A screening method for plastic-degrading fungi.

The growing amount of plastic waste requires new ways of disposal or recycling. Research into the biodegradation of recalcitrant plastic polymers is gathering pace. Despite some progress, these efforts have not yet led to technologically and economically viable applications. In this study, we show that respirometric screening of environmental fungal isolates in combination with scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy can be used to identify new strains with the potential for the degradation of plastic polymers. We screened 146 fungal strains, 71 isolated from car repair shops, an environment rich in long-chain hydrocarbons, and 75 isolated from hypersaline water capable of growing at high concentrations of NaCl. When grown in a minimal medium with no carbon source, some strains produced significantly more CO2 when a pure plastic polymer was added to the medium, some only at high salinity. A selection of these strains was shown by FTIR and Raman spectroscopy to alter the properties of plastic polymers: Cladosporium sp. EXF-13502 on polyamide, Rhodotorula dairenensis EXF-13500 on polypropylene, Rhodotorula sp. EXF-10630 on low-density polyethylene and Wickerhamomyces anomalus EXF-6848 on polyethylene terephthalate. Respirometry in combination with specific spectroscopic methods is an efficient method for screening microorganisms capable of at least partial plastic degradation and can be used to expand the repertoire of potential plastic degraders. This is of particular importance as our results also show that individual strains are only active against certain polymers and under certain conditions. Therefore, efficient biodegradation of plastics is likely to depend on a collection of specialized microorganisms rather than a single universal plastic degrader.

Biocontrol Potential of Sodin 5, Type 1 Ribosome-Inactivating Protein from Salsola soda L. Seeds.

Sodin 5 is a type 1 ribosome-inactivating protein isolated from the seeds of Salsola soda L., an edible halophytic plant that is widespread in southern Europe, close to the coast. This plant, known as 'agretti', is under consideration as a new potential crop on saline soils. Considering a possible defence role of sodin 5 in the plant, we report here its antifungal activity against different halophilic and halotolerant fungi. Our results show that sodin 5 at a concentration of 40 µg/mL (1.4 µM) was able to inhibit the growth of the fungi Trimmatostromma salinum (35.3%), Candida parapsilosis (24.4%), Rhodotorula mucilaginosa (18.2%), Aspergillus flavus (12.2%), and Aureobasidium melanogenum (9.1%). The inhibition observed after 72 h was concentration-dependent. On the other hand, very slight growth inhibition was observed in the fungus Hortaea werneckii (4.2%), which commonly inhabits salterns. In addition, sodin 5 showed a cytotoxic effect on the Sf9 insect cell line, decreasing the survival of these cells to 63% at 1.0 µg/mL (34.5 nM). Structural analysis of sodin 5 revealed that its N-terminal amino acid residue is blocked. Using mass spectrometry, sodin 5 was identified as a homologous to type 1 polynucleotide:adenosine glycosylases, commonly known as ribosome-inactivating proteins from the Amaranthaceae family. Twenty-three percent of its primary structure was determined, including the catalytic site.

Effect of Location, Disinfection, and Building Materials on the Presence and Richness of Culturable Mycobiota through Oligotrophic Drinking Water Systems.

Safe drinking water is a constant challenge due to global environmental changes and the rise of emerging pathogens-lately, these also include fungi. The fungal presence in water greatly varies between sampling locations. Little is known about fungi from water in combination with a selection of materials used in water distribution systems. Our research was focused on five water plants located in the Pannonian Plain, Slovenia. Sampled water originated from different natural water sources and was subjected to different cleaning methods before distribution. The average numbers of fungi from natural water, water after disinfection, water at the first sampling point in the water network, and water at the last sampling point were 260, 49, 64, and 97 CFU/L, respectively. Chlorination reduced the number of fungi by a factor of 5, but its effect decreased with the length of the water network. The occurrence of different fungi in water and on materials depended on the choice of material. The presence of the genera Aspergillus, Acremonium, Furcasterigmium, Gliomastix, and Sarocladium was mostly observed on cement, while Cadophora, Cladosporium, Cyphellophora, and Exophiala prevailed on metals. Plastic materials were more susceptible to colonization with basidiomycetous fungi. Opportunistically pathogenic fungi were isolated sporadically from materials and water and do not represent a significant health risk for water consumers. In addition to cultivation data, physico-chemical features of water were measured and later processed with machine learning methods, revealing the sampling location and water cleaning processes as the main factors affecting fungal presence and richness in water and materials in contact with water.

Degradation of polypropylene by fungi Coniochaeta hoffmannii and Pleurostoma richardsiae.

The urgent need for better disposal and recycling of plastics has motivated a search for microbes with the ability to degrade synthetic polymers. While microbes capable of metabolizing polyurethane and polyethylene terephthalate have been discovered and even leveraged in enzymatic recycling approaches, microbial degradation of additive-free polypropylene (PP) remains elusive. Here we report the isolation and characterization of two fungal strains with the potential to degrade pure PP. Twenty-seven fungal strains, many isolated from hydrocarbon contaminated sites, were screened for degradation of commercially used textile plastic. Of the candidate strains, two identified as Coniochaeta hoffmannii and Pleurostoma richardsiae were found to colonize the plastic fibers using scanning electron microscopy (SEM). Further experiments probing degradation of pure PP films were performed using C. hoffmannii and P. richardsiae and analyzed using SEM, Raman spectroscopy and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). The results showed that the selected fungi were active against pure PP, with distinct differences in the bonds targeted and the degree to which each was altered. Whole genome and transcriptome sequencing was conducted for both strains and the abundance of carbohydrate active enzymes, GC content, and codon usage bias were analyzed in predicted proteomes for each. Enzymatic assays were conducted to assess each strain's ability to degrade naturally occurring compounds as well as synthetic polymers. These investigations revealed potential adaptations to hydrocarbon-rich environments and provide a foundation for further investigation of PP degrading activity in C. hoffmannii and P. richardsiae.

Effects of Desiccation and Freezing on Microbial Ionizing Radiation Survivability: Considerations for Mars Sample Return.

Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.

Surviving in the Brine: A Multi-Omics Approach for Understanding the Physiology of the Halophile Fungus Aspergillus sydowii at Saturated NaCl Concentration.

Although various studies have investigated osmoadaptations of halophilic fungi to saline conditions, only few analyzed the fungal mechanisms occurring at saturated NaCl concentrations. Halophilic Aspergillus sydowii is a model organism for the study of molecular adaptations of filamentous fungi to hyperosmolarity. For the first time a multi-omics approach (i.e., transcriptomics and metabolomics) was used to compare A. sydowii at saturated concentration (5.13 M NaCl) to optimal salinity (1 M NaCl). Analysis revealed 1,842 genes differentially expressed of which 704 were overexpressed. Most differentially expressed genes were involved in metabolism and signal transduction. A gene ontology multi-scale network showed that ATP binding constituted the main network node with direct interactions to phosphorelay signal transduction, polysaccharide metabolism, and transferase activity. Free amino acids significantly decreased and amino acid metabolism was reprogrammed at 5.13 M NaCl. mRNA transcriptional analysis revealed upregulation of genes involved in methionine and cysteine biosynthesis at extreme water deprivation by NaCl. No modifications of membrane fatty acid composition occurred. Upregulated genes were involved in high-osmolarity glycerol signal transduction pathways, biosynthesis of ß-1,3-glucans, and cross-membrane ion transporters. Downregulated genes were related to the synthesis of chitin, mannose, cell wall proteins, starvation, pheromone synthesis, and cell cycle. Non-coding RNAs represented the 20% of the total transcripts with 7% classified as long non-coding RNAs (lncRNAs). The 42% and 69% of the total lncRNAs and RNAs encoding transcription factors, respectively, were differentially expressed. A network analysis showed that differentially expressed lncRNAs and RNAs coding transcriptional factors were mainly related to the regulation of metabolic processes, protein phosphorylation, protein kinase activity, and plasma membrane composition. Metabolomic analyses revealed more complex and unknown metabolites at saturated NaCl concentration than at optimal salinity. This study is the first attempt to unravel the molecular ecology of an ascomycetous fungus at extreme water deprivation by NaCl (5.13 M). This work also represents a pioneer study to investigate the importance of lncRNAs and transcriptional factors in the transcriptomic response to high NaCl stress in halophilic fungi.

Tracking gene expression, metabolic profiles, and biochemical analysis in the halotolerant basidiomycetous yeast Rhodotorula mucilaginosa EXF-1630 during benzo[a]pyrene and phenanthrene biodegradation under hypersaline conditions.

Polyaromatic phenanthrene (Phe) and benzo[a]pyrene (BaP) are highly toxic, mutagenic, and carcinogenic contaminants widely dispersed in nature, including saline environments. Polyextremotolerant Rhodotorula mucilaginosa EXF-1630, isolated from Arctic sea ice, was grown on a huge concentration range -10 to 500 ppm- of Phe and BaP as sole carbon sources at hypersaline conditions (1 M NaCl). Selected polycyclic aromatic hydrocarbons (PAHs) supported growth as well as glucose, even at high PAH concentrations. Initially, up to 40% of Phe and BaP were adsorbed, followed by biodegradation, resulting in 80% removal in 10 days. While extracellular laccase, peroxidase, and un-specific peroxygenase activities were not detected, NADPH-cytochrome c reductase activity peaked at 4 days. The successful removal of PAHs and the absence of toxic metabolites were confirmed by toxicological tests on moss Physcomitrium patens, bacterium Aliivibrio fischeri, human erythrocytes, and pulmonary epithelial cells (A549). Metabolic profiles were determined at the midpoint of the biodegradation exponential phase, with added Phe and BaP (100 ppm) and 1 M NaCl. Different hydroxylated products were found in the culture medium, while the conjugative metabolite 1-phenanthryl-ß-D-glucopyranose was detected in the medium and in the cells. Transcriptome analysis resulted in 870 upregulated and 2,288 downregulated transcripts on PAHs, in comparison to glucose. Genomic mining of 61 available yeast genomes showed a widespread distribution of 31 xenobiotic degradation pathways in different yeast lineages. Two distributions with similar metabolic capacities included black yeasts and mainly members of the Sporidiobolaceae family (including EXF-1630), respectively. This is the first work describing a metabolic profile and transcriptomic analysis of PAH degradation by yeast.

Shed Light in the DaRk LineagES of the Fungal Tree of Life-STRES.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

The Neurotropic Black Yeast Exophiala dermatitidis Induces Neurocytotoxicity in Neuroblastoma Cells and Progressive Cell Death.

The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits diverse indoor environments, in particular bathrooms, steam baths, and dishwashers. Here, we show that the selected strain, EXF-10123, is polymorphic, can grow at 37 °C, is able to assimilate aromatic hydrocarbons (toluene, mineral oil, n-hexadecane), and shows abundant growth with selected neurotransmitters (acetylcholine, gamma-aminobutyric acid, glycine, glutamate, and dopamine) as sole carbon sources. We have for the first time demonstrated the effect of E. dermatitidis on neuroblastoma cell model SH-SY5Y. Aqueous and organic extracts of E. dermatitidis biomass reduced SH-SY5Y viability by 51% and 37%, respectively. Melanized extracellular vesicles (EVs) prepared from this strain reduced viability of the SH-SY5Y to 21%, while non-melanized EVs were considerably less neurotoxic (79% viability). We also demonstrated direct interactions of E. dermatitidis with SH-SY5Y by scanning electron and confocal fluorescence microscopy. The observed invasion and penetration of neuroblastoma cells by E. dermatitidis hyphae presumably causes the degradation of most neuroblastoma cells in only three days. This may represent a so far unknown indirect or direct cause for the development of some neurodegenerative diseases such as Alzheimer's.

The extremely halotolerant black yeast Hortaea werneckii - a model for intraspecific hybridization in clonal fungi.

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 sea-water 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 ß-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.

Cystobasidium alpinum sp. nov. and Rhodosporidiobolus oreadorum sp. nov. from European Cold Environments and Arctic Region.

Over 80% of the Earth’s environments are permanently or periodically exposed to temperatures below 5 °C. Cold habitats harbour a wide diversity of psychrophilic and psychrotolerant yeasts. During ecological studies of yeast communities carried out in cold ecosystem in the Italian Alps, Svalbard (Norway, Arctic region), and Portugal, 23 yeast strains that could not be assigned to any known fungal taxa were isolated. In particular, two of them were first identified as Rhodotorula sp., showing the highest degree of D1/D2 sequence identity with Cystobasidum laryngis accounted to only 97% with the type strain (C. laryngis CBS 2221). The other 21 strains, exhibiting identical D1/D2 sequences, had low identity (97%) with Rhodosporidiobolus lusitaniae and Rhodosporidiobolus colostri. Similarly, ITS sequences of the type strains of the most closely related species (93⁻94%). In a 2-genes multilocus D1/D2 and ITS ML phylogenetic tree, the studied strains pooled in two well separated and supported groups. In order to classify the new 23 isolates based on phylogenetic evidences, we propose the description of two novel species Cystobasidium alpinum sp. nov. and Rhodosporidiobolus oreadorum sp. nov.

Synergistic Interactions in Microbial Biofilms Facilitate the Establishment of Opportunistic Pathogenic Fungi in Household Dishwashers.

Biofilms formed on rubber seals in dishwashers harbor diverse microbiota. In this study, we focussed on the microbial composition of bacteria and fungi, isolated from a defined area of one square centimeter of rubber from four domestic dishwashers and assessed their abilities to in vitro multispecies biofilm formation. A total of 80 isolates (64 bacterial and 16 fungal) were analyzed. Multiple combinations of bacterial isolates from each dishwasher were screened for synergistic interactions. 32 out of 140 tested (23%) four-species bacterial combinations displayed consistent synergism leading to an overall increase in biomass, in all experimental trails. Bacterial isolates from two of the four dishwashers generated a high number of synergistically interacting four-species consortia. Network based correlation analyses also showed higher co-occurrence patterns observed between bacterial members in the same two dishwasher samples, indicating cooperative effects. Furthermore, two synergistic four-species bacterial consortia were tested for their abilities to incorporate an opportunistic fungal pathogen, Exophiala dermatitidis and their establishment as biofilms on sterile ethylene propylene diene monomer M-class (EPDM) rubber and polypropylene (PP) surfaces. When the bacterial consortia included E. dermatitidis, the overall cell numbers of both bacteria and fungi increased and a substantial increase in biofilm biomass was observed. These results indicate a novel phenomenon of cross kingdom synergy in biofilm formation and these observations could have potential implications for human health.

Ecology of the Human Opportunistic Black Yeast Exophiala dermatitidis Indicates Preference for Human-Made Habitats.

Exophiala dermatitidis is an ascomycetous black yeast from the order Chaetothyriales. Its growth characteristics include the polymorphic life cycle, ability to grow at high and low temperatures, at a wide pH range, survival at high concentrations of NaCl, and survival at high UV and radioactive radiation. Exophiala dermatitidis causes deep or localized phaeohyphomycosis in immuno-compromised people worldwide and is regularly encountered in the lungs of cystic fibrosis patients. Regardless of numerous ecological studies worldwide, little is known about its natural habitat or the possible infection routes. The present review summarizes the published data on its frequency of occurrence in nature and in man-made habitats. We additionally confirmed its presence with culture-depending methods from a variety of habitats, such as glacial meltwater, mineral water, mineral-rich salt-pan mud, dishwashers, kitchens and different environments polluted with aromatic hydrocarbons. In conclusion, the frequency of its recovery was the highest in man-made indoor habitats, connected to water sources, and exposed to occasional high temperatures and oxidative stress.

Across the tree of life, radiation resistance is governed by antioxidant Mn2+, gauged by paramagnetic resonance.

Despite concerted functional genomic efforts to understand the complex phenotype of ionizing radiation (IR) resistance, a genome sequence cannot predict whether a cell is IR-resistant or not. Instead, we report that absorption-display electron paramagnetic resonance (EPR) spectroscopy of nonirradiated cells is highly diagnostic of IR survival and repair efficiency of DNA double-strand breaks (DSBs) caused by exposure to gamma radiation across archaea, bacteria, and eukaryotes, including fungi and human cells. IR-resistant cells, which are efficient at DSB repair, contain a high cellular content of manganous ions (Mn2+) in high-symmetry (H) antioxidant complexes with small metabolites (e.g., orthophosphate, peptides), which exhibit narrow EPR signals (small zero-field splitting). In contrast, Mn2+ ions in IR-sensitive cells, which are inefficient at DSB repair, exist largely as low-symmetry (L) complexes with substantially broadened spectra seen with enzymes and strongly chelating ligands. The fraction of cellular Mn2+ present as H-complexes (H-Mn2+), as measured by EPR of live, nonirradiated Mn-replete cells, is now the strongest known gauge of biological IR resistance between and within organisms representing all three domains of life: Antioxidant H-Mn2+ complexes, not antioxidant enzymes (e.g., Mn superoxide dismutase), govern IR survival. As the pool of intracellular metabolites needed to form H-Mn2+ complexes depends on the nutritional status of the cell, we conclude that IR resistance is predominantly a metabolic phenomenon. In a cross-kingdom analysis, the vast differences in taxonomic classification, genome size, and radioresistance between cell types studied here support that IR resistance is not controlled by the repertoire of DNA repair and antioxidant enzymes.

Aspergillus atacamensis and A. salisburgensis: two new halophilic species from hypersaline/arid habitats with a phialosimplex-like morphology.

Halophilic fungal strains isolated from historical wooden staircase in a salt mine in Austria, and from wall biofilm and soil of a cave in the Coastal Range of the hyperarid Atacama Desert in Chile were characterised and described newly as Aspergillus salisburgensis and Aspergillus atacamensis. Morphological characters including solitary phialides producing solitary conidia and conidia in chains and/or heads suggested affinity to Aspergillus subgenus Polypaecilum. Strains required salt for growth, grew optimally on media with 10-25% NaCl and at 15-28 °C. These values are similar to those observed for Aspergillus salinarus comb. nov. (Phialosimplex salinarum), while the ex-type strains of Aspergillus sclerotialis, Aspergillus chlamydosporus and Aspergillus caninus (all belonging to Aspergillus subgen. Polypaecilum) grew optimally at 0-5% NaCl and showed fastest growth at 28-37 °C. Phylogenetic analyses on the basis of rDNA sequences, RAPD-PCR fingerprint patterns, and cellobiohydrolase gene (cbh-I) polymorphism clustered the strains into three groups and supported their taxonomic recognition as A. salinarus, A. atacamensis and A. salisburgensis. On the basis of phylogenetic inferences, also Sagenomella keratitidis is newly combined as Aspergillus keratitidis and inferred as a species of Aspergillus subgenus Polypaecilum.

Salting of dry-cured meat - A potential cause of contamination with the ochratoxin A-producing species Penicillium nordicum.

Penicillium nordicum is a known contaminant of protein-rich foods and is primarily found on dry-cured meat products. It is an important producer of the mycotoxin ochratoxin A, which has nephrotoxic and cancerogenic activities. Recently a high number of P. nordicum strains was isolated from different dry-cured meat products from one of the Slovenian meat-processing plants. Since we have isolated P. nordicum in high counts also from Artic habitats, such as sea water and sea ice and due to its ability to grow well at low temperatures and at increased salinity, sea salt was suspected as the possible source of P. nordicum. In the present study contamination of meat products, air in the meat-processing plant and sea salt used for salting were analysed. When 50 g of salt sample from a sealed package was dissolved in sterile water and filtered, 12 colonies of P. nordicum were obtained on solid medium incubated at 15 °C, while a salt sample from an open vessel in the meat-processing area developed high, uncountable number of colonies. Amplified fragment length polymorphism analyses of P. nordicum isolates from different sources showed that contamination of meat products via salt was possible. Three selected isolates examined for extrolites all produced ochratoxin A. As contamination of dry-cured meat products with P. nordicum represents a potential health risk for consumers and workers in the meat-processing plants, salt should be taken into account as a potential cause of such contaminations.

Four new species of Emericella from the Mediterranean region of Europe.

Four new species of Emericella, E. discophora, E. filifera, E. olivicola and E. stella-maris, are proposed. Their new taxonomic status was determined applying a polyphasic taxonomic approach using phenotypic (morphology and extrolite profiles) and molecular (sequences of ITS, beta-tubulin and calmodulin genes) characters. Ascospores of E. stella-maris and E. olivicola have star-shape equatorial crests, those of E. filifera form long appendages that emerge radially from narrow stellate crests and those of E. discophora produce wide and entire, nonstellate equatorial crests. E. stella-maris originated from leaf litter in Tunisia and E. filifera from raisins in Argentina, and both of them also were found in hypersaline water of a saltern in Slovenia. E. olivicola was isolated from olives in Italy and E. discophora from soil in Spain. All listed species possess distinct extrolite profiles: E. stella-maris produced arugosin E, shamixanthone and the yet unelucidated metabolites glia 1-3; E. filifera produced shamixanthone and varitriols; E. discophora produced sterigmatocystin and versicolorins; E. olivicola produced numerous extrolites such as arugosin E, siderin, shamixanthone, sterigmatocystin, terrein, varitriols and aflatoxin B1, of which the latter was detected only in one of the two strains.

Novel 3'-phosphoadenosine-5'-phosphatases from extremely halotolerant Hortaea werneckii reveal insight into molecular determinants of salt tolerance of black yeasts.

The 3'-phosphoadenosine-5'-phosphatase encoded by HAL2 gene, is a ubiquitous enzyme required for the removal of the cytotoxic 3'-phosphoadenosine-5'-phosphate produced during sulfur assimilation in eukaryotes. Salt toxicity in yeast and plants results from Hal2 inhibition by sodium or lithium ions. Two novel HAL2-like genes, HwHAL2A and HwHAL2B, have been cloned from saltern-inhabited extremely halotolerant black yeast Hortaea werneckii. Expression of both HwHAL2 isoforms was differentially inducible upon salt. When the HwHAL2 genes were transferred from such a halotolerant species into the salt sensitive Saccharomyces cerevisiae, the resulting organism can tolerate 1.8M NaCl or 0.8M LiCl, the highest reported salt concentrations at which S. cerevisiae can grow. With genetic and biochemical validation we demonstrated the critical HwHal2B sequence motif--the META sequence--common only to Dothideales fungi, with evident effect on the HwHal2B-dependent salt tolerance. These results may have significance for biosaline agriculture in coastal environments.