The endophytic mycobiota of healthy twigs and the assemblage of species associated with twig lesions of Eucalyptus globulus and E. grandis planted at the same site in the central west region of Uruguay was examined. Twig segments from E. grandis and E. globulus were plated on 2% malt agar. The diversity of endophytes isolated was low, as was the number of host-specific species. E. globulus endophytes of healthy xylem were different from the colonizers of cracked lesions and healthy bark. Conversely, in E. grandis characterized by black spots, the main difference in species composition was between xylem and bark. Cytospora chrysosperma was isolated from healthy bark and symptomatic twigs of E. globulus, suggesting that symptomatic tissues, such as cracks produced under drought were favourable to saprotrophic expansion.

Responses of Rhizoctonia solani to spatial heterogeneity in sources of carbon, and associated translocation of carbon (C), were studied in a simple microcosm system comprising two discrete domains of agar gels separated on a glass slide and overlain with a porous membrane. Two arrangements of the gel pairs were used, one containing two equally large resources (representing ‘homogeneous’ conditions) and one containing a large and a negligible resource (representing ‘heterogeneous’ conditions). The nutrient sources were a standard mineral salt medium with or without glucose as sole C source. The fungus was inoculated onto one domain and growth responses determined by direct measurement of biomass. Translocation of C was quantified by use of 13C-enriched glucose. This substrate was either added to the agar at the outset, when studying newly developing colonies, or as a pulse into already established colonies. When growing in heterogeneous conditions, the fungus actively translocated C from a glucose-containing domain to sustain growth in the adjacent region lacking such a resource. In homogeneous conditions there was evidence of passive translocation (diffusion), but the fungus preferentially used local resource to maintain growth. Active translocation was only observed in newly growing colonies, whereas passive translocation occurred in both growing and established colonies. When the fungus was pulsed with a 13C-enriched glucose solution after 10 d growth, 2.5 times more 13C was taken up by the fungus grown in heterogeneous than homogeneous conditions, suggesting uptake exceeded local demands. In heterogeneous conditions, the total amount of 13C enriched glucose taken up by the fungus was independent of the location of the enriched glucose in the underlying medium. When the nylon membrane was replaced by Cellophane (an additional C source), degradation of the membrane and an increase in biomass occurred only in the heterogeneous system. The possible implications for these results in soil systems are discussed.

Phytophthora quercina, a new species associated with oak decline in Europe, has been assigned to Waterhouse's Group I of Phytophthora. The level of intraspecific variation and evidence of affinities to other Group I species and another, as yet unidentified, species from oak, were examined at the molecular level using four random ten-mer primers to amplify total DNA (RAPDs). Sequences and restriction fragment length polymorphisms of a 900 bp PCR product consisting of the ITS1 and ITS2 regions, and the 5.8S subunit of ribosomal DNA were also examined to estimate relatedness to a broader range of Phytophthora species. The RAPD banding patterns of ten isolates of P. quercina from eight sites in Germany, Hungary and Italy were almost identical and distinct from all the other species tested. Their ITS restriction fragment patterns were also identical, as were the ITS sequences of four selected isolates. Phylogenetic analysis of the ITS sequence data confirmed its unique position in this section of the genus which comprises P. quercina, another five Group I species, P. infestans (Group IV) and P. nicotianae (Group II). Isolates of P. quercina formed a distinct branch at the base of this clade showing no close affinities with any other species. Such data support morphological, physiological and pathological evidence that P. quercina is distinct, although it has some affinity with the other Group I species. The results support earlier reports that Waterhouse's groupings of morphospecies do not fully correspond to phylogenetic relationships indicated by molecular studies. The unidentified Phytophthora sp. 2 from oak was closely related to P. ilicis (Group IV); both were distinct on molecular criteria from all other species in the study.

During investigations of microfungi occurring on submerged plant material in tropical streams, seven species of Sporoschisma and one species of Sporoschismopsis were recorded. Three are newly described: Sporoschisma parcicuneatum, S. phaeocentri and Sporoschismopsis australiensis. Each of these species is described and illustrated in detail and a synopsis of remaining species of Sporoschismopsis is provided. Generic concepts and connections to teleomorphs are discussed and a key is provided to accepted species in both genera.

Gondwanan vegetation, and the Australian region in particular, is species rich for ectomycorrhizal fungi in epigeous and hypogeous forms with over 100 species recorded in small (1 ha) patches of forests. Distinguishing co-occurring ectomycorrhizal fungi as root associations in native (natural or wildlands) vegetation or plantations and discriminating them from other larger basidiomycetes, e.g. wood and leaf litter decomposer fungi, places large demands on molecular identification, especially if interspecific similarities and intraspecific variation occur in target sequences. One hundred and nine species of larger basidiomycetes from a single forest location were characterised by PCR–RFLP profiles of two genomic regions (nuclear rDNA ITS and mtLSU). Over one-third of the species for which multiple isolates were tested showed intraspecific variation in either one or both genomic regions. This remarkably high variation questions previous assumptions about intraspecific ITS sequence variation and highlights the value of integrated molecular and morphological databases including voucher specimens. It also emphasises the value of molecular investigations that use more than one genomic region. Interspecific similarities were common among the Cortinariaceae, especially in the ITS region. Discrimination of most Cortinariaceae species was achieved using variation in the mtLSU region in conjunction with the ITS. This new information raises the possibilities that the ITS sequence is more conserved and the mtLSU more variable than among species of the other 23 families. In the other families, interspecific ITS variation was greater and the mtLSU profiles grouped species within families. The high variation in the two genomic regions indicated possible differences in the fungal population structure between two adjacent, differently managed blocks of Eucalyptus marginata forest. The significance of this variation to ecology, biodiversity assessment and ecosystem management are discussed.

Mycosphaerella tasmaniensis is newly described from Mycosphaerella leaf blotch symptoms occurring on Eucalyptus nitens in Tasmania, Australia. Single ascospore cultures produced a Mycovellosiella anamorph, described here as M. tasmaniensis. Both states occurred together, as well as separately on leaf spots. Phaeophleospora epicoccoides (=Kirramyces epicoccoides) is commonly associated with leaf spots of Eucalyptus spp. in Australia. The teleomorph, Mycosphaerella suttoniae, previously known only from Indonesia, was also collected on E. grandis leaves from Australia. A Cylindrocladium leaf blight disease of young E. grandis trees in Indonesia was found to be associated with a new species of Calonectria. Calonectria multiseptata and its anamorph Cylindrocladium multiseptatum is newly described and distinguished from other species based on their larger, multi-septate ascospores and conidia.

Several independent molecular phylogenetic analyses have indicated that the genus Neolecta has a unique position within the Ascomycota. It is the only taxon outside the core-group of filamentous, ascoma-forming ascomycetes that also has the ability to form ascomata. Light and electron microscope studies indicate that hymenial structure and development in Neolecta spp. are unique. Ascogenous hyphae in N. vitellina branched repeatedly and successively to produce asci. Non-ascogenous hyphae were multinucleate, often with nuclei in pairs. Nuclear pairing was particularily prominent in the ascogenous hyphae. A basal septum delimited the dikaryotic ascus. Ascosporogenesis was initiated by nuclear fusion followed by a meiotic and mitotic division to form eight nuclei. The ascus apex was thin with an annular subapical thickening. Ascospores were forcibly released through a ‘split’ in the ascus apex. Woronin bodies were frequently associated with hyphal septa. Attempts to culture N. vitellina and to obtain molecular information from the type species, N. flavovirescens, were unsuccessful. However, N. flavovirescens showed several microscopic characters that indicated close relationships with the two other species in the genus, N. vitellina and N. irregularis. The position of Neolecta spp. within the Ascomycota is discussed.

The secondary metabolites, helvolic acid and cerulenin were detected in liquid culture of Sarocladiumoryzae, causal agent of sheath rot of rice, and also in diseased sheath tissues. These metabolites were examined by thin-layer chromatography (TLC), column chromatography, and by high-performance liquid chromatography (HPLC). Helvolic acid and cerulenin showed the Rf value of 0.49 and 0.61, respectively using TLC. Cerulenin produced two spots in the TLC chromatogram and produced two major peaks in the HPLC chromatogram due to its instability. External application of helvolic acid and cerulenin produced lesions similar to those caused by the fungus. When infiltrated both of these metabolites caused electrolyte leakage in susceptible cultivar, IR 36 and induced lower levels of leakges in White Ponni, the moderately resistant cultivar to S. oryzae. The concomitant production of helvolic acid and cerulenin by S. oryzae in liquid culture and in planta was confirmed and the role of helvolic acid and cerulenin on the virulence of S. oryzae and sheath rot disease of rice is discussed.

That the symbiotic fungus of leaf-cutting ants only occasionally produces the sexual phase makes their identification confusing. This has occurred so rarely, either in laboratory nests, or in unbalanced field nests, that the possibility of contamination of the fungal garden by other fungi cannot be disregarded. In this paper we describe the formation of several basidiomata in a healthy and free-living nest of the leaf-cutting ant Acromyrmex hispidus fallax, the cultivation in vitro of the sterile mycelia (isolated from the fungal garden) with their typical inflated tips, and the similarity of both forms confirmed by RAPD analysis of their genomic DNA. The fungus was identified as Leucoagaricus gongylophorus.

Most species of Ceratocystis sensu stricto are virulent pathogens of a wide variety of plants including forest and fruit trees, sweet potato, pineapple and sugar cane. Confusion exists regarding the taxonomy of the species in this genus. The aim of this study was to develop a rapid and reliable PCR-based RFLP identification method and to consider phylogenetic relationships among the better-known species of Ceratocystis. A 1·6 kb fragment within the ribosomal DNA operon was directly amplified from living fungal tissue, without extracting DNA. The amplified fragment included part of the small (SSU) and large (LSU) sub-unit rRNA genes, the 5·8S rRNA gene and the internal transcribed spacers (ITS) 1 and 2. The PCR fragments were digested with eighteen restriction enzymes. Four of these (Alu I, Dra I, Hae III and Rsa I) produced RFLPs that separated the species of Ceratocystis. The amplification products from the best-known species were sequenced, and the delimitation of taxa based on this phylogenetic analysis generally agreed with results of previous studies using isozymes and rDNA sequence analysis. This study provides an extended understanding of the relationships among species of Ceratocystis and will form a sound foundation for further taxonomic studies of the group.

In order to investigate the barotolerance of marine fungi and to elucidate their ecological role in the deep sea, high-pressure equipment was built and tested. Cultures of the marine yeasts Debaryomyces hansenii, Rhodotorula rubra and Rhodosporidium sphaerocarpum were inoculated into gas-permeable plastic foil bags and incubated in pressure vessels filled with hydraulic fluid that serves also as an oxygen reservoir. The equipment was used at temperatures from 7° to 34°C and pressures from 0·1 to 80·0 MPa. Comparison of five types of plastic foil showed effects on total cell number in batch culture. An exchange of the hydraulic fluid increased yield by replenishing oxygen, the growth-limiting factor. There were no detectable growth differences between buffered (TRIS, HEPES, imidazole, MES) and unbuffered media and thus unbuffered medium was used. Yeasts were best cultivated in an unbuffered sea-water medium (glucose–peptone–yeast extract) sealed in bags made from polyethylene foil. The fluorocarbon liquid FC-77 provided the best oxygen reservoir owing to its high oxygen and carbon dioxide solubility. An exchange of the hydraulic fluid is not necessary if sensitive growth determination methods are used. All marine yeasts cultivated under simulated deep sea conditions were able to grow at least up to a pressure of 20 MPa, with Rhodotorula rubra and Rhodosporidium sphaerocarpum growing at 40 MPa, corresponding to 4000 m depth. Thus, marine yeasts are able to grow under simulated deep sea conditions and may participate in the degradation of organic matter in the deep sea.

Strains of Ophiostoma ulmi, O. novo-ulmi subsp. americana, O. novo-ulmi subsp. novo-ulmi and O. himal-ulmi were examined for optional introns/insertions within the following mitochondrial genes: small subunit RNA gene (rns), large ribosomal subunit gene (rnl) and the cytochrome oxidase subunit I gene (coxI). Insertions were noted in the rns and coxI genes in strains of O. ulmi, the less aggressive species, but absent in strains of the more aggressive O. novo-ulmi subsp. americana. Strains of all species examined had a group I intron present in the U11 region of the mitochondrial-rnl gene. In all but two strains of O. novo-ulmi subsp. americana, this rnl-U11 intron was about 1.5 kb in length whereas a 2.6 kb version of this element was present in all strains representing O. ulmi, O. novo-ulmi subsp. novo-ulmi, and Ophiostoma himal-ulmi. Irrespective of size, this intron based on RNA folds is a class IA1 group I intron and it encodes a putative ORF for the rps3 ribosomal protein. The size variation of the rnl-U11 intron was examined in detail for two strains of O. novo-ulmi subsp. americana and sequence data suggests the presence of a complex ORF within the 2.6 kb version of this intron; here a homing endonuclease-like gene has been inserted in frame and fused to the carboxyl-terminus of the putative rps3 coding region. The mitochondrial optional introns/insertions in combination with nuclear markers might be useful in distinguishing among the various species and subspecies of the O. ulmi s. lat. complex.

A RAPD-PCR assay was developed and used to test for competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (I) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils; free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S in PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

Entomophaga maimaiga is a naturally occurring fungal pathogen specific to larvae of the gypsy moth, Lymantria dispar. E. maimaiga is thought to be native to Asia where its epizootics can suppress gypsy moth outbreaks. However, in the USA this beneficial fungal pathogen was not observed until 1989, although an isolate of E. maimaiga from Tokyo was released in Massachusetts to control gypsy moths as early as in 1910–1911, and another isolate from Ishikawa Prefecture in Japan was later released in 1985 and 1986 in New York and Virginia. Our objectives were to: (1) test the hypothesis that E. maimaiga populations in the USA have reduced genetic variability due to founder effects compared to the putative ancestral populations in Asia; (2) track the origin of the North American populations of this fungus; and (3) assess whether genetic differences among E. maimaiga isolates are correlated to morphological differences. We compared genetic diversity among 30 E. maimaiga isolates originating from seven states in the USA, five prefectures in Japan, one province of China and one region of far eastern Russia by AFLPs. Among 14 USA isolates, only ten polymorphic AFLP loci were found, whereas 56 polymorphic loci were found among 16 Asian isolates; 29 loci were polymorphic among 12 isolates from Japan alone. Average gene diversity (h¯) for the polymorphic loci was 0.223±0.005 for Asia (including Japan), 0.131±0.006 for Japan only, and 0.041±0.004 for the USA. Thus, native populations from Asia were more diverse than the USA populations. These results are consistent with the expectation of a population founded from a source population by a small number of individuals. Distance and parsimony analyses of AFLP data showed that the isolates from the USA formed one distinct clade that was most closely related to Japanese isolates collected outside the Tokyo area. No morphological variation of E. maimaiga from different geographical locations was detected.

The entomopathogenic hyphomycete Paecilomyces fumosoroseus is a promising candidate for biocontrol of economically important agricultural pests. Assessment of genetic relatedness of this species appears to be essential to gain insight into the monitoring of such biocontrol products. Intraspecific variation within the internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS) was studied in 48 isolates of P. fumosoroseus. These strains were isolated from different geographical and biological origins, more particularly from the silverleaf whitefly, Bemisia tabaci-argentifolii (Homoptera: Aleyrodidae), a major insect pest in field and greenhouse crops. PCR amplification of the ITS1-5.8S-ITS2 rDNA region followed by restriction analysis of the PCR products underlined the overall highly variable nature of this region. Digestion with the six endonucleases AluI, HaeIII, Hin6I, HpaII, NdeII, and SmaI allowed the separation of the isolates into three distinct groups. The group 1, representing 25 isolates, is composed of strains isolated only from the host B. tabaci-argentifolii. By contrast, the group 3 is more diffuse as it included strains from various insect host and geographical origins. Phylogenetic analysis of rDNA-ITS sequence data strongly supported the conclusions of the PCR-RFLP analysis and recognized three monophyletic groups within the P. fumosoroseus complex. The high level of polymorphism within the P. fumosoroseus species is discussed in the light of their biological origin.

From the alkali-extractable material obtained from cell walls of 44 species (49 strains) of Penicillium a water-soluble fraction (F1S) was isolated. The main component (F1S-S) of fraction F1S was purified by gel permeation chromatography through Sepharose CL6B, analysed chemically and its structure determined by 1H- and 13C-NMR. Galactose was the predominant neutral sugar in this fraction. Six different polysaccharidic structures were found and the species investigated were arranged into six groups according to these structures. These results illustrate the complexity of the cell wall components of Penicillium and add to existing information on the relatedness of species of the genus with Eupenicillium and Talaromyces. The results show that there are several types of cell wall in Penicillium.

Twenty-one Aspergillus strains representing different A. awamori, A. phoenicis and A. foetidus isolates were studied in order to explore the potential of a fast RFLP analysis to identify fungal strains. The patterns were compared with those characteristic for A. niger, A. tubingensis, A. carbonarius, A. japonicus and A. aculeatus represented by A. niger CBS 120.49, A. tubingensis NW 756, A. carbonarius CBS 111.26, A japonicus CBS 114.51 and A. aculeatus CBS 101.43 and also with those of the type strains of A. heteromorphus CBS 117.55 and A. ellipticus CBS 707.79.

Sma I digested chromosomal DNA revealed characteristic rDNA patterns after ethidium bromide staining which were used in combination with hybridization patterns of Pst I/Sal I double digested chromosomal DNA with well-defined probes. This allowed clear distinction of eight separate species within the Aspergillus sect. Nigri group. The probes used were a 0·9 kb fragment of the 28S rDNA from Agaricus bisporus, an internal fragment of the pkiA gene from A. nidulans, and the pelA gene from A. niger. All the strains examined including those indicated as A. awamori and A. phoenicis were shown to belong either to A. niger, A. tubingensis or a group representing isolates of A. foetidus varieties, amongst which A. foetidus var. acidus CBS 564.65 and A. foetidus var. pallidus CBS 565.65.

Two-dimensional gel electrophoresis was used to analyse stage-specific proteins from Phytophthora palmivora, a pathogen of cocoa and other economically important tropical crops. Approximately 1% of proteins appeared to be specific for each of the mycelial, sporangial, zoospore, cyst and germinated cyst stages of the life-cycle. Three proteins excised from protein gels of P. palmivora were identified as isoforms of actin by database searches to public libraries of Phytophthora infestans. The protein profiles of parallel samples of P. palmivora and P. infestans demonstrated that 30% of proteins precisely co-migrated suggesting that proteomics may be used to examine changes in the specific stages in the life cycles of Phytophthora spp.

One hundred and thirty four dikaryotic isolates of Ganoderma, morphologically determined as G. adspersum, G. annulare, G. applanatum, G. brownii, G. lobatoideum, G. lobatum, G. lucidum, G. oerstedii, G. resinaceum, and G. tornatum, were examined by horizontal PAGE for eight enzymatic activities. A total of 94 bands were analysed. The objective was to evaluate the relatedness among the specific taxa by analysing numerically the isoenzymic patterns. To discern fungal individuals somatic incompatibility tests were used. Within each enzyme system several allozyme phenotypes could be observed. The enzymic systems analysed failed to yield diagnostic bands for each morphologically defined group, except the AKP system for G. resinaceum. Some bands were shared by all the isolates studied. On the other hand, diagnostic bands at the species complex level were encountered.

The antifungal activity of flavone and flavanone was tested in three different concentrations, 0·5, 2 and 8 × 10−4 M, against 34 fungi. In general, flavone was the substance with the higher activity, and could at 2 × 10−4 M completely inhibit several fungi. No linear relationships between the concentration and the antifungal activity could be observed for some fungi. Chaetomium globosum was the only fungus which was totally inhibited by flavanone at 8 × 10−4 M. Several fungi did, however, show a higher inhibition rate at 2 × 10−4 M.