As part of research to implement methods for the identification of Ganoderma species, this paper describes the use of PCR coupled to restriction digestions, single-strand conformational polymorphism (SSCP) and direct sequencing to assay rDNA polymorphism in South American collections of Ganoderma. The aim of the present study was to determine whether morphologically defined groups could be discriminated by molecular markers and to examine ITS sequence variation. Nineteen isolates of subgenus Ganoderma representing seven morphological taxa, and thirty isolates of subgenus Elfvingia representing three taxa, were studied. ITS I and IT II regions of twenty-one isolates were sequenced and were cladistically analyzed. Seven additional sequences were downloaded from EMBL/Genbank for comparison. In general, agreement between groupings delimited by restriction patterns, SSCP of ITS I and ITS II was evident. Gene trees derived from ITS I, ITS II and from a combined data set, were obtained and compared. Agreement between molecular and morphological data was clear at the subgeneric level; however, at the specific level, this relationship was difficult to visualize.

Partial sequences of the mtLSU rDNA were obtained from the arbuscular mycorrhizal (AM) fungi Glomus proliferum (isolate DAOM 226389) and G. intraradices (isolates JJ291 and BEG75). The exon sequences of the two species showed regions of strong divergence. There was no evidence of intra-isolate sequence heterogeneity as it is found in variable regions of nuclear ribosomal genes of Glomeromycota. In G. intraradices JJ291, two introns were found in the partial LSU sequence. One of the introns contained an ORF for a putative site-specific homing endonuclease of the LAGLIDADG family. In G. intraradices BEG75, one of the introns was missing and the other had a DNA sequence distinct from JJ291. G. proliferum had no introns in the region sequenced. A PCR primer was designed to amplify the fragment of the mtLSU of a different, distinguishable G. intraradices genotype from colonized roots of a field sample. These mitochondrial gene sequences are the first reported from the phylum Glomeromycota. Our findings indicate that the intra-individual sequence heterogeneity of the Glomeromycota may be a peculiar feature of the nuclear genes. Therefore, mtLSU and its introns have the potential to be highly sensitive genetic markers for these fungi in the future.

Norway spruce needles were sampled from two series of stand areas located in southern Finland. Both series consisted of five sampling areas in mature managed stands and one in a mature virgin stand. The proportion of spruce varied from 8 to 100% of the basal tree area and the major other species were pubescent birch and Scots pine. From each sampling area (some of which consisted of several sites) 40 mature spruces were randomly chosen and healthy looking needles of the third age class were sampled from heights of 5–8 m and incubated on water agar for isolation of endophytic fungi.

The majority of isolates were identified by their combined fatty acid and sterol profiles (FAST-profiles) as Lophodermium piceae. Tiarasporella parca was less common and Rhizosphaera kalkhoffii, Sclerophoma pythiophila, Lirula macrospora and Thysanophora penicillioides occurred occasionally. For calculations of fungal diversity all isolates were classified by their FAST-profiles into 81 groups (=operational chemotaxonomic units) according to a defined upper variation limit, i.e. an upper FAST-profile mismatch limit.

The highest percentage of endophytically infected needles was found in pure spruce stands and dense virgin stands. Location of the stand, its proportion of spruce and total basal area of trees (i.e. tree density) explained 82% of the variation of the overall infection rate. The effect of location was probably a consequence of differences in air quality between the various sampling areas.

The endophyte diversity, expressed as the number of FAST-groups per 40 spruces of each sampling area, correlated positively and statistically significantly with the percentage of needles infected or with the proportion of spruce in the stand. The highest endophytic diversity, expressed as FAST-groups per tree, was found in pure spruce and mixed virgin stands.

Pycnidial hyperparasites of powdery mildew fungi, often used in biological control experiments, are currently considered as belonging to a single species, Ampelomyces quisqualis. However, RFLP analysis of the nuclear rDNA ITS region in a worldwide collection of Ampelomyces isolates showed the existence of seven RFLP groups. Cultural characters, e.g. growth rate of isolates, correlated with the exhibited restriction patterns. The results suggested that A. quisqualis should be regarded as a problematic species complex. The geographical distribution of genetically distinct isolates showed that (i) isolates belonging to the same RFLP group were present even in different continents in their fungal hosts, and (ii) isolates representing various RFLP groups were found in the same geographical region, in the same species of the Erysiphaceae and on the same host plants. The detected genetic differences could influence the applicability of various isolates in biological control.

The genetic structure of Pyrenophora teres, the causal agent of net blotch of barley, was examined in two fields 30 km apart in the south-western Cape of South Africa. The two fields respectively represented a net- and spot-type population, the two types being distinguished on the basis of symptom expression on differentially susceptible cultivars. The number of isolates sampled from each field was 36 for the net-type population and 29 for the spot-type population. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. Of the 40 10-mer random oligonucleotide primers screened, five produced scorable, reproducible DNA bands suitable for the determination of population structure. A total of 65 loci were produced of which 54 were polymorphic. Genetic analysis of bands produced by one of the primers has revealed single locus segregation in a mating between a net- and spot-type isolate, indicating that RAPD bands can be interpreted as alleles at genetic loci. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spot-type populations. Genetic diversity among the two populations was divided into within- (variation between sampling quadrants) and among population components using Nei's GST. A coefficient of genetic differentiation (GS) of 0.0149 was obtained between quadrants within populations while a coefficient (GT) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis of the two populations together with six progeny from a mating between a net- and spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the net-type population that did not cluster with the other net-type isolates clustered directly next to the cluster containing the sexual progeny. This isolate also contained a unique spot-type DNA band. This suggested that sexual recombination may be occurring between net- and spot-type isolates under field conditions.

Growth of A. ochraceus at 30°C was significantly influenced by changes in both solute (NaCl, glycerol) and matric potential (PEG 8000). Optimum growth rates were at approx. −10·0 MPa with the non-ionic solute, and between −3·5 (=0·975 aw) and 14·0 MPa (=0·90 aw) with the ionic solute in a sucrose-based minimal salts medium. The optimum matric potential for growth was at −7·0 MPa (=0·95 aw). Growth rates were influenced by whether colonies were grown directly on the agar medium or on cellophane overlays. Sclerotia were profusely formed on media >−1·5 MPa (=0·99 aw), only initials at −3·5 MPa, and none under optimum conditions for growth (−7·0 to −10·0 MPa). The contents of low and high mol. wt polyols (glycerol, erythritol, arabitol and mannitol) and sugars (glucose and trehalose) were quantified in whole colonies and conidia grown under different osmotic and matric potential conditions for the first time. There was a higher total amount of polyols (μg mg−1D.W..) in conidia than in whole colonies. In glycerol-amended osmotic media maximum total polyols were present at −4·5 (=0·965 aw) and −17·0 MPa (=0·88 aw) in mycelial colonies and conidia respectively, with glycerol and mannitol being predominant in both, with small amounts of erythritol also present in conidia. By contrast, in matrically-modified media, the high mol. wt mannitol was the major component (>75% of the total) with small amounts of arabitol and the low mol. wt erythritol and glycerol, regardless of matric potential. For sugars, trehalose was predominant in conidia (>90%) from osmotically-modified media with low levels of glucose present in both conidia and mycelial colonies at all potentials tested. By contrast, in matric media trehalose was the predominant sugar in both conidia and whole colonies. This study suggests that xerophilic fungi may use different mechanisms for overcoming osmotic and matric stress.

A glasshouse investigation was conducted to study the effectiveness of 11 arbuscular mycorrhizal (AM) fungi on the medicinal plant Coleus forskohlii. Coleus plants raised in presence of most of the AM fungi in polythene bags showed an increase in plant growth (height, number of branches and biomass), P, and forskolin contents over those grown in the absence of soil inoculation with AM fungi. The extent of growth, P, and forskolin status varied with the AM fungi used. Based on the plant biomass, P uptake and forskolin content per plant, Glomus bagyarajii was found to be the best AM symbiont for inoculating C. forskohlii, the next being Scutellospora calospora.

The family Porinaceae (Trichotheliales) is characterized by perithecial ascomata of ascohymenial origin. The phylogenetic position of this family in the system of ascomycetes has been uncertain and is investigated using mtSSU rDNA sequences. The dataset consists of lichenized representatives of major ascomycete lineages, including those that were previously suspected as relatives of Porinaceae, such as Pyrenulaceae. The dataset was subjected to a Bayesian phylogenetic analysis implementing a Metropolis Coupled Markov Chain Monte Carlo method. The analysis confirms previous classification of the apothecial Gomphillaceae near to Graphidaceae, and suggests that the pyrenocarpous Porinaceae are also close to Graphidaceae, Gyalectaceae, and Stictidaceae. The subclass Ostropomycetidae is here suggested to include the families Gomphillaceae, Gyalectaceae, Graphidaceae (incl. Thelotremataceae), Porinaceae, and Stictidaceae. A special type of hemiangiocarpous ontogeny of the ascomata is shared throughout the Ostropomycetidae, and the closed fruit bodies of Porinaceae are apparently a result of a neotenic ontogeny. This is associated with special hymenial characters: rather thin-walled narrow asci, and a different consistency of the hymenial gels.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts which can only be propagated in association with intact plants or root explants. Molecular study of these fungi has been hampered by the inability to grow them in vitro and to obtain protoplasts. With the advent of advanced gene transformation systems, the opportunity has arisen to use biolistic technology to introduce foreign DNA linked to molecular markers into these fungi. The biolistic transformation system chosen for the AMF was GUS-based as this system was shown to work effectively for ectomycorrhiza. In this study the plasmid pNOM102 was used for transformation of Gigaspora rosea and transient GUS gene expression was detected in 40–50% of spores by colorimetric and immunological based methodologies.

A fungus was discovered causing a progressive twig dieback on stems of Vaccinium vitis-idaea (lingonberry) in Oregon. Both morphological and molecular data suggest that the fungus belongs in Diaporthe/Phomopsis but is distinct from P. vaccinii, cause of a dieback and fruit rot of blueberry and cranberry (Vaccinium spp.). This fungus is described and illustrated as a new species, Phomopsis columnaris. It is distinguished from other species of Phomopsis by the distinctive conidiophores that consist of vertically aligned cells lining the base and sides of the conidiomata. Another species of Phomopsis described on Vaccinium, Phomopsis myrtilli, known from V. myrtillus, is redescribed and illustrated based on authentic herbarium material.

The Neighbour-Sensing model brings together the basic essentials of hyphal growth kinetics into a vector-based mathematical model in which the growth vector of each virtual hyphal tip is calculated by reference to the surrounding virtual mycelium. The model predicts the growth pattern of many hyphae into three spatial dimensions and has been used to simulate complex fungal fruit body shapes. In this paper we show how the Neighbour-Sensing model can simulate growth in semi-solid substrata like agar or soil, enabling realistic simulation of mycelial colonies of filamentous fungi grown in ‘Petri-dish style’ experimental conditions. Newly implemented capabilities in the model include: a measurement and logging system within the program that maintains basic statistics about the mycelium it is simulating, this facilitates kinetic experimentation; inclusion of ‘substrates’ in the data space causing positive or negative tropisms for the growing mycelium; a horizontal plane tropism that provides a way of simulating colonies growing in or on a substratum like agar or soil by imposing a horizontal constraint on the data space the cyberhyphal tips can explore; three categories of hypha – standard hyphae are those that start the simulation, leading hyphae can emerge from the colony peripheral growth zone to take on a leading role, and secondary hyphae are branches that can arise late, far behind the peripheral growth zone, when mature hyphal segments resume branching to in-fill the older parts of the colony. We show how the model can be used to investigate hyphal growth kinetics in silico in experimental scenarios that would be difficult or impracticable in vivo. We also show that the Neighbour-Sensing model can generate sufficiently realistic cord-like structures to encourage the belief that this model is now sufficiently advanced for parameters to be defined that simulate specific in silico cyberfungi. The potential utility of these cyberspecies is that they provide a means to model the morphogenetic effects of a variety of factors, from environmental and nutritional features to mutations, in experimentally realistic situations, offering a valuable addition to the experimental toolkit of all those interested in fungal growth and morphology.

To study the genetic variability in Glomus mosseae and Gigaspora margarita sequence similarities of ITS regions of rDNA were analysed from spores collected from a permanent pasture and from pot cultures. PCR amplification with the primers ITS1 and ITS4 was performed and products were cloned and sequenced. The sequences from single spores of G. mosseae and Gi. margarita confirmed that there is variation in the ITS region in a single spore. Phenetic analysis of sequences from both species supported the morphological identification which placed the species into two separate groups. Through the analysis of these sequences an estimate of genetic diversity was derived which clearly showed that the three field spores of G. mosseae were at least 2–5 times more genetically diverse than one single spore (field) and a pool of spores (pot culture) of Gigaspora margarita. This demonstrates that a high degree of variation exists in this natural population of G. mosseae.

The amplified fragment length polymorphism (AFLP) was used to study genetic variation in Pyrenophora teres causing net blotch of barley in Finland. The mean similarity was 93% between all isolates and a bit higher within two distinct populations based on 175 AFLP markers. Despite the high genetic similarity, 70 unique AFLP genotypes were identified among 72 isolates. Most of the genetic variation (68.5%) was observed within a field population and a smaller portion (30.3%) between them. Significant genetic differentiation (Fst=0.308, P<0.001) was identified between field populations. However only 1.2% of the variation was observed between mating types within a field and a lack of genetic differentiation (Fsc=0.017, P=0.087) was observed. The occurrence of the form of blotch (spot type, f. sp. maculata, or net type, P. teres f. sp. teres) was identified with specific PCR. All isolates were found to be of the net type. The existence of both mating types (MAT1 and MAT2) was identified for the first time in Finland and the ratio of the two mating types was almost 1[ratio ]1 in both locations. The evolutionary potential and the possibility of sexual reproduction of P. teres occurring in Finland are discussed.

Leaves of members of the Cruciferae influence the germination of the entomogenous fungus Metarhizium anisopliae. The percentage germination of conidia on reconstituted epicuticular waxes of oilseed rape, Chinese cabbage, and turnip was influenced by the solvent used, the plant species, and the age of the leaf. The epicuticular waxes contain a mixture of stimulatory and inhibitory compounds. Germination was more rapid and greater on the surfaces of dewaxed than intact leaves suggesting that epicuticular waxes contain fungistatic compounds and/or act as a barrier to the leaching of nutrients. Surface leachates and soluble extracts of leaves increased percentage germination of conidia and the virulence of M. anisopliae to the mustard beetle, Phaedon cochleariae. Plant extracts stimulated germination more than yeast or starch extract. Formulation of conidia in leachates or leaf extracts increased the virulence of M. anisopliae. Germination increased on insect cuticle that were pretreated with leaf extracts/leachates, suggesting that the insect cuticle will adsorb or sequester nutrients.

Several species of Mortierella (Mortierellales, Zygomycota) were examined for substances regulating their sexual reactions. Compounds isolated from both mated and single growing Mortierella strains were purified by thin layer chromatography. Some of these compounds showed UV absorbance-characteristics similar to those of trisporoids, a group of compounds involved in sexual regulation in Mucorales. A compound with a 4-dihydromethyltrisporate-like absorbance spectrum was detected. To test for the interspecific sexual responses typically induced by trisporoids, the compounds extracted from Mortierella spp. were tested against the Mucorales Mucor mucedo and Phycomyces blakesleeanus and were found to induce sexual reactions in both tester strains. A gene encoding 4-dihydromethyltrisporate dehydrogenase was identified in several Mortierella species and the activity of the gene product was shown using a histochemical assay. We suggest that the regulation of sexual processes by trisporoids is common to both Mucorales and Mortierellales and may be more widespread within the Zygomycota.

Phanerochaete is a genus of resupinate homobasidiomycetes that are saprophytic on woody debris and logs. Morphological studies in the past indicated that Phanerochaete is a heterogeneous assemblage of species. In this study the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA was used to test the monophyly of the genus Phanerochaete and to infer phylogenetic relationships of the 24 taxa studied. Maximum parsimony, maximum likelihood, and Bayesian analyses do not support the monophyly of the genus. However, a core group of species represented by Phanerochaete velutina, P. chrysosporium, P. sordida, P. sanguinea and others are closely related and group together in a clade. Other common Phanerochaete species including Phanerochaete rimosa, P. chrysorhiza, P. omnivora, P. avellanea, P. tuberculata, P. flava, and P. allantospora, however, do not cluster with the core Phanerochaete group.

In the field, the concentrations of several soluble carbohydrates in mycorrhizas of red pine varied seasonally. Fructose, glucose, sucrose and trehalose concentrations were negatively correlated with soil temperature, while myo-inositol and mannitol concentrations were positively correlated. The patterns for the concentrations of fungal carbohydrates (trehalose and mannitol) were consistent with their previously-reported functions. Trehalose may serve as a storage carbohydrate, accumulating during the winter when host carbohydrate is plentiful and when fungal growth is reduced. In contrast, mannitol may serve a translocatory role, increasing in concentration in warmer months when fungal growth is more rapid and when sporocarps are formed.

The carbohydrates of six species of ectomycorrhizal fungi grown at room temperature and at reduced temperatures were also analysed to determine the extent to which seasonal variation in concentrations of fungal carbohydrates of mycorrhizas may be due to fungal acclimation to temperature. Variation in carbohydrate concentration occurred both among fungal species and due to cooling treatment. The variation due to cooling was relatively small, generally less than the variation among species, and less than the seasonal variation observed in field-collected mycorrhizas. This suggests that seasonal variation in the fungal carbohydrate trehalose in mycorrhizas could possibly be due to shifts in host carbohydrate supply rather than to independent shifts by the fungi in response to temperature. Production of a distinct pattern of carbohydrate concentrations by different ectomycorrhizal fungi suggests that fungal carbohydrates may be of taxonomic significance. This was further demonstrated using discriminant analysis of fungal carbohydrates.

The ITS region including the 5.8S rRNA gene as well as the 5′ end of the 28S rRNA gene of hypogeous Pezizaceae and Tuberaceae were studied to clarify the generic placement of two southern African desert truffles, Terfezia pfeilii and Choiromyces echinulatus. The results show that neither species belongs in the genus to which it has been assigned on the basis of morphological characters. As expected, two Choiromyces spp. grouped close to the representative of the Tuberaceae (Tuber melanosporum). However, C. echinulatus diverged from the other Choiromyces species and emerged near members of the genus Terfezia, being even closer to that genus than T. pfeilii. Two new genera and new species combinations, Kalaharituber gen. nov. with K. pfeilii (syn. T. pfeilii) comb. nov. and Eremiomyces gen. nov. with E. echinulatus (syn. C. echinulatus) comb. nov. are therefore introduced to accomodate these taxa. Both genera are closely related to Terfezia, and thus are placed in the Pezizaceae.

The objective of this study was to assess the influence of clay minerals on the morphology of mycelial pellets produced under submerged conditions as a prelude to the development of biomineral sorbents for toxic metals. The macro- and microscopic morphology of fungal pellets of melanin-containing microfungi Cladosporium cladosporioides, C. herbarum and Humicola grisea grown in liquid clay-containing medium was studied using scanning electron microscopy. It was found that the inclusion of clay minerals (bentonite, palygorskite and kaolinite) in the liquid medium influenced size, shape and structure of the mycelial pellets produced. In general, a reduction of pellet size, an increase in the length of surface hyphae of the pellets (except for H. grisea), and a reduction in exopolymer production were observed with increasing clay mineral concentrations up to 5% (w/v). For C. herbarum, an increasing concentration of bentonite changed the normal pelleted growth form to diffuse star-like growth, which was also observed for C. cladosporioides in the presence of kaolinite. The presence of bentonite and kaolinite in the medium for H. grisea and palygorskyte for C. cladosporioides led to the formation of numerous bulbous hyphae in pellets. However, bentonite decreased such bulbous growth in C. herbarum compared to control pellets which grew as bulbous mycelium. The dynamics of C. cladosporioides growth and pellet formation in the presence of bentonite (0·5% (w/v)) was investigated. It was found that the clay particles were involved in the formation of pellet structure at all stages of fungal growth. The porosity of a growing pellet (evaluated as the ratio of surface hyphae length to pellet radius) increased during early growth (17–24 h) and then decreased up to the stationary phase. A general model of the structure of a fungal pellet grown in clay-containing medium is proposed. The pellets consist of three main layers: a central core, with densely packed mycelium aggregated with solid clay minerals or a matrix of clay/polysaccharides; a middle layer with looser mycelium mixed with clay mineral flakes; and an outer, or ‘hairy’ zone, with loose hyphae surrounded by clay mineral flakes. The relevance of these studies to fungal growth and morphology, and to the development of biomineral sorbents for metal removal is discussed.

The Amphisphaeriaceae (sensu lato) presently includes 36 genera and 23 synonyms and is a heterogeneous assemblage of ascomycetes. In this paper molecular and morphological data and teleomorph–anamorph connections are examined and confirm that the Amphisphaeriaceae (sensu stricto) should be confined to ten teleomorphic genera and their Pestalotia-like anamorphs. These include Amphisphaeria, Broomella, Discostroma, Ellurema, Griphosphaerioma and Neobroomella which are described and illustrated; and Blogiascospora, Lepteutypa, Paracainiella and Pestalosphaeria which are discussed.