Five species of pathogenic fungi belong to Neofabraea. One of these, N. krawtzewii (syn. N. populi), is responsible for bark lesions on poplar (Populus) trees. The other four species cause post-harvest bull's eye rot of pome fruits, and at least two of these also cause bark cankers on pome fruit trees. Morphological variation among these species is slight, and overlap in geographic range sometimes occurs. As a consequence, identification based on conventional criteria can be tenuous. PCR primers with putative species specificity were developed following genetic analysis of the β-tubulin gene for isolates of each of the five species of Neofabraea. PCR conditions required to achieve specificity of the primer sets were determined, and a multiplex PCR protocol was developed to optimize their diagnostic utility on apple fruits. A protocol with higher annealing temperatures in the initial PCR cycles followed by lower temperatures in later cycles gave complete species-specificity when the primer sets were used individually and in multiplex, resulting in successful detection of the pathogens from axenic culture and infected apple fruits.

Pythium oligandrum is a parasite of cultivated Agaricus bisporus. Infection results in significant yield reductions and a disease referred to as ‘black compost’. In this study, P. oligandrum isolates were isolated from New Zealand mushroom composts, and their ribosomal DNA internal transcribed spacer (ITS) regions were amplified using the polymerase chain reaction (PCR). ITS nucleotide sequences obtained from New Zealand P. oligandrum isolates were compared with those previously identified P. oligandrum isolates and 23 described Pythium species. Although New Zealand P. oligandrum isolates had high ITS nucleotide identity with internationally identified P. oligandrum, the order of nucleotides in some regions varied when compared with other Pythium species. These varied nucleotides within the ITS region were used to design PCR primers (P.OLIG.F1 and P.OLIG.R04) for the specific amplification of a 384-bp fragment from P. oligandrum DNA. P.OLIG.F1 and P.OLIG.R04 were used to identify a major source of P. oligandrum inoculation on a New Zealand mushroom farm. Application of this diagnostic test will assist farming strategies implemented to prevent future P. oligandrum outbreaks. Furthermore, results presented identify a need for species resolution between P. oligandrum and P. hydnosporum.

The abilities of isolates of Amanita alboverrucosa, A. conicoverrucosa, A. fuscosquamosa, A. nauseosa, A. ochrophylla, A. pyramidifera, A. roseolamellata, A. xanthocephala and six unidentified Amanita species from eastern Australian temperate sclerophyll forests to utilise a range of inorganic and organic nitrogen sources for growth was examined in axenic liquid cultures. All taxa utilised NH4+ and at least some amino acids readily, while biomass yields on NO3− and histidine were generally low. All taxa were able to utilise bovine serum albumin, but for most taxa biomass yields on this substrate were significantly lower than on NH4+. Significant intraspecific variation in biomass yield was observed on all substrates for taxa for which multiple isolates were screened. As a group, eastern Australian Amanita species thus have the potential to utilise nitrogen from a broad range of organic substrates and this might be important in the nitrogen nutrition of their ectomycorrhizal tree hosts in sclerophyll forests.

Species delimitation is still controversial in the Xerocomus chrysenteron complex. We have therefore established comprehensible and reliable species concepts based on statistical evaluation of morphological and ecological characters. We examined many collections from different geographical regions and different developmental stages within collections. Quantitative micromorphological characters (basidiospores, pileipellis end cells) were measured in statistically relevant numbers. The same material was used to generate 24 rDNA-LSU sequences, and the results of phylogenetic analyses clearly confirmed our species concepts: spore size and ornamentation, length of the pileipellis end cells and ‘pruinatus-hyphae’ are most valuable characters for the delimitation of species in this complex. Molecular data demonstrated that the X. chrysenteron complex is a monophyletic group. All the examined species (X. chrysenteron, X. cisalpinus, X. pruinatus, X. ripariellus, X. dryophilus, X. fennicus, X. porosporus, and X. rubellus) represent independent lineages. The faintly striate spores, a key character characterising species of section Striatulispori, probably evolved independently. In addition, the ‘pruinatus-hyphae’ have multiple origins, and truncate spore apices are derived at least twice. Xerocomus cisalpinus sp. nov. is characterised by striate spores, the presence of ‘pruinatus-hyphae’ and a pileipellis strongly reminiscent of X. chrysenteron. For reasons of discussion, microscopical data are presented on Boletellus episcopalis for the first time. Xerocomus fennicus (Boletellus) comb. nov. is proposed. We provide descriptions to all included taxa. Our results once more demonstrate that reliably identified and characterised voucher collections are the basic requirement for meaningful phylogenetic studies.

Rust is reported for the first time in North America on tansy (Tanacetum vulgare), a widespread and invasive Eurasian plant that was introduced into North America in the 17th century. Morphologically, the rust fungus corresponds exactly to Gäumann's description of Puccinia tanaceti, and to European specimens of P. tanaceti on T. vulgare. An inoculation study confirmed the narrow host specialization of P. tanaceti that Gäumann described, in that T. vulgare was successfully inoculated whereas four other species of the tribe Anthemidae of the Asteraceae were entirely resistant, i.e. Leucanthemum maximum (Shasta daisy), L. vulgare (ox-eye daisy), Artemisia absinthium (wormwood), and A. tridentata (sagebrush). This confirmation of a specialized P. tanaceti invalidates P. tanaceti s. lat. The latter complex has resulted from synonymies, and compilation of host records of rust fungi resembling P. tanaceti on at least 105 species representing 19 genera from six tribes of the Asteraceae from both New and Old Worlds.

Twenty-four isolates of Aspergillus sojae, A. parasiticus, A. oryzae and A. flavus, including a number that have the capacity to produce aflatoxin, have been compared using amplified fragment length polymorphisms (AFLPs). Based on analysis of 12 different primer combinations, 500 potentially polymorphic fragments have been identified. Analysis of the AFLP data consistently and clearly separates the A. sojae/A. parasiticus isolates from the A. oryzae/A. flavus isolates. Furthermore, there are markers that can be used to distinguish the A. sojae isolates from those of A. parasiticus, which form the basis for species-specific markers. However, whilst there were many polymorphisms between isolates within the A. oryzae/A. flavus subgroup, no markers could be identified that distinguish between the two species. Sequencing of the ribosomal DNA ITS (internal transcribed spacers) from selected isolates also separated the A. sojae/A. parasiticus subgroup from the A. oryzae/A. flavus subgroup, but was unable to distinguish between the A. sojae and A. parasiticus isolates. Some ITS variation was found between isolates within the A. oryzae/A. flavus subgroup, but did not correlate with the species classification, indicating that it is difficult to use molecular data to separate the two species. In addition, sequencing of ribosomal ITS regions and AFLP analysis suggested that some species annotations in public culture collections may be inaccurate.

About 15000 species of ascomycete fungi lack a known sexual state. For fungi with asexual states in the anamorph genera Embellisia, Ulocladium, and Alternaria, six species have known sexual states but more than 50 species do not. In sexual filamentous ascomycetes, opposite mating type information at the MAT1 locus regulates mating and the opposite mating type genes each have a clonal, non-recombining phylogenetic history. We used PCR to amplify and sequence fragments of the opposite mating type genes from three supposedly asexual species, A. brassicae, A. brassicicola and A. tenuissima. Each haploid fungal isolate had just one mating type, but both mating types were present in all the three species. We sequenced the ribosomal ITS regions for isolates of opposite mating type, for the three asexual species and four known related sexual species. In a phylogenetic analysis including other ITS sequences from GenBank®, the three asexual species were not closely related to any of the known sexual species. Isolates of opposite mating type but the same species had identical ITS sequences. During any period of asexual evolutionary history, lineages of each mating type would have had a separate evolutionary history and any ITS substitutions shared between isolates of opposite mating type would have had to accumulate by convergence. Allowing for varying substitution rates and assuming a Poisson distribution of substitutions, the probability that isolates of opposite mating type shared an ITS substitution through convergence was low. This suggests that isolates of opposite mating type of A. brassicae, A. brassicicola and A. tenuissima were exchanging substitutions through sexual or parasexual reproduction while the ITS was evolving. If sexuality was lost, it was lost after the period of evolutionary history represented by the shared substitutions.

The efficacies of three nematophagous fungi, Paecilomyces lilacinus, Plectosphaerella cucumerina and Pochonia chlamydosporia, for controlling potato cyst nematodes (PCN) as part of an Integrated Pest Management (IPM) regime were studied. The compatibility of the nematophagous fungi with commonly used chemical pesticides and their ability to compete with the soil fungi Rhizoctonia solani, Chaetomium globosum, Fusarium oxysporum, Penicillium bilaii and Trichoderma harzianum were tested in vitro. Paecilomyces lilacinus was the most successful competitor when the ability to grow and inhibit growth of an opposing colony at both 10 and 20 °C was considered. P. lilacinus also showed potential for control of the soil-borne fungal pathogen R. solani, releasing a diffusable substance in vitro which inhibited its growth and caused morphological abnormalities in its hyphae. Pochonia chlamydosporia was least susceptible to growth inhibition by other fungi at 20° in vitro, but the isolate tested did not grow at 10°. Plectosphaerella cucumerina was a poor saprophytic competitor. Radial growth of Paecilomyces lilacinus and Plectosphaerella cucumerina was slowed, but not prevented, when grown on potato dextrose agar incorporating the fungicides fenpiclonil and tolclofos-methyl, and was not inhibited by the addition of pencycuron or the nematicide oxamyl. Radial growth of Pochonia chlamydosporia was partially inhibited by all the chemical pesticides tested. The efficacy of Paecilomyces lilacinus as a control agent for R. solani was further investigated in situ. Treatment with P. lilacinus significantly reduced the symptoms of Rhizoctonia disease on potato stems in a pot trial. The effectiveness of P. lilacinus and P. cucumerina against PCN was also tested in situ. Three application methods were compared; incorporating the fungi into alginate pellets, Terra-Green® inoculated with the fungi and applying conidia directly to the tubers. Both formulations containing P. lilacinus and formulation mixtures alone, particularly alginate pellets, significantly reduced multiplication of PCN in soil. We conclude that P. lilacinus showed the greatest potential for use in combination with selected fungicides and nematicides as part of an IPM programme for the control of PCN, but further work is required to confirm whether it is effective against PCN in soil.

Sterile mycelia isolated from Pinus tabulaeformis were grouped into white morphotype strains based on cultural characteristics. Eighteen of the isolates were randomly selected and identified to various taxonomic levels based on nuclear ribosomal DNA (nrDNA) sequence analysis. The 5.8S gene and flanking internal transcribed spacer (ITS1 and ITS2) regions of nrDNA were amplified and sequenced. Phylogenetic analysis of the 5.8S gene sequences indicated that the white morphotype strains were Ascomycota. Further identification was achieved by means of sequence similarity comparison and phylogenetic analysis of the ITS regions. Results showed that strains WMS9 and WMS10 were Lophodermium species (Rhytismataceae), while strains WMS11, WMS13 and WMS18 were species of Rhytismataceae. Strains WMS2, WMS3, WMS4, WMS5 and WMS6 were identified to Rosellinia, strain WMS1 to Entoleuca, and strain WMS14 to Nemania (Xylariaceae). Strains WMS7, WMS8, WMS12, WMS15, WMS16 and WMS17 were xylariaceous species. The potential of using DNA sequence analysis in the identification of endophytic fungi is discussed.

Cercospora beticola strains with laboratory induced resistance to tetraconazole were compared with their parental WT sensitive strains to evaluate the effects of resistance on fitness and assess whether any change in the sterol biosynthetic pathway was associated to the reduced fungicide sensitivity. In vitro growth rate on agar media and pathogenicity were found to be negatively affected by resistance. The main functional sterols in C. beticola WT strains under investigation were ergosterol, brassicasterol and ergosta-7,22-dienol. Resistant strains showed the same qualitative sterol composition, ruling it out as, per se, a cause for resistance. On the basis of the sterols detected both in sensitive and resistant strains, a possible biosynthetic pathway to the three functional sterols is proposed. Tetraconazole treatment caused, in all sensitive strains, the immediate accumulation of 14α-methylated sterols, which, for inhibitor concentrations up to EC50 values, were, in order of abundance, 14α-methylergosta-8,24(28)-dien-3β,6α-diol, eburicol and obtusifoliol. However the data do not support a critical role of the 14-methyl-3,6-diol derivative in the growth arrest of C. beticola. As main difference between sensitive and resistant strains, the formers were found to accumulate higher amounts of 14α-methylated sterols. Although the data do not allow to establish a specific mechanism for resistance, some molecular mechanisms such as target site alterations and sterol biosynthetic pathway can be ruled out as a possible cause for reduced sensitivity.

Sequence analysis of the ribosomal DNA internal transcribed spacers (ITS) was used to establish phylogenetic relationships among 23 isolates of Pythium insidiosum, the etiological agent of pythiosis in mammals. The isolates were divided into three distinct clades that exhibited significant geographic isolation. Clade I consisted of isolates from North, Central, and South America, while clade II contained isolates from Asia and Australia. Also present in clade II was an isolate from a patient in the USA, but the origin of the infection may have been in the Middle East. Clade III was comprised of isolates from Thailand and the USA. All 23 P. insidiosum isolates were more closely related to each other than to any other Pythium species in this study. Additionally, all Pythium isolates formed a clade separate from both outgroup species, Phytophthora megasperma and Lagenidium giganteum. The ITS sequence results tend to support the existence of geographic variants or cryptic speciation within P. insidiosum. The sequence information obtained also provides an abundance of data for applications in the diagnosis of pythiosis and identification of P. insidiosum from clinical samples.

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.

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.

The effect of Trichoderma atroviride, T. harzianum, T. longibrachiatum, Clonostachys rosea and Bacillus subtilis isolates applied to wheat seeds against Rhizoctonia cerealis disease of seedlings was investigated under controlled greenhouse conditions. Most Trichoderma isolates significantly reduced the incidence of disease compared with the infected control. Bacillus subtilis was also effective against sharp eyespot, although less active than Trichoderma spp. Interactions between the antagonistic microorganisms and the cereal pathogenic fungus in dual culture experiments on agar growth medium were also studied. Almost all tested antagonists showed competitive activity against R. cerealis: inhibition of its mycelial growth and hyphal interaction. The production of extracellular β-N-acetylhexosaminidase, chitin 1,4-β-chitobiosidase, glucan 1,3-β-glucosidase and protease activity by the tested microorganisms in the presence of cell walls of R. cerealis was then determined. All isolates showed glucosaminidase and chitobiosidase activity. They also produced glucosidase activity, except B. subtilis, whereas only C. rosea, B. subtilis and one isolate of T. harzianum showed detectable levels of protease activity.

Rhodotorula mucilaginosa produces the siderophore rhodotorulic acid (RA) when grown in iron-limited conditions. R. mucilaginosa grew at rates between 0.10 and 0.19 h−1 in iron-restricted conditions, depending on the carbon source, and at 0.23 h−1 in iron-sufficient conditions. In bioreactors inoculated with iron-starved pre-cultures, initial specific growth rates in batch culture were dependent on the iron concentration. The critical dilution rate (Dcrit, at which steady state cultures cannot be sustained) in continuous cultures was also dependent on the iron concentration and was lower than μmax in batch culture. Sucrose was the best carbon source for RA production [287±11 μmol (g biomass)−1] and production could be further increased by supplementing the medium with the precursors acetate [460±13 μmol (g biomass)−1], ornithine [376±6 μmol (g biomass)−1], or both [539±15 μmol (g biomass)−1]. Citric acid was an effective suppresser of RA production. RA was produced in a growth rate dependent manner and was optimally produced at pH 6.5.

Didymella bryoniae (anamorph Phoma cucurbitacearum) is an ascomycete that causes gummy stem blight, a foliar disease that occurs on cucurbits in greenhouses and fields throughout the world. In a previous study using RAPD analysis, little genetic diversity was found among isolates of D. bryoniae from New York and South Carolina, USA. Here we report the use of amplified fragment length polymorphism (AFLP) analysis to assess the genetic variation within a worldwide collection of D. bryoniae. 102 field and greenhouse isolates from ten states in the USA (California, Delaware, Florida, Georgia, Indiana, Maryland, Michigan, Oklahoma, South Carolina, and Texas) and seven other countries (Australia, Canada, China, Greece, Israel, Sweden, and The Netherlands) were examined. Seven different AFLP primer-pair combinations generated 450 bands, of which 134 were polymorphic (30%). Using cluster analysis, two groups and a total of seven subgroups were delineated. Representative isolates varied in their virulence on muskmelon and watermelon seedlings, but the degree of virulence was not strongly associated with AFLP groupings. However, isolates from the northern USA grouped separately from isolates originating from the southern USA.

The synthesis of plant growth hormone auxin, indole-3-acetic acid (IAA), is not only confined to flowering plants. Yeasts and other fungi are also known to produce this hormone and in many cases at higher levels than plants. The main concern of this study was to determine the physical and chemical conditions necessary for optimal biosynthesis of this hormone by Lentinus sajor-caju. Glucose was determined to be superior to sucrose as carbon and energy source. The synthesis of IAA in a nitrogen-free medium or in a medium with low external phosphate was substantially reduced. Light exposed and non-agitated cultures grown in dark had also reduced levels of IAA compared to agitated cultures grown in dark. The highest (0.18 mg ml−1) IAA level was determined in cultures grown in glucose containing medium (pH 7.5) on a rotary shaker (150 rpm) at 30 °C in dark. The biological activity of IAA obtained from the extra-cellular culture of Lentinus sajor-caju was determined using oat coleptile growth test.

The mycoparasite Sphaerellopsis filum (teleomorph Eudarluca caricis) was applied simultaneously with Melampsora larici-epitea on to willow leaf discs using eight concentrations of conidia. Inoculum densities were quantified and the numbers of uredinia of the rust, pycnidia and conidia of S. filum and rust spores produced per leaf disc were measured 13 d after inoculation (first assessment). Higher S. filum inoculum densities resulted in more rust uredinia being infected, but did not reduce the number of uredinia produced. The ratios of infected rust pustules: S. filum conidia applied were in a range of 0.25–0.31 when less than 20 S. filum spores were inoculated on to a leaf disc (0.95 cm2). Suppressive effects of S. filum on rust spore production were more obvious in the second assessment, carried out 23 d after inoculation. Inoculum densities of S. filum were significantly (P<0.001) correlated with the frequency of uredinia infected (% variance accounted for [VAF]=85.8), the number of S. filum pycnidia (%VAF=81.4), S. filum spores produced (%VAF=72.3) and rust spore production (%VAF=48.6). Rust spore production was significantly (P<0.001) negatively correlated with the frequency of uredinia infected (%VAF=51.1), the number of S. filum pycnidia (%VAF=42.0) and the number of S. filum spores produced (%VAF=40.6). The best correlation was found between the number of pycnidia and the number of S. filum spores produced (%VAF=88.8).

Four group I introns, designated Bb1, Bb2, Bb3 and Bb4, were identified in the entomopathogenic hyphomycete Beauveria bassiana. Sequence analyses of these introns verified that they were invariably inserted at specific target sequences after conserved positions Ec2563, Ec2449, Ec2066 and Ec1921 of the large nuclear subunit (LSU) rDNA 3′-end. Secondary structure modelling confirmed that Bb1 and Bb3 belonged to subgroup IE while Bb2 and Bb4 belonged to subgroup IC1. Intron presence, distribution and size-variation were studied in a population of 125 B. bassiana strains using site-specific primers. Nucleotide sequences and secondary structures were compared and showed considerable variations usually at P1, P6 and P9 helices, but concomitantly, high homology between members of the same site-specific group. Intron distribution studies revealed that few (7.2%) strains were intron-less, most contained one (28%), two (48%) or three (16%) introns, while only one strain contained all four introns. Bb4-like introns (Ec1921) were the most abundant (86.4%), whereas the other three introns were evenly represented (ca 30%) in the B. bassiana population. Analysis of intron genotype distribution indicated a tenuous dependence upon geographic origin or insect host species. Phylogenetic analysis of all B. bassiana LSU introns and their close relatives from other entomopathogenic fungi showed a strong correlation between specific insertion sites and intron subgroups, fully supported by corresponding clades, suggesting common ancestry of the site specific LSU introns.

Development of specific DNA probes targeting rDNA internal transcribed spacer (ITS-1 or -2) sequences is described for the detection of strains representing uninucleate and binucleate Rhizoctonia spp. and Suillus bovinus. Discriminatory taxon/species-specific target sequences were identified following full length ITS sequence alignment of the test fungal sequences and those of other root associated pathogenic or mycorrhizal fungi. Both long (124–151 bp) and shorter (20–25 bp) probes were generated for assessment in Southern dot blot and liquid hybridization ITS capture-fragment length polymorphism assays. Fungal genomic DNA was presented as the target in dot blot protocols using the longer DIG (digoxigenin) labelled probes whilst the shorter 3′ biotin-labelled oligonucleotide probes were hybridized to PCR amplified full length ITS (ITS1-5.8S-ITS2) in both dot blot and liquid hybridization assays. The optimal hybridization temperatures for dot blot detection also produced maximal target specific signals in the liquid hybridization protocol. The latter protocol was found to be more discriminatory as target fungi were detected on the basis of combined probe hybridization-ITS capture and 5′ Cy-5 labelled ITS length polymorphism analysis (±5 bp) following denaturing sequencing gel electrophoresis in a ALFexpress DNA sequencer.