This Mycological Research News features: Non-lichenized ascomycete lineages are derived from lichenized ancestors; and Changes in Coprinus questioned.

The issue contains the Presidential Address given by Stefan Buczacki in December 2000 on Berkeley's legacy, and 15 original research papers.

A protocol for protoplast preparation in Rhizoctonia solani is presented and transformation reported. Four papers describe molecular variability in different fungi; mycorrhizal Pisolithus strains in Australia; Mycosphaerella species on Eucalyptus; Phytophthora infestans; and Sphaeropsis sapinea.

The fungus causing anthracnose of tobacco is shown to be Colletotrichum destructivum, and the infection process followed.

One biotype of Trichoderma harzianum causing damage in Agaricus bisporus beds is tolerant of inhibitory bacteria. An extracellular mutanase from this same species may have applications in oral hygiene, and a protease from Cryptococcus neoformans has been found to be a metallopetidase.

Two papers focus on fungal development: sclerotial formation in Sclerotinia sclerotiorum may be triggered by oxidative stress; and rhizomorphs of Armillaria luteobulbina formed in aerial and submerged culture are compared.

The assemblages of endophytic fungi in different species of mangroves differ.

Systematic papers in this issue: revise the corticioid Sistotremateae in the Patagonian Andes; describe a new genus in the Kickxellales; and describe a new species of Chionosphaera.

The following new scientific names are introduced: Myconymphaea gen. nov.; Chionosphaera cuniculicola, M. yatsukahoi, Sistotrema botryobasidioides, and S. globosa spp. nov.; C. phylacicola (syn. Fubulostilbum phylacicola) comb. Nov.

The current state of professional mycology in Britain is considered in relation to the intellectual legacy left by the founding father of British mycology, the Reverend M. J. Berkeley (1803–1889). Through a review of Berkeley's life and work, it is suggested that there are many unexpected parallels between British mycology today and in the nineteenth century. Although there are now a considerable number of professional mycologists, support for professional mycology and mycological education is considered to be inadequate and seems in some ways to have advanced little in the past century and a half. It is concluded that above all, the current poor level of support for whole organism studies is the greatest betrayal of Berkeley's legacy.

Optimization of protoplast release (202×104 ml−1) from a strain of Rhizoctonia solani anastomosis group three was achieved by treating 3 d old mycelia from broth cultures with Novozyme 234 for 3 h at 30 °C. The only other treatment, of many tested, that yielded any protoplasts (15·6×104 ml−1) was commercial lysing enzymes from Trichoderma harzianum. Various combinations of commercial enzyme preparations, variations of osmotic stabilisers, and mycelial age, were ineffective in raising the protoplast yield. Regeneration of colonies from protoplasts was maximal (83–87%) when protoplasts were incorporated in cooled agar containing 5% glucose, malt extract and yeast extract, supplemented with 0·8 or 1·0 M mannitol as osmotic stabiliser. Liquid culture broth and other potential osmotic protectants at various concentrations yielded fewer regenerating colonies (5% in broth and 13–75% in agar). The Rhizoctonia strain was highly sensitive to hygromycin B, so transformation of protoplasts was achieved with a polyethylene glycol-based procedure, using plasmid pES200 containing the bacterial hygromycin B phosphotransferase gene driven by a trpC promoter from Aspergillus nidulans. Transformation was achieved using the DNase inhibitor aurintricarboxylic acid and incubation temperatures below 10–12 °. It was confirmed by high stringency hybridization of DNA extracted from the transformants, using pES200 as a radiolabelled probe. The transformants grew slowly (25–35 mm diam after 14 d) then ceased growth, and never produced fast-growing sectors indicative of integrated plasmid DNA. Transformation was also achieved with pAXHY2 from Cryphonectria parasitica, and colonies reached 55–65 mm diam before they stopped growing. Southern blots confirmed the presence of non-integrated pAXHY2, but no fast-growing sectors were produced.

Fifty-three isolates of Pisolithus were obtained from various locations in central and eastern Australia. These, along with two isolates from south-east Asia and one from USA, were compared using ITS–RFLP and ITS sequencing analyses. Results of the RFLP analysis initially grouped the isolates into eight RFLP types. ITS sequences were obtained for at least one representative isolate from each RFLP type and compared by a neighbour-joining analysis with Pisolithus ITS sequences available in databases. The majority of isolates clustered into four groups within two major clades, each clade comprising isolates of similar basidiospore characteristics. Most Australian isolates corresponded with recent provisional descriptions of P. albus or P. marmoratus. One isolate (LJ30) had low sequence identity (61·6–78%) to the other isolates and probably represents a separate undescribed Australian species. Significant intraspecific variation was observed in ITS–RFLP profiles for the putative P. albus isolates, suggesting that the sole use of RFLP analysis in diversity assessment may overestimate Pisolithus species richness.

Mycosphaerella contains some of the most damaging foliar pathogens of Eucalyptus. Identification of the species has relied mainly on characters such as ascospore morphology and the mode of ascospore germination. Wide variation and overlap in morphological characters, especially ascospore shape and size, has lead to confusion in species concepts. This study examines the potential of RAPD–PCR to differentiate Mycosphaerella species that cause leaf diseases of Eucalyptus in Australia. RAPD–PCR was conducted on 39 isolates representing 10 species of Mycosphaerella collected from southern Australia. Primers were chosen mainly to distinguish between the two most common and damaging species in Australia, M. cryptica and M. nubilosa. The RAPD analysis revealed five groups within the 39 isolates, four of which represent distinct species: M. cryptica, M. gregaria, M. nubilosa and M. marksii. For these four species, the cluster analysis and RAPD patterns gave results that fitted well with classical taxonomic criteria. Variation within M. cryptica, M. nubilosa and M. marksii was low, even though the isolates within these species originated from diverse hosts and locations. The remaining species in this study grouped together and could not be easily separated using the RAPD data here.

Selected isolates of Phytophthora infestans from around England and Wales were fingerprinted using both RG57, a multi-locus RFLP probe, and Amplified Fragment Length Polymorphisms (AFLPs). The larger number of polymorphisms detectable with the AFLP method allowed resolution of several similar AFLP genotypes among isolates with identical RG57 fingerprints. However, some isolates with the same RG57 genotype had remarkably dissimilar AFLP genotypes, suggesting that there has been convergent evolution of some RG57 fingerprints. Also, some isolates with dissimilar RG57 fingerprints had similar or identical AFLP fingerprints. Both techniques distinguished isolates of mitochondrial DNA haplotype Ia from those of haplotype IIa. However, with AFLPs only, most of the isolates of A2 mating type were very similar and were distinguished from those of A1 mating type, suggesting that gene flow between A1 and A2 genotypes is limited and that sexual recombination is rare.

Sphaeropsis sapinea is an endophyte and latent pathogen of pines, assumedly introduced to the Southern Hemisphere along with its host. There are at least three recognised forms of S. sapinea that differ from each other morphologically and can also be separated based on molecular characteristics. Pinus radiata is a native to California but has been used extensively for afforestation in the Southern Hemisphere. For this study, populations of S. sapinea were collected from exotic P. radiata plantations in South Africa, South and Western Australia, Tasmania and New Zealand and from native P. radiata in California. The genotypic diversity of the populations was assessed and compared using vegetative compatibility tests. SSR markers were used to determine the morphotype of isolates from each vegetative compatibility group. All Californian isolates of S. sapinea were found to be of the ‘B’ morphotype, while all introduced isolates in the Southern Hemisphere were of the ‘A’ morphotype. The genotypic diversities of S. sapinea populations ranged from extremely low in Australia to very high in South Africa with New Zealand having an intermediate genotypic diversity. S. sapinea is an asexual fungus and, therefore, different genotypes in an exotic population represent separate introductions. Our results suggest there have been very few introductions into Australia and multiple introductions into South Africa. In addition, it appears that S. sapinea isolates from P. radiata in the Southern Hemisphere did not originate from native P. radiata, but rather the widely planted exotic P. radiata has acquired this fungal endophyte from other Pinus within the exotic environment.

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.

Green moulds caused by Trichoderma harzianum biotypes Th2 and Th4 are responsible for important economic losses on mushroom farms producing Agaricus bisporus, whereas other biotypes present in farms have only slight effects on the cultivated mushroom. Adaptation of Th2 and non-Th2 strains of Trichoderma to grow in compost prepared for mushroom cultivation was compared through extracellular enzyme activities and competition with bacteria isolated from compost. No specific differences between Th2 and non-Th2 strains were observed for the activities of the 17 secreted enzymes tested. Confrontations of 27 bacterial isolates with 4 Trichoderma strains revealed that the aggressive Th2 strains were affected by a lower number of bacterial isolates than the non-Th2 strains. Capacities of T. harzianum, biotype Th2, to colonise mushroom compost are not due to a specific ability to produce enzymes necessary for compost degradation but to their tolerance to the inhibitory effect of bacteria present in compost.

Trichoderma harzianum (CCM F-470) was found to produce large amounts of extracellular mutanase (0·33 U ml−1, 1·85 U mg protein−1) when grown aerobically on the optimized mutan medium in fermenter culture with an automatic pH control. The mutanase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The enzyme was recovered with a 94-fold increase in specific activity and a yield of 73%. The molecular weight of the purified enzyme proved to be 67 kDa, as estimated by SDS–PAGE, and to be 274 kDa, as determined by size-exclusion HPLC. These results indicate that the native mutanase is probably a tetramer protein. The isoelectric point was at 7·11, and the carbohydrate content in the purified enzyme was 4·42%. The pH and temperature optima were 5·5 and 40 °C, respectively. The enzyme remained stable over a pH range of 4·5–6·0 and up to 35 ° for 1 h. The values of Km and Vmax under standard assay conditions were 1·2×10−3 g ml−1 and 8·48×10−2 U mg−1, respectively.

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.

Proteolytic activities were detected in a cytosoluble 100000 g extract of Cryptococcus neoformans using 7-amido-4-methylcoumarin substrates. The main proteolytic activity cleaved the L-arginine-AMC substrate. This protease was purified by high performance liquid chromatography and had a molecular weight of 80 kDa as demonstrated by SDS-PAGE in the presence of reducing conditions. The enzyme was inhibited by EDTA and o-phenanthroline which are metallopeptidase inhibitors. The protease displayed high enzymatic activity between pH 5·8 and 7, and its pI was localised at pH 5·04. Furthermore, this metallopeptidase was not secreted in culture supernatants, but was able to degrade partially or totally some of the extracellular matrix components, as fibronectin or laminin, and host IgG.

The structure of rhizomorphs of Armillaria luteobubalina grown on agar and in liquid culture has been compared using light and scanning electron microscopy. They differed in the extent of their peripheral hyphae, amount of surface mucilage, degree of pigmentation, growth rate, and structure of their inner cortex. Otherwise their internal structure was similar. At maturity four distinct radial zones could be recognised: (1) surface layer of peripheral hyphae from which hyphae extended radially; (2) outer cortex; (3) inner cortex; and (4) central medulla. The A. luteobubalina rhizomorph can be interpreted as a relatively strong ‘tube' of hyphae with a variously developed surface, surrounding a medulla of gas space containing wefts of fluffy fine hyphae. Submerged rhizomorphs grew at much faster rates than aerial rhizomorphs. The central gas-filled cavity most likely facilitates diffusion of oxygen along the rhizomorph, allowing growth through anaerobic environments. There was no evidence for specialised translocatory hyphae such as vessel hyphae. A zone of swollen hyphae interspersed with narrow hyphae developed at the interface between the inner cortex and medulla in submerged rhizomorphs. These were alive and septate and gave the tissue a resemblance to vascular plant aerenchyma, rather than specialised translocatory tissue. Peripheral hyphae took up and hydrolysed the fluorescent probe carboxy-DFFDA (Oregon Green) and accumulated the hydrolysis product in the vacuoles, suggesting that they are permeable. In contrast penetration of the cortex by the probe was limited. These data support the view that in moist conditions these rhizomorphs can function as organs of absorption and play a role in nutrient uptake.

Seven dominant mangrove species of an estuarine mangrove forest in south India were studied for the presence of foliar endophytes. Mitosporic fungi, ascomycetes and sterile mycelia were isolated from the leaves of the mangrove hosts studied. Although many endophytes were common to more than one host, the endophyte assemblage of each mangrove species was dominated by different endophyte species.

The Sistotremateae of the southern forests of Argentina are reviewed. Fourteen species are reported, collected on Nothofagus spp. and Drymis winteri. Sistotrema botryobasidioides and S. globosa are described as new. S. athelioides, S. efibulatum s. lat., S. porulosum and S. sernanderi are reported for the first time from Argentina. A key to the 14 species now known in the area is provided.

Myconymphaea yatsukahoi gen. sp. nov. is described in the Kickxellaceae (Kickxellales, Zygomycetes). The fungus is characterised by unicellular sporocladia formed on apical vesicles of sporangiophores, conspicuously long sporangiospores and peculiar septal plugs with prominent protuberances. In addition, a key to all known genera of the Kickxellales is provided.

An undescribed species of the heterobasidiomycetous genus Chionosphaera is carried by the bark beetles Dryocoetus autographus, Hylurgops palliatus, Ips acuminatus, Ips sexdentatus, Ips typographus, Orthotomicus laricis, Pityogenes chalcographus, Pityokteines spinidens, and Polygraphus poligraphus infesting conifers in Europe (Abies alba, Larix decidua, Picea abies, and Pinus sylvestris). Chionosphaera cuniculicola sp. nov. differs morphologically from C. apobasidialis in the more slender basidiospores. The segregation of the two species is supported by comparison of partial sequences of the large subunit of the ribosomal gene. Isotype material of Chionosphaera lichenicola was re-examined. In contrast to C. cuniculicola and C. apobasidialis, C. lichenicola exhibits clamps at the septa. Fibulostilbum phylacicola is considered as a clamp-bearing species of Chionosphaera.