Two rapid, independent molecular assays have been developed for discriminating between the closely related rusts of hard pines, Cronartium flaccidum and Peridermium pini. A portion of the ITS region and a portion of the IGS region from the ribosomal RNA operon of the two organisms were amplified with the PCR. Amplifications were made using DNA extracted from aeciospores collected from unopened aecia, taking a small number of spores as a source of template DNA. Amplified fragments were subjected to RFLP or to SSCP analysis. Digests of the amplified products from the IGS1 region were electrophoresed on polyacrylamide gel and stained with ethidium bromide. Hinf I digestion of these fragments created polymorphic restriction profiles which allowed differentiation of the autoecious P. pini from the heteroecious C. flaccidum. Similar-sized DNA fragments representing the ITS2 region of the two rusts were denatured, subjected to electrophoresis as single strands on polyacrylamide gel under non-denaturing conditions and ‘silver stained’. The different mobility displayed by these short fragments revealed sequence polymorphism in the examined portion of the ITS region. This technique therefore represents an accurate and sensitive method for detection of base changes in given sequences of genomic DNA. A high level of homology was found between the two biotroph organisms in the loci screened. Results obtained in this trial indicate that PCR-RFLP and PCR-SSCP can be used as simple, speedy taxonomic tools for elucidating relationships among related organisms.

Two nematode-parasitic isolates of Harposporium are reported to form synanamorphs attributable to the Hirsutella. One is attributedto Harposporium anguillulae, the second is described as a new species, H. cerberi. H. cerberi also produced an arthroconidial synanamorph. In infection studies of nematodes, conidia typical of Harposporium proved to be capable of killing bacterial feeding nematodes (Rhabditis sp.) when ingested. Infective conidia of H. cerberi were located posterior to the oesophageal bulb, while those of H. anguillulae lodged above it. Hirsutella conidia were not observed to be infective or adhesive, and none of the spore forms was capable of infecting either of two species of stylet-bearing nematodes (Aphelenchus sp. and Meloidogyne hapla).

Analysis of quality-related parameters at the green leaf-stage and in orthodox-made tea revealed marked quality deterioration with increasing blister blight severity. Total phenols, catechin(s), total nitrogen, amino acids, and chlorophylls, as well as polyphenol oxidase activity, declined in tea shoots with increasing disease severity. Depending on disease intensity, orthodox tea made from infected shoots showed loss in leaf-style. Likewise, theaflavins, thearubigins, caffeine, and high polymerized substances as well as total liquor colour, brightness and briskness also exhibited marked progressive decline in tea made from shoots with increasing disease severity. Aroma components, particularly hexene-1-ol, phenyl acetaldehyde, linalool, methyl salicylate, geraniol, indole, β-ionone, nerolidol, and several unassigned components were also lower in disease affected teas. The magnitude of decline in various aroma constituents corresponded to disease intensity.

Twenty-six isolates representing six morphologically similar taxa of Tilletia occurring in the western U.S.A. were compared using randomly amplified polymorphic DNA (RAPD) markers and restriction fragment length polymorphisms (RFLP) of the 5·8 s rDNA and flanking ITS regions. Two distinct clusters were separated in the dendrogram based on the RAPD analysis. One cluster contained isolates of T. controversa, Apera interrupta-infecting isolates of a potentially new species of Tilletia, and isolates of the Bromus-infecting varieties bromi-tectorum and guyotiana of the T. fusca (=T. bromi) complex. The second cluster contained Vulpia-infecting isolates of T. fusca var. fusca. The separation of the Bromus-infecting varieties from the Vulpia-infecting variety of T. fusca was supported by distinct restriction digest phenotypes. The RAPD analysis was more sensitive than the RFLP analysis, and allowed separation of the Bromus-infecting varieties bromi-tectorum and guyotiana corresponding to host specificity shown in previous studies. The presence of genetically distinct populations of T. fusca var. fusca occurring on V. microstachys and V. octoflora is indicated by RAPD and RFLP analyses. Species status of T. togwatii, a bunt infecting Poa reflexa that was previously removed from the T. fusca complex, is also supported by RAPD and RFLP analyses.

The present study treats several fungi associated with leaf spots of Proteaceae from Africa, Australia and India. Leptosphaeria leucadendri (anamorph: Sclerostagonospora leucadendri) is described from Leucadendron leaves from Australia, while three previously described African taxa from Protea leaves are transferred to Phaeophleospora as P. abyssinicae, P. congestum and P. protearum. Trimmatostroma protearum and a Phyllosticta sp. are described from Protea leaves collected in South Africa and Australia, respectively. Cercospora agharkarii, which occurs on Grevillea, is redisposed to Pseudocercospora, while a new genus Pseudohendersonia, is proposed for P. proteae occurring on Protea leaves in South Africa.

More than 90 species are accepted in the xylariaceous genus Rosellinia. Thorough morphological characterisation of all known species will provide a taxonomic hypothesis that can be later tested by DNA sequence analysis. Exploratory analyses of ascospore morphology, shape of ascus apical structure, and anamorph genus suggest six morphological groups in the genus.

Two new thermotolerant species of Thielavia were isolated from Easter Island and India, after treating soil samples with 65% (v/v) ethyl alcohol: T. intermedia sp. nov. characterized by large, obovate, dark brown ascospores with a terminal germ pore; and T. arxii sp. nov. distinguished by the large, ellipsoidal to fusiform, brown ascospores with a prominent, lateral to medial germ pore. A phylogenetic study of the ITS-5.8S rDNA sequences of 18 species of Thielavia and Melanocarpus thermophilus was performed. This analysis revealed that T. intermedia could be the ancestor of the remaining species and that all the species tested were genetically closely related.

Four day/night temperature regimes (11/6, 14/9, 17/12, 20/15 °C), three photoperiods (0, 12, 24 h d−1), and four continuous temperature regimes (10, 13, 16, 19 °) were evaluated for their effects on growth, sporulation, and conidia discharge of two isolates of Valdensinia heterodoxa, a foliar pathogen of salal (Gaultheria shallon). All temperature regimes supported mycelial growth; however, growth rates were faster at higher temperatures. Sporulation and conidia discharge in both isolates was generally highest at 17/12 ° (day/night) or 16 ° constant compared with other temperature regimes. Photoperiods of 12 and 24 h d−1 resulted in faster growth and significantly greater sporulation and discharge compared with a continuous dark treatment. The study shows that temperature and light conditions have a moderate effect on mycelial growth but are crucial for sporulation of V. heterodoxa. Possible implications for the use of V. heterodoxa as a potential biological control agent for salal are discussed.

We compared the ability of five strains of the ericoid mycorrhizal fungus Hymenoscyphus ericae to utilise glutamine, ammonium or nitrate at high or low carbon (C) availability. The pattern of intraspecific variation in growth was affected by C availability. When C supply was high, growth differences between strains were explained by the total amount of nitrogen (N) taken up, suggesting variation in uptake kinetics. Under C-limiting conditions, strain differences were linked with their nitrogen use efficiency, implying intraspecific differences in N metabolism. The relationship between growth on glutamine and pH shifts in the media indicated that there was intraspecific variation in glutamine transporters. In addition, the correlation between pH changes and the amount of glutamine-N recovered as ammonium in the media indicated that there were intraspecific variations within the enzymatic pathways involved in glutamine metabolism. Our findings, compared with those of a previous study involving the same ericoid strains, draw attention to the temporal variation in nitrogen source utilisation by ericoid mycorrhizal fungi when maintained in axenic culture.

Two fruitbodies each of Russula messapica and Macowanites messapicoides, identified by morphological characters, were screened for polymorphism in the ITS and the IGS of their ribosomal DNA, as well as in mitochondrial rDNA by RFLP analysis of PCR-products. The two species were monomorphic for all markers with the exception of the IGS. This limited genetic differentiation indicates a close relationship between these two species. The overall correspondence of the RFLPs between R. messapica and M. messapicoides adds molecular evidence to the relatedness of Russula and Macowanites, and suggests the possibility that M. messapicoides is recently derived from R. messapica.

Comparative morphology, mating compatibility and extracellular laccase approaches were used to provide information on infraspecific variation within geographically separated collections of Marasmius androsaceus. Collections/cultures from North America and Europe were used for these analyses. While all utilized collections belonged to one morphological species, mating compatibility crosses revealed three intersterility groups. Extracellular laccase banding patterns confirmed genetic isolation of intersterility groups I and II.

Immunogold labelling and electron microscopy were used to investigate β-1,3-glucanase secretion by Claviceps purpurea during ergot disease of rye in situ. Molecular cytology allowed us to explore the hypothesis that this enzyme might degrade host phloem callose to maintain flow of assimilates for fungal nutrition and therefore β-1,3-glucanase could play a role in pathogenicity. An extracellular endo-β-1,3-glucanase was purified from axenic culture and an antibody was raised. Enzyme activity staining and immunoblotting showed that the antibody was monospecific for β-1,3-glucanase present in fungal protein populations. Mycelia printings of plate-grown cultures displayed spot-like and streaky immuno-signals suggesting a secretion of β-1,3-glucanase at hyphal tips and young hyphae. The enzyme was immuno gold localized predominantly in cell walls of mycelia from axenic culture. In Western blots of honeydew fractions, one β-1,3-glucanase was immunoreactive. In inoculated plants, immunogold labelling was found in all infection stages and limited to the host-pathogen interface. Gold labelling was detected over fungal protoplasts in vacuoles and in tubular-vesicular complexes and multivesicular bodies, which fused with the fungal plasma membrane, indicating that they are part of the secretion pathway. The labelling of the fungal secretory organelles and lack of labelling in any host area apart from the interface verified the fungal origin of β-1,3-glucanase immuno-detected in infected ovaries. Antigenic sites were located in external, subcuticular, penetrating and intercellular hyphae, indicating that the enzyme was secreted throughout the colonization process in planta. Intense labelling regularly associated with fungal cell walls extended into adjacent host walls, indicating a migration of the fungal β-1,3-glucanase into the host apoplast. The gold labelling over host periplasmic spaces showed that the enzyme reached the deposition sites of callose, pointing to an enzymatic suppression of putative plant defense reactions. The host phloem was colonized inter- and intracellularly. Hyphae penetrated into the pectic middle lamella of sieve plates and intense immuno-labelling for β-1,3-glucanase in this area supports a phloem unblocking hypothesis.

Aspergillus fumigatus is saprotrophic with an unusual ability to colonize the respiratory tract. The mechanisms that permit pathogenicity may have evolved to adapt the fungus to life as a saprobe. To define the nature of these adaptations and identify common themes in fungal pathogenesis to vertebrates, insects and plants, we compared A. fumigatus with a plant pathogen (Haematonectria haematococca) and an insect pathogen (Metarhizium anisopliae) in their abilities to degrade and utilize host-derived macromolecules (horse lung polymers, porcine mucin, hyaluronic acid, alfalfa cell walls and cockroach cuticle). Each fungus produced a similar range of proteases on mucin and lung polymers, and high levels of several glycosidic enzymes on mucin and plant cell walls, which contain inductive carbohydrate substrates. Following 18 h of growth by A. fumigatus at pH 4 or pH 8, the degradation of mucin carbohydrates and mucin protein were approximately 40% and 75% respectively, suggesting that the aspartyl proteases (produced at pH 4) and the subtilisin proteases (produced at pH 8) are more important than carbohydrases for degrading mucin. The highly glycosylated mucin residue remaining after 18 h growth resisted further degradation, in part due to bound sialic acid as A. fumigatus secretes a sulphatase but not sialidase. Hyaluronidase activity (an important virulence factor in bacteria) was not produced by A. fumigatus, M. anisopliae or H. haematococca, but each fungus secreted a range of other enzymes (phospholipase A2, phospholipase C, acid phosphatase, alkali phosphatase, phosphodiesterase and esterase) that are common toxic components of bacteria as well as reptilia and invertebrate venoms. Thus thermotolerant opportunists such as A. fumigatus may sustain themselves and cause disease in human hosts using depolymerases that are widely distributed in fungi and that provide them with the versatility to exploit many environments.

Mycelial interactions on Hagem agar between 156 isolates of Stereum sanguinolentum obtained from wounded Picea abies or from slash in 12 sample plots located in Sweden, Finland and Lithuania revealed 97 vegetative compatibility groups (VCGs). Of the 23 VCGs that consisted of two or more (maximum, 23) isolates, 13 came from different sample plots, and sometimes from two or all three countries, whereas 10 were confined to a single sample plot. Some variation was observed in cultural characteristics among isolates belonging to the same VCG. Within a sample plot compatible isolates were randomly distributed indicating that distances up to 180 m in a forest stand have little influence on spread of S. sanguinolentum VCGs. Proportions of compatible pairings decreased among isolates collected within the bigger spatial scale. Occurrence of compatibility and consequent grouping of the isolates into VCGs was more pronounced in Swedish populations then Finnish and Lithuanian populations.

A prerequisite for symbiotic phosphate transfer in an ectomycorrhizal (ECM) association is hypothesized to be conditions in the interface between both symbiotic partners, that either promote the release of inorganic phosphate (P) from the Hartig net into the interfacial apoplast and/or decrease the fungal reabsorption from this location. To get more information about conditions, which might be involved in the regulation of P efflux or P reabsorption, the effect of various external conditions on 33P-orthophosphate (33P) uptake or efflux by axenic cultures of the ECM basidiomycetes Hebeloma crustuliniforme, Amanita muscaria and Laccaria laccata was analysed. In short-time experiments the following external conditions were analysed: an external supply of (1) P in the preculture, (2) cations (0.1–100 mM K, 0.1–50 mM Na, Mg and Ca), and (3) carbohydrates (0.5–50 mM glucose, fructose or sucrose).

The P absorption was generally reduced in cultures previously supplied with an abundant P supply and with increased P concentrations in their tissues. The P uptake was also affected by an external supply of cations, whereas carbohydrates had only a slight effect. Compared to Na, Mg and Ca, the P absorption by H. crustuliniforme and L. laccata was increased by 0.1 mM K in the labelling solution but decreased after a supply of 100 mM K and then did not differ from the other cation treatments. Compared to other cations, an addition of 50 mM Ca led to a decrease of P absorption by A. muscaria, whereas 50 mM Mg increased the P uptake by H. crustuliniforme.

The P efflux from the fungi was affected by both the cation and carbohydrate concentration of the bathing solution. High concentrations of the monovalent cations K and Na (5 mM or 50 mM) in the bathing solution increased the P efflux by H. crustuliniforme (only Na) and L. laccata (K and Na), but had little effects on A. muscaria. By contrast, the same concentrations of the divalent cation Mg reduced the P efflux from all fungal basidiomycetes. The P efflux from hyphae of H. crustuliniforme and A. muscaria was also affected by the carbohydrate concentration in the bathing solution. Compared to the hexoses, glucose and fructose, the P efflux by both basidiomycetes was stimulated even by low concentrations of sucrose (0.5 mM) in the bathing solution. The effect of carbohydrates on P efflux especially from hyphae of H. crustuliniforme was clearly concentration-dependent and the maximal efflux rates were observed after a supply of 2.5 mM sucrose or fructose or 5 mM glucose in the bathing solution. A further rise of the carbohydrate concentration led to a progressive decrease of P efflux from hyphae of H. crustuliniforme. The results are discussed with respect to the transfer processes between both symbiotic partners in a mycorrhizal association and a possible regulation of these exchange processes.

Proliferation of intraradical hyphae of the arbuscular mycorrhizal fungus Glomus fistulosum was observed and measured in surface disinfected mycorrhizal maize root segments in an aseptic cultivation system. The maximum growth of hyphae proliferating from the root segments was observed at pH 6·3, and low concentrations of buffering substances stabilized the pH of the incubation medium (0·9 mm MES, 1 mm BIS-TRIS). The proliferation of intraradical hyphae was not influenced but the germination of chlamydospores of the mycorrhizal fungus was inhibited by an artificial inorganic soil solution at lowest dilutions. The percentage of root segments bearing proliferating hyphae and the growth of hyphae decreased with increasing concentration of phosphate in the incubation medium. All reduced sulphur-containing compounds tested (sulphite, dithionite and metabisulphite) suppressed the proliferation of hyphae completely.

PCR was used to amplify ribosomal internal transcribed spacer and 5·8 S DNA from type material of Rhizopogon colossus var. colossus, R. hawkerae, R. parksii, R. reticulatus and R. villosulus plus a number of collections belonging to the same section (Sect. Villosuli). Different restriction enzymes were used to digest these amplified rDNAs to find polymorphisms useful in identification. On the basis of these studies and our previous morphological observations, the conclusion is that the names proposed by Smith & Zeller for collections studied, as well as R. reticulatus, represent only one species, R. villosulus.

The fungi associated with diseased and healthy shoots of young, planted Betula pendula were studied at five sites in Scotland where crown die-back of birch is occurring. At each site, 30 diseased and 30 healthy shoots were sampled twice, with 1 yr-old shoots collected in May 2002, and 4–5-month-old shoots collected in September/October 2002. Overall, 34 different fungal species were recorded, with 92% and 60% of all diseased and healthy shoot pieces, respectively, colonised by at least one fungal species. The main colonisers of both diseased and healthy shoots were Melanconium bicolor, Discula betulina and Godronia urceolus, and the fungi most frequently found on diseased shoots only included Marssonina betulae and Fusarium avenaceum. The pathogenicity of these fungi was tested on seedlings of B. pendula and B. pubescens. Melanconium bicolor required a wound for lesion development on both birch species and caused larger lesions on B. pubescens than on B. pendula. Discula betulina caused lesions on stems and shoots of both birch species, including non-wounded current shoots. Marssonina betulae caused lesions and die-back of wounded and non-wounded current shoots of B. pendula seedlings. This fungus was not pathogenic on B. pubescens. Fusarium avenaceum caused restricted lesions on the main stems of both birch species. Godronia urceolus was not pathogenic on either birch species. Further work needs to focus on the role of these fungi in causing crown die-back of young, planted B. pendula in Scotland.

Auxarthron conjugatum (Onygenaceae) and Myxotrichum deflexum (Myxotrichaceae) are distantly related cleistothecial (gymnothecial) ascomycetes that form ascomata with strikingly similar peridia in which rigid, branched and anastomosed, thick-walled hyphae create a cage- or mesh-like enclosure (reticuloperidium). We tested the hypothesis that the reticuloperidium plays a role in dispersal mediated by arthropods by enclosing ascomata of these fungi together with flies from the family Sarcophagidae. Gymnothecia of both fungi were picked up easily when the stiff hairs of the flies impaled the ascomata by passing through the interhyphal spaces of the reticuloperidium. Ascospore release from the gymnothecia then occurred during grooming activities during which the limbs of the flies caught the ascoma appendages causing the peridium to be torn apart. This adaptation to arthropod morphology and behaviour is interpreted as the driving force behind the evolution of reticuloperidia in unrelated groups of cleistothecial ascomycetes.