The shelled grains of glutinous broomcorn millet, Panicum miliaceum, were used as solid substrate to prepare granular cultures of Zoophthora radicans, an entomophthoralean biocontrol agent against numerous insect pests. Steamed millet grains were inoculated by mixing 15 g millet grains (D.W.) with mashed pieces of half a 60-mm-dish colony in 3 ml modified Sabouraud dextrose broth and incubated at 15 °C and L[ratio ]D 12[ratio ]12 for up to 24 d. 20 grains were sampled at 3 d intervals from day six onwards and individually assessed for their sporulation capacity using a self-designed device for spore collection. The millet cultures after [ges ]12 d incubation produced 12.0–14.9×104 spores grain−1 during a 7 d period. The maximal sporulation capacity associated with the 21 d-old culture was about half of that of Z. radicans-killed Plutella xylostella larvae (28.7×104 spores cadaver−1), which individually were at least three times larger than the millet grains. Based on the time–concentration–mortality responses of second-instar P. xylostella larvae to Z. radicans in three independent bioassays, the spores ejected from the cultured millet grains, from the mycelial mats from liquid culture, and from larval cadavers displayed insignificant variations in infectivity to the host species, and yielded similar LC50 and LT50 estimates. Conclusively, the millet-based technology for production of granular cultures of Z. radicans was easy, inexpensive and highly efficient, and it could be superior to previous methods used in mass production of mycelium-based preparations of Entomophthorales since this new approach requires no special additives, drying, freezing and milling. This technology may suit to mass production of culturable but nutritionally fastidious entomopathogens from the Entomophthorales.

On the basis of all accessible records from the literature and our own field observations and collections, the ecology of the rare myxomycete Barbeyella minutissima is described. Analysis of these data, derived mainly from the Northern Ammergauer Alps (Germany) and the Appalachian Mountains (USA: West Virginia, North Carolina), permit the microhabitat requirements of this myxomycete and its ecological associations with other myxomycetes and bryophytes to be elucidated in some detail. Barbeyella minutissima appears to have a distribution centred in montane spruce-fir forests, where it is regularly associated with three other species of myxomycetes – Colloderma oculatum, Lamproderma columbinum, and Lepidoderma tigrinum. Several leafy liverworts are associated with Barbeyella. Particularly noteworthy is Nowellia curvifolia, which seems to be an indicator organism for this myxomycete. A world distribution map of Barbeyella minutissima is provided, along with conclusions relating to the putative range of the species.

Eleven enzymes from 139 specimens of thirteen Italian populations of Tuber magnatum were analysed with multilocus horizontal starch gel electrophoresis. The research was carried out to obtain insight into the genetic variability of this species across its geographic range. All the gene-enzyme systems scored appear to be fixed in homozygosity in accordance with what is hitherto known in the genus Tuber; nine seemed monomorphic, whereas two, that is MDH-1 and ME-2, showed three alleles each. These results indicated a self-reproductive system and the low genetic variability is in agreement with the restricted endemism of the white truffle. The distribution of the electrophoretic types is discussed as a basis for further molecular applications.

In an attempt to clarify the relationship between fungi classified as Ophiostoma minus, but of different geographic origins and mating systems, sequencing of the 5.8S and ITS 2 rDNA, and β-tubulin gene was carried out. The β-tubulin gene was highly informative, supporting the sub-division of O. minus into two groups based on geographic origin. Furthermore, isolates previously classified as O. pseudotsugae were confirmed as being clearly distinct from O. minus. However, sequencing did not reveal any polymorphisms between isolates with homothallic as compared to heterothallic mating systems. This was supported by crosses using methylbenzamidazole-2-yl carbamate nuclear markers which showed that hybridisation between isolates of different mating systems was possible. However, we propose that different mating systems may still signal a divergence of isolates of O. minus.

A fossil ascomycete was found attached to the thorax of a stalk eyed fly (Diopsidae: Prosphyracephala succini) in a fragment of Baltic amber. The fungus is assigned to the extant genus Stigmatomyces and described as S. succini sp. nov. This find is the first fossil record of the order Laboulbeniales. At the same time it constitutes the oldest record of a parasitic fungus on an insect. The palaeohabitat is discussed with regard to the find.

Genetic variability among isolates of the ectomycorrhizal fungus genus Pisolithus collected in Brazil, USA and France was examined, based on nucleotide sequence of ITS from rDNA genes and restriction polymorphism of the mitochondrial DNA (mtDNA). Alignment of the ITS sequences showed 97.0% homology among isolates from Brazil, 97.8% homology among isolates from USA and France and 75.7–78.5% among the isolates from Brazil and USA/France. These sequences were aligned with ITS sequences of Australian, Brazilian and Kenyan Pisolithus isolates described in the literature. A consensus tree generated by sequence data of all Pisolithus isolates showed the presence of a number of species among the isolates. The mtDNA analysis was carried out by total DNA restriction analysis with different enzymes (Dra I, EcoR I, EcoR V, Hha I–Xba I, Hind III and Rsa I) and probing with purified mtDNA labelled with fluorescein d-UTP. The size of the mitochondrial genome of isolates Pt 90A, Pt 185R and PF cleaved with EcoR I was estimated to be, approx. 40.5, 47.2 and 47.6 kb, respectively. Cluster analysis based on the mtDNA restriction fragments grouped the isolates into two distinct groups, which coincide with their host specificity and geographical origins. PCR-RFLP analysis of mitochondrial rDNA (mt rDNA) from 12 Pisolithus isolates did not show any polymorphism. These data corroborate previous results obtained in our laboratory with RAPD-PCR and rDNA PCR-RFLP markers and support recent taxonomic studies where a number of Pisolithus species were described.

Rhizoctonia solani AG-3 causes considerable yield loss in potato fields in eastern Canada and U.S.A. The accurate identification of AG-3 isolates is strategic prior to planting potatoes. To obtain a fast and reliable identification method, RAPD experiments were carried out to obtain specific genetic markers of AG-3 isolates. DNA from various isolates of R. solani was submitted to RAPD amplification using random 10 mers primers. One of the forty primers used yielded a 2·6 kbp fragment present in all isolates of AG-3. The specificity of this fragment was assessed by Southern blot analysis. It was partly sequenced and DNA primers were designed for PCR amplification experiments. A PCR-based restriction mapping method, using one restriction endonuclease, Xho I, was developed for specific detection of AG-3 isolates. Analysis of data showed that AG-3 isolates were distinct to other AGs R. solani. The detection method described here is very specific and reliable; it can be applied on plant tissue and soil infected with R. solani AG-3.

Arbuscular (AM) mycorrhizal fungi form symbiotic associations with the roots of plants that lead to increased growth and health of many plants. The agricultural application of mycorrhizas has remained difficult since AM fungi are obligate biotrophs that show only very limited growth in the absence of a host. As a first step in the molecular and genetic characterization of these fungi we have constructed genomic libraries of Glomus versiforme and Gigaspora margarita. We demonstrated that in addition to fungal genes these libraries contain clones derived from the genome of endosymbiotic bacteria that are present in fungal spores. A genomic clone encoding elongation factor 1 alpha (EF-1) was isolated from G. versiforme and to our knowledge this is the first time a low copy number gene was cloned from an AM fungus. The EF-1 promoter is highly active in mycorrhizal roots and will be an important tool for the expression of foreign genes in the fungus.

The genetic structure of five Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota) was investigated using codominant PCR–RFLP, allele specific amplification (ASA) markers, inter simple sequence repeat (ISSR) markers and mating studies. Sequence analyses of a subset of single spore isolates revealed sequence variation in four target sequences; internal transcribed spacer (ITS) and intergenic spacer (IGS1) of the nuclear ribosomal DNA, the translation elongation factor 1 alpha (efa) gene and the super oxide dismutase (sod) gene. No sequence variation was found in amplified portions of the mitochondrial large and small rRNA genes. Genotype distributions were mostly (90%) in accordance with Hardy–Weinberg expectations, and the nrDNA markers (ITS/IGS1), efa and sod were in most cases (87%) in linkage equilibrium, indicating an outcrossing reproductive mode, panmictic conditions and large population sizes of the fungus. Mating tests confirmed that F. rosea exhibits an outcrossing bipolar heterothallic mating system. Mating allele richness was high in two investigated populations. Phylogenetic analyses of ITS and IGS1 sequences from the five geographic populations revealed some geographic sub-structuring of the ITS sequences, but no sub-structuring of IGS1. The nrDNA (ITS/IGS1), efa and sod markers gave a low overall FST (0.013). The ISSR markers gave no clustering of the populations in UPGMA, and the between-population variance component was very low in AMOVA (0.4%), indicating a high level of gene flow.

A survey of Penicillium albocoremium was undertaken to identify potential taxonomic metabolite markers. One major and four minor metabolites were consistently produced by the 19 strains surveyed on three different media. Following purification and spectral studies, the metabolites were identified as the known protein farnesyl transferase inhibitors andrastin A (1) and barceloneic acid A (2) along with barceloneic acid B (3), barceloneic lactone (4), and methyl barceloneate (5). These compounds are significant taxonomic markers for P. albocoremium; moreover this is the first report of a methyl ester of a barceloneic acid being produced as a secondary metabolite. Tissue extracts created following pathogenicity trials involving P. albocoremium and Allium cepa confirmed the production of these five metabolites in planta. Barceloneic acid B was found to be biologically active against a P388 murine leukemia cell line.

Restriction fragment length polymorphisms (RFLPs) were identified among total DNA from clonal lines of Uncinula necator when cloned sequences of total U. necator DNA were used as probes. Four probes, pUnP14, pUnP27, pUnE21 and pUnE4, hybridized to multiple-copy sequences and, with the exception of pUnE4, detected genetic variation among clonal lines of U. necator. Clones pUnP14, pUnP27 and pUnE4 produced banding patterns that were stable for DNA extracted from different asexual generations of U. necator clonal lines over at least 15 months. In addition, clones were evaluated for species specificity. Clones pUnP27 and pUnE4 detected only U. necator sequences in total DNA from infected grapevine leaves. Clones pUnP14 and pUnE4 did not hybridize to total DNA from a range of fungi.

Genetic diversity in a sample of the Australian U. necator population was investigated; 15 genotypes were identified among 29 U. necator clonal lines examined. Genetic variation was detected in samples collected within micro-geographical areas, for example, different genotypes representing both mating types of U. necator were detected on a single plant of Vitis amurensis. RFLP analysis of the banding patterns produced using pUnP14, pUnP27 and pUnE21, identified two broad genetic groups, designated A and B. Analysis of DNA fragment patterns obtained using the polymerase chain reaction (PCR) and the plant intron splice junction (ISJ) primer R1 also supported the allocation of U. necator clonal lines into groups A and B.

Selenate-resistant (SelR) mutants of Botrytis cinerea were selected by plating conidia or mycelial plugs onto minimal medium amended with selenate and taurine. Ultraviolet irradiation of conidia to give 5% survival increased mutant yield twenty-fold. Mutants could be divided into three classes based on growth in the presence of selenate or chromate and on response to taurine in minimal media. Nitrate non-utilizing (Nit) mutants, generated as spontaneous sectors on minimal media amended with chlorate, behaved as nit1 mutants in growth tests (i.e. putatively defective in nitrate reductase apoenzyme). When nit1 mutants were paired on medium with nitrate as sole nitrogen source some pairings complemented, behaviour attributed to intragenic complementation. Selected crosses of SelR and nit1 mutants with wild-type strains gave 1[ratio ]1 segregation of both phenotypes and no evidence of linkage to either Mbc1 (benzimidazole resistance) or Daf1 (dicarboximide resistance) markers; loose linkage was confirmed between Mbc1 and Daf1. Strains showing the SelR phenotype may result from mutations in different genes; the genotypic symbol Sel 1 is allocated to one of these mutations. Four-point crosses involving the markers at Sel 1, nit1, Mbc1 and Daf1 generated progeny with all 16 genotypes. Both Sel1R and nit1 mutants were stable following subculture and retained pathogenicity in a bean seedling assay. Complementation was demonstrated between SelR and nit1 mutants selected from the same parent.

As one of the endangered breed of mycologists to have maintained an interest in oomycetes, I am compelled to defend the rationale in continuing to refer to these micro-organisms as fungi. This paper is stimulated by recent communications in which my mycological colleagues have, with apparent relish, pointed to what they perceive as inaccurate references to oomycetes as fungi. David Moore (1997) provides a prime example in his review of The Growing Fungus edited by N. A. R. Gow & G. M. Gadd (1995). Moore criticizes the editors for allowing so many pages of their book to be devoted to these micro-organisms, when ‘…Neurospora is probably more closely related to a cow's nose than it is to Saprolegnia’. While a phylogenetic chasm separates the oomycetes from other fungi, I shall argue that it is impractical to restrict the usage of the term fungus to those micro-organisms that qualify as members of the Phylum Fungi (Alexopoulos, Mims & Blackwell, 1996, provide a contemporary overview of fungal systematics). This argument should have been settled by a series of clear-sighted articles published after molecular genetic analyses confirmed the polyphyletic nature of the fungi (Christensen, 1990; Bruns, White & Taylor, 1991; Hawksworth, 1991; Barr, 1992). For example, Donald Barr (1992) suggested that oomycetes are fungi in the colloquial sense of the term and that it is the colloquial sense of the term that will continue to be most meaningful to mycologists. But since the message of these articles was apparently missed by many mycologists, I hope that this note will clarify a practical definition of the fungi that includes the oomycetes but which does not conflict with a natural classification of the Phylum Fungi that excludes them.

Extracts from entomopathogenic fungi of the genus Hypocrella (7 species) and its anamorph Aschersonia (11 species) were screened for cytotoxicity to Sf9 and C6/36 insect cells and L929, BHK(21)C13 and HepG2 mammalian cells. Cytotoxic extracts to insect cells (ID50's[les ]10 μg ml−1) but not to mammalian cells (ID50's[ges ]10 μg ml−1) conformed to the criteria of the project and were considered ‘lead’ extracts for further investigation. ‘Leads’ were found in two of the Hypocrella species: H. discoidea, and H. tamurai and in three of the Aschersonia species: A. samoensis, A. badia, and A. tamurai. Bioassay-guided fractionation of the cell extract of the fungus A. samoensis BCC 1393 led to the identification of two known anthraquinone dimers, (+)rugulosin (1) and skyrin (2) which showed selective toxicity towards insect cells. (+)Rugulosin (1) and skyrin (2) exhibited strong cytotoxic activity against the insect cell line Sf9 with respective ID50 values of 1.2 and 9.6 μg ml−1, but showed weak activity toward mammalian cells. This first report of (+)rugulosin (1) and skyrin (2) in A. samoensis is confirmed and demonstrated in another four strains of A. samoensis isolated in Thailand. The preferential cytotoxicity against Sf9 insect cells gives evidence that these insect-pathogenic fungi of the Hypocrella/Aschersonia group might be useful as an agent for pest control.

Five ophiostomatoid taxa have been found associated with the infructescences of Protea species, an ancient group of flowering plants endemic to South Africa. Two of these fungi are characterized by their unusual Knoxdavesia anamorphs and have been placed in Gondwanamyces. The three remaining species have Sporothrix anamorphs and have accordingly been accommodated in Ophiostoma. The phylogenetic relationships between the fungi associated with Protea spp. and other ophiostomatoid fungi in Ceratocystis and Ophiostoma are unknown. Large subunit ribosomal RNA sequence data was obtained for the fungi associated with Protea infructescences as well as for the type species of Ceratocystis and Ophiostoma. Both groups of ophiostomatoid fungi were phylogenetically distinct from either Ceratocystis or Ophiostoma, despite sharing morphological and physiological characters with these genera. The species of Ophiostoma associated with Protea infructescences group within the Ophiostomatales while species of Gondwanamyces group within the Microascales. Furthermore, the Ophiostoma spp. from Protea should reside in a separate genus and are a fascinating example of convergent evolution towards insect dispersal.

Twenty-seven Fusarium oxysporum isolates were studied by RAPD-PCR and isozyme analyses. Thirteen isolates were from barley and the rest from different hosts, most of which were dicotyledonous plants. All isolates could be distinguished from each other by RAPD-PCR analysis, and clustered into seven groups in NJ and parsimony consensus trees. Isozyme analysis detected polymorphism in five of the six enzymes and the isolates could be divided into 26 different electrophoretic groups. Five groups were supported by high branch support and bootstrap values in the approximate support tree of combined RAPD-PCR and isozyme data. These five groups were found also in NJ and parsimony consensus trees. The matrices from RAPD-PCR and isozyme data proved to be incongruent, but they did not totally disprove each other. Some correlation was found between geographical origin and phylogenetic relationships of isolates collected from barley. Representatives of the main clades of phylogenetic trees, were further studied by rDNA RFLP and rDNA sequence analyses, together with isolates of other Fusarium species. Isolates of F. oxysporum and F. avenaceum formed distinct groups in the phylogenetic analyses, except for two isolates of F. oxysporum which were grouped with isolates of F. redolens.

The identity of strains identified as Trichoderma aureoviride/Hypocrea aureoviridis was reconsidered. Trichoderma aureoviride was isolated originally from a specimen identified as H. aureoviridis and thus is H. aureoviridis. The morphological and molecular characters of most strains identified as T. aureoviride differ from those of the ex-type but are more typical of T. harzianum, a member of sect. Pachybasium. Molecular data do not support inclusion of T. aureoviride in sect. Trichoderma, nor was there strong phenotypic similarity between H. aureoviridis and H. rufa. In the ITS phylogeny the T. aureoviride ex-type and other collections of H. aureoviridis form a strongly supported clade that is separate from any other recognized section of Trichoderma. Hypocrea vinosa, which was originally included in the T. aureoviride aggregate species concept, is distinct from T. aureoviride, but closely allied with H. rufa/T. viride. Trichoderma aureoviride/H. aureoviridis is a rare species, restricted to the UK and the Netherlands. We redefine T. aureoviride, limiting it to strains with very slow growth rate, effuse conidiation, and the ITS-1 and 2 sequence type D.

The name Marchandiobasidium aurantiacum gen. sp. nov. is introduced for the teleomorph of Marchandiomyces aurantiacus. Dolipore septa and septal pore caps of the closely related Marchandiomyces corallinus are typical of the Ceratobasidiales, and the basidiomatal characters of Marchandiobasidium aurantiacum are reminiscent of those of the monotypic genus Waitea. Morphological, ultrastructural and molecular data suggest that Marchandiobasidium should not be included in Waitea, but should be treated as a distinct genus.

Pythium oligandrum is a parasite of cultivated Agaricus bisporus. Infection results in significant yield reductions and a disease referred to as ‘black compost’. In this study, P. oligandrum isolates were isolated from New Zealand mushroom composts, and their ribosomal DNA internal transcribed spacer (ITS) regions were amplified using the polymerase chain reaction (PCR). ITS nucleotide sequences obtained from New Zealand P. oligandrum isolates were compared with those previously identified P. oligandrum isolates and 23 described Pythium species. Although New Zealand P. oligandrum isolates had high ITS nucleotide identity with internationally identified P. oligandrum, the order of nucleotides in some regions varied when compared with other Pythium species. These varied nucleotides within the ITS region were used to design PCR primers (P.OLIG.F1 and P.OLIG.R04) for the specific amplification of a 384-bp fragment from P. oligandrum DNA. P.OLIG.F1 and P.OLIG.R04 were used to identify a major source of P. oligandrum inoculation on a New Zealand mushroom farm. Application of this diagnostic test will assist farming strategies implemented to prevent future P. oligandrum outbreaks. Furthermore, results presented identify a need for species resolution between P. oligandrum and P. hydnosporum.

Rabbit pellets collected from the field were colonized by Podospora pleiospora at the exclusion of other coprophilous fungi, suggesting antibiosis. In liquid culture, P. pleiospora produced sordarin (1); sordarin B (2), a new compound in which sordarose is replaced by rhamnose; hydroxysordarin (3); and sordaricin (4). The major compounds 1 and 2 exhibited minimum inhibitory concentrations of 0.5–2.5 μg ml−1 against the yeasts Nematospora coryli and Sporobolomyces roseus, but showed little or no activity against bacteria or coprophilous filamentous fungi. In liquid culture, the production of 1 and 2 together amounted to 2.7 μg ml−1, whereas in rabbit dung only 1 was produced at a similar concentration (2.3 μg g−1 fresh weight). The biosynthesis of these substances was unaffected by the presence of inoculum of other fungi tested (Sporobolomyces roseus or Penicillium claviforme) in liquid culture or on dung. Sordarin-type natural products are therefore synthesized by P. pleiospora at sufficiently high concentrations to account for antibiosis against yeasts, but not against filamentous fungi.