The ecologically and economically important arbuscular mycorrhizal (AM) fungi, crucial in the ecology and physiology of land plants, and the endocytobiotic fungus, Geosiphon pyriformis, are phylogenetically analysed by their small subunit (SSU) rRNA gene sequences. They can, from molecular, morphological and ecological characteristics, unequivocally be separated from all other major fungal groups in a monophyletic clade. Consequently they are removed from the polyphyletic Zygomycota, and placed into a new monophyletic phylum, the Glomeromycota.

The recognition of this monophyletic group, which probably diverged from the same common ancestor as the Ascomycota and Basidiomycota, gives these fungi their proper status, and provides a basis for a new and natural systematics of these fascinating, yet largely hidden organisms, with three new orders (Archaeosporales, Paraglomerales, Diversisporales) described herein. Additionally, several clades resolve at family level; their formal description is in progress.

In spite of the term ‘endophyte’ being employed for all organisms that inhabit plants, mycologists have come to use the term ‘fungal endophyte’ for fungi that inhabit plants without causing visible disease symptoms. The term refers only to fungi at the moment of detection without regard for the future status of the interaction. This paper is a review of literature on non-balansiaceous fungi involved in asymptomatic colonisations of plants. These fungal endophytes represent a continuum of fungi with respect to physiological status, infection modus, colonisation pattern, secondary metabolism, life-history strategy, and developmental and evolutionary stages, but also with respect to the fungal and host taxa involved in the symbioses.

We hypothesize that there are no neutral interactions, but rather that endophyte-host interactions involve a balance of antagonisms, irrespective of the plant organ infected. There is always at least a degree of virulence on the part of the fungus enabling infection, whereas defence of the plant host limits development of fungal invaders and disease. It is also hypothesized that the endophytes, in contrast to known pathogens, generally have far greater phenotypic plasticity and thus more options than pathogens: infection, local but also extensive colonisation, latency, virulence, pathogenity and (or) saprophytism. This phenotypic plasticity is a motor of evolution.

The number of known species of fungi is estimated as at least 74 K, but could be as much as 120 K with allowances for ‘orphaned’ species. Yet in 1990 the magnitude of fungal diversity was estimated ‘conservatively’ at 1·5 M species. This figure has been widely accepted as a working hypothesis, but subsequent estimates have ranged from 500 K to 9·9 M and the bases of these suggestions are analyzed. Additional data pertinent to the estimation of the number of fungal species on Earth that has become available since 1990 is discussed. Site inventories demonstrate the need for long-term (20 yr plus) intensive studies to determine the number of species in a site. Fresh data sets on fungus[ratio ]plant ratios and degrees of host specificity, especially from well-studied hosts in the tropics, are consistent with earlier estimates. The extent of novelty discovered in recent monographic generic revisions and studies of species in particular habitats varies from 0–96%. Allowances for cryptic species, now known to be widespread by incompatibility and molecular studies, could on their own justify an upward revision by a factor of at least five. To enable confidence in any overall estimate to be increased, more detailed studies, especially on particular sites in the tropics, are needed. The consensus of tropical and molecular mycologists in particular is that an increased estimate could be justified. However, it is prudent to retain 1·5 M as the current working hypothesis for the number of fungi on Earth while additional data to test it further accumulates.

In the continual search by both pharmaceutical and agricultural industries for new products, natural selection has been found to be superior to combinatorial chemistry for discovering novel substances that have the potential to be developed into new industrial products. Since natural products are adapted to a specific function in nature, the search for novel secondary metabolites should concentrate on organisms that inhabit novel biotopes. Endophytic fungi inhabit such a biotope. In the course of the last 12 years, we have isolated ∼ 6500 endophytic fungi from herbaceous plants and trees, screened them for biological activities, and have isolated and determined the structures of the biologically active compounds. Correlations were found between biological activity and biotope, e.g. a higher proportion of the fungal endophytes, in contrast to the soil isolates, inhibited at least one of the test organisms for antialgal and herbicidal activities. The substances isolated originated from different biosynthetic pathways: isoprenoid, polyketide, amino acid derivatives, and belonged to diverse structural groups: terpenoids, steroids, xanthones, chinones, phenols, isocumarines, benzopyranones, tetralones, cytochalasines, and enniatines. The potential role of the endophyte and its biologically active metabolites in its association with its host has been investigated. The fungal endophytes possess the exoenzymes necessary to colonize their hosts and they grow well in the apoplastic washing fluid of the host. When the roots of larch are colonized, the association with the host may be mutualistic, improving growth of the host and supplying the mycobiont with enough nourishment to extensively colonize the host's roots. The concentrations of some plant defence metabolites are lower than in the control when the host is infected with a pathogen than with an endophyte. We hypothesize that the interaction fungal endophyte–plant host is characterized by a finely tuned equilibrium between fungal virulence and plant defence. If this balance is disturbed by either a decrease in plant defence or an increase in fungal virulence, disease develops. Not only must the endophyte synthesize metabolites to compete first with epiphytes and then with pathogens in order to colonize the host, but presumably also to regulate metabolism of the host in their delicately balanced association. The utilization of a biotope such as that of the fungal endophyte is one aspect of intelligent screening, another very important one is the taxonomy of the fungus in order to avoid redundant structural isolations. It is not a random walk through a random forest. Many groups of fungi in different biotopes are waiting to be exploited.

Four different primer pairs were designed for the preferential PCR amplification of fungal SSU rDNA directly from environmental samples. Most of the amplification products obtained from a reference collection of 46 wood-decomposing fungi could be separated by denaturing gradient gel electrophoresis (DGGE) using 1650 bp rDNA fragments produced by primer pair FR1+NS1. A relatively high level of resolution was also achieved using 390 bp products amplified with primer pair FR1+FF390. In contrast, the separation of amplification products obtained by the remaining two primer pairs (FR1+FF700 and FR1+FF1100; 700 bp and 1100 bp, respectively) was inadequate when applied to our fungal collection. Differentiation between all the species tested was achieved by combined analysis of the rDNA fragments produced by primer pairs FR1+NS1 and FR1+FF390. The DGGE analysis of environmental samples collected from Norway spruce stumps showed that the analysis of fungal DNA extracted directly from wood was usually in accordance with the investigation of mycelial cultures isolated from the same decay column. In some cases, however, disagreement was observed, which suggests that these two fundamentally different techniques present different views about fungal diversity. The investigation of fungal species profiles directly from environmental samples using DGGE analysis of PCR-amplified SSU rDNA molecules can be used to improve the detection of fungal groups that are difficult to cultivate.

The phylogeny of 116 species and varieties of Pythium was studied using parsimony and phenetic analysis of the ITS region of the nuclear ribosomal DNA. The D1, D2 and D3 regions of the adjacent large subunit nuclear ribosomal DNA of half the Pythium strains were also sequenced and gave a phylogeny congruent with the ITS data. All the 40 presently available ex-type strains were included in this study, as well as 20 sequences of recently described species from GenBank. Species for which no ex-type strains were available were represented by either authentic strains (6), strains used in the 1981 monograph of the genus by van der Plaats-Niterink (33), or strains selected on morphological criteria (17). Parsimony analysis generated two major clades representing the Pythium species with filamentous or globose sporangia. A small clade of species with contiguous sporangia was found in between the two main clades. A total number of 11 smaller clades was recognized, which often correlated with host-type or substrate and in several cases with a subset of morphological characters. Many characters used in species descriptions, such as antheridium position, did not correlate with phylogeny. A comparison of the ex-type and representative strains with all ITS sequences of Pythium in GenBank revealed limited infraspecific variation with the exception of P. rostratum, P. irregulare, P. heterothallicum, and P. ultimum. The total number of species examined was 116 (including 60 ex-type strains). Twenty-six species had ITS sequences identical or nearly identical to formerly described species, suggesting possible conspecificity. The importance of comparing ITS sequences of putative new species to the now available ITS database in order to avoid unwarranted new species names being introduced.

Since secondary metabolites are involved in fungal-host interactions, those of endophytes and their hosts were studied to try to understand why endophytic infections remain symptomless. A screening of fungal isolates for biologically active secondary metabolites (antibacterial, antifungal, herbicidal) showed that the proportion of endophytic isolates that produced herbicidally active substances was three times that of the soil isolates and twice that of the phytopathogenic fungi. As markers for the plant defence reaction, the concentrations of certain phenolic metabolites were chosen. Those that differed in concentration were higher in the roots of plants infected with an endophyte than in those infected with a pathogen. The results presented here were regarded together with previous studies on other aspects of the plant defence response using dual cultures of plant host calli and endophytes, and of cell suspension cultures following endophytic as compared to pathogenic elicitation. The following hypothesis was developed: both the pathogen-host and the endophyte-host interactions involve constant mutual antagonisms at least in part based on the secondary metabolites the partners produce. Whereas the pathogen-host interaction is imbalanced and results in disease, that of the endophyte and its host is a balanced antagonism.

Within the basidiomycetes, the vast majority of known mycorrhizal species are homobasidiomycetes. It was therefore surprising when molecular and ultrastructural studies revealed a broad diversity of mycorrhizal associations involving members of the heterobasidiomycetous Sebacinaceae, fungi which, due to their inconspicuous basidiomes, have been often overlooked. To investigate the phylogenetic position of the Sebacinaceae within the basidiomycetes and to infer phylogenetic relationships within the Sebacinaceae, we made molecular phylogenetic analyses based on nuclear rDNA. We present a well-resolved phylogeny of the main lineages of basidiomycetes which suggests that the Sebacinaceae is the most basal group with known mycorrhizal members. Since more basal taxa of basidiomycetes consist of predominantly mycoparasitic and phytoparasitic fungi, it seems possible that a mycorrhizal life strategy, which was transformed into a saprotrophic strategy several times convergently, is an apomorphic character for the Hymenomycetidae. Mycorrhizal taxa of Sebacinaceae, including mycobionts of ectomycorrhizas, orchid mycorrhizas, ericoid mycorrhizas, and jungermannioid mycorrhizas, are distributed over two subgroups. One group contains species with macroscopically visible basidiomes, whereas members of the other group probably lack basidiomes. Sebacina appears to be polyphyletic; current species concepts in Sebacinaceae are questionable. Sebacina vermifera sensu Warcup & Talbot consists of a broad complex of species possibly including mycobionts of jungermannioid and ericoid mycorrhizas.

This wide spectrum of mycorrhizal types in one fungal family is unique. Extrapolating from the known rDNA sequences in Sebacinaceae, it is evident that there is a cosm of mycorrhizal biodiversity yet to be discovered in this group. Taxonomically, we recognise the Sebacinaceae as constituting a new order, the Sebacinales.

Siderophores are low molecular weight, iron-chelating ligands produced by nearly all microorganisms. Fungi synthesize a wide range of hydroxamate siderophores. This review considers the chemical and biological aspects of these siderophores, their distribution amongst fungal genera and their possible applications. Siderophores function primarily as iron transport compounds. Expression of siderophore biosynthesis and the uptake systems is regulated by internal iron concentrations. Transport of siderophores is an energy-dependent process and is stereoselective, depending on recognition of the metal ion coordination geometry. In addition to transporting iron, siderophores have other functions and effects, including enhancing pathogenicity, acting as intracellular iron storage compounds and suppressing growth of other microorganisms. Siderophores can complex other metals apart from iron, in particular the actinides. Because of their metal-binding ability there are potential applications for siderophores in medicine, reprocessing of nuclear fuel, remediation of metal-contaminated sites and the treatment of industrial waste.

The diversity, biological activity and secondary metabolite production of fungi associated with marine sponges were investigated in order to assess the potential of these fungi for the production of novel biologically active secondary metabolites. 681 fungal strains were isolated from 16 sponge species from six different locations, representing 13 genera of Ascomycota, 2 of Zygomycota, 37 of mitosporic fungi, and 37 strains of sterile mycelium. The spectrum of fungi from each location was dominated by a few genera of mainly mitosporic fungi that are also commonly encountered in terrestrial habitats.

A high proportion of culture extracts of sponge-associated fungi was biologically active in tests to detect antifungal, antialgal, and antibacterial activity, cytotoxicity and inhibition of reverse-transcriptase and tyrosine kinase. The corresponding chemistry was structurally diverse and related to that of terrestrial fungi. From the biologically active culture extracts of five fungal strains, nine pure compounds were isolated in addition to 18 previously reported.

The taxonomy of Metarhizium has been reassessed using sequence data and RAPD patterns from 123 isolates recognised as M. anisopliae, M. flavoviride or M. album. A high level of genetic diversity was found which was best resolved at the species/variety level by sequence data from the ITS and 28S rDNA D3 regions. RAPD patterns correlated closely with the sequence data and revealed a much greater degree of diversity useful for distinguishing strains within a variety. Ten distinct clades were revealed by the cladogram based on the combined sequence data set. Several major evolutionary lines were revealed, but the taxonomic relationships at the base of the tree are poorly resolved. The data support the monopoly of the M. anisopliae group, and recognise four clades within it. Two correspond with M. anisopliae var. anisopliae and M. anisopliae var. majus. The other two are described as new varieties based on their distinctive ITS sequence data: M. anisopliae var. lepidiotum and M. anisopliae var. acridum vars nov. M. album, M. flavoviride var. flavoviride and M. flavoviride var. minus are recognised and redefined according to ITS sequence data. Three clades represent two new varieties, M. flavoviride var. novazealandicum and M. flavoviride var. pemphigum vars nov., based on their distinct ITS sequence data. The third, with two isolates, has not been named pending further data.

The nature and mechanisms of successions of fungi in soil and plant litter are discussed, and an autecological study of a basidiomycete used to illustrate some methods of approach.

We isolated over 650 yeasts over a three year period from the gut of a variety of beetles and characterized them on the basis of LSU rDNA sequences and morphological and metabolic traits. Of these, at least 200 were undescribed taxa, a number equivalent to almost 30% of all currently recognized yeast species. A Bayesian analysis of species discovery rates predicts further sampling of previously sampled habitats could easily produce another 100 species. The sampled habitat is, thereby, estimated to contain well over half as many more species as are currently known worldwide. The beetle gut yeasts occur in 45 independent lineages scattered across the yeast phylogenetic tree, often in clusters. The distribution suggests that the some of the yeasts diversified by a process of horizontal transmission in the habitats and subsequent specialization in association with insect hosts. Evidence of specialization comes from consistent associations over time and broad geographical ranges of certain yeast and beetle species. The discovery of high yeast diversity in a previously unexplored habitat is a first step toward investigating the basis of the interactions and their impact in relation to ecology and evolution.

In 1993 a destructive new Phytophthora pathogen of riparian Alnus trees was discovered in the UK and subsequently shown to be present in other parts of Europe. The new Phytophthora comprised a group of emergent heteroploid hybrids, probably between P. cambivora and a species related to P. fragariae. These included a common, near tetraploid standard hybrid, the presumptive allopolyploid; and four scarcer major variant types with chromosome numbers intermediate between diploid and tetraploid, named the Swedish, Dutch, German and UK variants. The standard hybrid type is formally designated here as Phytophthora alni subsp. alni. The Swedish variant is designated as P. alni subsp. uniformis; and the Dutch, German and UK variants collectively as P. alni subsp. multiformis. The properties of the Dutch, German and UK variants within subsp. multiformis are informally described. The problems of designating emergent species hybrids under the International Code of Botanical Nomenclature and the reasons for the taxonomic choices made are discussed.

The internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal RNA gene repeat from Phytophthora species were amplified using the polymerase chain reaction and sequenced. Sequences from P. cambivora, P. cinnamomi, P. citricola, P. cryptogea, P. drechsleri, P. fragariae var. fragariae, P. fragariae var. rubi, P. megasperma var. megasperma and P. nicotianae were compared with published sequences and phylogenetic trees were produced. The resultant grouping of species generally agreed with groupings established using classical morphological criteria, primarily sporangial morphology. Amongst species with non-papillate sporangia two clades were evident, one consisting of P. fragariae, P. cambivora and P. cinnamomi and the other of P. megasperma, P. drechsleri and P. cryptogea. The latter three were placed in the tree between the non-papillate groups and the papillate and semi-papillate groups which formed three distinct clades. One group comprised P. citricola, P. citrophthora and P. capsici, another P. megakarya and P. palmivora and a third P. pseudotsugae, P. cactorum, P. idaei, P. nicotianae and P. infestans. More isolates of P. megasperma, P. drechsleri and P. cryptogea will need to be examined to settle more precisely the relationship of these species to the others. PCR amplification of ITS sequences using freeze-thawed mycelial scrapings from pure cultures growing on agar followed by digestion with restriction enzymes is a quick and easy way to compare and identify isolates without the need for laborious DNA extraction procedures. With improved technology, rapid automatic sequencing of PCR-amplified ITS regions is now possible and yields detailed information of relationships within the genus as well as allowing the design of species-specific PCR primers for diagnostic purposes.

Homobasidiomycetes display a variety of fruit body morphologies. Examples include gilled mushrooms, coral fungi, polypores and puffballs but also species with simple crust-like basidiomata, usually called corticioid fungi. The latter group has largely been neglected in recent studies of homobasidiomycete evolution. The major goal of the present study was to explore the impact that the addition of a wide selection of species with crust-like basidiomata would have on homobasidiomycete phylogeny. Two genes, 5.8S and 28S in the nuclear rDNA repeats, were sequenced and a data set with 178 taxa analysed using neighbour-joining and maximum parsimony methods. Support for clades was evaluated by bootstrap. Basal nodes generally received weak support and branching order for major clades remained largely unresolved. Twelve major groups were recovered and corticioid fungi make up a major or important constituent in most of them. Nine groups are strongly supported but support for euagarics and polyporoid clades is poor. Phlebioid fungi were in earlier studies merged with the polyporoid clade but are here identified as a separate clade. Athelia is allied with ectomycorrhizal genera, inter alia Piloderma and Amphinema, in a separate clade forming a sister group to the boletes. We conclude that corticioid fungi hold a considerable share of the phylogenetic diversity displayed by homobasidiomycetes, and should always be considered when phylogenetic studies of larger basidiomycetes are designed.

Fungal endophytes have been found in every plant species examined to date and appear to be important, but largely unquantified, components of fungal biodiversity. Endophytes are especially little known in tropical forest trees, where their abundance and diversity are thought to be greatest. Here, we explore the occurrence of endophytes in a broad diversity of woody, angiospermous taxa in a lowland, moist tropical forest in central Panamá. We use similarity indices to assess host preference and spatial heterogeneity of endophytes associated with two co-occurring, but distantly related, understorey tree species in two sites of that forest, and assess the utility of indices based on frequencies of morphospecies occurrence (Morisita-Horn index) and on presence-absence data (Sørensen’s index). We suggest that our understanding of fungal diversity will be enhanced by exploring ecological patterns underlying endophyte occurrence in host species, and discuss methods for assessing the proportion of fungal biodiversity represented by tropical endophytes.

Leptographium wingfieldii is a well-known fungal associate of the pine shoot beetle, Tomicus piniperda, in Europe. This fungus is pathogenic to pines and is an important cause of blue-stain in the sapwood of infested trees. Tomicus piniperda was first found in a Christmas tree plantation in Ohio, USA, 1992, but isolation of the fungi associated with these intercepted insects was not attempted. Fungal strains resembling L. wingfieldii were recently isolated from pines attacked by T. piniperda, Dendroctonus valens and Ips pini in the northeastern United States. These strains were morphologically similar to the ex-type and other reference strains of L. wingfieldii. Strains were also compared based on sequences of the partial ITS ribosomal DNA operon, β-tubulin and elongation factor 1-alpha (EF-1α) genes. Based on these DNA sequence comparisons, reference strains of European L. wingfieldii were conspecific with North American strains from pines attacked by T. piniperda, D. valens and I. pini. A single strain from Canada, collected in 1993 near the Ontario border with the USA, shortly after the discovery of T. piniperda in that area and tentatively identified as L. wingfieldii, was also included in this study. Its identification was confirmed, suggesting that L. wingfieldii has been present in this region and probably over the whole range of the insect's distribution for at least a decade. This represents the first record of L. wingfieldii associated with the introduced and damaging pine shoot beetle T. piniperda in North America. It shows that the fungus is well established and can become associated with other native bark beetles that attack stressed and/or dying trees. The occurrence and spread of this highly pathogenic fungus associated with North American bark beetles should be monitored.

The development of a transformation system is a significant technical hurdle in the study of many filamentous fungi. Recently Agrobacterium tumefaciens was shown to transform several filamentous fungi, but until now only one independent confirmation of this promising finding has been published. Fusarium circinatum (teleomorph Gibberella circinata) is an important pathogen of pine that has not been transformed previously. A. tumefaciens strain AGL1(pPK2) transformed three different isolates of F. circinatum with an efficiency of 2–150 transformants 10−5 conidia. The T-DNA was integrated into the genome and was stable through mitotic and meiotic cell divisions. Most F. circinatum transformants contained a single T-DNA copy. Half of the tested transformants also contained non-T-DNA vector sequences. These findings should facilitate future analysis of F. circinatum pathogenicity and stimulate wider use of this valuable transformation method in fungal research.

The phylogeny of Trichoderma and the phylogenetic relationships of its species was investigated by maximum parsimony analysis and distance analysis of DNA sequences from multiple genetic loci. 18S rDNA sequence analysis suggests that the genus Trichoderma evolved at the same time as Hypomyces and Fusarium and thus about 110 Myr ago. 28S rDNA sequence analysis shows that the genus Trichoderma is part of a monophyletic branch within the Hypocreaceae, which also includes Arachnocrea and Aphysiostroma in basal positions. Gene trees inferred from a combined analysis of the nuclear ribosomal internal transcribed spacer (ITS1 and 2), the D1 and D2 region of the 28S rDNA, the small subunit of the mitochondrial rDNA (mitSSU), the fifth and part of the sixth exon of translation elongation factor 2 (tef1), and a fragment of ech42 provide strong statistical support for a phylogeny consistent with the existence of four clades: clade A comprises species of Bissett's (1991) sect. Trichoderma but also T. hamatum, T. pubescens, T. asperellum, and T. strigosum; clade C comprises all the species contained in section Longibrachiatum as revised by Samuels et al. (1998), and clade D contains only T. aureoviride which is genetically most distant to all other species. Clade B, on the other hand, contains a large and taxonomically heterogeneous mixture of species, among which several subclades could be identified: subclade B1 containing H. lactea, H. citrina, H. citrina var. americana, H. lutea, and an unnamed T. sp. 1; subclade B2 containing T. stromaticum, and an unnamed T. sp. PPRI3559; subclade B3 containing T. fertile, T. oblongisporum, and H. hunua; subclade B5 containing T. polysporum, T. croceum, Hypocrea pilulifera, and T. minutisporum; and a larger subclade (B4), in which three strain clusters could be distinguished: one comprising T. harzianum, T. inhamatum, H. vinosa, and T. aggressivum, another one containing T. fasciculatum, T. longipile, and T. strictipile; and a third containing T. virens, T. flavofuscum, and T. crassum. The position of the remaining species of subclade B4 (T. spirale, T. cfr aureoviride, H. tawa, and T. tomentosum) was not resolved. A comparison of the topologies of the individual gene trees was concordant with the topology of the combined tree in most cases, but also revealed incongruent positions for a few species (T. oblongisporum, T. longipile, T. fasciculatum) which was most pronounced in the ITS1 and 2 tree. The results confirm the recent concept for sects Longibrachiatum and Trichoderma, indicate that the sects Hypocreanum and Pachybasium cannot be distinguished phylogenetically, and provide a first phylogenetic basis for dissection of the latter two sections.