Random Amplified Microsatellite (RAMS) markers were used to detect variation among Phlebiopsis gigantea isolates from seven European countries. The scoring of 26 bands revealed five markers common to all isolates and 21 variable markers, of which six occurred in only one strain. A considerable amount of variation was observed among the progeny isolates of single strains. The analysis of variable markers resulted in 75 different banding patterns among the 86 isolates studied. The high degree of polymorphism and heterozygosity observed in RAMS markers suggests that the amount of genetic variation within this fungus is considerable. The equal distribution of markers in strains from different locations and the lack of country-specific markers suggests that genetic differentiation among the populations is low. The lack of distinct groups of similar banding patterns supports the idea of P. gigantea being a true biological species consisting of a single intersterility group.

Production of the carcinogenic mycotoxins fumonisins has been reported in several Fusarium species, most of which are members of the Gibberella fujikuroi (Gf) complex. In this study, we examined 15 Fusarium species in the Gf complex and 12 other species for fumonisin production and the presence of fumonisin biosynthetic genes (FUM). Among the species within the Gf complex, fumonisin production was detected only in F. fujikuroi, F. globosum, F. proliferatum, F. nygamai, F. oxysporum and F. verticillioides. These five species include members of two of the three major clades delineated in the Gf complex. The FUM genes were detected in these same five species and in F. anthophilum, a member of the third clade. Among the species outside the Gf complex, fumonisin production and FUM genes were detected only in F. oxysporum. Phylogenetic analyses of nucleotide sequences from two FUM gene fragments inferred relationships similar but not identical to those inferred from previous analyses of other genes. The results indicate the FUM genes are discontinuously distributed in the Gf complex and that this distribution gives rise to the differences in the abilities of closely related Fusarium species to produce fumonisins.

Epichloë spp. (Clavicipitaceae) and their close asexual relatives, Neotyphodium spp., form systemic endophytic associations with Pooideae grasses. Interactions between Lolium and Festuca host grasses and fungal endophytes were examined in studies focusing on leaves of natural associations and also of plants into which the endophyte was introduced by seedling inoculation. Light microscopy as well as fungal isolation was used to locate the position of hyphae, while transmission electron microscopy was used to examine host/fungus interactions at the cellular level. These studies provide support for synchronised plant and endophyte growth. This characteristic pattern of growth was maintained when both Epichloë and Neotyphodium spp. were introduced by seedling inoculation into new host species. Hyphae, with few exceptions, grew rapidly as leaves grew and ceased when leaf growth ceased. This pattern of growth offers an explanation for the characteristic appearance of hyphae in leaf sheaths of host grasses; seldom branched and for all species other than N. occultans, aligned parallel to the leaf axis. Hyphal growth of a second group of endophytic fungi, referred to as p-endophytes, was not regulated in the same way, with growth continuing as leaves aged. This pattern of growth gives rise to high concentrations of branched ramifying hyphae in old leaf sheaths. Although host genotype did not effect the basic pattern of hyphal growth of Epichloë/Neotyphodium endophytes, it strongly influenced the concentration and distribution of hyphae throughout leaves. Examination by TEM revealed no evidence that penetration of hyphae into dense tissue was aided by the secretion of pectic enzymes that loosen the middle lamella connecting host cells. Instead it appeared that penetration occurred by hyphae physically pushing between cells. The absence of enzymatic loosening of the middle lamella during penetration could explain why hyphae of these endophytes apparently do not elicit host defence reactions in natural associations. However, subtle interactions between the hyphae of Epichloë/Neotyphodium and host cells in natural associations were observed.

The genetic structure within and between USA and European populations of the emerging phytopathogen Phytophthora ramorum was examined. Four primer combinations were used for amplified fragment length polymorphism (AFLP) fingerprinting of 67 USA isolates from California and Oregon, and 18 European isolates from Belgium, Germany, The Netherlands, Spain and the UK. In addition, three DNA regions (ITS, cox II, and nad 5) of additional Phytophthora species were amplified by polymerase chain reaction, sequenced, and analysed to provide better phylogenetic understanding of P. ramorum within the genus Phytophthora. AFLP banding patterns indicate that the 85 isolates form two distinct lineages within a monophyletic group, distinct from the closely related outgroup species P. lateralis. With the exception of two isolates from an Oregon nursery, European and USA isolates clustered separately within individual clades. The AFLP profiles also indicate that a single clonal lineage dominates the North American population, while the European population consists of an array of mainly unique, closely related AFLP types. Sequences from the three DNA regions were identical among all P. ramorum isolates, and phylogenetic analysis indicates that P. ramorum is closely related to P. lateralis and P. hibernalis.

A toxic protein, bassiacridin, was purified from a strain of the entomopathogenic fungus Beauveria bassiana isolated from a locust, using chromatographic methods. The final toxic fraction contained between 0.1 and 0.3% of the proteins of the crude extract. Bassiacridin showed no affinity for ion exchangers, was characterised as a monomer with a mol. wt of 60 kDa and an isoelectric point of 9.5, and exhibited β-glucosidase, β-galactosidase and N-acetylglucosaminidase activities. Injection of fourth instar nymphs of Locusta migratoria with the pure protein at relatively low dosage (3.3 μg toxin g body wt−1) caused a rate of mortality near to 50%. The effects of the crude and pure fractions were characterized at tissular and cellular levels. The formation of melanised spots on tracheae and air sacs and of melanised nodules in contact with the fat body was observed in injected locusts. Alterations of the fine structure of epithelial cells of tracheae, air bags, and integument were also revealed. The insecticidal protein showed a specific activity against locusts. Bassiacridin is different from the other macromolecular toxins of entomopathogenic fungi already described. Microsequencing of peptides generated by trypsic digestion of bassiacridin confirmed that it is a novel molecule and showed that it exhibits a probably limited similarity with a chitin binding protein from yeast.

Epichloë typhina is a biotrophic symbiont of grasses with its sexual state on immature host inflorescences. The ectophytic, presexual structure (stroma) of E. typhina produces spermatia and also serves as the female structure in mating. After fertilization there is proliferation of a dense mycelium which has been suggested to be heterokaryotic. Perithecia form in the thickened stroma. We investigated the possible formation of heterokaryons in matings and the role of ascospores in contagious spread of E. typhina. In almost all instances the transfer to a stroma of spermatia of opposite mating type leads to thickening of the stroma; however, ascospores were produced only if the parents were of the same mating population. Fertilization of part of a stroma by one spermatial parent often inhibited fertilization by another strain elsewhere on the stroma depending upon the spermatial isolates used. Tests for heterokaryon formation were performed by culturing stromata after matings that produced no ascospores, to avoid analysing meiotic products. In two instances the female was cultured, and in two the nuclear and mitochondrial haplotypes were primarily of the male. In another instance mitochondrial haplotypes and mitochondrial plasmids from the female became associated with the male nuclear haplotype. These results suggest that, following mating, male hyphae proliferate and heterokaryons may sometimes form and also proliferate. To test how ascospores mediate infection, inflorescences of uninfected perennial ryegrass plants were surrounded by fertilized stromata from which E. typhina ascospores were being ejected, seeds were collected and grown, newly infected progeny plants were identified, and isolates from five progeny plants were analysed genetically. As expected for ascospore progeny the rDNA haplotype of the stromal and spermatial parents had segregated, whereas all five isolates had the stromal profile of mitochondrial DNA. This observation demonstrated that ascospores mediate infection of new host plants.

Endophytic fungi were isolated from 7500 samples of wild Musa acuminata collected from five sites at Doi Suthep Pui National Park, Thailand during December 1998 to July 1999. Overall colonization rates from surface sterilized tissues were 56·5, 48·9, 48, 47·9 and 41·7% for the Medicinal Plant Garden, Ban Suthep, Queen Sirikit Botanic Garden, San Gu, and Montatarn waterfall sites respectively. Sixty-one different fungal taxa were isolated. Fewer isolates were recovered from younger than older samples. Xylariaceous taxa and Guignardia cocoicola were the most frequently isolated endophytes from leaves and were either absent or rare in midrib, petiole and pseudostem. Colletotrichum gloeosporioides, C. musae, Guignardia cocoicola, various sterile mycelia and xylariaceous spp. were common at all sites. The endophyte fungal communities at the five sites were found to differ. Deightoniella torulosa was the most frequent isolate at the Ban Suthep site and was either absent or rare at other sites. Colletotrichum species were most common in the midribs and petioles at all sites, while Pyriculariopsis parasitica and Dactylaria sp. were most common in the pseudostems. The endophyte communities isolated from M. acuminata in this study are compared with those from previous studies on tropical hosts. Several of the endophytes isolated are established pathogens of banana and provide support for the hypothesis that some endophytes are latent pathogens. The diversity of fungi on banana is discussed in relation to global estimates of numbers of fungus species.

Fungi developing on dung samples, from a wide range of locations and incubated in moist chambers, were recorded. Highly significant differences were found among the mycobiota of different dung types, from different latitudinal ranges, and collected at different seasons. Sheep, cattle, deer, rabbit, hare and grouse provided 86 % of the 425 samples. Highly significant differences in the community composition of the mycobiota of these six dung types were observed. Coprinus stercoreus was the commonest basidiomycete, and most frequent on sheep, cattle, deer and rabbit; C. miser was the only common species, of any taxonomic group, which showed no significant difference in its frequency on different dungs. Apothecial fungi comprised about a quarter of the records, with Ascobolus, Saccobolus and Thelebolus spp., Iodophanus carneus and Lasiobolus cuniculi the commonest. A. carletonii occurred uniquely, but frequently, on grouse. Perithecial species, especially of Schizothecium, Podospora, Coniochaeta and Sporormiella, accounted for ca 50% of all records. The grouse dung mycobiota was markedly different, with a lower species richness (mean 3.2 per sample), from that of the mammalian herbivores, which had means of 9–12 spp. per sample. In temperate latitudes more species were recorded on samples in winter. Species richness was greater in samples from lower latitudes. Significant differences in the composition of different dung types are discussed in relation to their fungal communities. The usefulness of studying the fungi of the dung microcosm as a means of rapid biodiversity assessment is considered, by comparing the results with those of a similar, but smaller, study 30 years earlier, and by examining the diversity of a sample from a subjectively poor habitat in Morocco. There was no indication of any major change in the coprophilous mycobiota that might be associated, e.g. with climatic change or changed farming practice over the last three decades. The Moroccan samples had a much lower diversity (80 spp./50 samples) than would be expected of samples from that latitude (146 spp./50 samples) based on the worldwide data set.

Leptosphaeria maculans (anamorph Phoma lingam), the ascomycete causing stem canker of crucifers, is a species complex that can be separated into at least seven distinct subgroups using a combination of biochemical and molecular criteria. In the present study sequences of the entire ITS region, including the 5.8S rDNA, of 38 isolates representing the seven subgroups, along with specimens from culture collections, were analysed, compared to those of closely related Leptosphaeria species, and the phylogeny inferred using parsimony and distance analyses. A well-supported clade encompassed all isolates of the seven subgroups along with L. conferta, a known saprobe of dried crucifer stems. The L. maculans isolates were further separated into two well-supported clades corresponding to L. maculans s. str. and the recently named L. biglobosa. Parsimony and distance analyses further separated groups within both species, usually corresponding to specific host plants or geographic origin, e.g. L. maculans ‘brassicae’ from cultivated Brassica, L. maculans ‘lepidii’, from Lepidium sp., L. biglobosa ‘brassicae’, from various Brassica species, L. biglobosa ‘thlaspii’ from Thlaspi arvense, L. biglobosa ‘erysimii’ from Erysimum sp., and L. biglobosa ‘canadensis’ mostly found in central Canada. The oldest L. maculans specimens maintained in international collections clustered with either L. maculans ‘brassicae’, L. biglobosa ‘brassicae’, or a still different group closely related to L. biglobosa ‘thlaspii’. The evolutionary relationships between the seven infraspecific groups are discussed in terms of phytopathological relevance and species isolation linked with specific life cycle, geographic isolation or host specificity.

Thirty-nine ectomycorrhizal isolates of Paxillus involutus, Pisolithus tinctorius, Suillus bovinus, S. luteus and S. variegatus were tested on cadmium, copper, nickel and zinc amended media to determine their in vitro tolerance, measured as inhibition of biomass production. Twenty-one isolates were from heavy metal polluted sites, whereas the others were from non-contaminated soils. There was a strong interspecific variation in metal tolerance. S. luteus, S. variegatus and P. tinctorius were more tolerant of Cu, Cd and Zn when compared with P. involutus, whereas the reverse was true for Ni. A high intraspecific heterogeneity in metal tolerance was also found. EC50 values for isolates originating from polluted sites were not statistically different from EC50 values for isolates originating from non-contaminated sites. The findings are discussed in relation to the potential benefits of ectomycorrhizal fungi in protecting their host plants from metal contamination.

Arbuscular mycorrhizal (AM) fungi, classified in the order Glomales, cannot easily be identified by hyphal morphology. PCR can be used successfully to identify the fungi within the host or soil. Some of the primers that have been used are based on a few sequences of a variable region in the SSU rRNA gene. To verify the specificity of primers of this type which have been used in earlier publications, and to provide data for future primer design, we analysed 51 partial Glomales SSU rRNA gene sequences from 33 glomalean isolates.

To distinguish contaminants from the AM fungi (AMF) all sequences were verified by phylogenetic analyses. Together with sequences from the EMBL database a set of 88 sequences from 58 isolates was analysed, comprising 39 species. The phylogenetic trees reveal at least three distinct well supported Glomus clades and other interesting relationships within the Glomales. Sequence data show that most of the primers published in earlier studies are not useful at the proposed taxonomic level. For example, the VANS1 primer site is not specific for the Glomales, and is homologous to fewer than half of the AMF sequences investigated. Primer regions that have been considered to be family-specific within the Glomales do not show strict specificity. Because clades in the phylogenetic trees fit well to several sequence patterns, priming sites for AMF are discussed and sequence data for future analyses provided.

The study of mould spores is of major importance as many fungi can cause considerable economic losses worldwide acting as plant pathogens or triggering respiratory diseases and allergenic processes in humans. Knowledge of spore production relationships to different altitudes or weather patterns can be applied in a more efficient and reliable use of pesticides or improving diagnosis and treatment of respiratory allergic diseases. In this way monitoring of Cladosporium cladosporioides, C. herbarum and Alternaria spp. airborne spores during 2002 was carried out by means of three LANZONI VPPS 2000 pollen traps located in areas of north-west Spain at various altitudes and with various weather patterns.

High spore counts were recorded in the late summer and early autumn, with a fairly similar hourly spore-count pattern, increasing the concentrations in the late evening (7–10 p.m.). High spore concentrations were detected in inland rural areas in front of coastal ones. As the continentality index increased, C. cladosporioides spore concentrations rose and Alternaria declined. C. herbarum concentrations increased with increasing height above sea level. The weather factor displaying the strongest positive correlation with mean daily spore counts was temperature. The optimal conditions for high airborne spore concentrations were recorded at temperatures ranging from 23–29 °C and RH values of around 80%, followed rapidly by rainfall in Vigo and Ourense and preceded by heavy rain two days prior to recording peak values in Trives.

Aseptic root exudates were collected from the liquid culture of roots of two host (Daucus carota and Lycopersicum esculentum) and one non-host plant (Beta vulgaris) of arbuscular mycorrhizal (AM) fungi. Exudate was also collected from maize (Zea mays FRB6) seedlings which were grown hydroponically under aseptic conditions. Exudate fractions of host roots stimulated hyphal branching behind any actively growing hyphal tip of three AM fungi tested (Gigaspora gigantea, G. rosea, and Glomus intraradices). Fractionation patterns obtained from C18 Sepak cartridges loaded with carrot root exudates isolated from roots grown under various phosphorus regimes, TLC analyses, and solubility properties of fractionated components, indicated a range of hydrophilic to hydrophobic hyphal branching stimulators. The 50/70% methanol fraction from a C18 cartridge induced hyphal branching patterns of G. gigantea that were dose dependent and were identical to those observed when germinated G. gigantea spores were grown with host roots in dual culture. Exudate fractions from B. vulgaris inhibited hyphal tip growth, but inhibited hyphal tips formed recovery branches which would allow continued fungal growth. These recovery hyphae were also formed when germinated G. gigantea spores were grown in dual culture with sugar beet roots. The recovery branches induced by non-host roots and the prolific branching induced by host roots have ecological implications.

Mycotoxins are secondary metabolites produced by many phytopathogenic and food spoilage fungi including Penicillium, Fusarium and Aspergillus. The toxicity and carcinogenicity of many of these mycotoxins, and their potential to contaminate foods and animal feedstuffs is a cause of serious concern globally, both from a food safety and food trade standpoint. Thus the rapid identification of mycotoxigenic fungi would be desirable, such that early intervention steps could be applied to help limit the amounts of contaminated materials, particularly cereals and cereal-based products, gaining access to the human food chain. With this in mind a number of PCR-based methodologies have been developed for the identification of mycotoxin biosynthetic genes in different fungal genera, together with assays developed using other genes or random amplification of polymorphic DNA (RAPD) methodologies for the identification of specific toxigenic fungi. In addition, reverse transcription (RT)-PCR, competitive PCR and Real Time quantitative PCR methodologies have also been developed for this purpose. The development of each of these techniques, their usefulness, limitations and adaptability will be discussed together with descriptions of specific examples where these techniques have been utilised in different experimental settings.

In order to integrate ustilaginomycetous anamorphs into the general phylogenetic system of Ustilaginomycetes, partial nuclear large subunit ribosomal DNA sequences of 56 teleomorphic and 19 anamorphic species of the Ustilaginomycetes were analysed. Maximum parsimony and neighbour joining confirm the new suprageneric system of Ustilaginomycetes and indicate that (i) the species of Pseudozyma represent anamorphs of Ustilaginales parasitizing grasses, (ii) Pseudozyma prolifica, the type of Pseudozyma, is very closely related to Ustilago maydis, (iii) Pseudozyma tsukubaënsis is probably synonymous with Ustilago spermophora, (iv) the species of Malassezia represent a group of its own within the Exobasidiomycetidae, (v) Tilletiopsis cremea, T. lilacina and T. washingtonensis belong to the Entylomatales and (vi) T. flava, T. fulvescens and T. minor are members of the Georgefischeriales. Like all Tilletiopsis species tested, T. albescens and T. pallescens are members of the Exobasidiomycetidae, but they cannot be ascribed to any of the known orders of this subclass. The description of the Malasseziales is emended.

Seven dominant mangrove species of an estuarine mangrove forest in south India were studied for the presence of foliar endophytes. Mitosporic fungi, ascomycetes and sterile mycelia were isolated from the leaves of the mangrove hosts studied. Although many endophytes were common to more than one host, the endophyte assemblage of each mangrove species was dominated by different endophyte species.

In order to investigate phylogenetic relationships of selected members of Peronosporaceae to the genera Pythium, Halophytophthora, Phytophthora, and Peronophythora, Bayesian analysis of partial sequences of the ITS1-5.8S-ITS2 region was performed. In addition, sequences of the complete ITS1-5.8S-ITS2 region were analysed for 101 collections belonging to the genera Peronospora, Hyaloperonospora, Perofascia, Pseudoperonospora, Phytophthora and Peronophythora, using Bayesian inference of phylogeny and maximum parsimony. The results confirm a close relationship of these genera. The strongly supported Peronosporaceae clade is located within the paraphyletic genus Phytophthora (including Peronophythora). Monophyly of the genera Pseudoperonospora and Hyaloperonospora are strongly supported, but monophyly of Peronospora s. str. can neither be confirmed nor rejected. Within Peronospora s. str., basic relationships often remain unclear; however, some groups form highly supported monophyletic clades. Peronospora species parasitising the same host families or orders are only partly resolved as monophyletic, indicating frequent host-jumping also between distantly related host families. All species inhabiting flowers of different host families form a strongly supported monophyletic group.

Extraradical hyphae of Glomus intraradices or G. claroideum were extracted from root free sand of two-compartment pot cultures and used to determine fungal phosphatase activity [p-nitrophenyl phosphate (p-NPP) hydrolysis]. Enzyme activity was assayed with respect to pH, temperature and different fractions of the hyphae (external soluble, wall-bound and internal phosphatases). The results showed an overall maximum enzyme activity at pH 5·2–5·6 for both fungi, with a possible secondary maximum at pH [ges ]8·8 for G. claroideum. Of the two fungi tested, G. intraradices had the highest external phosphatase activity in two experiments and the same activity in one experiment, reaching 184 μmol p-NPP hydrolysed mg−1D.W. h−1. Phosphatase activity at pH 5·2 decreased sharply with temperature, with 4·5 and 10·5% of the enzyme activity remaining at 5 °C relative to that at 37° for G. intraradices and G. claroideum, respectively. Separation of the phosphatase activity into external soluble, wall-bound and internal fractions revealed that up to 70% of the measured activity was associated with the hyphal wall, and the rest with internal structures. Exuded phosphatases were not found in measurable amounts. The implications of these results on possible hyphal utilization of organic P in soil are discussed.

Sphaeropsis sapinea is an opportunistic pathogen causing serious damage to conifers, pre-disposed by adverse environmental conditions or mechanical damage. Three different morphological forms of the fungus have been described and are commonly referred to as the A, B and C morphotypes. Isolates of the different morphotypes have also been separated based on differences in pathogenicity and molecular characteristics. These differences, however, overlap and have not been considered sufficiently robust to justify the description of separate taxa. The aim of this study was to consider relationships between isolates representing different S. sapinea morphotypes, using multiple gene genealogies inferred from partial sequences of six protein-coding genes and six microsatellite loci. Genealogies generated for the protein-coding genes and microsatellite loci were not congruent but both consistently grouped isolates representing the A and C morphotypes in separate but closely related clades. In contrast, isolates of the B morphotype grouped together in a clade that was equally different to the A and C morphotypes as it was to the clade encompassing isolates of Botryosphaeria obtusa. These results provide strong evidence to show that the B morphotype isolates are distantly related to S. sapinea and represent a discrete taxon, which we describe here as Diplodia scrobiculata sp. nov.