Three new species of Stenella, S. cassiicola, S. eugeniicola and S. rhododendricola, occurring on Cassia fistula, Eugenia heyniana and Rhododendron campanulatum, respectively, are described and illustrated.

Phytophthora megakarya is an important pathogen of cocoa in Africa. We used isozyme and RAPD markers to estimate the genetic diversity and structuring among 161 isolates, from the known distribution area of the fungus which corresponds to the cocoa belt in Ghana, Togo, Nigeria, Cameroon, Gabon and Sao Tome. Thirty six and 44 multilocus patterns were identified with isozymes and RAPDs, respectively. Patterns were separated into two highly differentiated genetic groups with both types of markers, one located in Central Africa and the other in West Africa. This distribution coincides with two major biogeographical domains which may reflect an ancient evolution of P. megakarya in this part of Africa. The genotypic diversity was lower in West Africa as compared to Central Africa. Inside Central Africa, isolates from Gabon and Sao Tome were highly differentiated based on RAPDs. Four intermediate marker patterns corresponding to isolates sampled near the border between Nigeria and Cameroon were putatively derived from genetic exchanges between the two major groups. The mating type determination permitted to confirm the high prevalence of A1 over A2. Although clonal multiplication seems to be the rule, indices of other reproduction means have been detected.

A field study was carried out in a remote broadleaved Fagus longipetiolata forest in Shaanxi province, China to study the natural local distribution of Lentinula edodes. Following spatial mapping, 24 fruit bodies were collected for tissue isolation into axenic culture. 24 genets distributed on fallen tree trunks within a distance of 120 m were identified and clustered into 7 groups using the unweighted pair-group method algorithm using data based on colony morphologies, abilities to degrade aromatic poly-R478 dye, somatic incompatibility reaction patterns and DNA fingerprints. Among the parameters used, the somatic incompatibility reaction, a polygenic phenotype, was the most differentiating, identifying 22 incompatible classes. Two sets of fruit bodies of different genets were so close together that they would otherwise have been described as aggregate fruits of presumed identical origin. Eighteen genets found on the same 5.6 m long tree trunk divided roughly into two clusters, matching their spatial distribution, and a nearby branch bore another distinct cluster. More heterogeneity was encountered between isolates the greater the distance separating them on the original site. Genets on the same tree trunk showed more compatible somatic reactions among themselves, and their DNA fingerprints showed higher similarity. Nevertheless, considering the totality of phenotypic characters, each fruit body is a genet in L. edodes. Such features are concluded to result from a reproductive strategy which depends on basidiospore dispersal. Within each cluster of isolates from the collection site genets seemed to have arisen from multiple sib-mating events. Thus, a cluster may represent a lineage of L. edodes. Individualism in L. edodes is based on a strong somatic incompatibility system. Strong competition from contaminating individuals arriving as air-borne basidiospores could explain decreased and fluctuating crop yields which are now frequently observed in later flushes from the outdoor wood log cultivation system. Further, it would also explain why multispore spawn is not favoured in artificial cultivation of this economically important edible mushroom.

The extracellular subtilisin-like protease activity of several fungal pathogens of nematodes, insects and plants was recorded after batch cultivation. Nematophagous and entomophagous isolates of Verticillium, Paecilomyces, Beauveria and Metarhizium produced Suc-(Ala)2-Pro-Phe-pNA hydrolysing enzymes that differed in serological properties based on Western blot analysis with polyclonal antibodies. The antibodies used were raised against the main subtilisin-like proteases from one species from each of the four genera. From each strain of V. chlamydosporium tested, between one and four isoforms with different pI values were separated by isoelectric focusing and visualized by enzymoblots. The occurrence of multiple isoforms with unique N-terminal sequences in single strains might suggest the presence of subtilisin gene families. Using as a probe a fragment of the Pr1 gene from M. anisopliae, RFLPs were observed that confirmed qualitative differences between subtilisin-like genes within and between species. In contrast to these pathogens of invertebrates, plant-pathogenic species of Verticillium did not produce subtilisin-like proteases, nor were homologous genes detected. Such activity, however, and the corresponding genes, were detected in species weakly pathogenic to plants, or saprotrophs.

The ITS2 of rDNA of eight strains representing five controversial species and a variety of Cunninghamella, i.e. C. bertholletiae, C. blakesleeana, C. elegans, C. phaeospora var. phaeospora, C. phaeospora var. multiverticillata, and C. polymorpha, were amplified through PCR. The PCR products were purified and their sequences determined. Sequence variations among the three strains of C. elegans and between the two strains each of C. bertholletiae and C. polymorpha are less than 5%, while those among the other three strains each representing a different taxon as well as among these strains and the above mentioned five strains representing two different species are 14–32%. Sequence variations between each of the eight Cunninghamella strains and Rhizopus stolonifer var. lyococcos, used as outgroup, are more than 70%. These results confirm the value of sequence analyses of ITS2 rDNA in the taxonomy of Cunninghamella. C. polymorpha is proven to be synonymous with C. bertholletiae, while the others are considered to be distinct taxa. Phylogenetic analyses of the sequence data by the neighbour-joining method generally reflect the systematic relationship of different species based on morphologic studies made by our research group. The three strains of C. elegans, and the two strains each of C. bertholletiae and C. polymorpha (a synonym of C. bertholletiae) are respectively clustered in the same clade with high confidence, while the strains representing the two varieties of C. phaeospora are also clustered in the same clade though with relatively low bootstrap value and long distance which supports the establishment of the distinct varieties.

Growth of A. ochraceus at 30°C was significantly influenced by changes in both solute (NaCl, glycerol) and matric potential (PEG 8000). Optimum growth rates were at approx. −10·0 MPa with the non-ionic solute, and between −3·5 (=0·975 aw) and 14·0 MPa (=0·90 aw) with the ionic solute in a sucrose-based minimal salts medium. The optimum matric potential for growth was at −7·0 MPa (=0·95 aw). Growth rates were influenced by whether colonies were grown directly on the agar medium or on cellophane overlays. Sclerotia were profusely formed on media >−1·5 MPa (=0·99 aw), only initials at −3·5 MPa, and none under optimum conditions for growth (−7·0 to −10·0 MPa). The contents of low and high mol. wt polyols (glycerol, erythritol, arabitol and mannitol) and sugars (glucose and trehalose) were quantified in whole colonies and conidia grown under different osmotic and matric potential conditions for the first time. There was a higher total amount of polyols (μg mg−1D.W..) in conidia than in whole colonies. In glycerol-amended osmotic media maximum total polyols were present at −4·5 (=0·965 aw) and −17·0 MPa (=0·88 aw) in mycelial colonies and conidia respectively, with glycerol and mannitol being predominant in both, with small amounts of erythritol also present in conidia. By contrast, in matrically-modified media, the high mol. wt mannitol was the major component (>75% of the total) with small amounts of arabitol and the low mol. wt erythritol and glycerol, regardless of matric potential. For sugars, trehalose was predominant in conidia (>90%) from osmotically-modified media with low levels of glucose present in both conidia and mycelial colonies at all potentials tested. By contrast, in matric media trehalose was the predominant sugar in both conidia and whole colonies. This study suggests that xerophilic fungi may use different mechanisms for overcoming osmotic and matric stress.

Four strains of Tuber borchii were grown in liquid medium containing glucose. Evaluation of growth patterns revealed a trend of exponential growth for only one strain. Subsequently, enzyme levels were assayed and polypeptide and mRNA electrophoretic profiles were analyzed. Although the four strains were found to have many properties in common such as the growth behaviours for three strains and the levels of some glycolytic enzymes, the polypeptide and mRNA electrophoretic patterns highlighted intraspecific variability.

Gel-based and spectrophotometric assays were used to demonstrate expression of a manganese-dependent peroxidase (MnP) activity in culture filtrates of the ectomycorrhizal basidiomycete Tylospora fibrillosa. PCR amplification using a primer pair specific for a 260 bp fragment from the 5′-end of a gene encoding an H3-like MnP isozyme in Phanerochaete chrysosporium, produced single amplification products of 260 bp from DNA extracted from two isolates of T. fibrillosa. The amplified fragment from T. fibrillosa had a 93·7% nucleotide overlap (over 201 bases) with a published sequence for a 260 base amplification product from the P. chrysosporium H3 MnP isozyme gene, strongly suggesting the presence of a homologous gene in the ectomycorrhizal fungus. The results are discussed in the context of lignin degradation by ectomycorrhizal fungi.

Phaeoramularia bauhiniae, P. marsdeniae and P. tephrosiicola sp. nov., on Bauhinia bahli, Marsdenia verticulata and Tephrosia hirta, respectively, are described, illustrated and compared with similar taxa. Keys have been provided to distinguish Phaeoramularia spp. on Asclepiadaceae and Fabaceae.

The macro- and micromorphology of 45 specimens and 19 holotypes of the subgenus Ganoderma (=G. lucidum complex) were examined, together with isoenzymic data. The objective was to evaluate relationships by analysing numerically morphological and isoenzymic data to discern the number of species occurring in southern South America, and their delimitation. A total of 73 isozymic bands and 26 morphological characters were considered. Spore ornamentation was studied under SEM. Nine taxa appear to be represented in the region (G. lucidum var. lucidum, G. multiplicatum, G. sessiliforme, G. praelongum, G. subincrustatum, G. platense, G. zonatum, G. sessile and G. tuberculosum) and the presence of another eight is doubtful (G. argillaceum, G. bibadiostraum, G. chaffangeonii, G. lucidum var. capense, G. lucidum var. dorsale, G. subamboinense var. laevisporum, G. resinaceum and G. tropicum). Three major cutis types were defined and illustrated. Although congruence between dendrograms derived from both data sets was low, some similar groupings were formed. Species descriptions and a key for identification are provided.

The objective of the study was to develop methods for the specific and rapid identification of Tuber spp. to aid ecological and taxonomical investigations of Tuber spp. and ectomycorrhizal fungi in general. Antisera raised against Tuber melanosporum, T. magnatum, Boletus edulis and Tricholoma matsutake and monoclonal antibodies produced against T. melanosporum were used to develop various immunological assays such as ELISA, Dot-blot and Western blot. A simple squash Dot-blot was used to visualize mycorrhizal infection of inoculated host plants grown in tube cultures and experimental truffières.

Most antisera reacted strongly with homologous antigens from ascomata or mycelia and mycorrhizal root-tips. Their specificity allowed differentiation between Tuber spp. and other ectomycorrhizal fungi. Differentiation among Tuber spp. was possible by Western blot but not by ELISA or Dot-blot assay. The results suggested that specificity of the antisera was confined to genus level. In two cases specificity was increased by absorption with cross-reactive antigens.

Eleven monoclonal antibodies were produced against T. melanosporum. Two were directed against proteinaceous material, the others probably against various carbohydrate determinants. Unfortunately they could not be used for differentially recognizing T. melanosporum as they all cross-reacted at least with extracts of four other ectomycorrhizal fungi.

Development of mushrooms is driven by genetic and epigenetic factors in a continuous interaction with the environment. It is assumed that each successive stage of morphogenesis depends on specific sets of signals arising at the appropriate time and place during the growth process. Morphogenetic dynamism proceeds in a time dimension through a cascade of signal-effect associations. Developmental errors may occur when such signals originate in the wrong place and/or at the wrong time. As a result various abnormalities such as ectopic tissues can develop and morphogenesis can be severely disturbed. Both endogenous genetic disturbances and exogenous factors can cause developmental errors. Lamellar dysplasia, which is a pore-like proliferation of the gills, forms an example; it may be induced experimentally. Both lamellar dysplasia and rosecomb disease of Agaricus bisporus result from endogenous genetic instability, whereas the developmental errors observed in wet bubble disease, which is caused by the infection of Mycogone perniciosa, originate from an exogenous factor. Morphogenesis normally leads to a symmetrical form of primordia of A. bisporus. Asymmetry is very frequently associated with an underlying pathological situation. Defining exact criteria of and sharp borderlines between normal and abnormal development seems infeasible. Fungi may readily tolerate morphogenetic imprecision. In this report, various macro- and microscopic features of normal and pathological development are illustrated; the dilemma of normal versus abnormal fruit body development has been discussed.

Adenylyl cyclase activity was detected in a mixed-membrane fraction from dormant spores of Phycomyces blakesleeanus. This enzymatic activity increased linearly as a function of protein concentration up to 300 μg of protein 100 μl−1 and 20 min incubation at 25°C. It used Mn2+ or Mg2+ indiscriminately as a cofactor, and the addition of both cations together did not have a synergistic effect. The crude enzyme showed a Kmapp for ATP of 0·25 mM, when measured in the presence of Mg2+. It was stable for 48 h at −20°, losing 25% of its activity after 72 h. The addition of 10 μM GTP to the enzymatic assay stimulated the adenylyl cyclase, whereas higher concentrations (500 μM) inhibited it. Cholera toxin and 25 μM forskolin caused a two-fold stimulation of the enzymatic activity. The calcium-calmodulin complex stimulated activity two-fold; this stimulation was inhibited by the anti-calmodulin drug trifluoperazine. The enzyme could not be solubilized by NaCl, but was partially solubilized with non-ionic detergents, indicating that the enzyme is an integral membrane protein. The detergent-solubilized enzyme only used Mg2+ as a divalent cation and was also stimulated by calcium-calmodulin, low concentrations of GTP, cholera toxin and forskolin, but was extremely unstable. These results suggest that the adenylyl cyclase present in dormant spores of Phycomyces blakesleeanus is an integral Type I-like membrane enzyme.

Germinating spore-balls of Urocystis hypoxis produce both unicellular basidia, crowned with 4–6 basidiospores, and what appear to be transversely septate, 3-celled, prostrate basidia with conjugation, through a conjugation tube, between top and bottom cells. This most unusual situation is discussed in relation to the taxonomy of smut fungi.

Multivariate analyses (cluster and correspondence) were used to assess the taxonomic structure of 34 isolates of Penicillium miczynskii scored for 95 binary characters. Six isolates of P. raistrickii and P. rolfsii were added solely for the purpose of comparison. The characters used were 38 micromorphological, 15 cultural and 42 secondary metabolite characters. With just one exception, allocation of isolates to clusters was equal in the separate analyses using morphological and secondary metabolite data. The combined data set, using 95 characters, resulted in clearly-defined clusters interpretable as species on the basis of the distribution of ex-type cultures. Four species were accepted: P. miczynskii, P. manginii, P. soppii and P. atrosanguineum. Two additional species, P. syriacum and P. chrzaszczii, represented only by ex-type cultures, were interpreted as a nomen ambiguum and a form of uncertain taxonomic status, respectively.

Brief descriptions, illustrations and a synoptic key are included to aid identification of the accepted species.

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.

A new species of Menisporopsis was collected from submerged wood in the Lam Tsuen River, Hong Kong. It differs from other species in having conidia with 5–6-setulae. The generic concept of Menisporopsis is briefly discussed and six species are accepted. A composite illustration of their conidial morphology is provided for comparison, and a synopsis of and a key to the genus are provided. The new species is described and illustrated with light and scanning electron micrographs.

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.

Phomopsis cucurbitae is a latent infection fungus which causes muskmelon fruit decay only after harvest. It can cause severe losses during fruit storage and marketing in the U.S.A., Japan and some Central American countries. Previous studies showed that P. cucurbitae produced polygalacturonase (PG) in both culture and muskmelon fruit tissue. In this study, a prominent PG isozyme (PG1) produced by P. cucurbitae in decayed fruit was purified to homogeneity by a procedure of extraction, ultrafiltration, preparative isoelectric focusing, anion exchange, and gel filtration chromatography. The isozyme had a primary endo-activity, a relative molecular weight of 54 kD according to SDS-PAGE, and a pI of 4·2 based on IEF-PAGE. The isozyme was active in a low pH range from 2 to 6, and was optimum at pH 5. Optimum temperature for the activity of PG1 was 40–45°C. The Km and Vmax on polygalacturonic acid were 44·7 μg ml−1 and 0·14 U μg−1 enzyme, respectively. The purified PG1 effectively macerated mature fruit tissue, suggesting that the isozyme may be involved in the pathogenesis of Phomopsis fruit rot of muskmelon.

The ability of the maize pathogen Fusarium subglutinans to metabolize benzoxazolin-2(3H)-one (BOA) and 6-methoxybenzoxazolin- 2(3H)-one (MBOA), important allelochemicals of Zea mays, was studied. Growth of mycelium was completely inhibited by 10 mM BOA and reduced by lower concentrations. In vitro the fungus completely degraded 1 mM concentrations of BOA and MBOA within 2 d. N-(2-hydroxyphenyl)-malonamic acid and N-(2-hydroxy-4-methoxyphenyl)-malonamic acid were identified as the main degradation products of these allelochemicals.