Coniothyrium minitans was successfully co-transformed with the uidA (β-glucuronidase) and the hygromycin-resistance (hph) genes. Both were under the control of the glyceraldehyde-3-phosphate promoter from Aspergillus nidulans. Hygromycin resistance was used as a selectable marker for transformation. In successive transformation experiments, transformation frequencies of up to 1000 transformants μg−1 of plasmid DNA were obtained for isolate A69. Of the ten monospore hygromycin-resistant cultures tested, nine also expressed the uidA gene. Expression of hph and uidA was stable in all transformants after several months of successive subculturing on non-selective medium, and after passage through a sclerotium of Sclerotinia sclerotiorum. Southern hybridization analyses showed all transformants carried multiple copies of each marker gene. When grown on PDA, the culture morphology of three of the transformants of (T×2, T×3 and T×4) was similar to the wild type. Four of the five transformants (T×3, T×4, T×21 and T×24) grew as well as the wild type on different media, and responded to changes in water potential in a similar manner to the wild type. All five transformants were equally parasitic on sclerotia of S. sclerotiorum compared with the wild type. Transformants T×3 and T×4 were the most similar to the wild type in biological characteristics and will be used in future studies. The results indicate that hph- and uidA-transformed strains of C. minitans will be useful for ecological studies on its survival and dissemination.

A new ascomycete Ascomauritiana lignicola gen. et sp. nov. is described and illustrated from a specimen collected on submerged wood in Mauritius. The taxon is compared with species of Arnium, Melanospora and Pustulipora, unitunicate ascomycetes having brown ascospores with germ pores and with species of Argynna, Caryospora, Lepidopterella and Zopfia. Ascomauritiana is characterized by large 5-septate, brown ascospores with polar germ pores and clavate, 4-spored asci that lack an apical ring and deliquesce early. Its taxonomic placement in the Ascomycetes incertae sedis is 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.

Scutellospora rubra is a new arbuscular mycorrhizal species trapped in a pot culture of rhizosphere soil from Eucalyptus dunnii. Spores are dark red-brown, 140–220 μm diam., and closely resemble those of S. heterogama in size, colour, and subcellular organization, but the outer layer of the spore wall is smooth rather than ornamented. Resolution of such species-level variation to properties of the spore wall is consistent with that measured among all other species of arbuscular mycorrhizal fungi studied to date.

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.

One characteristic feature of eukaryotic cells is the organization of special functions into membranous organelles such as vesicles, mitochondria, the Golgi apparatus or nuclei. The distribution and exact positioning is an active process and is crucial for the establishment of cell polarity. For molecular analysis of the processes, filamentous fungi are ideal model organisms because hyphae are extremely polarized cells which continuously extend at their tips. In this study a novel approach was used to isolate mutants in nuclear migration and hyphal morphology. Conidia of A. nidulans, in which nuclei were stained with the green fluorescent protein (GFP) were germinated and visually analysed in a fluorescence microscope. Strains with an abnormal nuclear distribution pattern or defects in polarized growth were isolated with a micro-manipulator and transferred to agar plates to form colonies. Approximately 70 mutants with a corresponding mutant phenotype were recovered, some of which are presented in this report.

Fusarium equiseti and F. pallidoroseum are frequently reported as secondary colonizers of plant tissues. In this study they were isolated from the embryos of weathered cottonseed. Most isolates tested produced equisetin, an antibiotic, when grown on potato dextrose agar, rice, surface-sterilized cottonseed, or autoclaved cottonseed. This is the first report of equisetin from F. pallidoroseum. Equisetin was extracted from cultures of F. equiseti and F. pallidoroseum with acetone and dichloromethane, and partially purified by TLC. Two epimers of equisetin, designated as EQ and epi-EQ, were separated by HPLC. EQ or epi-EQ at 2·5–10 μg ml−1 suppressed germination or inhibited growth of various monocotyledonous and dicotyledonous seed, when the seed were incubated at 30°C under aqueous shake conditions. The two epimers also inhibited the growth of young seedlings and caused necrotic lesions on the roots, cotyledons, and coleoptiles of tested plant seedlings. The results suggest that equisetin may be a pathogenic factor of F. equiseti and F. pallidoroseum on seed and seedling health of cotton and other plants.

Metarhizium anisopliae has potential as a biological control agent. Included among its hosts are certain insect pests of brassica crops. Brassica species produce isothiocyanates, some of which are known to be fungitoxic. In our study, isothiocyanates inhibited both germination and subsequent growth by M. anisopliae in vitro and its ability to infect insects. Conidia were more sensitive than the mycelium to these compounds, the most fungistatic of which were phenylethyl-, 2-chlorophenyl- and allyl-isothiocyanates. Appressorium production in vitro was suppressed by all isothiocyanates except allyl- and propyl-isothiocyanates, which appeared to stimulate appressorium formation. Phenylethyl- and 3-butenyl isothiocyanates, which are present in several of the plant hosts of Phaedon cochleariae, reduced the pathogenicity of M. anisopliae when inoculated insects were exposed to their vapours. These findings have implications for the efficacy of biocontrol of brassica pests by this fungus.

The phylogenetic relationships among fungi that cause spring dead spot disease of Bermuda grass in Australia and the United States were studied from nucleotide sequence data of the ITS region of the rDNA. High levels of sequence similarity were observed among Ophiosphaerella herpotricha, O. korrae and Leptosphaeria narmari. These species clustered into a distinct clade that was distant from other Leptosphaeria and Phaeosphaeria species. Based on sequence data and previous descriptions of the ascoma and ascospore characteristics of these species, we propose a taxonomic transfer of L. narmari to O. narmari. Oligonucleotide primers specific for O. narmari were developed from ITS1 and ITS2 regions to help identify non-sporulating isolates. These primers amplified all isolates of O. narmari, including those from the United States, but not those of O. herpotricha, O. korrae or other ectotrophic root-inhabiting fungi. This is the first report of O. narmari in North America. Two Group I introns were found in the small subunit rDNA of some isolates of O. korrae and O. narmari, but not O. herpotricha. A 425-nucleotide intron in O. narmari and O. korrae was inserted two nucleotides downstream from the ITS5 conserved primer sequence. These introns exhibited an 80% sequence identity. In addition, a second 431-nucleotide intron in O. narmari was similar in size and location to one in O. korrae.

Twelve wood decaying species of Phlebia and Phanerochaete were analysed by RFLP (restriction fragment length polymorphism) analysis of an 18S rRNA gene fragment and an ITS region. The data obtained by different restriction endonucleases were used to construct phenograms based on the UPGMA algorithm. PCR-RFLP within the 18S rRNA gene was sufficient to distinguish between Phlebia species but did not show variation among Phanerochaete spp. ITS region amplified from Phlebia spp. varied in length from 570–745 bp. The smallest ITS fragment was amplified from P. subcretacea and the longest from P. hydnoides. The size of ITS fragments amplified from Phanerochaete spp. was uniform (635 bp), except for the ITS from two P. sanguinea strains (690 bp). RFLP analysis within ITS amplified from Phanerochaete spp. distinguished between them. Results from PCR-RFLP of 18S rRNA and ITS strongly suggest that Phiebia gigantea is closely related to Phanerochaete. Morphological characteristics (lack of clamp connections in hymenium, well developed subiculum) further support this hypothesis.

The growth characteristics and uptake capacities for NO3− and NH4+, as well as activities of nitrogen assimilating-enzymes have been determined in Scleroderma verrucosum. Biomass, grown on agar or liquid MMN media, was higher on nitrate. When S. verrucosum was grown in the presence of equal amounts of ammonium and nitrate, uptake rate for NH4+ was considerably higher than that of NO3−. In addition, it was found that a limitation in ammonium concentration was a prerequisite for nitrate uptake. Nitrate reductase was stimulated when the fungus was transferred from ammonium to nitrate containing media, indicating that the enzyme was inducible by nitrate and repressed by ammonium. Glutamine synthetase and NAD-glutamate synthase activities were clearly detected in S. verrucosum, while the NADP-glutamate dehydrogenase was almost undetectable. This is consistent with the view that ammonium assimilation occurs through the GS/GOGAT cycle in S. verrucosum.

Phenotypes of species hybrids created from in vitro fusion of zoospores from Phytophthora nicotianae and P. capsici were characterized and compared. The species hybrids were created as part of a study of sources of genetic variation in populations of the parent species that are pathogenic over a similar range of plants. Four isolates of species hybrids proved to be similar to both P. capsici and P. nicotianae in relation to vegetative and reproductive morphologies. As in a previous study, DNA of P. capsici was detected more readily than that of P. nicotianae in all hybrid isolates. In the present study, DNA of P. nicotianae was detected in three of four hybrids by hybridization of RAPD-PCR products with species-specific DNA from P. nicotianae. By thermal denaturation analyses, DNA melting temperatures and GC contents of parent species and species hybrids were similar. The mean GC content of 47·2% was similar to GC contents reported for other Phytophthora spp. Additionally, the distributions of GC-rich regions of hybrids were more similar to the distribution in P. capsici than in P. nicotianae. By these molecular analyses, the hybrids were shown to be more similar to P. capsici than to P. nicotianae. Even though interspecific somatic fusion is likely to occur rarely under natural conditions, it could contribute to the genetic diversity of heterothallic species of Phytophthora.

Fifty-eight fungi from different physio-ecological groups were compared for their capacity to oxidize five polycyclic aromatic hydrocarbons (PAH) with three to five benzene rings (R), and produce the appertaining extracellular oxidoreductases in liquid culture. In 14 d, wood- and straw-associated basidiomycetes converted 19–90% of the original PAH compounds. The groups of wood- and straw-degrading, wood-degrading, terricolous, ectomycorrhizal, and mitosporic fungi converted PAH at a proportion of 100[ratio ]75[ratio ]34[ratio ]19[ratio ]18. All fungi preferred fluoranthene and pyrene (4R). Anthracene (3R) was preferred by wood-associated fungi. Phenanthrene (3R) and perylene (5R) were sufficiently converted by wood-associated and terricolous, but poorly by ectomycorrhizal and mitosporic fungi. Nevertheless, fungi that converted the entire set of PAH satisfactorily were found in all five groups. PAH conversion was correlated with the production of manganese peroxidase (r=0·98), peroxidase (r=0·89), and laccase (r=0·85), but not with monophenol monooxygenase (r=0·07). Mn(III) ions oxidized all PAH with preference to anthracene. Hydrogen peroxide converted PAH possibly by the products of Fenton's reaction. Limiting factors were shortages in peroxidases and H2O2. Gymnopilus sapineus and Agrocybe praecox converted top quantities of PAH in the absence of peroxidases, manganese peroxidases, and lignin peroxidases. The relative contributions of intra- and extracellular enzymes to the conversion of PAH and the possible role of the fungi in the long-term detoxification of soil xenobiotics are discussed.

A new genus of freshwater ascomycetes, Cataractisopora, with three new species: C. aquatica, C. appendiculata and C. viscosa, are described. These fungi occur on decaying wood and possess similar characteristics including coriaceous globose ascomata, wide septate paraphyses, cylindrical asci with a relatively massive refractive apical ring and ascospores with polar chambers and filamentous appendages. Appendage material accumulates within the polar chambers and, on release from the ascus, the ascospore appendage unfurls to form long threads. The appendage material is fibrillar at the ultrastructural level, and is derived from episporial projections at the ascospore tip. Although these three fungi are similar to species of Annulatascus, they are assigned to a new genus – Cataractispora based on their unique appendage ontogeny. Annulatascus bipolaris is also assigned to Cataractispora on the basis of its ascospore appendage ontogeny.

A new anamorph genus, Acarocybiopsis, with A. cubitaensis as type species, is described. It is characterized by an unusual combination of indeterminate synnemata having two kinds of hyphae and terminal head composed of a single conidium.

The influence exerted by the non-pathogenic binucleate Rhizoctonia (np-BNR) isolate 232-CG in stimulating plant defence reactions in young bean plants inoculated with the root rot fungus Rhizoctonia solani (AG-4) was examined using light and electron microscopy and further investigated by gold cytochemistry. Severe necrotic lesions on hypocotyles of diseased beans were observed, and the pathogen invaded the cortical tissue causing extensive damage including cell disorganization and cell wall degradation. In contrast, these host reactions were not seen in bean plants inoculated with the non-pathogenic BNR or in plants that were inoculated with BNR and subsequently challenge-inoculated with R. solani. Microscopic examination of hypocotyls inoculated with the non-pathogenic BNR, showed a different host reaction typical of plant defence reactions. In these samples, epidermal and outer cortical cells were impregnated with an electron-dense material. Histochemical assays of this material confirmed the substantial presence of phenols, pectic substances and suberin. Electron microscope observations clearly showed that in non-pathogenic BNR-inoculated plants, fungal cells were confined to the epidermal layer which was darkly stained. Gold cytochemistry confirmed the presence of pectic substances in the electron dense material. The possibility that pectic oligogalacturonides released after hydrolysis by the non- pathogenic BNR enzymes may act as elicitors of defence responses is discussed. The present ultrastructural observations corroborate that non-pathogenic BNR isolates may function as potential inducers of plant disease resistance.

The correct identity of the ergot strains Claviceps fusiformis PRL 1980 (ATCC 26245), C. fusiformis 129 and 59, and Pepty 695/ch-I, formerly described as Claviceps purpurea, was proven by means of RAPD, genomic DNA restriction patterns as well as by a literature search about the strain origin.

The phylogenetic relationships of two Meliola spp., one Meliolina sp., and 36 other ascomycetes were inferred from sequences of the nuclear small subunit ribosomal RNA gene. Phylogenetic analyses revealed that the Meliolaceae are members of the unitunicate Pyrenomycetes, and are not closely related to the bitunicate ascomycetes. The Meliolaceae have been hypothesised to be closely related to the Erysiphaceae, but the cladistic analyses showed that the two families are not closely related. The phylogenetic position of Meliolina is found amongst the bitunicate ascomycetes making it distantly related to the Meliolaceae.

Emericella indica sp. nov. and a peculiar morphological variant of E. quadrilineata are described and illustrated. The former is characterized by large, smooth, violet ascospores and the latter by apparently budding ascospores. Analysis of the sequences of the nuclear rDNA ITS region gene of these species and E. nidulans and E. variecolor support our concepts based on morphological criteria.

Several hyaline-spored coelomycetes placed in Ascochyta, Phoma and Phyllosticta have been recorded from spots on leaves and pods of soybean. Comparison of herbarium material, pathogenicity tests and in vitro studies have demonstrated that Phoma sojicola comb. nov. (syn. Ascochyta sojicola) was predominantly involved as a pathogen of soybean. The diagnostic characters and history of this fungus and other hyaline-spored coelomycetes occurring on soybean are described.