Many intimate associations between different species of ectomycorrhizal fungi are inferred on the basis of the consistent co-occurrence of their fruit bodies. Suillus bovinus and Gomphidius roseus, where the latter never occurs without the former, is one example. This association was examined with PCR identification and light microscopy. S. bovinus and G. roseus were unambiguously separated on the basis of RFLPs of the PCR-amplified ITS region of ribosomal DNA. Tuberculate mycorrhizas of Pinus sylvestris sampled under fruit bodies of G. roseus and S. bovinus were investigated and the majority were identified as mixed associations involving both G. roseus and S. bovinus. Tuberculate mycorrhizas, which macroscopically resemble the ones of Suillus species, contained typical chlamydospores of G. roseus and they had haustoria where G. roseus hyphae penetrated the cortical root cells. Pine seedlings collected near the fruit bodies of the two species were mainly colonised by S. bovinus. Mycelial rhizomorphs collected under the fruit bodies of G. roseus were identified as S. bovinus, while both fungal species were present at the base of G. roseus fruit bodies. The significance of these observations and the possibility that G. roseus acts as a parasite are discussed.

Chytridiomycota were isolated into pure culture from cool temperate and warm semi-arid soils of eastern Australia. In pure culture these fungi responded variably to the range of temperatures commonly recorded in their environment. All members of the Blastocladiales, Spizellomycetales and Chytridiales grew in culture at temperatures up to 30 °C. Some isolates from the Blastocladiales and Spizellomycetales continued to grow at or above 37 °. Some isolates of the Chytridiales grew up to but not beyond 35 °. All isolates in the Chytridiales were able to resume growth at 20 ° after brief exposure to temperatures higher than the maximum growth temperature, but were killed by exposure to higher temperatures for 7 d. Because in the natural soil habitat temperature may exceed the maximum for growth it may be a limiting factor that determines the distribution of chytrids in the soil.

Cladistic analysis of nucleotide characters derived from partial 18S rDNA sequences has been used to infer phylogenetic relationships among five Candida species that exist in nature strictly as intracellular gut endocytobionts of anobiid or cerambycid beetles. Concordant with their assumed taxonomic status, all five species resolve within Saccharomycetales. For both the anobiid-derived taxa, C. ernobii, C. karawaeiwii and C. xestobii, and the cerambycid-derived taxa, C. tenuis and C. rhagii, this phylogenetic position clearly discriminates Candida yeasts from the anobiid yeast-like endocytobionts in Symbiotaphrina which resolve within filamentous ascomycetes. The analyses provide no evidence for a discrete lineage of Candida endocytobionts within Saccharomycetales. Rather, each of the anobiid symbionts and the cerambycid-derived species resolve in separate clades. These data confirm the polyphyletic origins of intracellular symbioses between ascomycetes and Coleoptera and provide another example of convergent evolution in fungal-arthropod associations. The implications of the phylogenetic data for theories on the origins of endocytobiosis are discussed.

The foliage of Pinus strobus (eastern white pine), as that of all other conifers examined, is occupied by endophytic fungi, the most frequent of which is Lophodermium nitens. The number and extent of endophytic infections and the genetic relationship of individual isolates within living needles as well as their relationship to isolates from forest floor needles is unknown. To examine these and related questions, forest floor isolates and foliar endophytes from needle segments were obtained for ribosomal DNA sequencing and randomly amplified polymorphic DNA (RAPD) analysis. Molecular and morphological data were compared and infection frequency determined as a function of position along the needle. Ribosomal DNA sequences of foliar and ascospore isolates showed high levels of genetic similarity (>97% identity) for the internal transcribed spacer region. RAPD profiles were able to distinguish ascospore siblings from non-siblings, and also revealed that many needle isolates belonged to the same genotype as adjacent neighbour isolates, as would be expected from mycelial spread within the needle. Morphotype evaluation and RAPD profiles showed similar patterns: identical morphotypes grouped together and showed little or no genetic difference under RAPD analysis. Both morphological and molecular data indicated that the majority of infections were contained within 1 mm needle segments but could extend to about 4 mm in length. Infection frequency increased along the length of the needle from the proximal (shoot) end to the distal tip, with markedly higher rates in the distal quarter. Thus, endophytic infections of L. nitens in white pine needles consist of many localized, discrete infections, originating from ascospores and differentially distributed along the length of the needle. In the course of this work, it was found that GenBank accession no. AF203470 under the name Meloderma desmaszieresii appeared not to be that species but L. nitens on the basis of the ITS sequences.

An examination of microscopic and macroscopic vegetative reactions between paired isolated of Rhizoctonia solani AG-8 revealed that pairs of isolates that give a C2 anastomosis reaction at the microscopic level always gives a ’tuft’ reaction at the macroscopic level. Similarly, any pair that gives a C3 anastomosis reaction at the microscopic level always gives a ’merge’ reaction at the macroscopic level. Thus, macroscopic vegetative (’tuft type’) reactions can be used to predict the microscopic (anastomosis) reaction (and vice versa) between paired isolate of AG-8. This pattern also seems to apply to paired isolates of AG-3, AG-7 and AG-10 but not the other AGs of R. solani. The C2 anastomosis reaction is always observed when two isolates from different zymogram groups of R. solani AG-8 are paired. When isolates from within any of the five zymogram groups comprising AG-8 are paired, either a C2 or a C3 anastomosis reaction will result. Many field isolates of AG-8, when paired, give a C3 anastomosis reaction. We propose that a group of field isolates of R. solani that give a C3 anastomosis reaction be designated a ’vegetatively compatible population’ (VCP).

The distribution of the major components of the bolete mycobiota of South East Asia is considered and suggestions made as to their possible origin, mycogeography and relationships. The final number of species of boletes in South East Asia is likely to be well in excess of the number compiled in Corner’s 1972 monograph. Molecular information is incorporated into the theme where of interest.

Conidia of the entomopathogenic Metarhizium flavoviride were harvested 8, 10, 12 or 15 d after inoculation at 25 °C and then (as conidiated rice) dried rapidly (10–12% r.h. and 17–20° for 17 h to about 15–22% moisture content) or slowly (50–60% r.h. and 27° for 5 d to about 27–32% moisture content initially). The subsequent survival of these conidia in air-dry storage at 50° with 8·1% moisture content was then assessed. Conidia longevity (assessed by the duration of storage until conidia viability was reduced to 50%, p50) was maximal when conidia were harvested 10 d after inoculation, and was much greater following slow rather than rapid drying. The substantial beneficial effect of slow desiccation to subsequent conidia survival is consistent with that detected in other propagules in anhydrous biology, and is also of considerable practical utility for the biological control of insects by entomopathogenic fungi.

The sensitivity to UV-B (290–320 nm) radiation of common phylloplane yeasts from two contrasting UV-B environments was compared in the laboratory using mixtures of white light (PAR: 400–700 nm) and UV-B radiation from artificial lamp sources. Sporidiobolus salmonicolor, Rhodotorula mucilaginosa and Cryptococcus sp., the dominant yeasts on leaves of tea (Camellia sinensis), were isolated in Sri Lanka (SL), while Sporidiobolus sp. and Bullera alba, dominant on faba bean (Vicia faba), were isolated in the U.K. Dose responses were determined separately for each yeast. UV-B reduced colony forming units (due to cell mortality or inactivation) and colony size (due to reduced multiplication) of all yeasts. The LD50 values and doses causing 50% reduction of cells per colony were higher for SL isolates than U.K. isolates. Results indicated that each yeast is somewhat vulnerable to UV-B doses representative of its natural habitat. The relative insensitivity of SL isolates was shown when SL and U.K. isolates were irradiated simultaneously with the same dose of UV-B. Of the two U.K. yeasts, B. alba was significantly more sensitive than Sporidiobolus sp. to UV-B. Except for R. mucilaginosa from SL, all yeasts demonstrated some photorepair in the presence of white light. White light provided relatively little protection for the U.K. isolate of Sporidiobolus sp. although it allowed increased colony size.

The spectral responses of Sporidiobolus sp. (U.K.) and of B. alba (U.K.) were broadly similar. Wavelengths longer than 320 nm had no measurable effect on colony forming units. However, colony survival was significantly reduced at 310 nm and all shorter wavebands. No colonies were counted at 290 nm or below.

Various fungi interact with heavy metals (HM) differently regarding their binding ability and their tolerance. Besides the ability of the fungus to form an extensive extramatrical mycelium, its binding and transport properties are the most important factors in ameliorating the effects of HM toxicity on the mycorrhizal symbiosis. The tolerance of seven ectomycorrhizal fungi to elevated lead concentrations was tested using agar cultures and the results were compared to those from liquid culture. Critical toxic concentrations, where the growth of the mycelium was inhibited to 50%, were lower in liquid cultures (around 0·2 mm) in comparison to agar cultures (0·5–2 mm). The less tolerant fungi in agar culture were Amanita muscaria and Laccaria laccata, the most tolerant Lactarius piperatus, Pisolithus tinctorius and two strains of Suillus bovinus, DB83 from a lead polluted area and DB84 from a non-polluted area. Tolerance indices, based on the dry weight of mycelia from liquid culture, differ from the tolerance indices obtained from solid culture. L. laccata growth was limited to a lesser extent than other fungi and the growth of S. bovinus DB83 was strongly stimulated at a Pb concentration of 30 μm, which could indicate lower susceptibility of this strain to low lead concentrations. The uptake, transport and release of Pb were then studied with 210Pb tracer using Conway cells with the active tracer added to the inoculated medium in the centre compartment. The uptake of Pb by P. tinctorius (1·1% of the added activity) was much lower than the uptake by L. laccata and S. bovinus DB83 (6·2 and 5·4% of the added activity respectively). The same situation was reflected in the activity of the tracer transported in the mycelia. Further, the release of transported Pb from mycelia to the outer medium differs between L. laccata and S. bovinus DB83 mycelia, reaching 45 and 10% of the transported activity, respectively, indicating stronger binding of lead to S. bovinus DB83. The results of the tracer study are discussed with respect to various experiments with ectomycorrhizal seedlings and lead.

The effects of grazing by the collembolan Folsomia candida on mycelial foraging patterns of Hypholoma fasciculare growing from beech (Fagus sylvatica) wood block inocula in trays of non-sterile soil was investigated. The wood inocula differed in size, state of decay (time for which wood has been colonized: 2 yr, 1 yr, 6 and 3 months) and nutrient status (inocula colonized on malt agar or nutrient agar). Mycelia were most luxuriant, had greater hyphal coverage and extended more rapidly from 2 yr old than younger inocula, from 4 cm3 than 1 cm3 inocula, and from inocula colonized on malt extract agar rather than on distilled water agar. Grazing dramatically reduced coverage and extension, especially in the less luxuriant systems characterized by many fine hyphae and fewer mycelial cords. Grazing by collembola often resulted in points of more rapid outgrowth as cords with a fanned margin. Results are discussed in terms of fungal foraging strategies.

In the southeastern USA, Armillaria root rot disease on peach (Prunus persica) is caused by Armillaria tabescens, and to a lesser degree by A. mellea. Recent attempts to genetically characterize A. tabescens isolates using rDNA indicated the existence of heterozygosity in diploid isolates. In order to clarify this heterozygosity, DNA from stipe and single spore cultures of A. tabescens isolate SC.MF-1.01 was characterized using RFLP and sequence analysis. Direct sequencing of rDNA amplicons from diploid stipe tissue indicated heterozygosity in the IGS1 and ITS2 regions. IGS1 copies A and B, and ITS copies A and B segregated in a 1[ratio ]1 ratio in single spore progeny, whereby IGS1 copy A always segregated with ITS copy A and IGS1 copy B always segregated with ITS copy B. The results indicate the existence of divergent haplotypes in the two nuclei of SC.MF-1.01 diploid mycelium and a single locus for rDNA tandem repeats. Additional IGS1 copies, designated IGS1 copy C, D, and E, with variable AluI restriction sites were cloned from SC.MF-1.01 diploid mycelium indicating polymorphism within ribosomal tandem repeats in A. tabescens. IGS1 copies C, D, and E were found once each in 100 clones analyzed, and therefore seemed to be rare compared to copies A (44 clones) and B (53 clones). RFLP and sequence analysis of the ribosomal IGS1 and ITS1 regions in North American A. tabescens isolates indicated that heterozygosity is common in A. tabescens diploid mycelium. A PCR-based molecular technique was developed to distinguish A. tabescens from many other North American Armillaria species, including A. mellea.

A method for the early detection and diagnosis of common bunt, caused by Tilletia tritici, has been developed. The technique is based on a nested PCR amplification of DNA from T. tritici in infected plant inflorescence tissue at the stage of internode elongation. Primers amplifying a 212 bp fragment in T. tritici were designed on the basis of sequences in the ITS2 region in the ribosomal RNA genes. Disease diagnosis obtained with nested PCR amplification correlated well with the conventional disease diagnosis, which involves manual observation of disease symptoms at maturity. With the present method, wheat varieties can be screened for resistance towards common bunt at the first node stage. There was no variation between the ITS2 region from T. tritici, T. laevis, T. controversa, and T. bromi and the primers are therefore not species specific. The PCR diagnosis method has only been validated for common bunt caused by T. tritici, but it is likely also to be applicable to T. laevis and T. controversa.

Extracellular matrix (ECM) surrounding conidia and germlings of B. sorokiniana was studied using light microscopy (LM), scanning (SEM) and transmission electron microscopy (TEM). Conidial ECM surrounding dry-inoculated, ungerminated conidia was fluid-like and observed only using a cryo-preparation technique, suggesting that the material was water soluble. ECM enveloping germlings appeared fibrillar in LM, TEM and SEM but amorphous in cryo-SEM, indicating that the structure of the ECM is dependent on the water content of the matrix. Fibrillar ECM formed thread-like structures that extended over long distances on the substrate or towards neighbouring conidia and hyphae. TEM of germlings negatively stained with uranyl acetate revealed the presence of fungal fimbriae. The strong resemblance between the extending organization of fibrillar thread-like ECM structures and fimbriae suggested that fimbriae constitute a basic structural component of the ECM and serve as the aggregation centre for the other ECM components. Histochemical labelling revealed significant differences between ECM surrounding the fungus at different morphological stages. The germ tube ECM was labelled for both proteins and polysaccharides whereas germling ECM consisted of two layers: an inner rich in proteins and an outer composed mainly of polysaccharides. Furthermore, the newly released ECM localized on germ tubes and hyphal tips showed affinity for microspheres carrying any type of surface properties while hyphal ECM had affinity only for negatively charged microspheres. This together suggests that ECM after its release is subjected to structural changes.

There were two successive pandemics of Dutch Elm Disease (DED) in Europe, parts of Asia and North America in the last century, caused by two ascomycete fungal species, Ophiostoma ulmi and O. novo-ulmi. A third DED species, O. himal-ulmi, was later discovered in the Himalayas. For each of these three species, we now report on the cloning and analysis of a 2.2 kb sequence containing the coding region and 5′ and 3′ flanking sequences of the mating type B (MAT-B) gene, which is involved in the control of sexual compatibility. The amino acid sequence of the single protein encoded by the gene for each species contained a conserved DNA-binding motif called the high mobility group (HMG) box which showed significant sequence similarity to corresponding sequences in many ascomycete MAT-2 genes. Phylogenetic trees constructed from the MAT-B (renamed MAT-2) nucleotide and derived amino acid sequences showed distinct clades corresponding to the three Ophiostoma species and a clear separation of the O. novo-ulmi clade into the two subspecies americana and novo-ulmi. The 3′ flanking regions have been shown to contain variable numbers of repeated oligonucleotide sequences, the number of which is species-specific and readily distinguished by a simple PCR assay.

Isolates of Verticillium fungicola, collected over a period of 45 yr from various geographical sites, were analysed for their responses to four fungicides (benomyl, thiabendazole, chlorothalonil and diethofencarb), and for colony morphology, virulence and DNA polymorphisms. Three different colony morphologies were observed for this population of isolates including fluffy, dense and appressed. The majority of isolates had the appressed colony morphology. Most isolates exhibited high levels of virulence. The appearance in this population of both benomyl and thiabendazole resistance was correlated with the introduction of benomyl for use on mushrooms. Eighty-eight percent of the isolates were cross-resistant between benomyl and thiabendazole and 80% exhibited negatively correlated cross-resistance between benomyl and diethofencarb. Seventy percent of the isolates exhibited negatively correlated cross-resistance between thiabendazole and diethofencarb. The level of resistance to chlorothalonil was relatively high even prior to its introduction and overall the response to this fungicide appears to have varied little over the last 45 yr. DNA polymorphisms, as detected by randomly amplified polymorphic DNA (RAPD) analysis, divided the isolates into four groupings. Limited correlations could be drawn between RAPD groupings, fungicide response and geographical origin. All recently collected isolates (1993–5) were extremely similar in their response to the four fungicides and had appressed colony morphology, high virulence and were members of RAPD group four. The lack of variation in the recent isolates as compared to the older isolates indicates that the V. fungicola population may be becoming more homogeneous.

Alkali-extractable and water-soluble cell-wall polysaccharides were purified from cell walls of some species of Fusarium and Gibberella. Their structures were determined by chemical analysis and NMR. The polysaccharides consisted of a main chain of β-(1 → 6)-linked galactofuranose units almost fully branched at positions O-2 by single residues of glucopyranose or acidic chains containing glucuronic acid and mannose. Individual differences were found, concerning the proportion of neutral and acidic side chains. These polysaccharides showed major differences from those of Microdochium nivale, Plectosphaerella cucumerina, Fusarium ciliatum, F. aquaeductuum and F. cavispermum. Highly specific polyclonal antibodies were raised against this structure, which were used in immunocompetence and immunofluorescence experiments.

Phytophthora megakarya is a devastating oomycete pathogen that causes black pod disease in cacao. Phytophthora species produce a protein that has a similar sequence to the necrosis and ethylene inducing protein (Nep1) of Fusarium oxysporum. Multiple copies of NEP1 orthologs (PmegNEP) have been identified in P. megakarya and four other Phytophthora species (P. citrophthora, P. capsici, P. palmivora, and P. sojae). Genome database searches confirmed the existence of multiple copies of NEP1 orthologs in P. sojae and P. ramorum. In this study, nine different PmegNEP orthologs from P. megakarya strain Mk-1 were identified and analyzed. Of these nine orthologs, six were expressed in mycelium and in P. megakarya zoospore-infected cacao leaf tissue. The remaining two clones are either regulated differently, or are nonfunctional genes. Sequence analysis revealed that six PmegNEP orthologs were organized in two clusters of three orthologs each in the P. megakarya genome. Evidence is presented for the instability in the P. megakarya genome resulting from duplications, inversions, and fused genes resulting in multiple NEP1 orthologs. Traits characteristic of the Phytophthora genome, such as the clustering of NEP1 orthologs, the lack of CATT and TATA boxes, the lack of introns, and the short distance between ORFs were also observed.

Seven hundred and eighty two isolates corresponding to 52 fungal taxa have been obtained from 920 segments of healthy and symptomatic twigs of Eucalyptus grandis, incubated on media with water potential −0·138 MPa and −4·19 MPa. Only thirty taxa were isolated at frequencies of 2% and greater. The composition of species isolated from bark of healthy twigs incubated on media with both water potentials was similar, with Aureobasidium pullulans being dominant. Sistrotrema brikmannii was the main species isolated from xylem of healthy twigs at −0·138 MPa whereas Pleospora sp. and Sphaeropsis eucalyptus were the dominant species isolated from this material on medium at −4·19 MPa. Fusicoccum eucalypti and Cytospora chrysosperma were most commonly isolated from bark and xylem of symptomatic twigs. Correspondence analysis showed the fungal species associated with each tissue. Summer drought followed by frequent frost in early autumn may have incited wounds allowing fungal invasion of the bark of twigs.

The genera Hobsonia, Illosporium, and Marchandiomyces include several common lichenicolous (lichen-inhabiting) species that have been taxonomically linked in the past. These mitosporic genera are frequently misidentified because of a paucity of morphological characters, which has led to some confusion about their relationships. We examined the phylogenetic positions of species in these genera using complete sequences of the small subunit (SSU) and internal transcribed spacer (ITS) ribosomal DNA (rDNA), and portions of the mitochondrial (mt) SSU rDNA obtained from isolated cultures of freshly collected specimens. Phylogenetic analysis of the sequence data indicated that Marchandiomyces corallinus and M. aurantiacus are closely related members of a basidiomycete clade. In addition, the clade includes an undescribed non-lichenicolous species that is morphologically similar to M. corallinus. Another undescribed species with marchandiomyces-like sclerotia, appears to be in a separate basidiomycete clade. The three recognized species of Hobsonia are unrelated, with H. christiansenii appearing to be a hypocrealean ascomycete closely related to a sample of Illosporium carneum, H. santessonii an ascomycete of unknown affinity, and H. mirabilis a basidiomycete. The latter, a plant pathogen, is the type species of Hobsonia. The new generic names Illosporiopsis and Hobsoniopsis gens. nov. are described for the lichenicolous species H. christiansenii and H. santessonii, respectively, and the necessary new combinations made.

Eudarluca caricis is assumed to be a nonspecific mycoparasite of rust fungi. The evidence for its mycoparasitism has rested on constant association with uredinia. In this study, stable isotopes provided additional evidence of mycoparasitism, as E. caricis was enriched with 15N relative to its associated rust fungus, as were parasites and mycoparasites generally with respect to their hosts. Host specificity was directly tested in inoculations in the greenhouse. Isolates of E. caricis from Puccinia on two Eurasian grasses (i.e. Holcus lanatus and Phalaris arundinacaea) did not infect Melampsora on Populus that, in contrast, was successfully infected by a poplar isolate of E. caricis. An isolate from M. medusae on P. deltoides infected a significantly greater percentage of uredinia of M. medusae on P. deltoides than uredinia of M. occidentalis on P. trichocarpa. The host specificity of the three isolates was reflected in their divergence in a phylogenetic analysis based on ITS sequences. Interestingly, the analysis revealed that mycoparasites of rust and powdery mildew fungi have evolved from a common ancestor.