A molecular phylogeny of the Lecanora rupicola group is presented, based on ITS sequence analyses. The study includes saxicolous and corticolous members of the Lecanora rupicola group as well as other Lecanora species with pruinose apothecia. A phylogenetic hypothesis for species in Lecanora s. lat. and various other genera in Lecanoraceae, based on an alignment-free distance estimation technique, shows that the Lecanora rupicola group forms a monophyletic clade within Lecanoraceae. Affinities to the core group of Lecanora are not well supported, likewise the monophyly of Lecanora s. str. with other species groups in Lecanora, such as the lobate taxa (and Rhizoplaca) is not supported. A more detailed analysis involving Lecanora species with pruinose apothecial discs was carried out with model-based Bayesian Markov chain Monte Carlo (B/MCMC) tree sampling. The results suggest the monophyly of the Lecanora species that are characterized by the presence of chromones. Corticolous as well as saxicolous species are included. Lepraria flavescens is closely related to the Lecanora swartzii subgroup, and the new name Lecanora rouxii nom. nov. is introduced for that species. Other Lecanora species with pruinose discs are not closely related to the Lecanora rupicola group.

Thirty-one clonal isolates of U. necator were taken from diseased grapes from three different vineyards in France and Germany. Samples were collected in April from typical ‘flagshoot’ symptoms, and in July and August from the same plants. The mating-type and the sensitivity to the fungicide triadimenol were determined for all isolates. Techniques for obtaining and characterizing isolates are described. Two isolates in the first sampling and eight in the second were resistant to triadimenol, with resistance factors ranging from 3·4 to 11·8. All isolates in the first sampling and nine out of the 21 isolates in the second were mating-type (+), the remaining isolates were mating-type (−). Genetic variation between all isolates was assessed using the RAPD technique. Phenetic analysis based on the 364 RAPD fragments obtained revealed two very distinct groups, one group containing nine out of the 10 isolates from the first sampling, the second group containing all the remaining isolates. Isolates clustered in the first group displayed 58 RAPD fragments specific to them. These isolates did not exhibit 60 RAPD fragments present in all other isolates. Molecular and biological data suggested that isolates clustering in the two groups represent two different biotypes of U. necator, which are likely to be genetically isolated.

The phylogenetic relationships of the stem rot pathogens Sclerotium rolfsii and S. delphinii were examined, based on their rDNA ITS sequences. The ITS regions were cloned and sequenced to identify three distinct ITS types: r-1, r-2, and r-3. Two different ITS types exist within S. rolfsii and S. delphinii strains. Japanese strains and one strain of S. rolfsii from the USA contain types r-1 and r-2, whereas another strain from the USA and one from Chile have only one ITS type, r-2. S. delphinii strains have types r-1 and r-3. We discuss the implications of the common presence of ITS type r-1 for the taxonomy and evolution of this species complex.

The Amphisphaeriaceae is an important family of ascomycetes within the Xylariales. There has been, however, disagreement regarding the taxonomic placement of many genera within this family and whether it should be confined to ascomycetes producing Pestalotiopsis-like anamorphs. In this study, phylogenetic relationships among members of the Amphisphaeriaceae are investigated using partial sequences of the 28S rDNA. Molecular data provided further evidence to support the association of several coelomycetous genera with the ascomycetous Amphisphaeriaceae. Phylogenetic analyses also show that all ascomycetous genera possessing Pestalotiopsis-like anamorphs are monophyletic and confirm the anamorphic-teleomorphic connections of some. There is, however, insufficient evidence to support the restriction of Amphisphaeriaceae to genera, which produce Pestalotiopsis-like anamorphs, because the phylogenetic placement of Amphisphaeria umbrina is not fully resolved and its affinities with other members received low bootstrap support. The results also indicate that Iodosphaeria and Arecophila should be excluded from the Amphisphaeriaceae. The placement of Lanceispora in the Amphisphaeriaceae is doubtful. A broad concept of the family Amphisphaeriaceae is advocated until further data are available.

A medium-dense genetic linkage map of Lentinula edodes (shiitake) was constructed based on 203 amplified fragment length polymorphism (AFLP) markers and two mating type factors. The segregation of these markers was generated from 95 progeny of a single cross of two distantly related strains. The map consists of 11 linkage groups comprised of eight large (over 100 centimorgans (cM)) and three small (less than 100 cM) groups, and the total genetic distance of the map was 1956.7 cM. The average rate of physical size to genetic distance could be roughly estimated to be less than 18.4 kb cM−1, which is low compared to the values obtained for other filamentous fungi. Seventeen of the AFLP markers showed highly distorted segregation ratios (χ2 values >6.63; P<0.01), and many of these were mapped in LG II (6 markers) and IV (6 markers).

Fermentation processes for the biochemical reagents cerulenin and helvolic acid employ ‘Cephalosporium caerulens,’ an invalidly published designation that has been used for more than 40 years. However, its identity has never been critically examined because strains were unavailable from major culture collections. An authentic strain of ‘C. caerulens’, derived from the original strain KF-140, was recently found and compared to Sarocladium oryzae, another Acremonium-like fungus which also produces cerulenin and helvolic acid. Morphological comparisons, rDNA sequence data, and chromatography of secondary metabolites established that ‘C. caerulens’ and S. oryzae are conspecific. Sequence data from ribosomal DNA genes indicated S. oryzae belongs to the Hypocreales and is allied with members of the Ceratostomataceae, Scopinella species, Emericellopsis species and certain Acremonium-like anamorphs of uncertain familial relationships. At least two of the isolates of S. oryzae produced titres of cerulenin and helvolic acid similar to those of KF-140. This finding demonstrates that manufacture of cerulenin need not be limited to the original strain.

A new order is introduced (Jahnulales) to accommodate ascomycetes with stalked/sessile and dimorphic ascomata, hyphal stalk cells that are ca 40 μm wide, and ascospores that are unequally 2-celled with or without various types of appendages or sheaths. The order includes a single family, the Aliquandostipitaceae, and has affinities with the Pleosporales within the Dothideomycetes. Studies on freshwater ascomycetes in Egypt and Thailand yielded one new genus Patescospora, and Jahnula siamensiae sp. nov., and the new combination J. sunyatsenii (syn. Aliquandostipite sunyatsenii) is made based on molecular results.

The Lulworthiales consists of four genera: three that were removed from the Halosphaeriales, namely Lulworthia, Lindra, and Kohlmeyeriella; and Spathulospora, reassigned from the Spathulosporales. However, studies have shown that neither Lulworthia nor Lindra are monophyletic genera. This study was therefore undertaken to re-evaluate the genera of the Lulworthiales based on the SSU and LSU rDNA genes. Taxonomic revisions are proposed here for Lulworthia crassa, L. lignoarenaria, L. uniseptata and Lindra marinera: Lulworthia crassa is transferred into the genus Kohlmeyeriella; Lulwoidea gen. nov. is established for L. lignoarenaria; Lulwoana gen. nov. is established for L. uniseptata; and Lindra marinera is reduced to synonymy with L. thalassiae. Taxonomic descriptions are emended for the genus Lulworthia s. str., and for L. grandispora and Lindra thalassiae. A neotype is designated for Lulworthia grandispora.

The ability of three Australian Pisolithus species, discriminated on the basis of ITS sequence data, to utilize a range of inorganic and organic nitrogen sources was assessed in liquid axenic culture. Both intra-, and putative interspecific, variation in nitrogen source utilization was observed. Most isolates demonstrated a preference for NH4+ over NO3−, although some showed no significant preference for either inorganic source. All isolates utilized a range of amino acids. Species I isolates demonstrated a preference for acidic and/or neutral amino acids over basic acids, while species II and III isolates generally utilized amino acids poorly relative to species I. Although most isolates utilized BSA poorly, two species I isolates that had been maintained in axenic culture for > 10 y grew well on this substrate, suggesting possible changes in nitrogen utilization with extended storage in axenic culture.

Healthy-looking needles were randomly collected from Pinus mugo ssp. uncinata at two sites in the montane zone with different edaphic and microclimatic conditions and examined for the diversity of endophytic fungi. One site is on a mountain ridge, the other in a peat bog. On average, about 35–40% of the needles were colonized at both sites. Eleven species of fungi were isolated. Cenangium ferruginosum and Cyclaneusma minus dominated the endophytic mycobiota of the tree stand along the mountain ridge. C. ferruginosum and Lophodermium pinastri were the dominant species in the needles of the trees in the peat bog. C. ferruginosum and L. pinastri occurred mainly in the needle tip; C. minus occurred most frequently in the middle segment of the needles. The needle base was rarely colonized by these fungi. Endophyte colonization of trees along the mountain ridge increased significantly from the uppermost trees to the ones further down the ridge. No clear pattern of colonization was observed for trees in the peat bog except for the colonisation by L. pinastri, which showed a tendency to form infection centres. Species composition was constant within sites, but the frequency of colonization by C. minus and L. pinastri varied greatly among trees.

The development of the extraradical mycelium and auxiliary cells and spore production of Scutellospora reticulata in association with Ri T-DNA transformed carrot roots was followed under root-organ culture conditions. Extraradical mycelium development followed classical lag-exponential-plateau phases, with an additional late decline phase in number of auxiliary cells. Spore production started in parallel with a critical extraradical mycelium biomass produced, continued long after root growth ceased and during the late decline in auxiliary cells number. Isolated auxiliary cells were shown to exhibit hyphal re-growth, but not root colonization, either in situ or in vitro. These results showed that root and extraradical mycelium development were intimately associated in a sequence where both grew together during active root growth, followed during root aging by a period in which only the fungus developed. Spore production appeared dependent on a critical extraradical mycelium biomass and on the re-allocation of resources from both the intraradical mycelium and the auxiliary cells via the hyphal network.

Unlike previous reports that have shown that water soluble and volatile compounds from roots or root exudates play an important role in precolonization events during arbuscular mycorrhizal (AM) fungus-host root interactions (Bécard & Piché 1989, Giovannetti et al. 1993), the results shown here deal with particulate and viscous fractions isolated from host roots. Root caps and a slow sedimenting particulate fraction (SSPF) were rapidly isolated and separated from Ri T-DNA transformed carrot roots (D. carota) grown in liquid culture. In addition, border cells (BC) and mucilage were isolated from aseptically grown corn seedlings (Zea mays). Root caps, SSPF (composed mainly of small root cap fragments and some BCs), BCs, and mucilage all had an associated AM fungus hyphal branching stimulator. Root caps stored for 5 d at 4 °C appeared to either synthesize or slowly release the branching stimulator. Also, isolated root caps from roots grown in the absence of P contained more branch stimulating activity than those isolated from roots grown in the presence of P. Although the branching stimulation activity in particulate fractions was low compared to that of the exudate, the particulate fractions can stick to the root surface at considerable distances from the root tip. This may be significant during the infection and colonization of host roots at sites far removed from the primary location of exudation.

Phylogenetic relationships of termitophilic fungi were estimated with Bayesian as well as other phylogenetic methods from partial sequences of the nuclear encoded large subunit ribosomal DNA (nLSU-rDNA) and the mitochondrial encoded small subunit ribosomal DNA (mtSSU-rDNA). Sequences were obtained from basidiomes covering the morphological, taxonomical, and geographical span of termitophilic mushroom-forming fungi, and analysed together with sequences from termite nests and termite guts from most known genera of fungus growing termites from geographically diverse regions. Topologies of trees resulting from the combined analyses of the two ribosomal genes generally show no positive conflicts with those obtained from separate analyses. We show that termitophilic fungi constitute a strongly supported monophyletic group within lyophylloid species. The genera Sinotermitomyces and Podabrella are derived within Termitomyces, and do not form monophyletic groups. Identical sequences were frequently found among samples of basidiomes from the same continents and among fungi utilized by termites from the same continent. However, only two sequences were identical between basidiome samples and termite nest/gut samples suggesting fruiting species do not form a representative sample of termitophilic fungi. No sequences were identical between samples from Asia and Africa indicating some geographic differentiation between these continents.

Observations on cytological and ultrastructural changes in Plasmopara viticola oospores were carried out during the overwintering period. Three types of oospores were observed. Type I, characterized by a thin inner oospore wall (IOW), large lipid globules and two nuclei, was recovered only in samples collected in October. These oospores were considered to be immature. Maturation occurred during November and involved a noticeable increase in thickness of the IOW, fusion of nuclei, formation of an ooplast and break up of large lipid globules into smaller ones (type II oospores). A few oospores (type III) showed abnormal organization with very large lipid globules and less frequently discernible nuclei. IOW solubilization, dissolution of the ooplast and lipid globules and nuclear division were the first detectable events during oospore germination. Germinating oospores produce a germ tube which was terminated by a sporangium. In its young stage, the sporangium had a thick wall and an unusual multi-layered membrane. During this phase, nuclear divisions took place in the sporangium. While sporangium development progressed, the ribosome density in the cytoplasm decreased and mitochondria, initially roundish with evident cristae, became their usual tubular profile. The plasma membrane had a typical structure and storage organelles, such as finger print vacuoles and lipid globules, became more numerous in the cytoplasm. Larger vacuoles contained the flagella of differentiating zoospores.

Dry core rot of apple (DCR) and Alternaria black rot of citrus (ABR) have in the past respectively been ascribed to Alternaria alternata and A. citri. In recent years, however, it has been speculated that several other species of Alternaria could also be associated with these diseases. In an attempt to elucidate the identity of these taxa, 25 isolates associated with DCR, and 26 isolates associated with ABR were selected for molecular characterisation. Nucleotide sequences of 1116 sites including the histone gene section and the internal transcribed spacers (ITS 1 and 2) of the rRNA gene were determined for these isolates. The gene trees generated from the individual and combined data sets using maximum parsimony, maximum likelihood and neighbour-joining analysis methods distinguished five clades with strong bootstrap support, namely Alternaria sp., A. arborescens, A. infectoria, A. tenuissima, and a clade containing isolates variable in morphology, referred to as the Alternaria group. In the alignment of the combined ITS and histone data set, unique transition/transversion substitutions, as well as positional insertions and deletions were observed for each of the above clades. In addition, key sequences in the form of serial composing nucleotides in both the ITS and histone sections of the alignment were also discovered for the molecular identification of A. arborescens, A. infectoria and A. tenuissima. The final phylogeny also indicated that no host specificity existed among the species associated with these two post-harvest disease complexes. Contrary to the host specificity observed on leaf diseases of these hosts in the field, it appears that the post-harvest diseases are the result of adverse storage conditions and opportunism of different small-spored Alternaria spp.

The symbiotic phenotype of a lichen arises through the interaction and cooperation of two or more genetically unrelated partners. Ultrastructural and molecular methods were used to investigate the changes that take place during early stages of the lichenization process. The resynthesis of prethallus stages of Baeomyces rufus was studied by co-culturing under sterile conditions the isolated, axenically grown mycobiont and its green algal photobiont Elliptochloris bilobata. The lichenization process was monitored by SEM. One day after co-culture, symbionts were bound together by a newly secreted mucilage. By day 12, photobiont induced, morphological changes in the mycobiont were visible. Aerial hyphae grew around photobiont cells, showed a high frequency of branching and formed appressoria on the algal wall surface. By day 28, many photobiont cells were completely engulfed by hyphae and soredia-like clusters were observed. These morphological developments resemble lichenized structures formed in the natural lichen. cDNA-AFLP was used to investigate gene expression profiles on day 12 of co-culture. Differential gene expression patterns revealed that few genes were induced, and many fungal and algal genes seemed to be suppressed in the early stages of lichenization.

Microsporidia are intracellular parasitic microbes, which were once thought to be among early diverging eukaryotes, mainly because they lack mitochondria. However, this evolutionary scheme has been challenged by protein-based molecular phylogenetic studies, which suggest a more recent origin of the Microsporidia, which clustered Microsporidia with or within ‘true’ fungal lineages. However, most of these studies did not include sequences from basal fungal phyla (Chytridiomycota and Zygomycota), and unrepresentative taxon sampling could have resulted in misleading relationships being suggested. To further investigate the evolutionary origin of Microsporidia relative to Fungi, we performed molecular phylogenetic analyses with newly determined RPB1 (DNA-dependent RNA polymerase II largest subunit) and EF-1α (translation elongation factor I alpha) including sequences from basal fungi. Although the phylogenetic position of Microsporidia in the EF-1α tree still might be misplaced due to the unusually high rate of sequence divergence of the microsporidian EF-1α gene, the phylogenies recovered based on these two protein sequences do not provide strong evidences for a close relationship between Microsporidia and Fungi. Moreover, we have identified within the EF-1α genes a characteristic two amino acid deletion conserved in all fungal sequences currently available, whereas this deletion is absent in microsporidian sequences. These results argue against the view of Microsporidia as highly degenerate fungi, and whether Microsporidia and Fungi are sister taxa remains unresolved.

Cryphonectria cubensis is an important canker pathogen of plantation Eucalyptus spp. in tropical and sub-tropical areas of the world, including South Africa. It is best known on Eucalyptus spp., but it also occurs on Syzygium aromaticum (clove). In 1998, C. cubensis was found to cause cankers on the non-myrtaceous hosts Tibouchina urvilleana and T. lepidota in Colombia. In this study, we report on a similar canker disease that has recently been found in South Africa on T. granulosa, commonly grown as an ornamental tree. The identity of the pathogen was determined through morphological comparisons and phylogenetic analyses of ITS and β-tubulin gene sequences. The pathogenicity of the fungus was also tested on T. granulosa and E. grandis. Morphological as well as DNA sequence comparisons showed that the fungus on T. granulosa is the same as C. cubensis occurring on Eucalyptus spp. in South Africa. Pathogenicity tests on T. granulosa and E. grandis clones showed that the fungus from T. granulosa is able to cause cankers on both hosts.

Substantial polymorphism was detected between isolates from five populations of Alternaria brassicicola attacking Cakile maritima along the New South Wales coast of Australia, with a maximum of two genotypes being shared between population pairs. Of ten pair-wise population comparisons, six had no pathogen genotypes in common; only one genotype occurred five times, and most (93%) were found only once. Although an UPGMA based on Nei's measure of genetic distance separated the five populations, a cluster analysis using individual isolates failed to group them according to population, indicating significant gene flow. An analysis of molecular variance indicated ca 14% of the variation occurred between populations, representing moderate population differentiation over the spatial scale of the study. Tests of the relative contribution of clonality and sexual recombination indicated low, albeit significant levels of linkage disequilibrium in all populations. The level of linkage disequilibrium, and the high genotype diversity, provides support for the contention that a hitherto unidentified sexual stage might be a significant factor in the life-cycle of A. brassicicola.

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