Proteus anguinus is a neotenic cave salamander, endemic to the Dinaric Karst and a symbol of worl... more Proteus anguinus is a neotenic cave salamander, endemic to the Dinaric Karst and a symbol of world natural heritage. It is classified as “vulnerable” by the International Union for Conservation of Nature (IUCN) and is one of the EU priority species in need of strict protection. Due to inaccessibility of their natural underground habitat, scientific studies of the olm have been conducted mainly in captivity, where the amphibians are particularly susceptible to opportunistic microbial infections. In this report, we focused on the diversity of cultivable commensal fungi isolated from the skin of asymptomatic and symptomatic animals obtained from nature (20 specimens) and captivity (22 specimens), as well as from underground water of two karstic caves by direct water filtration and by exposure of keratin-based microbial baits and subsequent isolation from them. In total 244 fungal isolates were recovered from the animals and additional 153 isolates were obtained from water samples. Toge...
Heavily pigmented glacier ice algae Ancylonema nordenskiöldii and Ancylonema alaskanum (Zygnemato... more Heavily pigmented glacier ice algae Ancylonema nordenskiöldii and Ancylonema alaskanum (Zygnematophyceae, Streptophyta) reduce the bare ice albedo of the Greenland Ice Sheet, amplifying melt from the largest cryospheric contributor to eustatic sea-level rise. Little information is available about glacier ice algae interactions with other microbial communities within the surface ice environment, including fungi, which may be important for sustaining algal bloom development. To address this substantial knowledge gap and investigate the nature of algal-fungal interactions, an ex situ co-cultivation experiment with two species of fungi, recently isolated from the surface of the Greenland Ice Sheet (here proposed new species Penicillium anthracinoglaciei Perini, Frisvad and Zalar, Mycobank (MB 835602), and Articulospora sp.), and the mixed microbial community dominated by glacier ice algae was performed. The utilization of the dark pigment purpurogallin carboxylic acid-6-O-β-D-glucopyran...
The novel extremophilic yeast Rhodotorula frigidialcoholis, formerly R. JG1b, was isolated from i... more The novel extremophilic yeast Rhodotorula frigidialcoholis, formerly R. JG1b, was isolated from ice-cemented permafrost in University Valley (Antarctic), one of coldest and driest environments on Earth. Phenotypic and phylogenetic analyses classified R. frigidialcoholis as a novel species. To characterize its cold-adaptive strategies, we performed mRNA and sRNA transcriptomic analyses, phenotypic profiling, and assessed ethanol production at 0 and 23 °C. Downregulation of the ETC and citrate cycle genes, overexpression of fermentation and pentose phosphate pathways genes, growth without reduction of tetrazolium dye, and our discovery of ethanol production at 0 °C indicate that R. frigidialcoholis induces a metabolic switch from respiration to ethanol fermentation as adaptation in Antarctic permafrost. This is the first report of microbial ethanol fermentation utilized as the major energy pathway in response to cold and the coldest temperature reported for natural ethanol production....
For a long time, halotolerant yeasts were known exclusively as contaminants of food preserved wit... more For a long time, halotolerant yeasts were known exclusively as contaminants of food preserved with high concentrations of salt or sugar. Their presence in natural thalassohaline hypersaline environments was unknown until 2000, when they were first reported to be active inhabitants of man-made solar salterns in Slovenia. Since then, they have been described on the surface of halophytic plants, in salt mines, in cold and temperate saline lakes, in brine and bittern of different solar salterns on three continents and in MgCl2-dominated waters of the Dead Sea. Yeasts in these environments can be described as halotolerant, extremely halotolerant and even extremely chaotolerant. The dominant representatives are different ascomycetous black yeast species, mainly of the genera Hortaea and Phaeotheca; non-melanised ascomycetous yeasts from the genera Candida, Debaryomyces, Meyerozyma, Metschnikowia, Pichia and Yarrowia; and basidiomycetous yeasts from the genera Bulleromyces, Cryptococcus, Cutaneotrichosporon, Papiliotrema, Rhodosporidium, Rhodotorula, Solicoccozyma, Sterigmatomyces and Vishniacozyma. Until the discovery and description of indigenous saltern mycobiota, the physiological and molecular mechanisms relating to salt tolerance in eukaryotic microorganisms were studied using salt-sensitive Saccharomyces cerevisiae as the model organism. Nowadays, most studies focus on halotolerant yeast species like Debaryomyces hansenii, Aureobasidium pullulans and Hortaea werneckii, which have been isolated globally from natural hypersaline environments and can tolerate up to 10%, 15% and 30% NaCl, respectively. Studies of halotolerant yeasts at the molecular level continue to unravel the complexity of the adaptations needed for yeasts to cope with the problems of ion toxicity and low water activity that are characteristic of hypersaline environments.
1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been com... more 1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been commonly drawn from halotolerant strains and few studies in basidiomycete fungi. These studies have been conducted in settings where cells are subjected to stress, either hypo-or hyperosmotic, which can be a confounding factor in describing physiological mechanisms related to salinity. (2) Methods: We have studied transcriptomic changes in Aspergillus sydowii, a halophilic species, when growing in three different salinity conditions (No NaCl, 0.5 M, and 2.0 M NaCl). (3) Results: In this fungus, major physiological modifications occur under high salinity (2.0 M NaCl) and not when cultured under optimal conditions (0.5 M NaCl), suggesting that most of the mechanisms described for halophilic growth are a consequence of saline stress response and not an adaptation to saline conditions. Cell wall modifications occur exclusively at extreme salinity, with an increase in cell wall thickness and lamellar structure, which seem to involve a decrease in chitin content and an augmented content of alfa and beta-glucans. Additionally, three hydrophobin genes were differentially expressed under hypo-or hyperosmotic stress but not when the fungus grows optimally. Regarding compatible solutes, glycerol is the main compound accumulated in salt stress conditions, whereas trehalose is accumulated in the absence of salt. (4) Conclusions: Physiological responses to salinity vary greatly between optimal and high salt concentrations and are not a simple graded effect as the salt concentration increases. Our results highlight the influence of stress in reshaping the response of extremophiles to environmental challenges.
Aspergillus section Restricti together with sister section Aspergillus (formerly Eurotium) compri... more Aspergillus section Restricti together with sister section Aspergillus (formerly Eurotium) comprises xerophilic species, that are able to grow on substrates with low water activity and in extreme environments. We adressed the monophyly of both sections within subgenus Aspergillus and applied a multidisciplinary approach for definition of species boundaries in sect. Restricti. The monophyly of sections Aspergillus and Restricti was tested on a set of 102 isolates comprising all currently accepted species and was strongly supported by Maximum likelihood (ML) and Bayesian inferrence (BI) analysis based on β-tubulin (benA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) loci. More than 300 strains belonging to sect. Restricti from various isolation sources and four continents were characterized by DNA sequencing, and 193 isolates were selected for phylogenetic analyses and phenotypic studies. Species delimitation methods based on multispecies coalescent model were employed on DNA sequences from four loci, i.e., ID region of rDNA (ITS + 28S), CaM, benA and RPB2, and supported recognition of 21 species, including 14 new. All these species were also strongly supported in ML and BI analyses. All recognised species can be reliably identified by all four examined genetic loci. Phenotype analysis was performed to support the delimitation of new species and includes colony characteristics on seven cultivation media incubated at several temperatures, growth on an osmotic gradient (six media with NaCl concentration from 0 to 25 %) and analysis of morphology including scanning electron microscopy. The micromorphology of conidial heads, vesicle dimensions, temperature profiles and growth parameters in osmotic gradient were useful criteria for species identification. The vast majority of species in sect. Restricti produce asperglaucide, asperphenamate or both in contrast to species in sect. Aspergillus. Mycophenolic acid was detected for the first time in at least six members of the section. The ascomata of A. halophilicus do not contain auroglaucin, epiheveadride or flavoglaucin which are common in sect. Aspergillus, but shares the echinulins with sect. Aspergillus.
Cellular Origin, Life in Extreme Habitats and Astrobiology
... Page 426. 410 NINA GUNDE-CIMERMAN ET AL. ... The latter was obtained from sea water, sea and ... more ... Page 426. 410 NINA GUNDE-CIMERMAN ET AL. ... The latter was obtained from sea water, sea and glacier ice. In hypersaline waste water ponds in Israel it represeted one of the two dominant microorganisms (Lahav et al., 2002). ...
Saprobic Cladosporium isolates morphologically similar to C. sphaerospermum are phylogenetically ... more Saprobic Cladosporium isolates morphologically similar to C. sphaerospermum are phylogenetically analysed on the basis of DNA sequences of the ribosomal RNA gene cluster, including the internal transcribed spacer regions ITS1 and ITS2, the 5.8S rDNA (ITS) and the small subunit (SSU) rDNA as well as β-tubulin and actin gene introns and exons. Most of the C. sphaerospermum-like species show halotolerance as a recurrent feature. Cladosporium sphaerospermum, which is characterised by almost globose conidia, is redefined on the basis of its ex-neotype culture. Cladosporium dominicanum, C. psychrotolerans, C. velox, C. spinulosum and C. halotolerans, all with globoid conidia, are newly described on the basis of phylogenetic analyses and cryptic morphological and physiological characters. Cladosporium halotolerans was isolated from hypersaline water and bathrooms and detected once on dolphin skin. Cladosporium dominicanum and C. velox were isolated from plant material and hypersaline water. Cladosporium psychrotolerans, which grows well at 4 °C but not at 30 °C, and C. spinulosum, having conspicuously ornamented conidia with long digitate projections, are currently only known from hypersaline water. We also newly describe C. salinae from hypersaline water and C. fusiforme from hypersaline water and animal feed. Both species have ovoid to ellipsoid conidia and are therefore reminiscent of C. herbarum. Cladosporium langeronii (= Hormodendrum langeronii) previously described as a pathogen on human skin, is halotolerant but has not yet been recorded from hypersaline environments.
The Cladosporium herbarum complex comprises five species for which Davidiella teleomorphs are kno... more The Cladosporium herbarum complex comprises five species for which Davidiella teleomorphs are known. Cladosporium herbarum s. str. (D. tassiana), C. macrocarpum (D. macrocarpa) and C. bruhnei (D. allicina) are distinguishable by having conidia of different width, and by teleomorph characters. Davidiella variabile is introduced as teleomorph of C. variabile, a homothallic species occurring on Spinacia, and D. macrospora is known to be the teleomorph of C. iridis on Iris spp. The C. herbarum complex combines low molecular distance with a high degree of clonal or inbreeding diversity. Entities differ from each other by multilocus sequence data and by phenetic differences, and thus can be interpreted to represent individual taxa. Isolates of the C. herbarum complex that were formerly associated with opportunistic human infections, cluster with C. bruhnei. Several species are newly described from hypersaline water, namely C. ramotenellum, C. tenellum, C. subinflatum, and C. herbaroides. Cladosporium pseudiridis collected from Iris sp. in New Zealand, is also a member of this species complex and shown to be distinct from C. iridis that occurs on this host elsewhere in the world. A further new species from New Zealand is C. sinuosum on Fuchsia excorticata. Cladosporium antarcticum is newly described from a lichen, Caloplaca regalis, collected in Antarctica, and C. subtilissimum from grape berries in the U.S.A., while the new combination C. ossifragi, the oldest valid name of the Cladosporium known from Narthecium in Europe, is proposed. Standard protocols and media are herewith proposed to facilitate future morphological examination of Cladosporium spp. in culture, and neotypes or epitypes are proposed for all species treated.
Physics and Chemistry of the Earth, Parts A/B/C, 2003
Little is known about fungal diversity in extremely cold regions. Low temperatures induce the for... more Little is known about fungal diversity in extremely cold regions. Low temperatures induce the formation of ice crystals and therefore also the creation of low water activity (a w ). These are the dominant factors in external chemistry that influence microbial biota in cold regions. Therefore, we have used selective low water activity media plus low incubation temperatures for the isolation of fungi from an Arctic environment. In comparison with the highest values of colony forming units (CFU) obtained on mesophilic media, considerably higher fungal CFU per litre of water were detected on low a w media, ranging from 1000 to 3000 l À1 in seawater, 6000 to 7000 l À1 in melted sea ice and up to 13,000 l À1 in melted glacier ice. The dominant taxa were ascomycetous and basidiomycetous yeasts, melanized fungi, mainly represented by the genera Cladosporium and Aureobasidium plus different species of the genus Penicillium. Preliminary taxonomic analyses revealed several new species and varieties. Further characterisations are needed to determine whether this diversity is due to geographic isolation, ecological conditions or independent evolutionary origin.
The aim of the present study was to investigate indigenous fungal communities isolated from extre... more The aim of the present study was to investigate indigenous fungal communities isolated from extreme environments (hypersaline waters of solar salterns and subglacial ice), for the production of metabolic compounds with selected biological activities: hemolysis, antibacterial, and acetylcholinesterase inhibition. In their natural habitats, the selected fungi are exposed to environmental extremes, and therefore the production of bioactive metabolites was tested under both standard growth conditions for mesophilic microorganisms, and at high NaCl and sugar concentrations and low growth temperatures. The results indicate that selected halotolerant and halophilic species synthesize specific bioactive metabolites under conditions that represent stress for non-adapted species. Furthermore, adaptation at the level of the chemical nature of the solute lowering the water activity of the medium was observed. Increased salt concentrations resulted in higher hemolytic activity, particularly within species dominating the salterns. The appearance of antibacterial potential under stress conditions was seen in the similar pattern of fungal species as for hemolysis. The active extracts exclusively affected the growth of the Gram-positive bacterium tested, Bacillus subtilis. None of the extracts tested showed inhibition of acetylcholinesterase activity.
Cryptococcus species (Basidiomycota) were isolated as the predominant yeast from glacial biomes o... more Cryptococcus species (Basidiomycota) were isolated as the predominant yeast from glacial biomes of both Patagonia (Argentina) and the Svalbard archipelago (Norway). For a selected group of Cryptococcus belonging to Tremellales, assimilative profile, production of extracellular hydrolytic enzymes and ribosomal DNA internal transcribed spacer and large subunit (D1/D2) sequences were analysed. Cryptococcus victoriae, which was originally described from Antarctica, was the most frequently found species at both locations. High variability within the species was observed and described at the genotypic and phenotypic levels, two newly described species were found in both Patagonia and Svalbard: Cryptococcus fonsecae and Cryptococcus psychrotolerans. Two other new species were found only in Patagonia: Cryptococcus frias and Cryptococcus tronadorensis. Three additional new taxa were found, but they are not named as they were only represented by single isolates.
The Wallemiomycetes includes three species of molds from the genus Wallemia . These fungi are ada... more The Wallemiomycetes includes three species of molds from the genus Wallemia . These fungi are adapted to environments of high osmotic stress, contaminate various foods, cause respiratory disease, and have an unusual mode of asexual reproduction. Wallemia was recently proposed as a new class based on 18S ribosomal RNA gene sequences to accommodate the isolated position of the clade in the Basidiomycota. We analyzed the phylogenetic position of the Wallemiomycetes using 3451 nucleotide characters of the 18S, 25S, and 5.8S ribosomal RNA genes and 1282 amino acid positions of rpb1, rpb2, and tef1 nuclear protein-coding genes across 91 taxa. Different gene regions and methods of phylogenetic inference produce mildly conflicting placements of the Wallemiomycetes. Parsimony analyses of nrDNA data suggest that the Wallemiomycetes is an early diverging lineage of Basidiomycota, occupying a basal position near the Entorrhizomycetidae. Ultrastructural data, some Bayesian analyses, and amino ac...
Until recently, it was believed that microbial communities at high salinities are dominated exclu... more Until recently, it was believed that microbial communities at high salinities are dominated exclusively by Archaea and Bacteria and one eukaryotic species, the alga Dunaliella salina. Recently, it became evident that melanized fungi, so far described only in the crystallization pond of Adriatic salterns within the season of salt production, can be considered as a new group of eukaryotic halophiles. They were represented by black, yeast-like hyphomycetes: Hortaea werneckii, Phaeotheca triangularis, Trimmatostroma salinum, Aureobasidium pullulans, together with phylogenetically closely related Cladosporium species. In the present study, the distribution of the melanized fungal population in five different evaporating ponds in the Adriatic salterns wcovering the entire salinity range (3-30% NaCl)x was followed throughout the year. It appeared in three peaks, at 5-8%, 10-20% and 18-25% NaCl, which correlated primarily with high nitrogen values. At the highest environmental salinities, melanized fungi represented 85-100% of the total isolated mycobiota, but with lowering salinities they were partially replaced by non-melanized fungi and, at the end of the season, with NaCl concentrations below 5%, they were detected only occasionally. Melanized fungi have been isolated from hypersaline waters on three continents, indicating that they are present globally in hypersaline waters of man-made salterns.
Summary For a long time halotolerant and halophilic fungi have been known exclusively as contamin... more Summary For a long time halotolerant and halophilic fungi have been known exclusively as contaminants of food preserved with high concentrations of either salt or sugar. They were first reported in 2000 to be active inhabitants of hypersaline environments, when they were found in man-made solar salterns in Slovenia. Since then, they have been described in different salterns and salt lakes on three continents. The mycobiota that inhabit these natural hypersaline environments are composed of phylogenetically unrelated halotolerant, extremely halotolerant, and halophilic fungi, which are represented not only by species previously known only as food contaminants, but also by new and rare species. The dominant representatives are different species of black yeast-like and related melanized fungi of the genus Cladosporium, different species within the anamorphic Aspergillus and Penicillium, and the teleomorphic Emericella and Eurotium, certain species of non-melanized yeasts, and Wallemia ...
Lichenized fungi usually develop complex, stratified morphologies through an intricately balanced... more Lichenized fungi usually develop complex, stratified morphologies through an intricately balanced living together with their algal partners, but several species are known to form only more or less loose associations with algae. These borderline lichens are still little explored although they could inform us about early stages of lichen evolution. We studied the association of the extremely halotolerant fungus Hortaea werneckii with the alga Dunaliella atacamensis, discovered in a cave in the Atacama Desert (Chile), and with D. salina, common inhabitant of saltern brines. D. atacamensis forms small colonies, in which cells of H. werneckii can be frequently observed, while such interaction has not been observed with D. salina. As symbiotic interactions between Dunaliella and Hortaea have not been reported, we performed a series of co-cultivation experiments to inspect whether these species could interact and develop more distinct lichen-like symbiotic structures. We set up co-cultures...
The polymorphic black yeast Hortaea werneckii (Capnodiales, Ascomycota) is extremely halotolerant... more The polymorphic black yeast Hortaea werneckii (Capnodiales, Ascomycota) is extremely halotolerant (growth from 0 to 30% [w/v] NaCl) and has been extensively studied as a model for halotolerance in Eukaryotes for over two decades. Its most frequent sources are hypersaline environments and adjacent seawater habitats in temperate, subtropical and tropical climates. Although typically saprobic, H. werneckii can also act as a commensal coloniser on human skin, causing tinea nigra on hands and soles. Here, we report that addition of NaCl to culture media expands the growth range of H. werneckii to 37°C, which explains its colonisation of human skin, with its increased salinity. The morphological and physiological plasticity/ versatility of H. werneckii indicate that a species complex might be involved. This was investigated in this polyphasic taxonomic analysis based on the global diversity of H. werneckii strains collected from hypersaline environments, and from humans and animals. Analysis of D1/ D2domains of 28S and internal transcribed spacer rDNA revealed 10 and 17 genotypes, respectively, that were not always compliant. The genotypes have global distributions. Human and environmental strains with the same genotypes are intermingled. Due to the limited number of phylogenetically informative characters in the ribosomal DNA dataset, the partial genes encoding for β-tubulin (BTB) and mini-chromosome maintenance protein (MCM7) were also sequenced. The use of these genes was hampered by ambiguous sequences obtained by Sanger sequencing, as a consequence of the diploid and highly heterozygous genome of many H. werneckii strains. Analysis of the BTB and MCM7 genes showed that in some cases two copies of the gene from the same genome are positioned in distant phylogenetic clusters of the intraspecific gene tree. Analysis of whole-genome sequences of selected H. werneckii strains generally confirmed the phylogenetic distances estimated on the basis of ribosomal genes, but also showed substantial reticulation within the phylogenetic history of the strains. This is in line with the hypothesis that the diploid genomes of H. werneckii were formed by hybridizations, which have sometimes occurred between relatively divergent strains.
Proteus anguinus is a neotenic cave salamander, endemic to the Dinaric Karst and a symbol of worl... more Proteus anguinus is a neotenic cave salamander, endemic to the Dinaric Karst and a symbol of world natural heritage. It is classified as “vulnerable” by the International Union for Conservation of Nature (IUCN) and is one of the EU priority species in need of strict protection. Due to inaccessibility of their natural underground habitat, scientific studies of the olm have been conducted mainly in captivity, where the amphibians are particularly susceptible to opportunistic microbial infections. In this report, we focused on the diversity of cultivable commensal fungi isolated from the skin of asymptomatic and symptomatic animals obtained from nature (20 specimens) and captivity (22 specimens), as well as from underground water of two karstic caves by direct water filtration and by exposure of keratin-based microbial baits and subsequent isolation from them. In total 244 fungal isolates were recovered from the animals and additional 153 isolates were obtained from water samples. Toge...
Heavily pigmented glacier ice algae Ancylonema nordenskiöldii and Ancylonema alaskanum (Zygnemato... more Heavily pigmented glacier ice algae Ancylonema nordenskiöldii and Ancylonema alaskanum (Zygnematophyceae, Streptophyta) reduce the bare ice albedo of the Greenland Ice Sheet, amplifying melt from the largest cryospheric contributor to eustatic sea-level rise. Little information is available about glacier ice algae interactions with other microbial communities within the surface ice environment, including fungi, which may be important for sustaining algal bloom development. To address this substantial knowledge gap and investigate the nature of algal-fungal interactions, an ex situ co-cultivation experiment with two species of fungi, recently isolated from the surface of the Greenland Ice Sheet (here proposed new species Penicillium anthracinoglaciei Perini, Frisvad and Zalar, Mycobank (MB 835602), and Articulospora sp.), and the mixed microbial community dominated by glacier ice algae was performed. The utilization of the dark pigment purpurogallin carboxylic acid-6-O-β-D-glucopyran...
The novel extremophilic yeast Rhodotorula frigidialcoholis, formerly R. JG1b, was isolated from i... more The novel extremophilic yeast Rhodotorula frigidialcoholis, formerly R. JG1b, was isolated from ice-cemented permafrost in University Valley (Antarctic), one of coldest and driest environments on Earth. Phenotypic and phylogenetic analyses classified R. frigidialcoholis as a novel species. To characterize its cold-adaptive strategies, we performed mRNA and sRNA transcriptomic analyses, phenotypic profiling, and assessed ethanol production at 0 and 23 °C. Downregulation of the ETC and citrate cycle genes, overexpression of fermentation and pentose phosphate pathways genes, growth without reduction of tetrazolium dye, and our discovery of ethanol production at 0 °C indicate that R. frigidialcoholis induces a metabolic switch from respiration to ethanol fermentation as adaptation in Antarctic permafrost. This is the first report of microbial ethanol fermentation utilized as the major energy pathway in response to cold and the coldest temperature reported for natural ethanol production....
For a long time, halotolerant yeasts were known exclusively as contaminants of food preserved wit... more For a long time, halotolerant yeasts were known exclusively as contaminants of food preserved with high concentrations of salt or sugar. Their presence in natural thalassohaline hypersaline environments was unknown until 2000, when they were first reported to be active inhabitants of man-made solar salterns in Slovenia. Since then, they have been described on the surface of halophytic plants, in salt mines, in cold and temperate saline lakes, in brine and bittern of different solar salterns on three continents and in MgCl2-dominated waters of the Dead Sea. Yeasts in these environments can be described as halotolerant, extremely halotolerant and even extremely chaotolerant. The dominant representatives are different ascomycetous black yeast species, mainly of the genera Hortaea and Phaeotheca; non-melanised ascomycetous yeasts from the genera Candida, Debaryomyces, Meyerozyma, Metschnikowia, Pichia and Yarrowia; and basidiomycetous yeasts from the genera Bulleromyces, Cryptococcus, Cutaneotrichosporon, Papiliotrema, Rhodosporidium, Rhodotorula, Solicoccozyma, Sterigmatomyces and Vishniacozyma. Until the discovery and description of indigenous saltern mycobiota, the physiological and molecular mechanisms relating to salt tolerance in eukaryotic microorganisms were studied using salt-sensitive Saccharomyces cerevisiae as the model organism. Nowadays, most studies focus on halotolerant yeast species like Debaryomyces hansenii, Aureobasidium pullulans and Hortaea werneckii, which have been isolated globally from natural hypersaline environments and can tolerate up to 10%, 15% and 30% NaCl, respectively. Studies of halotolerant yeasts at the molecular level continue to unravel the complexity of the adaptations needed for yeasts to cope with the problems of ion toxicity and low water activity that are characteristic of hypersaline environments.
1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been com... more 1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been commonly drawn from halotolerant strains and few studies in basidiomycete fungi. These studies have been conducted in settings where cells are subjected to stress, either hypo-or hyperosmotic, which can be a confounding factor in describing physiological mechanisms related to salinity. (2) Methods: We have studied transcriptomic changes in Aspergillus sydowii, a halophilic species, when growing in three different salinity conditions (No NaCl, 0.5 M, and 2.0 M NaCl). (3) Results: In this fungus, major physiological modifications occur under high salinity (2.0 M NaCl) and not when cultured under optimal conditions (0.5 M NaCl), suggesting that most of the mechanisms described for halophilic growth are a consequence of saline stress response and not an adaptation to saline conditions. Cell wall modifications occur exclusively at extreme salinity, with an increase in cell wall thickness and lamellar structure, which seem to involve a decrease in chitin content and an augmented content of alfa and beta-glucans. Additionally, three hydrophobin genes were differentially expressed under hypo-or hyperosmotic stress but not when the fungus grows optimally. Regarding compatible solutes, glycerol is the main compound accumulated in salt stress conditions, whereas trehalose is accumulated in the absence of salt. (4) Conclusions: Physiological responses to salinity vary greatly between optimal and high salt concentrations and are not a simple graded effect as the salt concentration increases. Our results highlight the influence of stress in reshaping the response of extremophiles to environmental challenges.
Aspergillus section Restricti together with sister section Aspergillus (formerly Eurotium) compri... more Aspergillus section Restricti together with sister section Aspergillus (formerly Eurotium) comprises xerophilic species, that are able to grow on substrates with low water activity and in extreme environments. We adressed the monophyly of both sections within subgenus Aspergillus and applied a multidisciplinary approach for definition of species boundaries in sect. Restricti. The monophyly of sections Aspergillus and Restricti was tested on a set of 102 isolates comprising all currently accepted species and was strongly supported by Maximum likelihood (ML) and Bayesian inferrence (BI) analysis based on β-tubulin (benA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) loci. More than 300 strains belonging to sect. Restricti from various isolation sources and four continents were characterized by DNA sequencing, and 193 isolates were selected for phylogenetic analyses and phenotypic studies. Species delimitation methods based on multispecies coalescent model were employed on DNA sequences from four loci, i.e., ID region of rDNA (ITS + 28S), CaM, benA and RPB2, and supported recognition of 21 species, including 14 new. All these species were also strongly supported in ML and BI analyses. All recognised species can be reliably identified by all four examined genetic loci. Phenotype analysis was performed to support the delimitation of new species and includes colony characteristics on seven cultivation media incubated at several temperatures, growth on an osmotic gradient (six media with NaCl concentration from 0 to 25 %) and analysis of morphology including scanning electron microscopy. The micromorphology of conidial heads, vesicle dimensions, temperature profiles and growth parameters in osmotic gradient were useful criteria for species identification. The vast majority of species in sect. Restricti produce asperglaucide, asperphenamate or both in contrast to species in sect. Aspergillus. Mycophenolic acid was detected for the first time in at least six members of the section. The ascomata of A. halophilicus do not contain auroglaucin, epiheveadride or flavoglaucin which are common in sect. Aspergillus, but shares the echinulins with sect. Aspergillus.
Cellular Origin, Life in Extreme Habitats and Astrobiology
... Page 426. 410 NINA GUNDE-CIMERMAN ET AL. ... The latter was obtained from sea water, sea and ... more ... Page 426. 410 NINA GUNDE-CIMERMAN ET AL. ... The latter was obtained from sea water, sea and glacier ice. In hypersaline waste water ponds in Israel it represeted one of the two dominant microorganisms (Lahav et al., 2002). ...
Saprobic Cladosporium isolates morphologically similar to C. sphaerospermum are phylogenetically ... more Saprobic Cladosporium isolates morphologically similar to C. sphaerospermum are phylogenetically analysed on the basis of DNA sequences of the ribosomal RNA gene cluster, including the internal transcribed spacer regions ITS1 and ITS2, the 5.8S rDNA (ITS) and the small subunit (SSU) rDNA as well as β-tubulin and actin gene introns and exons. Most of the C. sphaerospermum-like species show halotolerance as a recurrent feature. Cladosporium sphaerospermum, which is characterised by almost globose conidia, is redefined on the basis of its ex-neotype culture. Cladosporium dominicanum, C. psychrotolerans, C. velox, C. spinulosum and C. halotolerans, all with globoid conidia, are newly described on the basis of phylogenetic analyses and cryptic morphological and physiological characters. Cladosporium halotolerans was isolated from hypersaline water and bathrooms and detected once on dolphin skin. Cladosporium dominicanum and C. velox were isolated from plant material and hypersaline water. Cladosporium psychrotolerans, which grows well at 4 °C but not at 30 °C, and C. spinulosum, having conspicuously ornamented conidia with long digitate projections, are currently only known from hypersaline water. We also newly describe C. salinae from hypersaline water and C. fusiforme from hypersaline water and animal feed. Both species have ovoid to ellipsoid conidia and are therefore reminiscent of C. herbarum. Cladosporium langeronii (= Hormodendrum langeronii) previously described as a pathogen on human skin, is halotolerant but has not yet been recorded from hypersaline environments.
The Cladosporium herbarum complex comprises five species for which Davidiella teleomorphs are kno... more The Cladosporium herbarum complex comprises five species for which Davidiella teleomorphs are known. Cladosporium herbarum s. str. (D. tassiana), C. macrocarpum (D. macrocarpa) and C. bruhnei (D. allicina) are distinguishable by having conidia of different width, and by teleomorph characters. Davidiella variabile is introduced as teleomorph of C. variabile, a homothallic species occurring on Spinacia, and D. macrospora is known to be the teleomorph of C. iridis on Iris spp. The C. herbarum complex combines low molecular distance with a high degree of clonal or inbreeding diversity. Entities differ from each other by multilocus sequence data and by phenetic differences, and thus can be interpreted to represent individual taxa. Isolates of the C. herbarum complex that were formerly associated with opportunistic human infections, cluster with C. bruhnei. Several species are newly described from hypersaline water, namely C. ramotenellum, C. tenellum, C. subinflatum, and C. herbaroides. Cladosporium pseudiridis collected from Iris sp. in New Zealand, is also a member of this species complex and shown to be distinct from C. iridis that occurs on this host elsewhere in the world. A further new species from New Zealand is C. sinuosum on Fuchsia excorticata. Cladosporium antarcticum is newly described from a lichen, Caloplaca regalis, collected in Antarctica, and C. subtilissimum from grape berries in the U.S.A., while the new combination C. ossifragi, the oldest valid name of the Cladosporium known from Narthecium in Europe, is proposed. Standard protocols and media are herewith proposed to facilitate future morphological examination of Cladosporium spp. in culture, and neotypes or epitypes are proposed for all species treated.
Physics and Chemistry of the Earth, Parts A/B/C, 2003
Little is known about fungal diversity in extremely cold regions. Low temperatures induce the for... more Little is known about fungal diversity in extremely cold regions. Low temperatures induce the formation of ice crystals and therefore also the creation of low water activity (a w ). These are the dominant factors in external chemistry that influence microbial biota in cold regions. Therefore, we have used selective low water activity media plus low incubation temperatures for the isolation of fungi from an Arctic environment. In comparison with the highest values of colony forming units (CFU) obtained on mesophilic media, considerably higher fungal CFU per litre of water were detected on low a w media, ranging from 1000 to 3000 l À1 in seawater, 6000 to 7000 l À1 in melted sea ice and up to 13,000 l À1 in melted glacier ice. The dominant taxa were ascomycetous and basidiomycetous yeasts, melanized fungi, mainly represented by the genera Cladosporium and Aureobasidium plus different species of the genus Penicillium. Preliminary taxonomic analyses revealed several new species and varieties. Further characterisations are needed to determine whether this diversity is due to geographic isolation, ecological conditions or independent evolutionary origin.
The aim of the present study was to investigate indigenous fungal communities isolated from extre... more The aim of the present study was to investigate indigenous fungal communities isolated from extreme environments (hypersaline waters of solar salterns and subglacial ice), for the production of metabolic compounds with selected biological activities: hemolysis, antibacterial, and acetylcholinesterase inhibition. In their natural habitats, the selected fungi are exposed to environmental extremes, and therefore the production of bioactive metabolites was tested under both standard growth conditions for mesophilic microorganisms, and at high NaCl and sugar concentrations and low growth temperatures. The results indicate that selected halotolerant and halophilic species synthesize specific bioactive metabolites under conditions that represent stress for non-adapted species. Furthermore, adaptation at the level of the chemical nature of the solute lowering the water activity of the medium was observed. Increased salt concentrations resulted in higher hemolytic activity, particularly within species dominating the salterns. The appearance of antibacterial potential under stress conditions was seen in the similar pattern of fungal species as for hemolysis. The active extracts exclusively affected the growth of the Gram-positive bacterium tested, Bacillus subtilis. None of the extracts tested showed inhibition of acetylcholinesterase activity.
Cryptococcus species (Basidiomycota) were isolated as the predominant yeast from glacial biomes o... more Cryptococcus species (Basidiomycota) were isolated as the predominant yeast from glacial biomes of both Patagonia (Argentina) and the Svalbard archipelago (Norway). For a selected group of Cryptococcus belonging to Tremellales, assimilative profile, production of extracellular hydrolytic enzymes and ribosomal DNA internal transcribed spacer and large subunit (D1/D2) sequences were analysed. Cryptococcus victoriae, which was originally described from Antarctica, was the most frequently found species at both locations. High variability within the species was observed and described at the genotypic and phenotypic levels, two newly described species were found in both Patagonia and Svalbard: Cryptococcus fonsecae and Cryptococcus psychrotolerans. Two other new species were found only in Patagonia: Cryptococcus frias and Cryptococcus tronadorensis. Three additional new taxa were found, but they are not named as they were only represented by single isolates.
The Wallemiomycetes includes three species of molds from the genus Wallemia . These fungi are ada... more The Wallemiomycetes includes three species of molds from the genus Wallemia . These fungi are adapted to environments of high osmotic stress, contaminate various foods, cause respiratory disease, and have an unusual mode of asexual reproduction. Wallemia was recently proposed as a new class based on 18S ribosomal RNA gene sequences to accommodate the isolated position of the clade in the Basidiomycota. We analyzed the phylogenetic position of the Wallemiomycetes using 3451 nucleotide characters of the 18S, 25S, and 5.8S ribosomal RNA genes and 1282 amino acid positions of rpb1, rpb2, and tef1 nuclear protein-coding genes across 91 taxa. Different gene regions and methods of phylogenetic inference produce mildly conflicting placements of the Wallemiomycetes. Parsimony analyses of nrDNA data suggest that the Wallemiomycetes is an early diverging lineage of Basidiomycota, occupying a basal position near the Entorrhizomycetidae. Ultrastructural data, some Bayesian analyses, and amino ac...
Until recently, it was believed that microbial communities at high salinities are dominated exclu... more Until recently, it was believed that microbial communities at high salinities are dominated exclusively by Archaea and Bacteria and one eukaryotic species, the alga Dunaliella salina. Recently, it became evident that melanized fungi, so far described only in the crystallization pond of Adriatic salterns within the season of salt production, can be considered as a new group of eukaryotic halophiles. They were represented by black, yeast-like hyphomycetes: Hortaea werneckii, Phaeotheca triangularis, Trimmatostroma salinum, Aureobasidium pullulans, together with phylogenetically closely related Cladosporium species. In the present study, the distribution of the melanized fungal population in five different evaporating ponds in the Adriatic salterns wcovering the entire salinity range (3-30% NaCl)x was followed throughout the year. It appeared in three peaks, at 5-8%, 10-20% and 18-25% NaCl, which correlated primarily with high nitrogen values. At the highest environmental salinities, melanized fungi represented 85-100% of the total isolated mycobiota, but with lowering salinities they were partially replaced by non-melanized fungi and, at the end of the season, with NaCl concentrations below 5%, they were detected only occasionally. Melanized fungi have been isolated from hypersaline waters on three continents, indicating that they are present globally in hypersaline waters of man-made salterns.
Summary For a long time halotolerant and halophilic fungi have been known exclusively as contamin... more Summary For a long time halotolerant and halophilic fungi have been known exclusively as contaminants of food preserved with high concentrations of either salt or sugar. They were first reported in 2000 to be active inhabitants of hypersaline environments, when they were found in man-made solar salterns in Slovenia. Since then, they have been described in different salterns and salt lakes on three continents. The mycobiota that inhabit these natural hypersaline environments are composed of phylogenetically unrelated halotolerant, extremely halotolerant, and halophilic fungi, which are represented not only by species previously known only as food contaminants, but also by new and rare species. The dominant representatives are different species of black yeast-like and related melanized fungi of the genus Cladosporium, different species within the anamorphic Aspergillus and Penicillium, and the teleomorphic Emericella and Eurotium, certain species of non-melanized yeasts, and Wallemia ...
Lichenized fungi usually develop complex, stratified morphologies through an intricately balanced... more Lichenized fungi usually develop complex, stratified morphologies through an intricately balanced living together with their algal partners, but several species are known to form only more or less loose associations with algae. These borderline lichens are still little explored although they could inform us about early stages of lichen evolution. We studied the association of the extremely halotolerant fungus Hortaea werneckii with the alga Dunaliella atacamensis, discovered in a cave in the Atacama Desert (Chile), and with D. salina, common inhabitant of saltern brines. D. atacamensis forms small colonies, in which cells of H. werneckii can be frequently observed, while such interaction has not been observed with D. salina. As symbiotic interactions between Dunaliella and Hortaea have not been reported, we performed a series of co-cultivation experiments to inspect whether these species could interact and develop more distinct lichen-like symbiotic structures. We set up co-cultures...
The polymorphic black yeast Hortaea werneckii (Capnodiales, Ascomycota) is extremely halotolerant... more The polymorphic black yeast Hortaea werneckii (Capnodiales, Ascomycota) is extremely halotolerant (growth from 0 to 30% [w/v] NaCl) and has been extensively studied as a model for halotolerance in Eukaryotes for over two decades. Its most frequent sources are hypersaline environments and adjacent seawater habitats in temperate, subtropical and tropical climates. Although typically saprobic, H. werneckii can also act as a commensal coloniser on human skin, causing tinea nigra on hands and soles. Here, we report that addition of NaCl to culture media expands the growth range of H. werneckii to 37°C, which explains its colonisation of human skin, with its increased salinity. The morphological and physiological plasticity/ versatility of H. werneckii indicate that a species complex might be involved. This was investigated in this polyphasic taxonomic analysis based on the global diversity of H. werneckii strains collected from hypersaline environments, and from humans and animals. Analysis of D1/ D2domains of 28S and internal transcribed spacer rDNA revealed 10 and 17 genotypes, respectively, that were not always compliant. The genotypes have global distributions. Human and environmental strains with the same genotypes are intermingled. Due to the limited number of phylogenetically informative characters in the ribosomal DNA dataset, the partial genes encoding for β-tubulin (BTB) and mini-chromosome maintenance protein (MCM7) were also sequenced. The use of these genes was hampered by ambiguous sequences obtained by Sanger sequencing, as a consequence of the diploid and highly heterozygous genome of many H. werneckii strains. Analysis of the BTB and MCM7 genes showed that in some cases two copies of the gene from the same genome are positioned in distant phylogenetic clusters of the intraspecific gene tree. Analysis of whole-genome sequences of selected H. werneckii strains generally confirmed the phylogenetic distances estimated on the basis of ribosomal genes, but also showed substantial reticulation within the phylogenetic history of the strains. This is in line with the hypothesis that the diploid genomes of H. werneckii were formed by hybridizations, which have sometimes occurred between relatively divergent strains.
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