This contribution of Mycological Research News features : Why are mushroom caps mushroom-shaped? An obituary for Noel F. Robertson (1923–1999), a former President of the British Mycological Society is also included in the issue.

This issue contains 15 original research papers. Fast-tracked is a major molecular phylogenetic study of Russula species in Europe with a comparison of proposed infrageneric classification systems. Molecular studies also include a technique for in situ PCR detection and identification, consider the detection of Neonectria galligena, variation and infection patterns in endophytic Lophodermium nitens, and compare a newly discovered Paraphaeosphaeria with other species of the genus. Incompatability studies between strains of Monascus purpureus have demonstrated the occurrence of different compatability groups in the species.

Ingestion of ectomycorrhizal Rhizopogon fungal spores by small mammals is explored and their subsequent effectiveness assessed. Fungi able to grow in TNT-contaminated soil have been isolated and their biotransformation ability compared. The α-amylase production in Thermomyces lanuginosus and the proteins involved has been investigated. Trichoderma koningii and T. virens protoplast fusants have been made and their biocontrol effectiveness against Rhizoctonia solani studied.

A technique for the visualization of ectomycorrhizal rhizomorphs by scanning confocal microscopy has been developed, and the use of different fluourescent stains for the detection of soil fungi is compared.

Taxonomic papers consider Mycosphaerella species in Queensland, new species of Anthostomella on Juncus and Spartina, and of Paraphaeosphaeria on Yucca.

The following new scientific names are introduced: Anthostomella spissitecta, A. torosa, Mycosphaerella cuttsiae, M. cyperi, M. dodonaeae, M. glochidionis. M. khayae, M. melaleucoides, M. morindae, M. platylobii, M. queenslandica, M. wollemiae, M. xylomeli, and Paraphaeosphaeria barrii spp. nov.; M. escalloniae (syn. Sphaerella escalloniae) comb. nov.

Species in the large mushroom genus Russula are important ecologically as ectomycorrhizal fungi and economically as comestibles. Most infrageneric classification schemes of this genus have originated in Europe, but because of nomenclatural history and an evolving suite of characters these systems remain largely incongruent. Using ribosomal DNA sequences for 87 species representing all infrageneric taxa described from Europe, the phylogenetic position and relationships among these species were examined. Cladistic analysis of the ITS1, 5·8S, and ITS2 regions showed a cluster of five to six small to large clades basal in the topology and one large apical clade arising from the deeper nodes, none of which has been previously recognized in toto at the subgeneric level. Two of these groups, the Compactae and Pallidosporinae, which have been previously recognized as subsections of section Compacta, did not appear to be closely related. Bootstrap support and Bremer decay values indicated that collapse of the tree into monophyly at the deeper nodes would result in two large groups which are consistent with the classical subgeneric concept of the Eurussulae and a narrowed Compactae. The topology confirmed some previously described infrageneric taxa at the section level including the Tenellae and Heterophyllae and at the subsection level including the Cupreinae, Laricinae, Lilaceinae, Integroidinae, Violaceinae, Sphagnophilinae, Viridantinae, Emeticinae, Subvelatae, Pallidosporinae, and portions of the Polychromae and Sardoninae. The molecular analysis also indicated many interesting new combinations or relationships not previously conceived. Mapping of characters such as spore print colour, taste, and presence of acid resistant incrustations, which have been used to define infrageneric taxa in Russula, onto the phylogeny identified interesting patterns consistent with hypotheses regarding plesiomorphic and apomorphic characters. However, because of potential loss or reversal of character states, this analysis did not support their unequivocal use in infrageneric classification.

PCR and DNA sequence analysis have become standard tools for identification, detection and phylogenetic analysis of fungi. A large number of species are incapable of growth in the laboratory, making the preparation of pure DNA problematical. The amplification of DNA samples from impure material is subject to misinterpretation if more than one species is present. To overcome this problem, we designed an in situ PCR technique that links PCR amplification to the light microscopic image. The amplified tissue is stained, thus confirming which morphotype has been amplified. The PCR product can then be sequenced. We tested the technique on fixed Blumeria graminis spores and mycelia using primers derived from the sequence of the gene encoding the catalytic subunit of protein kinase A (bka1). This is the first report of in situ PCR on phytopathogenic fungal material. This technique allows positive confirmation of the origin of genes cloned from obligate pathogenic fungi and could be adapted for use on any samples containing mixed fungal species.

A pair of primers specific for Neonectria galligena were designed from comparisons of the internal transcribed spacer (ITS) regions 1 and 2 of 32 isolates of diverse origins with sequences from 7 other nectriaceous species: ‘Nectria’ ditissima, N. coccinea, N. coccinea var. faginata, N. punicea, N. fuckeliana, N. cinnabarina, and N. radicicola. Comparisons were also made with sequences from several commonly encountered fungal endophyte species of apple wood, in particular Fusarium lateritium. Under stringent PCR conditions, primers Ch1 and Ch2 amplify a 412 bp fragment from N. galligena DNA nectriaceous but not from other nectriaceous species, fungal apple endophyte species, rosaceous, or beech host tissues. The identity of PCR products from total DNA extracts from woody tissues was confirmed by Southern hybridization. A 500 bp heterologous internal standard incorporating identical primer recognition sites was generated for use as an internal positive control for individual PCR assays to identify false negatives due to the presence of high levels of co-precipitated PCR inhibitory compounds. Further adaptation of this approach allowed for semi-quantitative competitive PCR enabling the determination of target fungal levels in infected lignified tissues.

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.

Intraspecific heterogeneity among 27 Asian monoconidial isolates and four ATCC ex-type cultures of Monascus purpureus from red rice and sufu was revealed as mycelial compatibility groups (MCGs) using mycelial reactions of 496 mycelial parings. Among 88 pairs, at least three kinds of mycelial incompatible reactions were found at the interaction zone of confronting colonies as: (1) closely forming but not overlapping colonies; (2) sparsely forming aerial mycelia; and (3) thick mycelial band followed by mycelial deterioration of one partner. A mycelial-free zone was not found in any interaction. Compatible interactions were evidenced by freely intermingled hyphae with no apparent inhibition of growth of either partner. At least six MCGs were found based on compatible reactions. MCG1 and MCG2 consisted of 11 isolates, mostly from Thailand. Also, MCG1 contained two ATCC ex-type cultures, ATCC16365 (from Indonesia) and ATCC26264 (from Taiwan). In addition to nine Thai isolates, MCG2 included T9·1 isolate from China and isolate ATCC16427. Furthermore, four small MCGs of M. purpureus were identified. MCG3 (isolates A4·8 & TT4·2) and MCG4 (isolates 10 & M6) were comprised of isolates from Thailand. MCG6 consisted of Japanese isolate M22, and ATCC16360. Isolate ATCC16360 was of unknown origin and previously had been identified as M. anka. MCG5 consisted of Japanese isolate M20 and two Thai isolates, M13 & M14. The result indicated the Thai isolates of MCG1 (16·1, T8·6, TT2·3, TT5·1, M1, M2, M3, M7 & SR) were the same clone as those isolates from Indonesia and Taiwan. Also, the Thai isolates of MCG2 (4, 8, 11, TT1·1, M4, M8, M15, M17 & M18) along with the Chinese isolate T9·1 and ATCC16427 were from the same genetic origin. This study was correlated to those of RAPD analysis of genetic variation within a collection of Monascus spp. isolated from red rice and sufu. M. purpureus isolates used as a starter culture of red rice production among Asian countries were from a rather restricted genetic pool, e.g. China, Taiwan and Japan, although the isolates of M. purpureus studied were moderately genetically diverse.

The foliage of Pinus strobus (eastern white pine), as that of all other conifers examined, is occupied by endophytic fungi, the most frequent of which is Lophodermium nitens. The number and extent of endophytic infections and the genetic relationship of individual isolates within living needles as well as their relationship to isolates from forest floor needles is unknown. To examine these and related questions, forest floor isolates and foliar endophytes from needle segments were obtained for ribosomal DNA sequencing and randomly amplified polymorphic DNA (RAPD) analysis. Molecular and morphological data were compared and infection frequency determined as a function of position along the needle. Ribosomal DNA sequences of foliar and ascospore isolates showed high levels of genetic similarity (>97% identity) for the internal transcribed spacer region. RAPD profiles were able to distinguish ascospore siblings from non-siblings, and also revealed that many needle isolates belonged to the same genotype as adjacent neighbour isolates, as would be expected from mycelial spread within the needle. Morphotype evaluation and RAPD profiles showed similar patterns: identical morphotypes grouped together and showed little or no genetic difference under RAPD analysis. Both morphological and molecular data indicated that the majority of infections were contained within 1 mm needle segments but could extend to about 4 mm in length. Infection frequency increased along the length of the needle from the proximal (shoot) end to the distal tip, with markedly higher rates in the distal quarter. Thus, endophytic infections of L. nitens in white pine needles consist of many localized, discrete infections, originating from ascospores and differentially distributed along the length of the needle. In the course of this work, it was found that GenBank accession no. AF203470 under the name Meloderma desmaszieresii appeared not to be that species but L. nitens on the basis of the ITS sequences.

Mature basidiomes of the hypogeous ectomycorrhizal fungi Rhizopogon vinicolor and R. truncatus were fed to three species of mycophagous mammals in captivity. Spores which passed through the digestive tract of the mammals were isolated from faecal pellets. The metabolic activity and mycorrhizal effectiveness of these spores was assessed. Flouricene-diacetate (a vital stain) was used to assess spore metabolic activity. Mycorrhizal effectiveness was determined by inoculating Douglas-fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa) seedlings with spore slurries. The same assays were performed on spores isolated from uneaten basidiomes. Spores from R. vinicolor were viable after passage through the gut of each mammal species but the digestive process did not substantially enhance or detract from the ability of spores to form ectomycorrhizas relative to spores from uneaten basidiomes. Spores recovered from faeces of Californian red-backed vole (Clethrionomys californicus) and Townsend's chipmunk (Tamias townsendii) had significantly higher metabolic activity than spores from uneaten basidiomes and those recovered from northern flying squirrel (Glaucomys sabrinus) faeces. Spores from eaten and uneaten basidiomes of R. truncatus failed to show any signs of metabolic activity and largely failed to form ectomycorrhizas on seedlings. The reasons for this are unclear but we suggest that germination of spores from this fungus may depend upon other factors. Our findings add to the growing body of literature demonstrating that spores of at least some species of hypogeous fungi remain viable after passage through the digestive tract of mycophagous animals.

Several Trichoderma virens (syn. Gliocladium virens) strains have good biocontrol activity against Rhizoctonia solani on cotton but lack some of the commercially desirable characteristics found in other Trichoderma species. In an attempt to combine these desirable characteristics, we used a highly effective biocontrol strain of T. virens in protoplast fusions with a strain of T. koningii, which had good storage qualities, but little biocontrol efficacy. All fusants were morphologically similar to one of the parental species. However, when compared to the morphologically similar T. koningii parent, two fusants showed significantly better biocontrol activity against R. solani on cotton. In addition, one T. virens-like fusant gave significantly less control than the T. virens parent. Fusants also differed from the morphologically similar parent in the production of secondary metabolites. One fusant was obtained which maintained biocontrol activity during storage for up to a year.

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.

Seventy-one strains of fungi belonging to 53 species of Zygomycota and mitosporic fungi were isolated from soils containing different levels of 2,4,6-trinitrotoluene (TNT). Three moderately contaminated samples (500 ppm) and an uncontaminated control contained a rich diversity of species whereas few fungi were isolated from a heavily contaminated sample (1000 ppm). Most isolates were capable of biotransforming TNT into aminodinitrotoluenes and azoxy-dimers, although they differed widely in their tolerance of TNT. Spore germination was generally more strongly inhibited by TNT than hyphal growth. Among the Zygomycota, Absidia and Cunninghamella were highly TNT-tolerant and attacked the substrate even when this had precipitated at 1000 ppm, whereas 12 of the 14 Mortierella isolates were intolerant of TNT above 50 ppm. Among the mitosporic fungi, Acremonium, Cylindrocarpon, Gliocladium and Trichoderma but not Penicillium species were highly tolerant of TNT and had a high capacity to biotransform this compound. Large variations in these properties were determined between species and even strains belonging to the same species, and between different isolates from the same soil sample. Populations of soil microfungi therefore show a high natural variation of TNT tolerance and biotransformation ability, irrespective of any previous long-term exposure to this xenobiotic.

The thermophilic fungus Thermomyces lanuginosus was cultivated in shake-flasks for up to 120 h with low-molecular-weight dextran as carbon source. Maximal biomass was attained after 48 h of growth whereas extracellular α-amylase activity was highest in the stationary phase with maximum activity after 96 h of cultivation. A similar pattern was found for total extracellular protein. Pulse-labeling of proteins showed that α-amylase and other unidentified proteins were de novo synthesised in the stationary phase. Using specific primers with sequences from α-amylase from T. lanuginosus, RT-PCR analysis showed that α-amylase transcription did not start until late in the growth phase and reached a maximum more than 24 h after maximal biomass was obtained.

A method for the observation of the three-dimensional structure of intact ectomycorrhizal rhizomorphs is described. The method is based on a combination of clearing the material with KOH followed by staining with congo red and subsequent imaging under a laser scanning confocal microscope (LSCM). The images obtained are of a much higher three-dimensional resolution than those obtained previously by use of conventional light microscopical techniques. The structure of highly differentiated and undifferentiated rhizomorphs is described. Applications of the method are briefly discussed.

Eleven new species of the ascomycete genus Mycosphaerella from Queensland, Australia, are described, illustrated and where necessary compared with closely related members recorded in the same host family. The new species described are: M. cuttsiae, M. cyperi, M. dodonaeae, M. glochidionis, M. khayae, M. melaleucoides, M. morindaeM. platylobii, M. queenslandica, M. wollemiae, and M. xylomeli. With the exception of M. queenslandica, which is found on Dianella, all of the other species occur on host genera indicated by the specific epithet of the fungus. Apart from M. cyperi, the other species are all associated with necrotic leaf spots. Additionally, Sphaerella escalloniae is transferred to Mycosphaerella as M. escalloniae comb. nov., and M. aequatoriensis and M. pericampyli are newly reported from Australia.

All 39 marine species described so far from Spartina spp. are listed. Anthostomella torosa sp. nov. is described from culms of Juncus roemerianus in North Carolina, and A. spissitecta sp. nov. is described from leaf sheaths of saltmarsh Spartina alterniflora in the USA (Connecticut, Florida, North Carolina, Rhode Island) and from S. densiflora in Argentina (Buenos Aires). Both species occur close to the rhizome and are classified as obligate marine fungi. The new species are compared with similar taxa, and a table comprises characteristics of the nine known aquatic Anthostomella species.

Paraphaeosphaeria barrii, a new species of the genus found on Agavaceae, is described from leaves of Yucca schidigera collected in Baja California, Mexico. The Coniothyrium anamorph is briefly described from cultured ascospores. This species was found to be most closely related to P. conglomerata, P. filamentosa, and P. quadriseptata all of which are from Agavaceae, produce 3 or 4 septate ascospores and pigmented, non-septate conidia from holoblastic, percurrently proliferating conidiogenous cells.