This contribution of Mycological Research News features : New generic concepts in Verticillium sect. Prostrata, and The sterigmata of Volucrisporium: a clarification.

This issue includes 17 papers. The first describes a technique for genetically transforming ectomycorrhizal basidiomycetes. Molecular studies, often in association with other approaches, are applied to reassess species concepts in Polymyxa, explore the relationships of Melanospora and similar genera, examine lineages in violet root rot fungi, filament-beaked and other Alternaria species, Nectria galligena and N. coccinea var. faginata in North America, Phialophora gregata f. sp. sojae, the pine rusts Cronartium flaccidum and Peridermium pini, Rosellinia necatrix isolates from avocado, Claviceps purpurea populations on Spartina, Colletotrichum species on Hevea.

A major paper examines secondary metabolite profiles of 153 Alternaria isolates grown under standard conditions and assesses them in relation to morphological and RAPD data.

In addition, protein expression after low temperature exposure associated with fruit body formation in Flammulina velutipes is documented, the use of natural products to control sapstaining fungi reviewed, and the aerodynamics of conidia in Erynia neoaphidis and some other entomophthoralean fungi studied.

A new genus of mycorrhizal Cortinariaceae is described, and the Coemansia spiralis complex revised.

The following new scientific names are introduced: Anamika gen. nov.; Coemansia bainieri nom. nov.; A. indica, and C. linderi spp. nov.; and Polymyxa graminis f. sp. colombiana, f. sp subtropicalis, f. sp. tepida, f. sp. temperata, and f. sp. tropicalis f. spp. nov.

A technique was developed for transforming the ectomycorrhiza-forming basidiomycetes Suillus bovinus, Hebeloma cylindrosporum, and Paxillus involutus based on Agrobacterium tumefaciens-mediated T-DNA transfer. The selection marker employed was the Shble gene conferring resistance to phleomycin under control of the Schizophyllum commune GPD promoter and terminator. Transformants from all three investigated species were shown by PCR to contain the GPDScP-Shble-GPDScT construct, although the fate of the foreign DNA (integrated vs episomal, single-copy vs multi copy) could not be determined. The mycorrhiza formed between S. bovinus Bler transformants and Pinus sylvestris did not reveal any differences from those formed with untransformed Suillus bovinus.

A region of the nuclear ribosomal DNA containing the internal transcribed spacer 1 (ITS1), the 5·8S DNA and the internal transcribed spacer 2 (ITS2) was sequenced in 12 Polymyxa graminis and P. betae isolates, with particular emphasis on P. graminis from peanut clump-infested areas of the Indian subcontinent and West-Africa. Four different sequences were obtained from the seven isolates on sorghum or pearl millet, which differed from four sequences previously published for Polymyxa species and obtained for P. graminis isolates on barley, oat and wheat originating from temperate areas in Europe and America (two distinct sequences), for isolates on rice from Colombia and for P. betae isolates on sugar beet from several origins. The sequence variations concerned mainly the composition and length of ITS1 and ITS2 regions. Phylogenetic trees built with the distinct sequences currently known for Polymyxa spp. using parsimony, maximum likelihood and neighbour-joining methods separated P. betae from P. graminis. Within P. graminis, the hierarchy of the clustering partially matched the host range and geographical origin of the isolates. These results confirm the great diversity within P. graminis that has already been observed in host range and temperature requirements studies, and provide new arguments for considering several taxa within the species. On the basis of the ecological requirements and rDNA sequences of distinct P. graminis isolates, five special forms are proposed: P. graminis f. sp. temperata, P. graminis f. sp. tepida, P. graminis f. sp. subtropicalis, P. graminis f. sp. tropicalis and P. graminis f. sp. colombiana.

Species of Melanospora are parasitic on or closely associated with other fungi. The morphological characters traditionally used to place Melanospora and the Ceratostomataceae in Sordariales do not agree with their placement in our phylogenetic study. The nuclear encoded small subunit ribosomal DNA (SSU nrDNA) grouped four species of Melanospora and several allied genera and species in Hypocreales, an order rich in mycoparasites. An analysis based on characters derived from deduced amino acids from the second largest subunit of the RNA polymerase II (RPB2) gene further supports the close relationship of Melanospora and Hypocreales. The nuclear encoded large subunit ribosomal DNA (LSU nrDNA) phylogeny inferred six groups for 40 species in Hypocreales and allied genera (Bionectria, Claviceps, Hypocrea, Nectria, and Niesslia). Four species of Melanospora, including the type, and two Sphaerodes species formed a basal clade. Scopinella was placed in a distinct clade. Two other allies of Melanospora, Syspastospora and Persiciospora, were closely related to or within the Hypocrea and Nectria groups, respectively. The study argues for the reclassification of taxa that previously were placed in the Sordariales.

Genetic relationships among the violet root rot fungi were studied by hyphal anastomosis and sequencing of the ITS-5·8S rDNA regions. Twelve isolates of Helicobasidium mompa, one isolate each of H. purpreum and H. compactum, two isolates of Helicobasidium sp., eight isolates of Rhizoctonia crocorum, and three isolates of R. violacea anastomosed in most combinations, resulting in the death of fused cells and adjacent cells. Phylogenetic analyses of the ITS-5·8S rDNA regions separated 31 violet root rot fungal isolates into five lineages: H. mompa from Japan and Korea; R. crocorum from Europe and New Zealand; H. purpureum, R. crocorum, and R. violacea from Europe; Helicobasidium sp. from Japan; and H. compactum from the USA. Within each lineage there was no more than one nucleotide substitution, and similarity ranged from 81·8% to 98·2% between lineages. The discrepancy between the results from the two methods are discussed in terms of the biological species concept in the complex.

Internal transcribed regions (ITS1 and ITS2) of 11 different fungi, i.e. 8 species of Alternaria, Nimbya gomphrenae, Stemphylium vesicarium, and Ulocladium botrytis, were sequenced. Their phylogenetic relationships with another 36 species in Pleosporaceae in GenBank were analyzed by parsimony and distance methods. These two methods positioned filament-beaked Alternaria as a monophyletic group discrete from the other members in genus Alternaria. A hypothesis proposing that the filament-beaked speices of Alternaria evolved a particular evolutionary route distinguishable from other species based on molecular evidence, the unique traits of morphological adaptation for liberation and their subsistent strategies has been discussed.

Correct morphological identification of Alternaria is important and demands a combination of modern standardised methods and up-to-date literature. The production of secondary metabolites has previously been used as a means of identification and classification. In this study, 153 fungal isolates belonging to the genus Alternaria were examined. They were grown under standardised conditions and subjected to morphological and chemical examination. All isolates were grouped according to their three-dimensional sporulation pattern on potato carrot agar and their colony colour on dichloran rose bengal yeast extract sucrose agar (DRYES). After extraction, all isolates were analysed by a high performance liquid chromatograph equipped with a diode array detector and the resulting metabolite profiles were subjected to multivariate statistic analyses. The analyses of metabolite profiles showed that the isolates could be divided into three major species-groups that were morphologically identifiable as the A. infectoria species-group, the A. arborescens species-group and the A. tenuissima species-group. The A. infectoria species-group is chemically very different from both the A. arborescens and the A. tenuissima species-groups with only a few metabolites in common. None of the 35 A. infectoria species-group isolates produced any known metabolites and all had white or greyish white colonies on DRYES. The A. arborescens species-group and the A. tenuissima species-group, shared most of the known metabolites and had colonies of various shades of green on DRYES. One cluster of isolates belonging to the A. tenuissima species-group was able to produce tentoxin, which has not been reported previously from any A. tenuissima isolate. The results suggest that each species-group contains several taxa and these taxa need to be formally described before species specific metabolite profiles can be established.

Genetic variability was investigated in North American populations of the tree pathogenic fungi Nectria galligena and N. coccinea var. faginata. Genetic diversity was estimated by analysing RAPD and ribosomal DNA polymorphisms. The number of haplotypes was the maximum value in N. galligena (51), whereas 17 different haplotypes were detected among the 23 isolates of N. coccinea var. faginata studied. The frequency of polymorphic loci was greater in N. galligena (92%) than in N. coccinea var. faginata (35%). The estimate of genotypic diversity was a maximum in N. galligena while it represented only 59% of the maximum value for N. coccinea var. faginata. Gene diversity was also higher in the former (21%) than in the latter (6%). Moreover, four distance indexes calculated from the same genetic distance matrix gave similar results and showed that distances among isolates of N. galligena were four to nearly six times larger than distances among N. coccinea var. faginata isolates. An analysis of molecular variance based on genetic distances among haplotype frequencies confirmed that host origin (beech vs other species) was not the source of the genetic variability in N. galligena since less than 1% of the variability was attributable to the host factor. Overall, our results are consistent with the hypothesis that N. galligena is a pathogen native to North America while N. coccinea var. faginata may be a recent introduction.

A microsatellite-enriched library was constructed from genomic DNA of Phialophora gregata f. sp. sojae, the causal agent of soybean brown stem rot. Specific oligonucleotide primer pairs were designed to flank individual microsatellite loci, and used in PCR to detect polymorphisms among 118 contemporary and archival isolates of P. gregata from several countries, which represent two distinct, but sympatric populations delineated by rDNA genotypes. In general, low levels of genetic variation were observed and manifested in two ways. First, the number of polymorphic loci was low. Among 24 microsatellite loci screened, four polymorphic loci were detected, and two of them were located on the same clone. Second, the number of alleles in the polymorphic loci was low. Only two alleles were observed in each polymorphic locus. Alleles of the polymorphic microsatellite loci were invariant within, but different between, the two populations of P. gregata. From all evidence available, P. gregata f. sp. sojae exhibits several genetic features characteristic of clonal genetic structure, namely widespread distribution and stability of identical genotypes, absence of recombinant genotypes, and correlation of independent genetic traits including three sets of molecular markers, defoliating and nondefoliating pathotypes, and differential pathogenic fitness.

Cronartium flaccidum and Peridermium pini are rust fungi occurring on two-needle hard pines. According to previous molecular and morphological analyses, they are very closely related despite differences in their life-cycles. In this study we showed that although a low level of genetic differentiation occurs among populations of both P. pini and C. flaccidum, there is no overall differentiation between the two rusts, and in this respect they resemble a single taxon. We also observed evidence for linkage disequilibrium occurring between different alleles of separate loci of Peridermium pini suggesting that its population structure would be clonal. This suggests that strains of P. pini would have originated as asexual or self-fertilizing host range mutants of C. flaccidum.

The Claviceps purpurea population colonising British Spartina salt marsh stands is characterised by unusually long cylindrical conidia (average 9·5–12 μm) and sclerotia floating on the water surface. RAPD, AFLP and rDNA comparison defined these isolates as the third genetically distinct homogenous population (G3) of C. purpurea. The same morphological and genetical markers were found also in S. alterniflora isolates from Spartina from the USA. All G3 isolates belonged to a chemotype producing ergocristine and ergocryptine. In phylogenetic trees based on rDNA and AFLP, a G1 population from fields and meadows appeared as the sister clade to the one formed by G3 (Spartina) and G2 (wet and shady habitats), both with floating sclerotia and elongated conidia. British stands of S. anglica were probably colonised by isolates introduced from America, rather than by isolates from species of neighbouring biotopes.

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.

The influences of temperature on the fruit body induction of Flammulina velutipes were investigated. The fruit body could be induced after reducing of the ambient temperature under complete darkness, but could not be induced after light irradiation without temperature reduction. Fruit bodies formed under complete darkness elongated without pileus formation (pinhead fruit body). Protein expression patterns were investigated by two-dimensional electrophoresis, and 22 protein spots were found to be newly expressed in the mycelia and fruit bodies after reducing of the ambient temperature, but few protein spots were newly expressed in the mycelia after light irradiation without temperature reduction. Four of the spots had identical N-terminal sequences to polypeptides, Pf1, Pf3, Pfd3, and Pf6 which are previously sequenced as polypeptides expressed specifically during fruit body formation of F. velutipes.

Colonisation of Pinus radiata sapwood by sapstain fungi results in a permanent discoloration of the sap and is one of the major problems associated with P. radiata timber production in New Zealand. No fungicides are available that can provide a reliable level of control of sapstain for the extended time period required to export timber. A 4-year survey, commencing in 1996 revealed that the most common species which cause sapstain on radiata wood are Sphaeropsis sapinea (syn. Diplodia pinea), and two species of Ophiostoma (O. floccosum and O. ips). A total of 21 different species that cause sapstain have been identified to date. Two approaches for the control of sapstain are presented in this paper. The first relies on the ability of microorganisms to inhibit the growth of sapstain species. Such an ability has been demonstrated in laboratory trials on wood chips or wood blocks for 116 isolates of the 848 fungal and bacterial isolates initially considered. Six of the most effective isolates were further tested on sawn timber or debarked logs in field trials. Isolates of Trichoderma spp. and Trichothecium roseum were found to control sapstain as well as the standard fungicide treatments. T. harzianum was significantly better in controlling sapstain in the internal tissues of debarked logs than the fungicide. The second approach is based on inhibition of the sapstain causing fungi by secondary metabolites produced by plants or microorganisms. Massoialactone, produced by various fungi including Trichoderma spp., showed good antisapstain activity in laboratory and field trials. Twenty-nine essential oils from different plant species inhibited O. piceae in the laboratory. The most efficient oils were found to contain high levels of oxygenated monoterpenes. Oxygenated alcohol or phenolic monoterpenes proved to be extremely active against sapstain fungi preventing discoloration of sapwood for up to 9 months. A method of control for sapstain based on pine oil derivatives containing some of these compounds is now being developed for commercial use.

The aerodynamic diameter, da, of conidia produced in vivo and in vitro by the entomopathogenic fungus Erynia neoaphidis were estimated using an impaction method. The estimated values of da for conidia produced in vivo were smaller than those produced in vitro: in vivo the values of da for primary and secondary conidia were between 16 and 18 μm (equivalent fall speed, Vs, 0·8–1·0 cm s−1); for in vitro produced conidia da values were between 28 and 31 μm (Vs, 2·4–3 cm s−1). For conidia produced by field collected cadavers the value of da, was estimated to be similar to that for conidia produced in vivo in the laboratory. The aerodynamic diameter of primary conidia of Conidiobolus obscurus (strain X39) and Zoophthora radicans (strain NW250) produced in vivo were also measured using the same method. The values of da for these two species were 45 and 17 μm (Vs, 6·2 and 0·9 cm s−1) respectively. Implications for dispersal of E. neoaphidis are discussed. The physical diameters of the test spores were measured microscopically and compared with the aerodynamic diameters.

A somatic incompatibility system was demonstrated in Rosellinia necatrix (anamorph Dematophora necatrix) through barrage formation in pairings among mass fungal isolates from infected avocado trees in commercial orchards in southern Spain, as well as among single ascospore isolates derived from perithecia formed in infected roots. Results revealed the occurrence of high diversity in somatic incompatibility in the R. necatrix population in the study. The occurrence of R. necatrix perithecia in naturally infected avocado roots in the field suggests that such a diversity might originate from sexual reproduction. However, the possibility that other parasexual mechanisms demonstrated in this fungus may contribute to the genetic diversity can not be discounted.

Anamika gen. nov. is described from southern India with one species, A. indica sp. nov. The genus is characterized by spores with a strongly cavernous ornamentation due to cavernae in the epitunica (perispore), and the possession of cheilo-, pleuro-, and caulocystidia. It seems to be associated with Hopea species (Dipterocarpaceae). Molecular data suggest a close relationship to Hebeloma and Naucoria, but indicate that it represents an independent line of evolution.

The possibility that the Coemansia spiralis complex contains three species is discussed on the basis of six descriptions. The name C. spiralis is retained for the fungus first described with the appropriate generic placement and incorporates C. nantahalensis. The second species, for which preserved material is not available, is named as C. bainieri nom. nov., and C. linderi sp. nov. is the third species.