The aim of this study was to clarify the reproductive mode in the wood-rotting fungus Phellinus nigrolimitatus, using molecular markers and pairing experiments of cultural isolates. A 511 bp segment of the translation elongation factor 1a (EF1a) gene and the internal and intergenic transcribed spacer sequences (ITS and IGS) of the nuclear ribosomal DNA (nrDNA), were amplified by PCR from somatic cultures derived directly from basidiomes and single spore cultures of the fungus. Sequence analyses of the partial EF1a sequence (511 bp) and ITS (732 bp) of single spore cultures gave five and fourteen variable sites, respectively. Five somatic isolates yielded double ITS sequences, which suggested the existence of different ITS types within the same isolate. Restriction digestion of the partial EF1a and the complete ITS and IGS (3·4±0·2 kbp) sequences with HinfI and HaeIII gave restriction fragment length polymorphisms (RFLPs) among the isolates. The sum of fragments exceeded the lengths of the initial PCR product in several somatic isolates, which indicated the existence of EF1a and nrDNA heterotypes within isolates. Segregation of ITS and IGS RFLPs in single spore isolates suggested heterokaryosis in somatic isolates of P. nigrolimitatus. The polymorphic EF1a and ITS restriction sites were used as codominant markers in a test for Hardy–Weinberg expectations. The molecular data suggests a predominant outcrossing reproductive mode in P. nigrolimitatus. Results from cultural pairing experiments with single spore isolates corresponded poorly with the molecular data, suggesting that laboratory pairing experiments do not necessarily provide reliable information of the mode of reproduction in natural populations of fungi.

Lichens are typically formed by the association of two partners. The fungus (mycobiont) usually comprises most of the lichen, while the photosynthetic algal or cyanobacterial partner is referred to as the photobiont. Many lichens contain more than one photobiont and other associates, the lichenicolous fungi, may also be involved. The spectrum of interactions is fascinating and ranges from mutualism, through commensalism, to situations where one, or sometimes more, of the associates is doomed by the antagonistic activities of an invading fungus. Furthermore, these interactions are dynamic over time. Many mycologists assume that once a lichen ascospore has contacted a suitable photobiont, or once a dual propagule such as a soredium, isidium or lichen fragment has landed on an appropriate surface, a lichen thallus will develop. Accepted dogma is that the thallus will grow to form a mature lichen surviving for tens, if not hundreds or even thousands of years. These are over-simplifications; in fact there is war in the world of lichens. Following a brief consideration of parasitism and other forms of symbiosis, this war will be illustrated under the headings of algal slaves, exploiters of two kingdoms, alien invaders, cosy niche seekers and take-over specialists. It is hoped that this review will encourage mycologists to study these interactions which, as yet, remain largely unexplored.

A data set with 1551 fungal sequences of the small subunit ribosomal RNA has been analysed phylogenetically. Four animal sequences were used to root the tree. The parsimony ratchet algorithm in combination with tree fusion was used to find most parsimonious trees and the parsimony jackknifing method was used to establish support frequencies. The full-length consensus tree, of the most parsimonious trees, is published and jackknife frequencies above 50% are plotted on the consensus tree at supported nodes. Until recently attempts to find the most parsimonious trees for large data sets were impractical, given current computational limitations. The parsimony ratchet in combination with tree fusion was found to be a very efficient method of rapid parsimony analysis of this large data set. Parsimony jackknifing is a very fast and efficient method for establishing group support. The results show that the Glomeromycota are the sister group to a monophyletic Dikaryomycota. The majority of the species in the Glomeromycota/Dikaryomycota group have a symbiotic lifestyle – a possible synapomorphy for a group ‘Symbiomycota’. This would suggest that symbiosis between fungi and green plants evolved prior to the colonization of land by plants and not as a result of the colonization process. The Basidiomycotina and the Ascomycotina are both supported as monophyletic. The Urediniomycetes is the sister group to the rest of the Basidiomycotina successively followed in a grade by Ustilaginomycetes, Tremellomycetes, Dacrymycetales, Ceratobasidiales and Homobasidiomycetes each supported as monophyletic except the Homobasidiomycetes which are left unsupported. The ascomycete node begins with a polytomy consisting of the Pneumocystidomycetes, Schizosaccharomycetes, unsupported group with the Taphrinomycetes and Neolectales, and finally an unnamed, monophyletic and supported group including the Saccharomycetes and Euascomycetes. Within the Euascomycetes the inoperculate euascomycetes (Inoperculata) are supported as monophyletic excluding the Orbiliomycetes which are included in an unsupported operculate, pezizalean sister group together with Helvellaceae, Morchellaceae, Tuberaceae and others. Geoglossum is the sister group to the rest of the inoperculate euascomycetes. The Sordariomycetes, Dothideomycetes, Chaetothyriomycetes and Eurotiomycetes are each highly supported as monophyletic. The Leotiomycetes and the Lecanoromycetes both appear in the consensus of the most parsimonious trees but neither taxon receives any jackknife support.

Catalase and ascorbate peroxidase enzymatic activities were examined during the interaction between Nicotiana tabacum and the arbuscular mycorrhizal Glomus mosseae. Transient enhancements of both enzymatic activities were detected in the inoculated plant roots coinciding in time with the stage of appressoria formation in the root surface. The analysis of free salicylic acid content in roots revealed that the increases in enzymatic activities were coincident in time with the accumulation of SA in inoculated roots. These data indicate that the first reaction of the root cells to the invasion of arbuscular mycorrhizal fungi is a defence response.

About 35 Trichoderma species are currently recognised on the basis of morphological and molecular characters. Besides the role of a few of these species in biotechnology, several seem to play prominent roles in soil ecosystems. With a goal of investigating global biodiversity in Trichoderma, we report on the occurrence of Trichoderma spp. in Russia (Moscow and Ural areas), Siberia (Krasnoyarsk area) and the Himalayan mountains – areas from which no Trichoderma isolates are so far available. The ITS 1 and 2 sequence of the rDNA cluster of the 75 isolates obtained was compared with that of ex-type strains and taxonomically established isolates of Trichoderma. Thirty-nine isolates were positively identified as T. atroviride, T. virens, T. hamatum, T. asperellum, T. koningii and T. oblongisporum. A further 26 isolates yielded six closely related ITS1/2 sequence types, which are highly similar yet different from the ex-(neo)type strains of T. harzianum and T. inhamatum. Some of these genotypes (i.e. 1 and 2a) occurred only in Russia/Siberia, whereas others (2b, 3, 4 and 5) were found only in the Himalayas. RAPD analysis was consistent with these genotypes, and revealed genetic homogeneity even between strains from widely separated areas. Parsimony analysis placed these five genotypes, together with T. harzianum, T. inhamatum and the mushroom-aggressive T. harzianum ‘biotype 2’ in a large, unresolved ‘harzianum’ clade. Ten isolates were not safely alignable within known species, and five of them may be undescribed taxa: one isolate from 2700 m elevation in the Himalayas, which clustered in parsimony analysis at a basal position in section Longibrachiatum; and four isolates, displaying two closely related sequence types, forming a separate clade with T. stromaticum. The five remaining isolates also exhibited three unique ITS1 and 2 sequence patterns, but parsimony analysis placed them into the unresolved ‘harzianum’ clade, and their relationship to T. harzianum is thus unclear. The study shows that molecular screening of uninvestigated geographic areas can lead to the identification of isolates with new ITS1 and ITS2 sequence patterns, some of which may be new taxa. It also reveals that T. harzianum is at present the genetically most diverse member of the genus.

Sudden oak death has been an emerging disease problem in coastal California and has caused significant losses in forest ecosystems in some regions of the state. The causal agent of this disease has been described as Phytophthora ramorum with two other less aggressive species, P. nemorosa and P. pseudosyringae, recovered from some symptomatic plants. The phylogenetic relationship of these species with other members of the genus was examined by sequence alignment of 667 bp of the mitochondrially-encoded cytochrome oxidase II gene and the nuclear encoded rDNA internal transcribed spacer region. P. ramorum was most closely related to P. hibernalis and P. lateralis in trees from both regions, although the specific relationship among species differed depending on the tree. In the cox II tree these species were on a single clade with P. lateralis basal to a group containing P. ramorum and P. hibernalis. On the maximum parsimony ITS tree P. ramorum was most closely affiliated with P. lateralis and in the same clade as P. hibernalis, but with maximum likelihood analysis P. ramorum was basal to a grouping of P. hibernalis and P. lateralis. While bootstrap support was strong for the grouping of these species together, it was not for determining the relationship among them. In contrast to the cox II tree, the clade containing these three species grouped with P. cryptogea, P. drechsleri, P. erythroseptica, and P. syringae in the ITS tree. Since the same isolates of these species were used for both the cox II and ITS sequence analysis, this difference in species grouping suggests either a differential rate of evolutionary divergence for these two regions, incorrect assumptions about alignment of ITS sequences, or different evolutionary histories of the regions under study. Analysis of combined cox II and ITS data sets gave trees where the relationships among these species were the same as for the ITS tree alone, although the results of the partition homogeneity test (P=0.072) suggest caution should be used in interpretation of this data. All analyses supported a close relationship between P. ilicis, P. nemorosa and P. pseudosyringae, although the analysis did not clarify the evolutionary relationships among these three species. Interestingly, these three species had a unique 6 bp deletion in the cox II gene just before the termination codon. While there was some similarity in phylogenetic grouping of these species and morphological characteristics, this was not consistent across all comparisons in the genus. Data would suggest that P. ramorum, P. nemorosa and P. pseudosyringae are phylogenetically distinct new species and not the result of interspecific hybridization.

Traditional taxonomy of aquatic hyphomycetes has been based on conidial morphology and development. Since the predominantly tetraradiate and sigmoid forms are due to convergent evolution, they are often phylogenetically non-informative. The comparison of nuclear small-subunit ribosomal DNA sequences of 30 species (22 new, eight previously published) assigned 22 to Leotiomycetes, four to Dothideomycetes, three to Sordariomycetes, and one to Orbiliomycetes. Eight species of Anguillospora were distributed among the Leotiomycetes, Dothideomycetes, and Orbiliomycetes. All three anamorphs connected with Massarina were assigned to the Pleosporales, however, Clavariopsis aquatica and Tumularia aquatica separated from Anguillospora longissima. The nSSU rDNA sequences of several species were identical (e.g. Anguillospora crassa and A. furtiva), suggesting the need to include less conservative genes for resolving such differences.

Genetic variability within 69 biocontrol isolates of Trichoderma, obtained from different geographical locations and culture collections and selected as biocontrol agents, was studied. Sequence data, obtained from the ITS1 region of rDNA and a fragment of the translation elongation factor 1 (tef1) gene, were used in a phylogenetic analysis. Phylograms showing similar topologies were generated using alignments containing the ITS1 region or a portion of the tef1 gene. 21 distinct ITS1 sequence types and 17 distinct tef1 sequence types were identified among the 69 isolates. More than 50% of the potential biocontrol strains were grouped within Trichoderma sect. Pachybasium; of these, 81% were grouped within the cluster that included the ex-type strains of T. harzianum and T. inhamatum, and 16% were grouped with T. virens. Within T. sect. Trichoderma, which included 36% of the 69 strains, 56% were grouped with T. asperellum, and 24% with T. viride, T. atroviride or T. koningii. Only 10% of the strains studied were located in T. sect. Longibrachiatum.

The diversity and vertical transmission of double-stranded (ds) RNA in Helicobasidium mompa and Rosellinia necatrix was examined by electrophoresis and Northern hybridization. These two fungi share the similar niche as root rot pathogens of trees in forests and orchards, and had diverse dsRNAs. The detection frequency of dsRNA in both fungi was different; in H. mompa, 68.4% (132 out of 193 MCGs; mycelial compatibility groups) had dsRNA, whereas 20.9% (53 out of 254 MCGs) in R. necatrix. dsRNA banding patterns and Northern blot analyses revealed the presence of various dsRNA elements in both fungi. Hyphal tip isolation was mostly unsuccessful to remove dsRNA with some exceptions. Sexual reproduction functioned to remove dsRNA in both fungi since dsRNA was not detected from single sexual spore cultures. Possible explanations for the difference in the detection frequency of dsRNA are discussed in terms of the differences in their sexual reproduction and other factors.

Accurate identification of small-spored Alternaria spp. is challenging because of morphological plasticity under non-standard conditions and the common misapplication of the name A. alternata to a variety of morphologically distinct taxa. A system used by some authors of naming phytotoxigenic alternarias as pathotypes of A. alternata has further clouded the meaning and usefulness of this specific epithet. Two hundred and sixty isolates of small-spored Alternaria, primarily from fruit substrates, were segregated into morphological groups and then subjected to RAPD-PCR analysis using total genomic DNA and three different primers. When cultured under defined conditions, the pattern of sporulation observed at 50 × magnification was predictive of genetic relatedness as determined by cluster analysis of RAPD fragment patterns. In RAPD analyses, morphological groups or species were resolved as distinct branches of the dendrogram: Alternaria gaisen (= A. kikuchiana, A. alternata Japanese pear pathotype, group 2), A. longipes (= A. alternata tobacco pathotype, group 5), the ‘tenuissima’ group (group 5), the arborescens group (group 3) and the ‘infectoria’ group (group 6). Isolates in groups for which there were only a few representatives clustered as a branch. Analysis of RAPD fragment patterns confirmed that when Alternaria isolates are cultured and observed under defined conditions, their phenotypic plasticity is minimized and valid taxonomic separations can be made upon morphological characteristics. We conclude that A. alternata, A. gaisen, A. longipes and other small-spored saprotrophic or perthotrophic taxa are recognizable as morphologically distinct taxa. We suggest that the ‘pathotype’ system of naming small-spored Alternaria taxa confers no predictive value relative to observable morphological and genetic characters, and should be abandoned.

Xylaria species are common endophytes in tropical plants. It is not known, however, whether transmission of Xylaria occurs horizontally or vertically, whether individual Xylaria strains have wide host ranges or are host-specific, or how they are dispersed. We compared frequency of Xylaria endophytes in leaves and seeds of two tree species in Puerto Rico, Casuarina equisetifolia (Australian pine) and Manilkara bidentata (ausubo). These trees were chosen because they differ markedly in morphology, habitat, distribution, and origin. In C. equisetifolia Xylaria was significantly more frequent in leaves than in seeds. Xylaria was isolated from seeds of trees in inland parks, but never from seeds of trees growing on beaches. This suggests that vertical transmission of Xylaria may be possible but is not necessary for infection. In M. bidentata, Xylaria was isolated from 97% of leaves but was never isolated from seeds, suggesting that transmission is entirely horizontal. Seedlings raised in a greenhouse far from other M. bidentata trees had a level of Xylaria infection as high as seedlings in the forest, suggesting that inocula can come from other sources and endophytic strains are not host-specific.

The widespread occurrence of vegetative incompatibility in fungi and the high level of incompatibility gene polymorphisms in fungal populations means that generally parents in a cross will be vegetatively incompatible. In Neurospora crassa the mating type locus even functions as a vegetative incompatibility locus assuring vegetative incompatibility in a cross. In this paper we have tested the effect of vegetative incompatibility on regulating transmission of mitochondrial plasmids and nuclear genes in sexual interactions of N. crassa. In the absence of mating type vegetative incompatibility between the parents, transmission of plasmids from the conidial paternal parent was found to be approximately ten times higher than in normal crosses. Control experiments in which conidia of a contaminating plasmid bearing strain of similar mating type as (and fully vegetatively compatible with) the established maternal culture were added together with the presumed paternal conidia (opposite mating type) showed plasmid transmission to ascospores. Since trichogynes do not fuse with conidia of the same mating type, it may be concluded that plasmids from the contaminating strain entered the maternal tissue by somatic fusion. The fate of conidial nuclei in such sexual interactions has been investigated as well, both under conditions of vegetative compatibility and incompatibility between the strains. These experiments demonstrated that conidia that are vegetatively compatible to the established protoperithecial strain manage to get access to the resources of the maternal culture and initiate new fruiting bodies. This type of nuclear parasitism was never observed when vegetatively incompatible conidia were used. We propose that vegetative incompatibility may function in sexual crosses to protect unfertilised cultures from looting of maternal resources.

The 18S rDNA data base for mainstream Peronosporomycetes has been substantially increased. The sub-class divide between the Peronosporomycetidae and the Saprolegniomycetidae is supported. The deep clade separation of the Leptomitales within the Peronosporomycetidae is reinforced and support is provided for divergences within the Phytophthora and Pythium lines. A new family for the Leptolegnia lineage is proposed from the Saprolegniaceae sensu lato.

Culture media influenced the germination of conidia, appressorial development and mycelial growth of Metarhizium anisopliae. Although the addition of KCl to Sabouraud dextrose agar (SDAM) significantly reduced germination of the conidia of three isolates in vitro and lowered germination and appressorial development on the cuticles of Myzus persicae and Meligethes aeneus, conidia grown on SDAM and minimal medium (MM) were more aggressive than conidia derived from SDA or yeast extract agar. No relationship was found between either germination or appressorial development and LT50 values, suggesting that virulence depends on the speed of infection. Calcofluor staining and lectin binding showed that culture conditions also influenced the adhesion of Metarhizium anisopliae to insect cuticles through alteration of surface carbohydrates such as 1-4-β-glucans. Dark, pigmented conidia from MM bound less lectins and calcofluor whereas the paler conidia from the other media fluoresced more intensely. Conidia from SDAM fluoresced 20-fold brighter and adhered more readily to insect cuticle compared with conidia derived from MM. Although inocula from both media showed elevated pathogenic activity against aphid and pollen beetle, MM-grown conidia were more aggressive since high mortality was induced by comparatively fewer conidia. When fluorescent intensity of calcofluor-stained conidia was compared to the number of conidia adhering to the insect cuticles, the relationship was significant, indicating that this dye has practical implications for the assessment of fungal strains in screening programmes for commercial development.

The hydrophobin SC4 was isolated from the medium of a dikaryon from Schizophyllum commune with disrupted SC3 genes. Although not glycosylated, its biophysical properties were similar to those of SC3. As the hydrophobins SC3 from S. commune and ABH1 and ABH3 from Agaricus bisporus, SC4 self-assembled at hydrophilic-hydrophobic interfaces into an SDS insoluble amphipathic film with a typical rodlet structure at its hydrophobic face, and also proved to be a powerful surfactant. Similar rodlet structures were observed in the fruiting body plectenchyma. By immunodetection SC4 could be localized lining air channels within this tissue. A similar localization was found for the ABH1 hydrophobin in fruiting bodies of A. bisporus. Probably, these hydrophobin coatings prevent collapse of air channels allowing efficient gas exchange even under wet conditions.

The colonisation intensity and composition of the mycorrhizal community in the aquatic plants Lobelia dortmanna and Littorella uniflora were studied. The mycorrhizal fungi were characterised by fungal specific nested PCR and sequencing using the 5′-end of the LSU rDNA as target. For this, primers for the clade of Acaulospora, the clade including Glomus mosseae and G. intraradices and the clade containing G. etunicatum and G. claroideum were used.

The nested PCR products were screened for different sequence types using single stranded conformation polymorphism (SSCP) and representatives for each type were sequenced. A phylogenetic analysis of the sequences showed two phylotypes of Acaulospora, one phylotype within the clade of G. etunicatum/G. claroideum and five within the G. mosseae/G. intraradices clade. The colonisation intensity was comparable to that seen in typical grassland vegetation. The neutral lipid fatty acid 16[ratio ]1ω5 was seen to be indicative of mycorrhizal colonisation with concentrations up to 35 nmol mg−1 root DW, which indicates that the fungi are active.

Phialocephala fortinii is a common root endophytic fungus with a wide geographic distribution and little, if any, host specificity. Little is known about its habitat specificity, although there is evidence to suggest that high water tables may restrict the occurrence of P. fortinii in wetlands. We tested this hypothesis by determining the distribution of P. fortinii along a sand dune – wetland complex. Isolates of P. fortinii, identified on the basis of cultural and morphological characteristics, were obtained from the roots of vascular plants across the moisture gradient. Three ‘cultural groups’ were recognized among these isolates. Thirty-three of these isolates were compared among themselves and to strains of known identity using PCR/RFLP analysis of the ITS region and a portion of the 28S subunit of rDNA. The restriction digest profiles of all isolates were identical to those of P. fortinii for 4 restriction enzymes. DNA sequences, from a subset of these strains, showed a low percent sequence divergence confirming the reliability of the RFLP data. The same analyses were done with two strains of Leptodontidium orchidicola, a culturally similar root endophyte, to ensure that this taxon was not among the transect isolates. DNA data showed a clear difference between P. fortinii and L. orchidicola but did not discriminate among cultural groups. Thus, P. fortinii showed no habitat specificity and occurred in both xeric and hydric sites. RFLP profiles and ITS sequences showed little variation among isolates of P. fortinii and among the isolates of L. orchidicola.

Rhizophlyctis rosea was found in 44% of 59 soil samples from national parks, urban reserves and gardens, and agricultural lands of eastern New South Wales, Australia. As some of the soils are periodically dry and hot, we examined possible mechanisms that enable survival in stressful environments such as agricultural lands. Air-dried thalli of R. rosea in soil and pure cultures of R. rosea, two isolates of Allomyces anomalus, one isolate of Catenaria sp., one of Catenophlyctis sp. and one of Spizellomyces sp. recovered following incubation at 90 °C for two days. Powellomyces sp. recovered following incubation at 80 °. Sporangia of all seven fungi shrank during air-drying, and immediately returned to turgidity when rehydrated. Some sporangia of R. rosea released zoospores immediately upon rehydration. These data indicate that some Chytridiomycota have resistant structures that enable survival through periodic drying and high summer temperatures typical of soils used for cropping. Eleven Chytridiomycota isolated from soil did not survive either drying or heat. Neither habitat of the fungus nor morphological type correlated with the capacity to tolerate drying and heat.

Strains of Colletotrichum species derived from diseased strawberry plants from a wide geographical range were studied using mitochondrial and ribosomal DNA RFLPs, and acetyl and propionyl esterase isoenzymes. Two major species aggregates were detected, centred on C. acutatum and C. gloeosporioides respectively, with significant further subdivision. There were apparent discrepancies in the hierarchical nesting of some taxon groups based on the different molecular techniques. Strains assigned to C. acutatum fell into several rDNA RFLP groups, but there was less variation in mtDNA RFLP band patterns. There appears to be at least one probably clonal population in the U.S.A. which is also present in Europe, and a less well-defined series of groups which are at least sometimes sexually reproducing. Strains assigned to C. fragariae were found not to have distinct rDNA band patterns from the teleomorph linked strains studied, which had been referred to as C. gloeosporioides. They did vary in this respect from C. gloeosporioides associated with Citrus, its type host. It was, therefore, concluded that all the strains studied with cylindrical conidia should be placed within C. fragariae, which is confirmed as separate from C. gloeosporioides and recognized as a holomorphic taxon. Nevertheless, a separate asexually reproducing infraspecific group was distinguishable using mtDNA information. An epitype is designated for C. fragariae.

Black mould lesions were caused by Alternaria alternata in 76% of 228 tomato fruit with characteristic sunken black lesions collected from fields of processing tomatoes in California. Analysis of 29 RAPD primers revealed a high level of genetic diversity among the 69 isolates tested. Two major phenetic groups (Group 1 with 55 isolates and Group 2 with 14) were identified independently by PCA and by UPGMA of Jaccard similarity coefficients. Only 34 of 137 RAPD markers were monomorphic for all isolates and the genetic similarity between the two groups was 50%. Co-infection of black mould lesions by genetically distinct strains of A. alternata occurred in two of 10 isolates tested. There was no evidence for geographic clustering of isolates with high levels of genetic similarity, suggesting that isolates are widely dispersed across California. Only one isolate was identified which also caused stem canker disease on a susceptible tomato cv., suggesting that these strains play a minor role in causing black mould on processing tomatoes in California. This isolate and two other known stem canker isolates were clustered together with 11 other isolates in Group 2. Group 2-specific bands were also identified in a survey of seven additional isolates known to produce host-specific toxins.