Interactions of Fungi and Algae from the Greenland Ice Sheet.

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-ß-D-glucopyranoside (C18H18O12) by the two fungi was also evaluated in a separate experiment. P. anthracinoglaciei was capable of utilizing and converting the pigment to purpurogallin carboxylic acid, possibly using the sugar moiety as a nutrient source. Furthermore, after 3 weeks of incubation in the presence of P. anthracinoglaciei, a significantly slower decline in the maximum quantum efficiency (Fv/Fm, inverse proxy of algal stress) in glacier ice algae, compared to other treatments, was evident, suggesting a positive relationship between these species. Articulospora sp. did uptake the glycosylated purpurogallin, but did not seem to be involved in its conversion to aglycone derivative. At the end of the incubation experiments and, in conjunction with increased algal mortality, we detected a substantially increasing presence of the zoosporic fungi Chytridiomycota suggesting an important role for them as decomposers or parasites of glacier ice algae.

Bioactivity of volatile organic compounds by Aureobasidium species against gray mold of tomato and table grape.

Aureobasidium strains isolated from diverse unconventional environments belonging to the species A. pullulans, A. melanogenum, and A. subglaciale were evaluated for Volatile Organic Compounds (VOCs) production as a part of their modes of action against Botrytis cinerea of tomato and table grape. By in vitro assay, VOCs generated by the antagonists belonging to the species A. subglaciale showed the highest inhibition percentage of the pathogen mycelial growth (65.4%). In vivo tests were conducted with tomatoes and grapes artificially inoculated with B. cinerea conidial suspension, and exposed to VOCs emitted by the most efficient antagonists of each species (AP1, AM10, AS14) showing that VOCs of AP1 (A. pullulans) reduced the incidence by 67%, partially confirmed by the in vitro results. Conversely, on table grape, VOCs produced by all the strains did not control the fungal incidence but were only reducing the infection severity (< 44.4% by A. pullulans; < 30.5% by A. melanogenum, and A. subglaciale). Solid-phase microextraction (SPME) and subsequent gas chromatography coupled to mass spectrometry identified ethanol, 3-methyl-1-butanol, 2-methyl-1-propanol as the most produced VOCs. However, there were differences in the amounts of produced VOCs as well as in their repertoire. The EC50 values of VOCs for reduction of mycelial growth of B. cinerea uncovered 3-methyl-1-butanol as the most effective compound. The study demonstrated that the production and the efficacy of VOCs by Aureobasidium could be directly related to the specific species and pathosystem and uncovers new possibilities for searching more efficient VOCs producing strains in unconventional habitats other than plants.

Phylogeny of xerophilic aspergilli (subgenus Aspergillus) and taxonomic revision of section Restricti.

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 ß-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.

Environmental and bioclimatic factors influencing yeasts and molds distribution along European shores.

The present study employed data collected during the Mycosands survey to investigate the environmental factors influencing yeasts and molds distribution along European shores applying a species distribution modelling approach. Occurrence data were compared to climatic datasets (temperature, precipitation, and solar radiation), soil datasets (chemical and physical properties), and water datasets (temperature, salinity, and chlorophyll-a concentration) downloaded from web databases. Analyses were performed by MaxEnt software. Results suggested a different probability of distribution of yeasts and molds along European shores. Yeasts seem to tolerate low temperatures better during winter than molds and this reflects a higher suitability for the Northern European coasts. This difference is more evident considering suitability in waters. Both distributions of molds and yeasts are influenced by basic soil pH, probably because acidic soils are more favorable to bacterial growth. Soils with high nitrogen concentrations are not suitable for fungal growth, which, in contrast, are optimal for plant growth, favored by this environment. Finally, molds show affinity with soil rich in nickel and yeasts with soils rich in cadmium resulting in a distribution mainly at the mouths of European rivers or lagoons, where these metals accumulate in river sediments.

Antibacterial and antimycotic activities of Slovenian honeys.

In the present study, Slovenian honey samples produced from different floral sources are evaluated for their antibacterial and antifungal properties. The peroxide contribution to antibacterial activity is also determined. Minimum inhibitory concentration (MIC) of the honeys was assessed against four bacterial species (Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus) and against eight fungal species (Aspergillus niger, Aureobasidium pullulans, Candida albicans, Candida parapsilosis, Candida tropicalis, Cladosporium cladosporioides, Penicillium chrysogenum and Rhodotorula mucilaginosa). Honey at concentrations between 1% and 50% (v/v) were tested. Although all of the bacterial species were inhibited by the different honey samples, the chestnut and pasture honeys showed the highest antibacterial activities. The antifungal activities were concentration-dependent, with five (Aureobasidium pullulans, Candida parapsilosis, Candida tropicalis, Cladosporium cladosporioides, Rhodotorula mucilaginosa) inhibited only at honey concentrations greater than 50%. The fungi Aspergillus niger, Candida albicans and Penicillium chrysogenum were not inhibited by any of the tested honeys, even at the highest concentrations. The lowest MICs seen were 2.5% (v/v) for the chestnut, fir and forest honeys against Staphylococcus aureus, and 10.0% (v/v) for the chestnut and pasture honeys against Cladosporium cladosporioides. The non-peroxide action of chestnut honey was tested against Escherichia coli. The MIC of the catalase-treated chestnut honey was 50% (v/v). The antibacterial effect of Slovenian honeys is mostly due to peroxide action. These data support the concept that Slovenian honeys are effective antibacterials and antifungals, and can thus be applied for medicinal purposes.

Novel Antarctic yeast adapts to cold by switching energy metabolism and increasing small RNA synthesis.

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. R. frigidialcoholis increased its diversity and abundance of sRNAs when grown at 0 versus 23 °C. This was consistent with increase in transcription of Dicer, a key protein for sRNA processing. Our results strongly imply that post-transcriptional regulation of gene expression and mRNA silencing may be a novel evolutionary fungal adaptation in the cryosphere.

Unusual fungal niches.

Fungi are found in all aerobic ecosystems, colonizing a diversity of substrates and performing a wide diversity of functions, some of which are not well understood. Many spices of fungi are cosmopolitan and generalists or habitats. Unusual fungal niches are habitats where extreme conditions would be expected to prevent the development of a mycobiota. In this review we describe five unusual fungal habitats in which fungi occupy poorly understood niches: Antarctic dry valleys, high Arctic glaciers, salt flats and salterns, hypersaline microbial mats and plant trichomes. Yeasts, black yeast-like fungi, melanized filamentous species as well as representatives of Aspergillus and Penicillium seem to be dominant among the mycobiota adapted to cold and saline niches. Plant trichomes appear to be a taxa. The advent of new sequencing technologies is helping to elucidate the microbial diversity in many ecosystems, but more studies are needed to document the functional role of fungi in the microbial communities thriving in these unusual environments.

Mycosands: Fungal diversity and abundance in beach sand and recreational waters - Relevance to human health.

The goal of most studies published on sand contaminants is to gather and discuss knowledge to avoid faecal contamination of water by run-offs and tide-retractions. Other life forms in the sand, however, are seldom studied but always pointed out as relevant. The Mycosands initiative was created to generate data on fungi in beach sands and waters, of both coastal and freshwater inland bathing sites. A team of medical mycologists and water quality specialists explored the sand culturable mycobiota of 91 bathing sites, and water of 67 of these, spanning from the Atlantic to the Eastern Mediterranean coasts, including the Italian lakes and the Adriatic, Baltic, and Black Seas. Sydney (Australia) was also included in the study. Thirteen countries took part in the initiative. The present study considered several fungal parameters (all fungi, several species of the genus Aspergillus and Candida and the genera themselves, plus other yeasts, allergenic fungi, dematiaceous fungi and dermatophytes). The study considered four variables that the team expected would influence the results of the analytical parameters, such as coast or inland location, urban and non-urban sites, period of the year, geographical proximity and type of sediment. The genera most frequently found were Aspergillus spp., Candida spp., Fusarium spp. and Cryptococcus spp. both in sand and in water. A site-blind median was found to be 89 Colony-Forming Units (CFU) of fungi per gram of sand in coastal and inland freshwaters, with variability between 0 and 6400 CFU/g. For freshwater sites, that number was 201.7 CFU/g (0, 6400 CFU/g (p = 0.01)) and for coastal sites was 76.7 CFU/g (0, 3497.5 CFU/g). For coastal waters and all waters, the median was 0 CFU/ml (0, 1592 CFU/ml) and for freshwaters 6.7 (0, 310.0) CFU/ml (p < 0.001). The results advocate that beaches should be monitored for fungi for safer use and better management.

Fungal and bacterial diversity of Svalbard subglacial ice.

The composition of fungal and bacterial communities in three polythermal glaciers and associated aquatic environments in Kongsfjorden, Svalbard was analysed using a combination of cultivation and amplicon sequencing. 109 fungal strains belonging to 30 mostly basidiomycetous species were isolated from glacial samples with counts up to 103 CFU/100 ml. Glaciozyma-related taxon and Phenoliferia psychrophenolica were the dominant species. Unexpectedly, amplicon sequencing uncovered sequences of Chytridiomycota in all samples and Rozellomycota in sea water, lake water, and tap water. Sequences of Malassezia restricta and of the extremely halotolerant Hortaea werneckii were also found in subglacial habitats for the first time. Overall, the fungal communities within a glacier and among glaciers were diverse and spatially heterogenous. Contrary to this, there was a large overlap between the bacterial communities of different glaciers, with Flavobacterium sp. being the most frequently isolated. In amplicon sequencing Actinobacteria and Proteobacteria sequences were the most abundant.

Expression of fatty-acid-modifying enzymes in the halotolerant black yeast Aureobasidium pullulans (de Bary) G. Arnaud under salt stress.

Multiple tolerance to stressful environmental conditions of the black, yeast-like fungus Aureobasidium pullulans is achieved through different adaptations, among which there is the restructuring of the lipid composition of their membranes. Here, we describe three novel genes encoding fatty-acid-modifying enzymes in A. pullulans, along with the levels of their mRNAs under different salinity conditions. High levels of Delta(9)-desaturase and Delta(12)-desaturase mRNAs were seen at high salinities, which were consistent with an increased desaturation of the fatty acids in the cell membranes. Elevated levels of elongase mRNA were also detected. Surprisingly, increases in the levels of these mRNAs were also seen following hypo-osmotic shock, while hyperosmotic shock had exactly the opposite effect, demonstrating that data that are obtained from up-shift and down-shift salinity studies should be interpreted with caution.

HMG-CoA reductase is regulated by environmental salinity and its activity is essential for halotolerance in halophilic fungi.

The activity and level of HMG-CoA reductase (HMGR) were addressed in halophilic fungi isolated from solar saltpans. Representative fungi belonging to the orders Dothideales, Eurotiales and Wallemiales have a specific pattern of HMGR regulation, which differs from salt-sensitive and moderately salt-tolerant yeasts. In all of the halophilic fungi studied, HMGR amounts and activities were the lowest at optimal growth salinity and increased under hyposaline and hypersaline conditions. This profile paralleled isoprenylation of cellular proteins in H. werneckii. Inhibition of HMGR in vivo by lovastatin impaired the halotolerant character. HMGR may thus serve as an important molecular marker of halotolerance.

Adaptation of extremely halotolerant black yeast Hortaea werneckii to increased osmolarity: a molecular perspective at a glance.

Halophilic adaptations have been studied almost exclusively on prokaryotic microorganisms. Discovery of the black yeast Hortaea werneckii as the dominant fungal species in hypersaline waters enabled the introduction of a new model organism to study the mechanisms of salt tolerance in eukaryotes. Its strategies of cellular osmotic adaptations on the physiological and molecular level revealed novel, intricate mechanisms to combat fluctuating salinity. H. werneckii is an extremely halotolerant eukaryotic microorganism and thus a promising source of transgenes for osmotolerance improvement of industrially important yeasts, as well as in crops.

Redefinition of Aureobasidium pullulans and its varieties.

Using media with low water activity, a large numbers of aureobasidium-like black yeasts were isolated from glacial and subglacial ice of three polythermal glaciers from the coastal Arctic environment of Kongsfjorden (Svalbard, Spitsbergen), as well as from adjacent sea water, sea ice and glacial meltwaters. To characterise the genetic variability of Aureobasidium pullulans strains originating from the Arctic and strains originating pan-globally, a multilocus molecular analysis was performed, through rDNA (internal transcribed spacers, partial 28 S rDNA), and partial introns and exons of genes encoding beta-tubulin (TUB), translation elongation factor (EF1alpha) and elongase (ELO). Two globally ubiquitous varieties were distinguished: var. pullulans, occurring particularly in slightly osmotic substrates and in the phyllosphere; and var. melanogenum, mainly isolated from watery habitats. Both varieties were commonly isolated from the sampled Arctic habitats. However, some aureobasidium-like strains from subglacial ice from three different glaciers in Kongsfjorden (Svalbard, Spitsbergen), appeared to represent a new variety of A. pullulans. A strain from dolomitic marble in Namibia was found to belong to yet another variety. No molecular support has as yet been found for the previously described var. aubasidani. A partial elongase-encoding gene was successfully used as a phylogenetic marker at the (infra-)specific level.

Phylogeny and ecology of the ubiquitous saprobe Cladosporium sphaerospermum, with descriptions of seven new species from hypersaline environments.

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 beta-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 degrees C but not at 30 degrees 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.

Yeast diversity in hypersaline habitats.

Thus far it has been considered that hypersaline natural brines which are subjected to extreme solar heating, do not contain non-melanized yeast populations. Nevertheless we have isolated yeasts in eight different salterns worldwide, as well as from the Dead Sea, Enriquillo Lake (Dominican Republic) and the Great Salt Lake (Utah). Among the isolates obtained from hypersaline waters, Pichia guilliermondii, Debaryomyces hansenii, Yarrowia lipolytica and Candida parapsilosis are known contaminants of low water activity food, whereas Rhodosporidium sphaerocarpum, R. babjevae, Rhodotorula laryngis, Trichosporon mucoides, and a new species resembling C. glabrata were not known for their halotolerance and were identified for the first time in hypersaline habitats. Moreover, the ascomycetous yeast Metschnikowia bicuspidata, known to be a parasite of the brine shrimp, was isolated as a free-living form from the Great Salt Lake brine. In water rich in magnesium chloride (bitterns) from the La Trinitat salterns (Spain), two new species provisionally named C. atmosphaerica - like and P. philogaea - like were discovered.

Xerotolerant mycobiota from high altitude Anapurna soil, Nepal.

Xerophilic and xerotolerant microfungi were isolated from soil samples collected in Anapurna Mountains, Nepal, at altitudes from 3000 to 5400 m. The total numbers and proportions of xerotolerant and psychrophilic strains in comparison with mesophilic mycobiota were determined by using different enumeration, selective media and four isolation methods. The most extreme xerophilic fungi were taxonomically identified as belonging to the genera Eurotium and Aspergillus. The low water activity of the soil due to dry climate and frequent binding of water in ice crystals favors a high proportion of xerotolerant fungal species. The correlation between xerotolerant and psychrophilic fungi was observed.

A hydroxymethylglutaryl-CoA reductase inhibitor synthesized by yeasts.

Screening of different yeast species showed that they are able to synthesize hydroxymethylglutaryl-CoA (HMGCoA) reductase inhibitors. Crude methanol extracts and the purified inhibitors from Pichia labacensis and Candida cariosilignicola were tested for their biological activity on the solubilized microsomal HMGCoA reductase from Chinese hamster ovary cells. Identification of the inhibitors was studied by thin layer chromatography, high pressure liquid chromatography and mass spectroscopy.

Pleurotus fungi produce mevinolin, an inhibitor of HMG CoA reductase.

Fungi of the genus Pleurotus were shown to produce the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor mevinolin. Crude methanol extracts and the purified inhibitor from three different species, P. sapidus, P. saca and P. ostreatus, were tested using the solubilized microsomal HMGCoA reductase from Chinese hamster ovary cells. The identity of the inhibitor was also confirmed by thin layer chromatography, high pressure liquid chromatography and mass spectroscopy.

Salt stress affects sterol biosynthesis in the halophilic black yeast Hortaea werneckii.

Hortaea werneckii is a black yeast recently isolated from salterns in Slovenia. Some of the adaptations of halophilic microorganisms to increased salinity and osmolarity of the environment are alterations in membrane properties. By modulating the fluidity, sterols play an important role as a component of eukaryotic biological membranes. We studied the regulation of sterol biosynthesis in H. werneckii through the activity and amount of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG R), a key regulatory enzyme in the biosynthesis of sterols. We found some differences in the characteristics of HMG R and in its regulation by different environmental salinities in H. werneckii when compared to the mesophilic baker's yeast, Saccharomyces cerevisiae. Our results suggest that halophilic black yeast regulates sterol biosynthesis through HMG R in a different way than mesophiles, which might be a consequence of the different ecophysiology of halophilic black yeasts. From this perspective, H. werneckii is an interesting novel model organism for studies on salt stress-responsive proteins as well as on sterol biosynthesis in eukaryotes.

Dishwashers--a man-made ecological niche accommodating human opportunistic fungal pathogens.

Habitats in human households may accommodate microorganisms outside the common spectrum of ubiquitous saprobes. Enrichment of fungi that may require specific environmental conditions was observed in dishwashers, 189 of which were sampled in private homes of 101 towns or communities. One-hundred-two were sampled from various localities in Slovenia; 42 from other European countries; 13 and 3 from North and South America, respectively; 5 from Israel; 10 from South Africa; 7 from Far East Asia; and 7 from Australia. Isolation was performed on samples incubated at 37°C. Species belonging to genera Aspergillus, Candida, Magnusiomyces, Fusarium, Penicillium and Rhodotorula were found occasionally, while the black yeasts Exophiala dermatitidis and Exophiala phaeomuriformis (Chaetothyriales) were persistently and most frequently isolated. Sixty-two percent of the dishwashers were positive for fungi, and 56% of these accommodated Exophiala. Both Exophiala species are known to be able to cause systemic disease in humans and frequently colonize the lungs of patients with cystic fibrosis. We conclude that high temperature, high moisture and alkaline pH values typically occurring in dishwashers can provide an alternative habitat for species also known to be pathogenic to humans.