using parsimony, likelihood, and Bayesian analyses of complete sequences of the nuclear small subunit and internal transcribed spacers ribosomal DNA, and a portion of the nuclear large subunit ribosomal DNA. These DNA sequences were obtained from isolated cultures of freshly collected specimens. Significant Bayesian posterior probabilities, as well as maximum likelihood and parsimony analyses, indicate that the new lignicolous species is closely related to M. corallinus, the type species of Marchandiomyces. In most analyses these two species are monophyletic with the lichenicolous M. aurantiacus, although this relationship is not strongly supported. Since M. lignicola is more closely related to M. corallinus than to M. aurantiacus, either a transition to the lignicolous habit occurred recently within an ancestral lichenicolous group or, more likely, transition to the lichenicolous habit arose recently and in parallel from an ancestral lignicolous habit.

The wood of one Picea abies stump, including its roots (1·3 m in length), was sliced into 2 cm thick discs. The stump originated from a thinning conducted 7 yr prior to the investigation in a 30 yr old spruce stand planted on previous farmland that became heavily infected by Heterobasidion annosum. After incubation of the wood discs, interaction zones were observed on the surfaces and H. annosum was isolated from the areas between the zones and from the zone lines, resulting in 296 isolates. The isolates were tested with somatic incompatibility to detect 35 different genets. Of the 27 genets colonising the upper part of the stump (excluding the roots), 12 had proceeded into the roots. Seven genets found in the roots were not found in the upper part of the stump and one genet isolated from an interaction zone on the top of the stump was not found elsewhere in the stump or in the roots. Two of the genets had grown into root contact with other trees.