Dnabarcoder: An open-source software package for analysing and predicting DNA sequence similarity cutoffs for fungal sequence identification.

The accuracy and precision of fungal molecular identification and classification are challenging, particularly in environmental metabarcoding approaches as these often trade accuracy for efficiency given the large data volumes at hand. In most ecological studies, only a single similarity cutoff value is used for sequence identification. This is not sufficient since the most commonly used DNA markers are known to vary widely in terms of inter- and intraspecific variability. We address this problem by presenting a new tool, dnabarcoder, to predict local similarity cutoffs and measure the resolving powers of a biomarker for sequence identification for different clades of fungi. It was shown that the predicted similarity cutoffs varied significantly between the clades of a recently released ITS DNA barcode data set from the CBS culture collection of the Westerdijk Fungal Biodiversity Institute. When classifying a large public fungal ITS data set-the UNITE database-against the barcode data set, the local similarity cutoffs assigned fewer sequences than the traditional cutoffs used in metabarcoding studies. However, the obtained accuracy and precision were significantly improved. Our study showed that it might be better to extract the ITS region from the ITS barcodes to optimize taxonomic assignment accuracy. Furthermore, 15.3, 25.6, and 26.3% of the fungal species of the barcode data set were indistinguishable by full-length ITS, ITS1, and ITS2, respectively. Except for these indistinguishable species, the resolving powers of full-length ITS, ITS1, and ITS2 sequences were similar at the species level. Nevertheless, the complete ITS region had a better resolving power at higher taxonomic levels.

Redefining common endophytes and plant pathogens in Neofabraea, Pezicula, and related genera.

Species in Neofabraea, Pezicula, and related genera have been reported as saprobes, plant pathogens or endophytes from a wide range of hosts. The asexual morphs of Neofabraea and Pezicula had been placed in Cryptosporiopsis, now a synonym of Pezicula, while Neofabraea was also linked to Phlyctema. Based on morphology and molecular data of the partial large subunit nrDNA (LSU), the internal transcribed spacer region with intervening 5.8S nrDNA (ITS), partial ß-tubulin region (tub2), and the partial RNA polymerase II second largest subunit region (rpb2), the taxonomy and phylogenetic relationships of these fungi were investigated. Five new species were described in Pezicula based on morphology, while a further eight unnamed phylogenetic lineages revealed further diversity in the genus. Based on these results, the generic concept of Neofabraea was also emended. Phlyctema, which was previously associated with Neofabraea, formed a distinct clade, separate from Neofabraea s. str. Two new neofabraea-like genera, Parafabraea and Pseudofabraea were proposed, along with one new combination in Neofabraea s. str. To stabilise the application of these names, an epitype was designated for Pe. carpinea, the type species of Pezicula, and for N. malicorticis, the type species of Neofabraea.

The rise of Ramularia from the Mycosphaerella labyrinth.

In this study we aimed to resolve the Ramularia endophylla species-complex by applying a polyphasic approach involving morphology and multi-gene phylogeny. Eleven partial genes were targeted for amplification and sequencing for a total of 81 isolates representing R. endophylla s. lat. and 32 isolates representing 11 Ramularia species that were previously linked to a Mycosphaerella sexual morph in literature. A Bayesian phylogenetic analysis, as well as a parsimony analysis, was performed on a combined five-locus dataset and the resulting trees showed significant support for three species within the complex, including the previously described R. endophylla and R. vizellae, and one novel species, Ramularia unterseheri. A parsimony analysis was also separately performed with mating-type gene sequences (MAT1-1-1 and MAT1-2-1) and the resulting tree topologies were in accordance with that of the multigene analysis. A bibliographic review of the proposed links between Ramularia spp. and their purported Mycosphaerella sexual morphs is also presented, confirming six connections in Ramularia. In spite of more than 10 000 species having been described in Mycosphaerella, the majority is shown to belong to other genera, suggesting that the taxa identified as Mycosphaerella in much of the plant pathology literature needs to be revisited.

MycoBank gearing up for new horizons.

MycoBank, a registration system for fungi established in 2004 to capture all taxonomic novelties, acts as a coordination hub between repositories such as Index Fungorum and Fungal Names. Since January 2013, registration of fungal names is a mandatory requirement for valid publication under the International Code of Nomenclature for algae, fungi and plants (ICN). This review explains the database innovations that have been implemented over the past few years, and discusses new features such as advanced queries, registration of typification events (MBT numbers for lecto, epi- and neotypes), the multi-lingual database interface, the nomenclature discussion forum, annotation system, and web services with links to third parties. MycoBank has also introduced novel identification services, linking DNA sequence data to numerous related databases to enable intelligent search queries. Although MycoBank fills an important void for taxon registration, challenges for the future remain to improve links between taxonomic names and DNA data, and to also introduce a formal system for naming fungi known from DNA sequence data only. To further improve the quality of MycoBank data, remote access will now allow registered mycologists to act as MycoBank curators, using Citrix software.

Cutaneous phaeohyphomycosis caused by Paraconiothyrium cyclothyrioides.

Paraconiothyrium cyclothyrioides is a recently described coelomycetous fungal species. We present a case in a renal transplant patient with chronic skin lesions of the lower extremities caused by P. cyclothyrioides. Treatment with posaconazole led to complete resolution of the lesions. P. cyclothyrioides should be considered an opportunistic human pathogen in immunocompromised patients.

Correlated evolution of senescence and ephemeral substrate use in the Sordariomycetes.

Evolutionary theory predicts that senescence--a decline in reproduction and survival with increasing age--can evolve as a trade-off between investment in reproduction on one side and in somatic maintenance and repair on the other. The ecology of a species is crucial because it provides the external causes of death that determine the statistical limit to a species' lifespan. Filamentous fungi are generally believed to be nonsenescent, and there are indeed spectacular examples of very old fungal individuals in nature. However, some fungi utilize ephemeral resources, and therefore, senescence is expected to have evolved, like in the coprophilic Podospora anserina, the only well-studied filamentous fungus with intrinsic senescence. Here, we hypothesize that rapid senescence is more common in fungi than generally believed and that the phylogenetic distribution of senescence correlates with ecology. We collected lifespan data for a set of Sordariomycetes and constructed phylogenies based on several nuclear sequences. Several of the strains were from the CBS culture collection, originally isolated from various substrates, some of which ephemeral. In addition, we isolated new strains from short-lived substrates. Senescence was observed throughout the phylogeny. Correlation tests support the hypothesis that in the Sordariomycetes, senescence is a trait that has arisen in response to ephemeral substrates, and that it has evolved repeatedly and independently along the phylogeny.

Systematic reappraisal of species in Phoma section Paraphoma, Pyrenochaeta and Pleurophoma.

Sequence data from the 18S nrDNA (SSU) and 28S nrDNA (LSU) regions of isolates of Phoma section Paraphoma were compared with those of representative isolates of the morphologically similar anamorph genera Pleurophoma and Pyrenochaeta and of the type species of Phoma sections Phoma, Pilosa and Plenodomus. Phoma section Paraphoma was found to be highly polyphyletic within the Pleosporales and only distantly related to Phoma section Phoma. The genus Paraphoma, which is based on Paraphoma radicina, is reintroduced in the Phaeosphaeriaceae with two additional taxa. The new genera Setophoma and Neosetophoma, type species Setophoma terrestris comb. nov. and Neosetophoma samarorum comb. nov., are introduced and represent species that are closely related to Paraphoma but differ based on morphological characters and molecular phylogeny. Phoma coonsii is transferred to genus Chaetosphaeronema that also belongs to the Phaeosphaeriaceae. Pyrenochaetopsis gen. nov. is introduced to accommodate the type species Pyrenochaetopsis leptospora comb. nov., as well as several other species formerly accommodated in Phoma and Pyrenochaeta. Pyrenochaetopsis is closely related to Pyrenochaeta and classified in the Cucurbitariaceae. Pleurophoma cava is transferred to genus Pyrenochaeta. The new genera elucidate the confusing taxonomy of species in genera Phoma, Pyrenochaeta and Pleurophoma and recognize monophyletic genera with distinct teleomorph affinities.

Pyrenochaeta keratinophila sp. nov., isolated from an ocular infection in Spain.

The new coelomycete Pyrenochaeta keratinophila, isolated from corneal scrapings of a case of keratitis in Spain, is described and illustrated. This fungus is morphologically characterized by grey-olivaceous to greenish olivaceous colonies, scarce pycnidial setae placed mainly near the ostiole and production of phialoconidia from the aerial mycelium. The latter feature is unknown in any other species of the genus Pyrenochaeta. Sequencing of the ITS rDNA region of this clinical strain confirmed this proposal and revealed its close genetic relationship with the Leptosphaeriaceae.

DNA phylogeny reveals polyphyly of Phoma section Peyronellaea and multiple taxonomic novelties.

Species of the anamorph genus Phoma are commonly isolated from a wide range of ecological niches. They are notoriously difficult to identify due to the paucity of morphological features and the plasticity of these when cultivated on agar media. Species linked to Phoma section Peyronellaea are typified by the production of dictyochlamydospores and thus have additional characters to use in taxon delineation. However, the taxonomy of this section is still not fully understood. Furthermore the production of such chlamydospores also is known in some other sections of Phoma. DNA sequences were generated from three loci, namely ITS, actin, and 3-tubulin, to clarify the phylogeny of Phoma taxa that produce dictyochlamydospores. Results were unable to support section Peyronellaea as a taxonomic entity. Dictyochlamydospore formation appears to be a feature that developed, or was lost, many times during the evolution of Phoma. Furthermore, based on the multigene analyses, five new Phoma species could be delineated while a further five required taxonomic revision to be consistent with the genetic variation observed.

New pyrenochaeta species causing keratitis.

We report a new fungus as an agent of fungal keratitis in a diabetic woman. The fungal etiology was established by classic microbiology and PCR following 3 months of antibacterial therapy. The morphological features of the isolate and sequence analysis of the internal transcribed spacer region indicate a new species of Pyrenochaeta (Coelomycetes).

Molecular phylogeny of Phoma and allied anamorph genera: towards a reclassification of the Phoma complex.

The present generic concept of Phoma is broadly defined, with nine sections being recognised based on morphological characters. Teleomorph states of Phoma have been described in the genera Didymella, Leptosphaeria, Pleospora and Mycosphaerella, indicating that Phoma anamorphs represent a polyphyletic group. In an attempt to delineate generic boundaries, representative strains of the various Phoma sections and allied coelomycetous genera were included for study. Sequence data of the 18S nrDNA (SSU) and the 28S nrDNA (LSU) regions of 18 Phoma strains included were compared with those of representative strains of 39 allied anamorph genera, including Ascochyta, Coniothyrium, Deuterophoma, Microsphaeropsis, Pleurophoma, Pyrenochaeta, and 11 teleomorph genera. The type species of the Phoma sections Phoma, Phyllostictoides, Sclerophomella, Macrospora and Peyronellaea grouped in a subclade in the Pleosporales with the type species of Ascochyta and Microsphaeropsis. The new family Didymellaceae is proposed to accommodate these Phoma sections and related anamorph genera. The present study demonstrated that Phoma radicina, the type species of Phoma sect. Paraphoma and Phoma heteromorphospora, the type species of Phoma sect. Heterospora can be assigned to the Phaeosphaeriaceae and Leptosphaeriaceae respectively.

Eucalyptus microfungi known from culture. 1. Cladoriella and Fulvoflamma genera nova, with notes on some other poorly known taxa.

A study of microfungi associated with living Eucalyptus leaves and leaf litter revealed several novel and interesting taxa. Cladoriella eucalypti gen. et sp. nov. is described as a Cladosporium-like genus associated with litter collected in South Africa, while Fulvoflamma eucalypti gen. et. sp. nov. is newly described from leaf litter collected in Spain. Beta-conidia are newly reported for species of Pestalotiopsis, namely Pestalotiopsis disseminata in New Zealand, and a Pestalotiopsis sp. from Colombia. Satchmopsis brasiliensis is reported from litter in Colombia and Indonesia, while Torrendiella eucalypti is reported from leaf litter in Indonesia, and shown to have a Sporothrix-like anamorph. Leptospora rubella is reported from living Eucalyptus leaves in Colombia, where it is associated with leaf spots of Mycosphaerella longibasalis, while Macrohilumeucalypti is reported from leaf spots of Eucalyptus in New Zealand.

Phoma Basal Rot of Romaine Lettuce in California Caused by Phoma exigua: Occurrence, Characterization, and Control.

Beginning in 2000 and continuing into 2004, a previously undescribed disease caused significant damage to romaine lettuce (Lactuca sativa) in the coastal Salinas Valley of California. Symptoms were brownish black, sunken cavities on the crown and upper taproot. Cavities were firm, lacked signs of fungal growth, and resulted in cracking and weakening of the crown. Affected plants were stunted, uneven in growth, and unmarketable. Crisphead lettuce also developed these symptoms, although disease incidence was always lower than that for romaine. A fungus was consistently isolated from cavities. Using morphological features and molecular methods, the fungus was identified as Phoma exigua. Pathogenicity of isolates from romaine and crisphead lettuce was demonstrated on both of these lettuce types. Replicated field studies showed that azoxystrobin, boscalid, and cyprodinil + fludioxonil fungicides effectively controlled the disease. This is the first report of a crown disease caused by P. exigua in the United States. A similar disease, Phoma basal rot on greenhouse grown lettuce in the United Kingdom, had been previously observed, but no report has been published.

Mycosphaerella punctiformis revisited: morphology, phylogeny, and epitypification of the type species of the genus Mycosphaerella (Dothideales, Ascomycota).

Mycosphaerella punctiformis, the type species of the genus Mycosphaerella, is epitypified by material collected on Quercus robur in The Netherlands. The teleomorph is described in planta, and the Ramularia anamorph, for which the new name R. endophylla is proposed, and the Asteromella spermatial state are characterized in vitro. Sequence data of the nuclear ribosomal DNA are presented and analyzed together with other Mycosphaerella spp. with Ramularia and several other anamorphs. Several strains originating from Quercus, Acer and Tilia showed diverging ITS sequences, indicating that the M. punctiformis complex may comprise more than a single phylogenetic species, but this could not be confirmed by the analysis of our dataset. An endophytic phase is established for the first time in the life-cycle of M. punctiformis, as the species was repeatedly isolated from surface sterilized green healthy leaves of Quercus robur in summer at the epitype locality.

Systematic reappraisal of Coniella and Pilidiella, with specific reference to species occurring on Eucalyptus and Vitis in South Africa.

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-alpha gene (EF 1-alpha) 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:width > 1.5), in contrast to the dark brown conidia of Coniella (avg. length:width < or = 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.

Phylogenetic analyses of Septoria species based on the ITS and LSU-D2 regions of nuclear ribosomal DNA.

The phylogenetic relationships of 17 selected Septoria spp. (eight with a known Mycosphaerella teleomorph), Phloeospora ulmi (teleomorph M. ulmi) and 18 additional taxa (10 with a Mycosphaerella teleomorph) were inferred from ITS and D2-LSU nrDNA sequences. In total, 10 anamorph genera associated with Mycosphaerella were represented. Intraspecific variation in ITS was limited in Septoria, with the exception of three strains that all were identified as S. rubi but originated from different Rubus spp. and probably belong to different species of Septoria. The results of D2 region sequencing confirmed Mycosphaerella sense lato (including Davidiella and Eruptio) as monophyletic. The cereal pathogen Septoria tritici, which is closely related to S. passerinii as found in ITS analysis, clusters with Ramularia spp. in the D2 analyses, distant from the other Septoria spp. The pathogens S. apiicola, S. linicola and S. populicola cluster in a major clade containing Phl. ulmi, and other Septoria spp. and Cercospora spp. Short branch lengths in this clade suggest a very recent evolution. Septoria castaneicola and S. pyricola also might represent relatively distant lineages. Both analyses of the regions indicated that Septoria is not monophyletic within Mycosphaerella and that conidiomatal structure (acervulus, pycnidium) has little value for predicting phylogenetic relatedness. As a consequence, the separation between the acervular Phloeospora and pycnidial Septoria is untenable. The loss of the teleomorph most likely has occurred several times in the evolutionary history of Mycosphaerella.

Phylogeny and taxonomy of root-inhabiting Cryptosporiopsis species, and C. rhizophila sp. nov., a fungus inhabiting roots of several Ericaceae.

Three Cryptosporiopsis species have thus far been isolated from roots of woody plants. A fourth species, which was recently isolated from roots of Calluna vulgaris, Erica tetralix, Vaccinium vitis-idaea, and V. myrtillus in The Netherlands, is described here as new. Sporulation on the natural substratum has not been observed and the morphological description of this fungus is therefore based on characters expressed on oatmeal and malt extract agars. The phenotypic characters indicated a close relationship with the other root-inhabiting species of Cryptosporiopsis and species of the associated teleomorph genus Pezicula. This relationship was confirmed by phylogenetic analyses using sequence data of the 5.8S nuclear rDNA and flanking internal transcribed spacers. In order to facilitate recognition of this possibly under-recognized category of root inhabitants, a key to the root-inhabiting Cryptosporiopsis species based on characters in vitro is given.