The Nitschkiaceae has been placed in the Coronophorales or the Sordariales in recent years. Most recently it was accepted in the Coronophorales and placed in the Hypocreomycetidae based on sequence data from large subunit nrDNA. To confirm and corroborate the taxonomic placement and monophyly of the Coronophorales, additional taxa representing the diversity of the group were targeted for phylogenetic analysis using partial sequences of the large subunit nrDNA (LSU). Based on molecular data, the Coronophorales is found to be monophyletic and its placement in the Hypocreomycetidae is maintained. The order is a coherent group with morphologies that include superficial, often turbinate, often collabent ascomata that may or may not contain a quellkorper and asci that are often stipitate and at times polysporous. Three species with accepted Nitschkia names, together with Fracchiaea broomeiana and Acanthonitschkea argentinensis, comprise the paraphyletic nitschkiaceous complex. Two new families, Chaetosphaerellaceae and Scortechiniaceae fams nov., are described for the clades containing Chaetosphaerella and Crassochaeta and the taxa having a quellkorper (Euacanthe, Neofracchiaea and Scortechinia) respectively. The Bertiaceae is accepted for the clade containing Bertia species. Three new species are described: Bertia tropicalis, Lasiobertia portoricensis, and Nitschkia meniscoidea spp. nov.

The molecular diversity of Diaporthe helianthi (anamorph Phomopsis helianthi), the causal agent of sunflower stem canker, was studied in 16 isolates of different geographic origin using nuclear and mitochondrial markers. PCR products corresponding to the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal RNA gene, and to the mitochondrial atp6 gene were sequenced. The ITS1 and ITS2 sequences were compared with those of Phomopsis spp. and Diaporthe spp. obtained from databases. The diversity in the region surrounding the atp6 gene was also studied by restriction analysis using four enzymes. The analyses revealed a marked diversity within the sunflower-isolated strains, which appear to belong to phylogenetically unrelated groups. Noticeably, all the isolates collected in France and in the former Yugoslavia, where severe epiphytotics of sunflower stem canker are frequently reported, showed high similarity to each other forming a clade which clearly differentiated from all other ones within the genus Phomopsis. Conversely, all the isolates collected in Italy, where, despite favourable environmental conditions, the incidence of the disease is low, were only distantly related to the former group and showed sequence similarity with other previously established phylogenetic clades within the Phomopsis/Diaporthe complex.

Many small (temporary) collections of fungi maintained by plant pathologists during their research receive inadequate attention to ensure stability. Maintaining collections of fungi in pure and viable conditions, minimising physiological and morphological changes is, however, a necessity. The objective of this study was to find preservation techniques for three Rosellinia isolates used in our plant pathogenic research. Various inert and nutritious carriers, solid as well as liquid, were used to test their suitability for conserving these Rosellinia isolates. Different cryoprotectants, cooling rates and thawing rates were tested to optimise liquid nitrogen storage procedures. Survival and/or growth rate were assessed over time. Rosellinia bunodes was the most difficult to store with survival not exceeding six to nine months using traditional storage methods in mineral oil and silica gel. Storage of Rosellinia necatrix and Rosellinia pepo was successful for periods up to at least 16 months in several carriers and for up to two years for R. necatrix in silica gel. Storage in liquid nitrogen proved no problem for R. necatrix or R. pepo with a 100% survival in all cases, although radial growth rates after recuperation were affected by cryoprotectant and thawing rates. Storage of R. bunodes was more difficult and survival as well as growth rates were affected by cryoprotectant and thawing rates. Cooling rates did not affect radial growth in any of the isolates. The results showed that development of a generalised procedure for storage of our Rosellinia species was not possible and successful storage protocols had to be developed for individual isolates.

We analysed 40 isolates of species Armillaria. borealis, A. cepistipes, A. gallica, A. mellea, A. ostoyae and A. tabescens, mostly collected in the Czech Republic, by PCR-RFLP of the ITS rRNA genes using the restriction endonucleases AluI, HinfI and MboI. Restriction fragments were analysed by ion-exchange high performance liquid chromatography which proved to be more useful informative, and less time-consuming than classical electrophoresis on agarose gel. The HPLC method enabled detection of some heterozygous strains. HinfI discriminated between all six species. Ten isolates were sequenced to confirm changes in restriction sites found by restriction analysis. Cluster analysis based on the restrictions patterns of restriction endonucleases AluI and HinfI divided the analysed species into three groups. The first and the most distant group contained all A. mellea isolates, the second group was formed by A. tabescens and the third group contained species A. borealis, A. cepistipes, A. gallica and A. ostoyae. The A. tabescens group was very homogenous regardless of the origin of isolates (Czech Republic, France and Finland).

The relative complexity measure (RCM) is a new approach to evaluate relatedness of DNA sequences which eliminates the requirement to align sequences prior to analysis, a step required with standard reference methods. The value of the RCM approach to generate distance matrices for use in phylogenetic analysis of organisms has not been determined. This study compared RCM with the algorithmic and tree searching reference methods for phylogenetic analysis using fungal sequences. Sequences of the cytochrome b gene and the 18S rDNA gene were obtained from the GenBank database to determine feasibility of this method for phylogenetic relatedness. The RCM approach was also used to construct a phylogenetic tree using internal transcribed spacer (ITS) sequences from 23 medically relevant fungal species. The robustness of the RCM and reference approaches was determined by comparing the topology of seven medically relevant fungi within the phylogenetic trees generated after progressive removal of 10, 20, 30, 40 and 50% of the nucleotide bases from either the 5′ or 3′ end of the three genomic target sequences. The results demonstrated that the RCM method was equivalent to the reference methods for construction of phylogenetic trees from cytochrome b and 18S rDNA gene sequences. The phylogenetic tree constructed using the ITS sequence generated no contradictory topology. The RCM generated trees retained the appropriate topology after removal of up to 50% of the cytochrome b sequence, 40% of the ITS sequence, and 30% of the 18S gene target sequence. Comparatively, the reference methods failed to maintain topology after only a 10% sequence deletion for each genomic target. The results showed the RCM to be a reliable and robust computational approach for use in the construction of fungal phylogenetic trees without the requirement for prior sequence alignment.

Datronia caperata, a basidiomycete fungus, is one of the dominant polypore species found in neotropical mangrove forest fragments, where it is locally specialized on Laguncularia racemosa. We examined the genetic structure of D. caperata populations from four Panamanian mangrove forests using AFLP markers. Using five primer pair combinations, 145 loci were detected, 98.6% of which were polymorphic. Each of the populations showed a high degree of genetic diversity (Nei's h ranging from 0.146 to 0.223). Results from minimum spanning trees and Mantel tests showed little evidence for small-scale spatial structure within sites. A significant amount of total genetic variation was partitioned among populations (ΦST=0.21) separated by 10s to 100s of km, a considerably greater amount than has been detected in other mushroom and wood-decaying fungi sampled at equal or greater geographic distances. These results suggest that despite production of copious basidiospores capable of long distance dispersal, some homobasidiomycete fungi may be susceptible to genetic isolation due to habitat fragmentation.

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.

The phytopathogen Rhynchosporium alismatis, occurring on Alisma, Sagittaria and other genera in the Alismataceae, is under investigation as a mycoherbicide for alismataceous weeds in Australian rice crops. The type species of Rhynchosporium, R. secalis, belongs in the Helotiales while the phylogenetic relationships of R. alismatis were unknown. To identify fungi related to R. alismatis, the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1, 5.8S rRNA gene, ITS2) of 56 isolates was sequenced and compared to those available in databases. Analysis of ITS sequences revealed close relationships between R. alismatis and the teleomorph genus Plectosphaerella, as well as several anamorphic fungi which were primarily species of Verticillium. Rhynchosporium alismatis and Plectosphaerella cucumerina clustered together with 98% bootstrap support. Morphological comparisons supported this relationship indicating that R. alismatis and the anamorphic genus Plectosporium are congeneric. Rhynchosporium alismatis is transferred to Plectosporium, a name proposed for conservation.

We developed a method for separating physiologically active intraradical hyphae of arbuscular mycorrhizal (AM) fungi from mycorrhizal roots, allowing the hyphae to be used for physiological and biochemical experiments. In the present study, the phosphate efflux from the intraradical hyphae in vitro was examined in relation to hyphal phosphatase activity. Onion seedlings (Allium cepa) were planted in the soil inoculated with Gigaspora margarita. Six weeks after transplanting, the intraradical hyphae were isolated from the mycorrhizal roots using plant cell-wall digestion enzymes. The hyphae were incubated briefly at 25 °C in a buffer solution (50 mM Tris/HCl, pH 7.4), then incubated for 2 h and gently shaken with various inhibitors. Phosphate efflux, the amount of phosphate released to the buffer, was analysed by EnzChek phosphate assay kit. Hyphal phosphatase activity was stained histochemically and the proportion of phosphatase-active arbuscules was examined for each inhibitor. Phosphate effluxes were to some degree reduced by all inhibitors used, while the phosphatase inhibitor, BeSO4, greatly reduced the efflux. The degree of inhibition in the arbuscular phosphatase by each chemical was closely correlated to the decrease in the phosphate efflux. These results suggest that hyphal phosphatase may be partially involved in the phosphate efflux process from intraradical hyphae.

Specimens of Daldinia (Xylariaceae) from around the world possessing large ascospores were studied for teleomorphic and anamorphic morphological characters and compared with authentic material of D. grandis. A culture made from a specimen of D. grandis collected from Ecuador produced stromatic structures, but no conidiogenous structures referable to known xylariaceous anamorphs were observed. D. grandis is reconsidered and viewed as a species of warmer climates in the Americas. Three new species are recognised from new combinations of anamorphic and teleomorphic characters: (1) D. novaezelandiae sp. nov. from New Zealand, a fungus obviously related to D. bakeri, which is also reported from that country; (2) D. dennisii sp. nov., from Australia and New Zealand, of which two varieties are erected; and (3) D. loculatoides sp. nov., from the UK and Canada with affinities to D. loculata. The type of Sphaeria durissima was identified as D. loculata. Notes on further Daldinia spp. are included.

The taxonomic status and relationship of Tricholoma sulphureum and the similar T. bufonium were investigated using different sets of characters. These included morphological data on fruit bodies, ecological and chorological data, and analysis of the sequence data obtained for the ITS of basidiomes of different ecological and geographic origin. Moreover, the ectomycorrhizas formed by T. bufonium on Abies alba and Quercus sp. were characterised, and anatomical features compared with those of T. sulphureum mycorrhizas on coniferous and broad-leaved host trees. Our results revealed extensive ITS variation in members of the T. sulphureum group, but this variation was not correlated with morphology, ecology, or geographical distribution. We conclude that T. bufonium cannot be maintained as an autonomous taxon and should be treated as an infraspecific variant of T. sulphureum.

Diplodia pinea (syn. Sphaeropsis sapinea) is a well-known latent pathogen of Pinus spp. with a worldwide distribution. As such, this fungus is native where pines are endemic in the northern hemisphere and it has been introduced into all countries of the Southern Hemisphere where pines are exotic. The newly described D. scrobiculata (formerly known as the B morphotype of D. pinea) is thought to have a much more limited distribution. D. scrobiculata was first reported as an endophyte and weak pathogen of P. banksiana, where it was found to coexist with D. pinea. Diplodia scrobiculata is now known to have a much broader distribution in Northern America and Europe. In this study, seven Simple Sequence Repeat (SSR) markers were used to evaluate genetic diversity and gene flow between populations of D. scrobiculata. Results indicate a strong geographic isolation between populations of D. scrobiculata from different regions in North America, with unique alleles fixed in the different populations. The data fits the isolation by distance model indicating limited dispersal. Geographic isolation in combination with isolation by distance suggests prolonged reproductive isolation. Intensive collections of endophytes from native P. radiata in California have yielded only D. scrobiculata and not the significantly more pathogenic D. pinea. SSR analysis of three populations of D. scrobiculata from native P. radiata identified many shared alleles among the populations and moderate to high gene flow between them. The three Californian populations are distant and distinct from populations of D. scrobiculata from elsewhere. Under stress conditions, P. radiata is known to be very susceptible to D. pinea in plantations in the Southern Hemisphere. Native P. radiata is currently experiencing severe stress due to pitch canker caused by Fusarium circinatum. Such stress would provide ideal conditions for an associated outbreak of D. pinea. Thus, it is critical to prevent the movement of D. pinea into the last remaining native stands of P. radiata.

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

The genus Pilidiella, including its teleomorphs in Schizoparme, has a cosmopolitan distribution and is associated with disease symptoms on many plants. In the past, conidial pigmentation has been used as a character to separate Pilidiella (hyaline to pale brown conidia) from Coniella (dark brown conidia). In recent years, however, the two genera have been regarded as synonymous, the older name Coniella having priority. To address the generic question, sequences of the internal transcribed spacer region (ITS1, ITS2), 5.8S gene, large subunit (LSU) and elongation factor 1-α gene (EF 1-α) were analysed to compare the type species of Pilidiella and Coniella. All three gene regions supported the separation of Coniella from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by having species with hyaline to pale brown conidia (avg. length[ratio ]width >1.5), in contrast to the dark brown conidia of Coniella (avg. length[ratio ]width [les ]1.5). Pilidiella diplodiella, which is a pathogen associated with white rot of grapevines, was shown to be an older name for C. petrakii. To delineate species in the P. diplodiella species complex, isolates were also compared based on histone (H3) gene sequences. Analyses derived from these sequence data separated P. diplodiella from a newly described species, P. diplodiopsis. The new species P. eucalyptorum sp. nov. is proposed for isolates formerly treated as C. fragariae and associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens sp. nov. is newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. A key based on morphological characteristics is provided to separate the taxa treated in this study.

The life-cycle of many plant-pathogenic fungi, especially those infecting aerial plant organs, contains several specific developmental stages. If these are sufficiently distinct in their physiology from vegetative hyphal growth, they present potential targets for non-fungitoxic plant protectants. The present review identifies such targets especially in the pre-penetration stages of the infection cycle of Magnaporthe grisea and other fungi infecting from air-borne spores. Examples of non-toxic natural products with activity against spore germination, attachment, appressorium formation, appressorium maturation and penetration of the host surface are given. In contrast, no substances selectively active against in planta growth or sporulation appear to be known. The selective activity of numerous secondary metabolites against specific infection stages without accompanying toxicity against vegetatively growing hyphae indicates a direction for the development of future natural product-derived fungicides which are more easily degraded in the environment and possess fewer non-target effects. Such substances are produced by many saprotrophic and endophytic fungi in pure culture. The paucity of data on the production of biologically active substances in natural situations limits the interpretation of their ecophysiological significance for the producer.

A Bayesian analysis of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences was used to infer phylogenetic relationships of 14 Physconia species. The analysis supports the monophyly of the genus. Three well supported clades can be distinguished within Physconia: the series griseae, venustae and pulverulentae. The relationships of these clades, however, is not resolved with confidence. Cortical characters are re-evaluated on the basis of the phylogenetic hypothesis. Anatomical features of the upper cortex are only diagnostic above the species level for two special forms of two-layered cortices, while morphological characters, such as lower surface and rhizine-type are characteristic for distinct clades. P. venusta and P. perisidiosa are not separated in this analysis, but populations of P. muscigena, and European and North American samples of P. americana are clearly distinct and the monophyly of both P. americana and P. muscigena s. lat. is rejected on the basis of a Bayesian hypothesis testing.

DsRNAs were detected in 36 of 49 Monilinia fructicola isolates from stone fruit orchards in New Zealand. The dsRNA profiles were highly variable, even between isolates from a single tree. Comparison of pathogenicity on detached fruit, in vitro growth rate, and sporulation of 14 isolates showed no obvious correlation with presence of dsRNAs. Partially purified extracts from four isolates were examined for the presence of virus-like particles by transmission electron microscopy. One isolate contained 45 nm isometric particles similar in appearance to totiviruses and partitiviruses. A second isolate contained 200–250×25 nm rigid rods similar in appearance to the plant pathogenic tobraviruses and furoviruses. This is the first report of the presence of viral-like agents in the brown rot fungus Monilinia fructicola.

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.

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

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

Types of asexual sporulation in 17 Claviceps species and the closely related Corallocytostroma ornicopreoides were revised in relation to the phylogeny of clavicipitaceous fungi. We observed: (1) enteroblastic conidiation from branched phialidic conidiophores typical of the genus (anamorph Sphacelia) in all species including Corallocytostroma; (2) widespread and often sequential formation of terminal holoblastic secondary conidia on tapering hyphae arising from sphacelial macroconidia; and (3) in addition to sphacelial conidiation, sympodial holoblastic conidiation of the Ephelis-type in cultures of C. zizaniae and in both the culture and sphacelial tissue of C. citrina. Secondary conidiation was not found in C. purpurea, C. citrina and C. sorghicola. During sphacelial fructification, most species produced macroconidia and microconidia. Only macroconidia formed in planta underwent secondary conidiation whereas microconidia did not germinate at all. In C. phalaridis, the formation of holoblastic 2–3 celled appendaged conidia was observed, similar to that of Aciculosporium and Neoclaviceps. In dendrograms based on ITS1-5.8S rDNA-ITS2 sequences, genera and species with appendaged conidia grouped on a highly supported clade with ancestral Corallocytostroma. The clade was placed inside a group of tropical species of Claviceps, without any relationship to Balansiae.