A New Genus of the Microascaceae (Ascomycota) Family from a Hypersaline Lagoon in Spain and the Delimitation of the Genus Wardomyces.

The Saladas de Sástago-Bujaraloz is an endorheic and arheic complex of lagoons located in the Ebro Basin and protected by the Ramsar Convention on Wetlands. Due to the semi-arid climate of the region and the high salinity of their waters, these lagoons constitute an extreme environment. We surveyed the biodiversity of salt-tolerant and halophilic fungi residents of the Laguna de Pito, a lagoon belonging to this complex. Therefore, we collected several samples of water, sediments, and soil of the periphery. Throughout the study, we isolated 21 fungal species, including a strain morphologically related to the family Microascaceae. However, this strain did not morphologically match any of genera within this family. After an in-depth morphological characterization and phylogenetic analysis using a concatenated sequence dataset of four phylogenetically informative molecular markers (the internal transcribed spacer region (ITS) of the nuclear ribosomal DNA (nrDNA); the D1-D2 domains of the 28S gene of the nuclear ribosomal RNA (LSU); and a fragment of the translation elongation factor 1-alpha (EF-1α) and the ß-tubulin (tub2) genes), we established the new genus Dactyliodendromyces, with Dactyliodendromyces holomorphus as its species. Additionally, as a result of our taxonomic study, we reclassified the paraphyletic genus Wardomyces into three different genera: Wardomyces sensu stricto, Parawardomyces gen. nov., and Pseudowardomyces gen. nov., with Parawardomyces ovalis (formerly Wardomyces ovalis) and Pseudowardomyces humicola (formerly Wardomyces humicola) as the type species of their respective genera. Furthermore, we propose new combinations, including Parawardomyces giganteus (formerly Wardomyces giganteus) and Pseudowardomyces pulvinatus (formerly Wardomyces pulvinatus).

Diversity of Rock-Inhabiting Fungi in Tarragona Province, Spain.

Rock-inhabiting fungi (RIF) are usually extremely tolerant or extremophilic, as they can survive on natural and artificial rocks despite being exposed to stressful conditions. RIF have serious negative effects on the appearance and cohesion of rocky substrates, causing the alteration and decomposition of building materials, but also on human and animal health, as they can act as opportunistic pathogens. Their identification is therefore of great importance, especially in urban areas. In the present study, culturing techniques for isolating fungi, and a polyphasic taxonomic approach to their identification, were used to assess the diversity of micromycetes that darken the surfaces of buildings in various villages and cities in Tarragona Province (Spain). Sixty-four species of RIF belonging to forty-one genera were identified, including a new genus (Coccodomyces) and the following six new fungal species: Coccodomyces pleiosporus, Exophiala caementiphila, Exophiala multiformis, Neocatenulostroma spinulosum, Neodevriesia longicatenispora, and Paradevriesia holothallica. Thus, we have established that building materials are ecological niches where a high biodiversity of RIF can develop.

Hybrid De Novo Whole-Genome Assembly, Annotation, and Identification of Secondary Metabolite Gene Clusters in the Ex-Type Strain of Chrysosporium keratinophilum.

Chrysosporium is a polyphyletic genus belonging (mostly) to different families of the order Onygenales (Eurotiomycetes, Ascomycota). Certain species, such as Chrysosporium keratinophilum, are pathogenic for animals, including humans, but are also a source of proteolytic enzymes (mainly keratinases) potentially useful in bioremediation. However, only a few studies have been published regarding bioactive compounds, of which the production is mostly unpredictable due to the absence of high-quality genomic sequences. During the development of our study, the genome of the ex-type strain of Chrysosporium keratinophilum, CBS 104.66, was sequenced and assembled using a hybrid method. The results showed a high-quality genome of 25.4 Mbp in size spread across 25 contigs, with an N50 of 2.0 Mb, 34,824 coding sequences, 8002 protein sequences, 166 tRNAs, and 24 rRNAs. The functional annotation of the predicted proteins was performed using InterProScan, and the KEGG pathway mapping using BlastKOALA. The results identified a total of 3529 protein families and 856 superfamilies, which were classified into six levels and 23 KEGG categories. Subsequently, using DIAMOND, we identified 83 pathogen-host interactions (PHI) and 421 carbohydrate-active enzymes (CAZymes). Finally, the analysis using AntiSMASH showed that this strain has a total of 27 biosynthesis gene clusters (BGCs), suggesting that it has a great potential to produce a wide variety of secondary metabolites. This genomic information provides new knowledge that allows for a deeper understanding of the biology of C. keratinophilum, and offers valuable new information for further investigations of the Chrysosporium species and the order Onygenales.

Amesia hispanica sp. nov., Producer of the Antifungal Class of Antibiotics Dactylfungins.

During a study of the diversity of soilborne fungi from Spain, a strain belonging to the family Chaetomiaceae (Sordariales) was isolated. The multigene phylogenetic inference using five DNA loci showed that this strain represents an undescribed species of the genus Amesia, herein introduced as A. hispanica sp. nov. Investigation of its secondary metabolome led to the isolation of two new derivatives (2 and 3) of the known antifungal antibiotic dactylfungin A (1), together with the known compound cochliodinol (4). The planar structures of 1-4 were determined by ultrahigh performance liquid chromatography coupled with diode array detection and ion mobility tandem mass spectrometry (UHPLC-DAD-IM-MS/MS) and extensive 1D and 2D nuclear magnetic resonance (NMR) spectroscopy after isolation by HPLC. All isolated secondary metabolites were tested for their antimicrobial and cytotoxic activities. Dactylfungin A (1) showed selective and strong antifungal activity against some of the tested human pathogens (Aspergillus fumigatus and Cryptococcus neoformans). The additional hydroxyl group in 2 resulted in the loss of activity against C. neoformans but still retained the inhibition of As. fumigatus in a lower concentration than that of the respective control, without showing any cytotoxic effects. In contrast, 25″-dehydroxy-dactylfungin A (3) exhibited improved activity against yeasts (Schizosaccharomyces pombe and Rhodotorula glutinis) than 1 and 2, but resulted in the appearance of slight cytotoxicity. The present study exemplifies how even in a well-studied taxonomic group such as the Chaetomiaceae, the investigation of novel taxa still brings chemistry novelty, as demonstrated in this first report of this antibiotic class for chaetomiaceous and sordarialean taxa.

Novel Freshwater Ascomycetes from Spain.

Freshwater ascomycetes are a group of fungi of great ecological importance because they are involved in decomposition processes and the recycling of organic matter in aquatic ecosystems. The taxonomy of these fungi is complex, with representatives in several orders of the phylum Ascomycota. In the present study, we collected ninety-two samples of plant debris submerged in freshwater in different locations in Spain. The plant specimens were placed in wet chambers and developed several fungi that were later isolated in pure culture. A main phylogenetic tree using the nucleotide sequences of D1-D2 domains of the 28S nrRNA gene (LSU) was built to show the taxonomic placement of all our fungal strains, and, later, individual phylogenies for the different families were built using single or concatenated nucleotide sequences of the most suitable molecular markers. As a result, we found a new species of Amniculicola that produces a coelomycetous asexual state, a new species of Elongatopedicellata that produces an asexual state, a new species of Neovaginatispora that forms both sexual and asexual states in vitro, and the sexual states of two species of Pyrenochaetopsis, none of which have been reported before for these genera. In addition, we describe a new species of Pilidium characterized by the production of copper-colored globose conidiomata, and of Pseudosigmoidea, which produces well-developed conidiophores.

First Report of an Invasive Infection by Cephalotrichum gorgonifer in a Neutropenic Patient with Hematological Malignancy under Chemotherapy.

The etiological agents of infrequent invasive fungal infections (IFI) are difficult to identify on the species level using classic morphological examination. We describe the first case of an IFI caused by Cephalotrichum gorgonifer in a neutropenic patient with a hematological malignancy and put it on the map as a new causative agent of IFI. Case report, microbiological findings and description of the etiological agent. A 60-year-old man was diagnosed with mantle cell lymphoma. A CT scan confirmed the presence of lung infiltrates located at the right upper lobe. Histological examination of one of the nodules showed a large number of narrow septate hyphae with acute-angle branching and irregular round cell morphology; vessels walls appeared infiltrated, proving an angioinvasive pulmonary IFI. Sample culture resulted positive and molecular identification proved the presence of Cephalotrichum gorgonifer. Voriconazole was used for 12 months and the patient did not report any complications or side effects. Complete remission of lymphoma was achieved later by the time chemotherapy, radiotherapy, and radioimmunotherapy consolidation were completed. We recommend the inclusion of Cephalotrichum gorgonifer in the list of opportunistic pathogens causing mycoses in neutropenic hematological patients with suspected mould-related IFI.

New Dothideomycetes from Freshwater Habitats in Spain.

The Dothideomycetes are a class of cosmopolitan fungi that are present principally in terrestrial environments, but which have also been found in freshwater and marine habitats. In the present study, more than a hundred samples of plant debris were collected from various freshwater locations in Spain. Its incubation in wet chambers allowed us to detect and to isolate in pure culture numerous fungi producing asexual reproductive fruiting bodies (conidiomata). Thanks to a morphological comparison and to a phylogenetic analysis that combined the internal transcribed spacer (ITS) region of the nrDNA with fragments of the RNA polymerase II subunit 2 (rpb2), beta tubulin (tub2), and the translation elongation factor 1-alpha (tef-1) genes, six of those strains were identified as new species to science. Three belong to the family Didymellaceae: Didymella brevipilosa, Heterophoma polypusiformis and Paraboeremia clausa; and three belong to the family Phaeosphaeriaceae:Paraphoma aquatica, Phaeosphaeria fructigena and Xenophoma microspora. The finding of these new taxa significantly increases the number of the coelomycetous fungi that have been described from freshwater habitats.

Morinagadepsin, a Depsipeptide from the Fungus Morinagamyces vermicularis gen. et comb. nov.

The new genus Morinagamyces is introduced herein to accommodate the fungus Apiosordaria vermicularis as inferred from a phylogenetic study based on sequences of the internal transcribed spacer region (ITS), the nuclear rDNA large subunit (LSU), and partial fragments of ribosomal polymerase II subunit 2 (rpb2) and ß-tubulin (tub2) genes. Morinagamyces vermicularis was analyzed for the production of secondary metabolites, resulting in the isolation of a new depsipeptide named morinagadepsin (1), and the already known chaetone B (3). While the planar structure of 1 was elucidated by extensive 1D- and 2D-NMR analysis and high-resolution mass spectrometry, the absolute configuration of the building blocks Ala, Val, and Leu was determined as -l by Marfey's method. The configuration of the 3-hydroxy-2-methyldecanyl unit was assigned as 22R,23R by J-based configuration analysis and Mosher's method after partial hydrolysis of the morinagadepsin to the linear derivative compound 2. Compound 1 showed cytotoxic activity against the mammalian cell lines KB3.1 and L929, but no antimicrobial activity against the fungi and bacteria tested was observed, while 2 was inactive. Compound 3 was weakly cytotoxic against the cell line L929, but did not show any antimicrobial activity.

New Coelomycetous Fungi from Freshwater in Spain.

Coelomycetous fungi are ubiquitous in soil, sewage, and sea- and freshwater environments. However, freshwater coelomycetous fungi have been very rarely reported in the literature. Knowledge of coelomycetous fungi in freshwater habitats in Spain is poor. The incubation of plant debris, from freshwater in various places in Spain into wet chambers, allowed us to detect and isolate in pure culture several pycnidia-producing fungi. Fungal strains were phenotypically characterized, and a phylogenetic study was carried out based on the analysis of concatenated nucleotide sequences of the D1-D2 domains of the 28S nrRNA gene (LSU), the internal transcribed spacer region (ITS) of the nrDNA, and fragments of the RNA polymerase II subunit 2 (rpb2) and beta tubulin (tub2) genes. As a result of these, we report the finding of two novel species of Neocucurbitaria, three of Neopyrenochaeta, and one of Pyrenochaetopsis. Based on the phylogenetic study, we also transferred Neocucurbitaria prunicola to the genus Allocucurbitaria. This work makes an important contribution to the knowledge of the mycobiota of plant debris in freshwater habitats.

Xanthothecium peruvianum isolated from human stratum corneum: A case report, characterisation and short review that suggest emendation of Arachnomyces peruvianus.

From stratum corneum samples of a palmar eczema, a fungus was isolated that developed white colonies with a yellowish dark reverse, suggestive of dermatophytes. The isolate produced numerous chlamydospores and sparse aleuroconidia, was resistant to cycloheximide, grew well on human stratum corneum samples and was positive in tests for urease production and hair perforation, but no dermatophyte could be identified. After several weeks, cleistothecia with delicate asci and disc-shaped ascospores were formed, suggesting Arachnomyces spp. The analyses of the ribosomal ITS and LSU (D1/D2 domains) nucleotide sequences proved a good match with the ex-type strain of Xanthothecium peruvianum (family Onygenaceae, order Onygenales), and LSU sequence showed 99% similarity with Arachnomyces glareosus. This is the first report of X. peruvianum isolated from human skin. The description of our isolate provides new information about this species and proposes its transfer to the genus Arachnomyces with the subsequent emendation of the description of Arachnomyces peruvianus. Morphologically and physiologically it mimics dermatophytes and other species of the genus Arachnomyces. Although the clinical situation did not suggest any relevance for A. peruvianus as a primary pathogen, this fungus may act as a secondary pathogen under suitable conditions due to its keratinolytic capacity.

Saksenaea erythrospora, an emerging mucoralean fungus causing severe necrotizing skin and soft tissue infections - a study from a tertiary care hospital in north India.

BACKGROUND: Saksenaea erythrospora is an emerging and recently described pathogenic fungus mainly causing invasive cutaneous infections. Globally, very few human cases, caused by S. erythrospora, have been reported. In India, among the genus Saksenaea, S. vasiformis was the only reported pathogenic species, until recently when a case of fungal rhinosinusitis was reported to be caused by S. erythrospora. We observed five human cases of necrotizing skin and soft tissue infections caused by S. erythrospora following traumatic implantation over 1-year study period. METHODS: The study was conducted for a year observing the causative role of Saksenaea species in primary cutaneous necrotizing infections. The clinical entities were diagnosed by both microbiological and histopathological examination of the skin biopsies. The final identification of fungal strains was done by comparing internal transcribed spacer (ITS) and D1-D2 domains of the LSU (larger subunit) of the nuclear ribosomal RNA (rRNA) sequences with those of type strains of the different species of Saksenaea. RESULTS: Out of total 23 cases of necrotizing skin and soft tissue infections, 5 were caused by S. erythrospora. Intramuscular injection into the gluteal region was the predisposing factor in four patients, while upper limb involvement, following medicated adhesive tape application, was seen in one patient. All patients were treated with liposomal amphotericin B (LAMB) along with extensive debridement of necrotic tissues. Four patients responded well however one died. CONCLUSION: Saksenaea erythrospora is an emerging mucoralean fungus isolated in India among patients undergoing inadvertent I/M injections entailing necrotizing fasciitis at the local site.

Fungal necrotizing fasciitis, an emerging infectious disease caused by Apophysomyces (Mucorales) / Fascitis necrotizante fúngica, una enfermedad infecciosa emergente causada por Aphophysomyces (Mucorales)

Background: The mucoralean fungi are emerging causative agents of primary cutaneous infections presenting in the form of necrotizing fasciitis. Aims: The aim of this study was to investigate a series of suspected necrotizing fasciitis cases by Apophysomycesspecies over one-year period in a northern Indian hospital. Methods: The clinical details of those patients suspected to suffer from fungal necrotizing fasciitis were recorded. Skin biopsies from local wounds were microscopically examined and fungal culturing was carried out on standard media. The histopathology was evaluated using conventional methods and special stains.Apophysomyces isolates were identified by their morphology and by molecular sequencing of the internal transcribed spacer (ITS) region of the ribosomal genes. Antifungal susceptibility testing was carried out following EUCAST guidelines and treatment progress was monitored. Results: Seven patients were found to be suffering from necrotizing fasciitis caused by Apophysomyces spp. Six isolates were identified as Apophysomyces variabilis and one as Apophysomyces elegans. Five patients had previously received intramuscular injections in the affected area. Three patients recovered, two died and the other two left treatment against medical advice and are presumed to have died due to their terminal illnesses. Posaconazole and terbinafine were found to be the most active compounds against A. variabilis, while the isolate of A. elegans was resistant to all antifungals tested. Conclusions: Apophysomyces is confirmed as an aggressive fungus able to cause fatal infections. All clinicians, microbiologists and pathologists need to be aware of these emerging mycoses as well as of the risks involved in medical practices, which may provoke serious fungal infections such as those produced byApophysomyces (AU)

Antecedentes: Los hongos mucorales son agentes emergentes causantes de infecciones cutáneas primarias presentes en forma de fascitis necrotizante. Objetivos: La finalidad de este estudio fue la de investigar una serie de infecciones sugestivas de fascitis necrotizante causadas por alguna de las especies de Apophysomyces a lo largo de un año en un hospital del norte de la India. Métodos: Se obtuvieron los datos de todos los pacientes con sospecha de fascistis necrotizante. Las biopsias de piel de la zona afectada fueron cultivadas en medios de cultivos estándar y se evaluaron histopatológicamente mediante tinciones convencionales y específicas para hongos. Los aislamientos de Apophysomyces fueron identificados morfológicamente y mediante la secuenciación del espaciador intergénico ribosomal (ITS). La sensibilidad antifúngica se determinó mediante el método EUCAST y la evolución de los pacientes fue monitorizada. Resultados: Se encontraron siete pacientes con fascitis necrotizante causada por especies de Apophysomyces. Seis aislamientos fueron identificados como Apophysomyces variabilis y uno como Apophysomyces elegans. Cinco pacientes habían recibido previamente inyecciones intramusculares en el área afectada. Tres pacientes se recuperaron, dos fallecieron y de los dos restantes no se tiene seguimiento médico, aunque presumiblemente fallecieron debido a que padecían enfermedades terminales. El posaconazol y la terbinafina fueron los compuestos más activos frente a A. variabilis, mientras que el único aislamiento deA. elegans fue resistente a todos los antifúngicos ensayados. Conclusiones: Se confirma que Apophysomyces es un hongo agresivo capaz de causar infecciones con desenlace fatal. Clínicos, microbiólogos y patólogos deben ser conscientes de los riesgos de estas micosis emergentes y de que determinadas prácticas médicas puedan provocar infecciones fúngicas graves como las producidas por Apophysomyces (AU)

Fungal necrotizing fasciitis, an emerging infectious disease caused by Apophysomyces (Mucorales).

BACKGROUND: The mucoralean fungi are emerging causative agents of primary cutaneous infections presenting in the form of necrotizing fasciitis. AIMS: The aim of this study was to investigate a series of suspected necrotizing fasciitis cases by Apophysomyces species over one-year period in a northern Indian hospital. METHODS: The clinical details of those patients suspected to suffer from fungal necrotizing fasciitis were recorded. Skin biopsies from local wounds were microscopically examined and fungal culturing was carried out on standard media. The histopathology was evaluated using conventional methods and special stains. Apophysomyces isolates were identified by their morphology and by molecular sequencing of the internal transcribed spacer (ITS) region of the ribosomal genes. Antifungal susceptibility testing was carried out following EUCAST guidelines and treatment progress was monitored. RESULTS: Seven patients were found to be suffering from necrotizing fasciitis caused by Apophysomyces spp. Six isolates were identified as Apophysomyces variabilis and one as Apophysomyces elegans. Five patients had previously received intramuscular injections in the affected area. Three patients recovered, two died and the other two left treatment against medical advice and are presumed to have died due to their terminal illnesses. Posaconazole and terbinafine were found to be the most active compounds against A. variabilis, while the isolate of A. elegans was resistant to all antifungals tested. CONCLUSIONS: Apophysomyces is confirmed as an aggressive fungus able to cause fatal infections. All clinicians, microbiologists and pathologists need to be aware of these emerging mycoses as well as of the risks involved in medical practices, which may provoke serious fungal infections such as those produced by Apophysomyces.

Leiothecium cristatum sp. nov. and Aspergillus posadasensis sp. nov., two species of Eurotiales from rainforest soils in South America.

We describe two novel fungi isolated from soil samples collected in Northern Argentina and belonging to the family Aspergillaceae of the order Eurotiales: Leiothecium cristatum sp. nov. and Aspergillus posadasensis sp. nov. Leiothecium cristatum sp. nov., represented by the ex-type strain FMR 11998(T) ( = CBS 134260(T) = NBRC 109843(T)), is distinguishable morphologically from the type species of the genus, Leiothecium ellipsoideum, by the presence of irregular reticulate ascospores with two prominent equatorial crests, and Aspergillus posadasensis sp. nov., represented by the ex-type strain FMR 12168(T) ( = CBS 134259(T) = NBRC 109845(T)), is differentiated from Aspergillus acanthosporus, the nearest species phylogenetically, by its non-sclerotioid ascomata and a lack of an asexual stage on all culture media tested. The taxonomic proposals are supported by the analysis of the sequences of the internal transcribed spacer region, the D1-D2 domains of the 28S rRNA gene, the fragments of the RNA polymerase II largest subunit, and the putative chaperonin complex related to TCP-1, ß-tubulin and calmodulin genes.

Genus Hamigera, six new species and multilocus DNA sequence based phylogeny.

Genus Hamigera was erected for Talaro-myces species that make asci singly instead of in chains. Initially it contained two species, H. avellanea and H. striata. We describe six new species in the genus, H. fusca, H. inflata, H. insecticola, H. pallida, H. paravellanea and H. terricola. Merimbla ingelheimensis is a distinct anamorphic species in the Hamigera clade. None of our DNA sequence data (BT2, calmodulin, ITS, 1su rDNA, RPB2, Tsr1 and Mcm7) supported the placement of H. striata in the same clade as H. avellanea, thus we accepted Talaromyces striatus. In addition to Hamigera species we examined the phylogenetic disposition of Warcupiella spinulosa, Penicillium megasporum, Penicillium arenicola and Merimbla humicoloides. Despite nominal similarity of some of these species to Merimbla, none of these species are part of the Hamigera clade and M. humicoloides is placed in Penicillium to have a monophyletic genus Hamigera.