First Report of Globisporangium (Pythium) mastophorum causing Damping-off / Root Rot on Parsley in Slovenia.

Pythium-like species cause damping-off symptoms of various hosts, including umbelliferous crops. In April 2023, parsley plantlets (Petroselinum crispum), showing stunted growth, yellowing, decayed roots and damping-off, were obtained from a nursery in central Slovenia, where parsley was grown in polystyrene trays in a greenhouse. Nearly 30% of plants were symptomatic. Sampled roots of ten plants contained ornamented oogonia (avg. 33.3 ± 1.4 µm in diam) with conical projections (5.2 ± 0.5 µm long) (Figure S1 A, B) in microscopically analyzed squash mounts. The pathogen was isolated from root pieces treated for surface disinfection with 0.5% sodium hypochlorite for 30 s, and washed with sterile water. Four 1-2 mm root pieces were taken from each of 10 plants, plated on the selective medium P5ARP, and incubated at 21 °C. Mycelia emerging from root pieces were transferred to carrot piece agar (CPA). Twenty-two equally looking oomycetous colonies were obtained; all sampled plants were infested. Oogonia formed by all colonies were similar to those observed on decayed roots and suggested that Globisporangium (Pythium) mastophorum is the causal disease agent. Analyses of partial ß-tubulin (Kroon et al. 2004) and mitochondrial cytochrome c oxidase I (COI) gene sequences (Robideau et al. 2011) confirmed the identification. Obtained COI (Genbank accession number OR725417) sequence was 100% identical to that from G. mastophorum strain CBS 375.72 (EU350523), whereas the ß-tubulin sequence (OR725416) corresponded to 99.6 % pairwise identity (KJ595502). Further, pathogenicity of an obtained isolate was tested on 4 wk-old curly leaf (cv. Petra F1) parsley. Half of a 7 d-old CPA culture, consisting of mycelium and oogonia, was finely cut and mixed with ca 50 ml of nonsterile commercial substrate (Potgrond H, AGRO-FertiCrop) in each of six 400 ml pots. Pots were filled with ca 300 ml additional substrate, into which 5 parsley seedlings were planted. Control plants were treated equally but with sterile CPA. Plantlets were watered with sterile tap water and held at ambient light conditions and temperature (night 18 °C - day 23 °C). After 14 d, inoculated plants started wilting and yellowing and showed stunted growth. After 21 d, roots were severely decayed and the seedlings damped-off (Figure S1 C). Four pieces each from 10 decayed roots were plated. Thirty-one pieces revealed pythium-like colonies. Obtained isolates were morphologically identical to the strain used for inoculation and identified as G. mastophorum. Control plants developed no foliar or root symptoms and no pythium-like species was obtained. Agricultural advisors observed occurrence of parsley damping-off also in other nurseries in Slovenia what may lead to spreading the pathogen to parsley in production fields and private gardens. The case emphasizes the need for implementing phytosanitary measures in order to eliminate primary inoculum. Reports from field-infected plants showed that G. mastophorum is a pathogen of parsley in Australia (Petkowski et al. 2013) and the USA (Tsuchida et al. 2018), and celery in the Czech Republic (Safránková and Holková 2017). Others isolated G. mastophorum from parsley in The Netherlands (online database of the Westerdijk Fungal Biodiversity Institute, strain CBS 243.86). However, the here described case is, to the best of our knowledge, one of the rare documentations of damping-off due to G. mastophorum in Europe (Safránková and Holková 2017) and the first in Slovenia. Funding: The work was funded by the Ministry of Agriculture, Forestry and Food of Slovenia, and Slovenian Research and Innovation Agency (ARIS Programs P4-0431 and P4-0072).

First report of Colletotrichum incanum causing leaf spots on common bean in Europe (Slovenia).

A Phaseolus vulgaris L. leaf showing necrotic spots was collected in an experimental bean field in central Slovenia in August 2021. The field contained diverse common bean lines sourced from genebank collections, with each line represented by 10 plants. While symptomatic leaves were seen across various lines, the reported species derived exclusively from a Huasca Huallaga Colorado plant (single-seed descent, USDA accession PI153714, doi: 10.18730/H7P9N), a Peruvian landrace. After incubating the leaf for 2 d at ambient temperature in a moist chamber, setose acervuli developed producing curved, distally tapering and proximately truncated conidia. Single-spore cultures developed equally-shaped conidia measuring 14.5-21.5 (avg. 18.5) × 3-4 (avg. 3.5) µm (n=60) on corn meal agar when mounted in lactic acid. Obtained morphological characters and sequences of the partial actin (GenBank accession, OR208162), beta-tubulin (OR208164), and histone 3 (OR208165) gene identified the isolate as Colletotrichum incanum H.-C. Yang, J.S. Haudenshield & G.L. Hartman. Sequences were identical to those from CBS 133485 (= NRRL 62592, IL6A), ex-type strain of C. incanum (KC110823, KC110814, and KC110796). Partial sequences of the chitin synthase (CHS) gene (OR208163), not available for the ex-type strain, was identical to sequences of other C. incanum strains reported from China (KP145539, ON189040, and OQ613679-OQ613686) or differed in two nucleotide positions (OL471268 and OL471269). The strain from Slovenia was deposited in the CBS biobanks of the Westerdijk Fungal Biodiversity Institute (Utrecht, The Netherlands) as CBS 150848. Pathogenicity of the strain was tested by spraying ca. 3×105 conidia as a watery spore suspension onto each leaf of 6 greenhouse-grown and 3 wk-old common bean plantlets (cv. KIS Amand). Nonsterile commercial substrate (Potgrond H, AGRO-FertiCrop) was used and natural light conditions at ambient temperatures (18-23°C) applied. Sterile water was sprayed on 6, equally grown negative control plants. Treated plants showed small brownish spots after 3 wks similar to those described by Yang et al. (2014) on soybean. Setose acervuli formed within 5 days after detached leaves were incubated in moist chambers. No acervuli formed on negative control plants. Conidia re-isolated from these acervuli and obtained cultures were morphologically identical to originally obtained conidia and cultures and those used for performing the pathogenicity test. Anthracnose is an important disease of common bean attributed to various races of C. lindemuthianum (Sacc. & Magnus) Briosi & Cavara (Nunes et al. 2021). Reporting an additional agent potentially able to cause diseases in common bean and so far not known to occur in Europe is of high relevance as the various genetic bean lines used in Europe may show alternative susceptibility levels to it. However, symptoms caused by C. incanum seem to be less severe as those caused by C. lindemuthianum and the species belongs to the C. spaethianum species complex, whose members have so far not been considered as pathogens of economic importance (Talhinhas & Baroncelli 2021). Yang et al. (2014) based C. incanum on isolates from soybean petioles (USA) and associated it with common bean by re-identifying strain ATCC 64682 obtained by Tu (1990) in Canada. Database queries revealed that it was encountered also on sugar beet (USA; Hanson et al. 2023) and on various crop hosts in China (e.g., chili; Diao et al. 2017), but not in Europe. The work was funded by the Ministry of Agriculture, Forestry and Food and conducted as part of research programs P4-0072 and P4-0431, financed by the Slovenian Research and Innovation Agency ARIS, and the Horizon 2020 project INCREASE funded by the European Union.

Fifty Aureobasidium pullulans genomes reveal a recombining polyextremotolerant generalist.

The black yeast Aureobasidium pullulans is a textbook example of a generalistic and ubiquitous fungus thriving in a wide variety of environments. To investigate whether A. pullulans is a true generalist, or alternatively, whether part of its versatility can be attributed to intraspecific specialization masked by cryptic diversification undetectable by traditional phylogenetic analyses, we sequenced and analysed the genomes of 50 strains of A. pullulans from different habitats and geographic locations. No population structure was observed in the sequenced strains. Decay of linkage disequilibrium over shorter physical distances (<100 bp) than in many sexually reproducing fungi indicates a high level of recombination in the species. A homothallic mating locus was found in all of the sequenced genomes. Aureobasidium pullulans appears to have a homogeneous population genetics structure, which is best explained by good dispersal and high levels of recombination. This means that A. pullulans is a true generalist that can inhabit different habitats without substantial specialization to any of these habitats at the genomic level. Furthermore, in the future, the high level of A. pullulans recombination can be exploited for the identification of genomic loci that are involved in the many biotechnologically useful traits of this black yeast.

Occultifur mephitis f.a., sp. nov. and other yeast species from hypoxic and elevated CO2 mofette environments.

An inventory of culturable yeasts from the soil and water of natural CO2 springs (mofettes) in northeast Slovenia is presented. In mofettes, CO2 of geological origin reaches the soil surface causing temporarily and spatially stable hypoxic environments in soil and water. In total, 142 yeast strains were isolated and identified from high CO2 and control meadow soil, meadow ground-water, forest pond and stream water. All water locations showed below-ground CO2 release. They were assigned to six basidiomycetous yeast genera (six species) and 11 ascomycetous genera (18 species). All ascomycetous yeasts, with the exception of Debaryomyces hansenii, were able to grow under elevated CO2 and fermented glucose. Candida sophiae-reginae, Pichia fermentans and Candida vartiovaarae were the dominating species in meadow and forest high CO2 exposed water. Meyerozyma guilliermondii and Wickerhamomyces anomalus predominated in high CO2 exposed soils. Using high dilution plating of a mofette soil sample, four strains of an unknown basidiomycetous species were isolated and are here newly described as Occultifur mephitis based on molecular phylogenetic and phenotypic criteria. The type strain of Occultifur mephitis is EXF-6436T[CBS 14611=PYCC 7049, LT594852 (D1/D2), KX929055 (ITS)]. An additional three isolated strains are EXF-6437 (LT594853, KX929056), EXF-6473 (LT594863, KX929057) and EXF-6482 (LT594867, KX929054), as well as a strain reported from previous studies isolated from a leaf of Cistus albidus in Portugal (CBS 10223=PYCC 6067), EU002842 (D1/D2), KY308183 (ITS).

Genome sequencing of four Aureobasidium pullulans varieties: biotechnological potential, stress tolerance, and description of new species.

BACKGROUND: Aureobasidium pullulans is a black-yeast-like fungus used for production of the polysaccharide pullulan and the antimycotic aureobasidin A, and as a biocontrol agent in agriculture. It can cause opportunistic human infections, and it inhabits various extreme environments. To promote the understanding of these traits, we performed de-novo genome sequencing of the four varieties of A. pullulans. RESULTS: The 25.43-29.62 Mb genomes of these four varieties of A. pullulans encode between 10266 and 11866 predicted proteins. Their genomes encode most of the enzyme families involved in degradation of plant material and many sugar transporters, and they have genes possibly associated with degradation of plastic and aromatic compounds. Proteins believed to be involved in the synthesis of pullulan and siderophores, but not of aureobasidin A, are predicted. Putative stress-tolerance genes include several aquaporins and aquaglyceroporins, large numbers of alkali-metal cation transporters, genes for the synthesis of compatible solutes and melanin, all of the components of the high-osmolarity glycerol pathway, and bacteriorhodopsin-like proteins. All of these genomes contain a homothallic mating-type locus. CONCLUSIONS: The differences between these four varieties of A. pullulans are large enough to justify their redefinition as separate species: A. pullulans, A. melanogenum, A. subglaciale and A. namibiae. The redundancy observed in several gene families can be linked to the nutritional versatility of these species and their particular stress tolerance. The availability of the genome sequences of the four Aureobasidium species should improve their biotechnological exploitation and promote our understanding of their stress-tolerance mechanisms, diverse lifestyles, and pathogenic potential.

Osmoadaptation strategy of the most halophilic fungus, Wallemia ichthyophaga, growing optimally at salinities above 15% NaCl.

Wallemia ichthyophaga is a fungus from the ancient basidiomycetous genus Wallemia (Wallemiales, Wallemiomycetes) that grows only at salinities between 10% (wt/vol) NaCl and saturated NaCl solution. This obligate halophily is unique among fungi. The main goal of this study was to determine the optimal salinity range for growth of the halophilic W. ichthyophaga and to unravel its osmoadaptation strategy. Our results showed that growth on solid growth media was extremely slow and resulted in small colonies. On the other hand, in the liquid batch cultures, the specific growth rates of W. ichthyophaga were higher, and the biomass production increased with increasing salinities. The optimum salinity range for growth of W. ichthyophaga was between 15 and 20% (wt/vol) NaCl. At 10% NaCl, the biomass production and the growth rate were by far the lowest among all tested salinities. Furthermore, the cell wall content in the dry biomass was extremely high at salinities above 10%. Our results also showed that glycerol was the major osmotically regulated solute, since its accumulation increased with salinity and was diminished by hypo-osmotic shock. Besides glycerol, smaller amounts of arabitol and trace amounts of mannitol were also detected. In addition, W. ichthyophaga maintained relatively small intracellular amounts of potassium and sodium at constant salinities, but during hyperosmotic shock, the amounts of both cations increased significantly. Given our results and the recent availability of the genome sequence, W. ichthyophaga should become well established as a novel model organism for studies of halophily in eukaryotes.

Genome and transcriptome sequencing of the halophilic fungus Wallemia ichthyophaga: haloadaptations present and absent.

BACKGROUND: The basidomycete Wallemia ichthyophaga from the phylogenetically distinct class Wallemiomycetes is the most halophilic fungus known to date. It requires at least 10% NaCl and thrives in saturated salt solution. To investigate the genomic basis of this exceptional phenotype, we obtained a de-novo genome sequence of the species type-strain and analysed its transcriptomic response to conditions close to the limits of its lower and upper salinity range. RESULTS: The unusually compact genome is 9.6 Mb large and contains 1.67% repetitive sequences. Only 4884 predicted protein coding genes cover almost three quarters of the sequence. Of 639 differentially expressed genes, two thirds are more expressed at lower salinity. Phylogenomic analysis based on the largest dataset used to date (whole proteomes) positions Wallemiomycetes as a 250-million-year-old sister group of Agaricomycotina. Contrary to the closely related species Wallemia sebi, W. ichthyophaga appears to have lost the ability for sexual reproduction. Several protein families are significantly expanded or contracted in the genome. Among these, there are the P-type ATPase cation transporters, but not the sodium/ hydrogen exchanger family. Transcription of all but three cation transporters is not salt dependent. The analysis also reveals a significant enrichment in hydrophobins, which are cell-wall proteins with multiple cellular functions. Half of these are differentially expressed, and most contain an unusually large number of acidic amino acids. This discovery is of particular interest due to the numerous applications of hydrophobines from other fungi in industry, pharmaceutics and medicine. CONCLUSIONS: W. ichthyophaga is an extremophilic specialist that shows only low levels of adaptability and genetic recombination. This is reflected in the characteristics of its genome and its transcriptomic response to salt. No unusual traits were observed in common salt-tolerance mechanisms, such as transport of inorganic ions or synthesis of compatible solutes. Instead, various data indicate a role of the cell wall of W. ichthyophaga in its response to salt. Availability of the genomic sequence is expected to facilitate further research into this unique species, and shed more light on adaptations that allow it to thrive in conditions lethal to most other eukaryotes.

The mycobiota of the salterns.

Solar salterns are constructed as shallow multi-pond systems for the production of halite through evaporation of seawater. The main feature of salterns is the discontinuous salinity gradient that provides a range of well-defined habitats with increasing salinities, from moderate to hypersaline. These present one of the most extreme environments, because of the low levels of biologically available water and the toxic concentrations of ions. Up to the year 2000, hypersaline environments were considered to be populated almost exclusively by prokaryotic microorganisms till fungi were reported to be active inhabitants of solar salterns. Since then, numerous fungal species have been described in hypersaline waters around the world. The mycobiota of salterns is represented by different species of the genus Cladosporium and the related meristematic melanized black yeasts, of non-melanized yeasts, of the filamentous genera Penicillium and Aspergillus and their teleomorphic forms (Eurotium and Emericella), and of the basidiomycetous genus Wallemia. Among these, two species became new model organisms for studying the mechanisms of extreme salt tolerance: the extremely halotolerant ascomycetous black yeast Hortaea werneckii and the obligate halophilic basidiomycete Wallemia ichthyophaga.

From Glaciers to Refrigerators: the Population Genomics and Biocontrol Potential of the Black Yeast Aureobasidium subglaciale.

Apples are affected by numerous fungi known as storage rots, which cause significant losses before and after harvest. Concerns about increasing antimicrobial resistance, bans on various fungicides, and changing consumer preferences are motivating the search for safer means to prevent fruit rot. The use of antagonistic microbes has been shown to be an efficient and environmentally friendly alternative to conventional phytopharmaceuticals. Here, we investigate the potential of Aureobasidium subglaciale for postharvest rot control. We tested the antagonistic activity of 9 strains of A. subglaciale and 7 closely related strains against relevant phytopathogenic fungi under conditions simulating low-temperature storage: Botrytis cinerea, Penicillium expansum, and Colletotrichum acutatum. We also investigated a selection of phenotypic traits of all strains and sequenced their whole genomes. The tested strains significantly reduced postharvest rot of apples at low temperatures caused by B. cinerea, C. acutatum (over 60%), and P. expansum (about 40%). Several phenotypic traits were observed that may contribute to this biocontrol capacity: growth at low temperatures, tolerance to high temperatures and elevated solute concentrations, and strong production of several extracellular enzymes and siderophores. Population genomics revealed that 7 of the 15 strains originally identified as A. subglaciale most likely belong to other, possibly undescribed species of the same genus. In addition, the population structure and linkage disequilibrium of the species suggest that A. subglaciale is strictly clonal and therefore particularly well suited for use in biocontrol. Overall, these data suggest substantial biological control potential for A. subglaciale, which represents another promising biological agent for disease control in fresh fruit. IMPORTANCE After harvest, fruits are often stored at low temperatures to prolong their life. However, despite the low temperatures, much of the fruit is lost to rot caused by a variety of fungi, resulting in major economic losses and food safety risks. An increasingly important environmentally friendly alternative to conventional methods of mitigating the effects of plant diseases is the use of microorganisms that act similarly to probiotics-occupying the available space, producing antimicrobial compounds, and consuming the nutrients needed by the rot-causing species. To find a new microorganism for biological control that is particularly suitable for cold storage of fruit, we tested different isolates of the cold-loving yeast Aureobasidium subglaciale and studied their phenotypic characteristics and genomes. We demonstrated that A. subglaciale can significantly reduce rotting of apples caused by three rot-causing molds at low temperatures and thus has great potential for preventing fruit rot during cold storage.

New Insights into Antibacterial and Antifungal Properties, Cytotoxicity and Aquatic Ecotoxicity of Flame Retardant PA6/DOPO-Derivative Nanocomposite Textile Fibers.

The aim of this study was to evaluate the antibacterial and antifungal activity, cytotoxicity, leaching, and ecotoxicity of novel flame retardant polyamide 6 (PA6) textile fibers developed by our research group. The textile fibers were produced by the incorporation of flame-retardant bridged 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) derivative (PHED) in the PA6 matrix during the in situ polymerization process at concentrations equal to 10 and 15 wt% (PA6/10PHED and PA6/15PHED, respectively). Whilst the nanodispersed PHED provided highly efficient flame retardancy, its biological activity led to excellent antibacterial activity against Escherichia coli and Staphylococcus aureus, as well as excellent antifungal activity against Aspergillus niger and Candida albicans. The results confirmed leaching of the PHED, but the tested leachates did not cause any measurable toxic effect to the duckweed Lemna minor. The in vitro cytotoxicity of the leached PHED from the PA6/15PHED sample was confirmed for human cells from adipose tissue in direct and prolonged contact. The targeted biological activity of the organophosphinate flame retardant could be beneficial for the development of PA6 textile materials with multifunctional properties and the low ecotoxicity profile, while the PHED's leaching and cytotoxicity limit their application involving the washing processes and direct contact with the skin.

Stress-Tolerant Yeasts: Opportunistic Pathogenicity Versus Biocontrol Potential.

Stress-tolerant fungi that can thrive under various environmental extremes are highly desirable for their application to biological control, as an alternative to chemicals for pest management. However, in fungi, the mechanisms of stress tolerance might also have roles in mammal opportunism. We tested five species with high biocontrol potential in agriculture (Aureobasidiumpullulans, Debayomyceshansenii, Meyerozymaguilliermondii, Metschnikowiafructicola, Rhodotorulamucilaginosa) and two species recognized as emerging opportunistic human pathogens (Exophialadermatitidis, Aureobasidiummelanogenum) for growth under oligotrophic conditions and at 37 °C, and for tolerance to oxidative stress, formation of biofilms, production of hydrolytic enzymes and siderophores, and use of hydrocarbons as sole carbon source. The results show large overlap between traits desirable for biocontrol and traits linked to opportunism (growth under oligotrophic conditions, production of siderophores, high oxidative stress tolerance, and specific enzyme activities). Based on existing knowledge and these data, we suggest that oligotrophism and thermotolerance together with siderophore production at 37 °C, urease activity, melanization, and biofilm production are the main traits that increase the potential for fungi to cause opportunistic infections in mammals. These traits should be carefully considered when assessing safety of potential biocontrol agents.

Genomic Evidence of Recombination in the Basidiomycete Wallemia mellicola.

One of the most commonly encountered species in the small basidiomycetous sub-phylum Wallemiomycotina is Wallemiamellicola, a xerotolerant fungus with a widespread distribution. To investigate the population characteristics of the species, whole genomes of twenty-five strains were sequenced. Apart from identification of four strains of clonal origin, the distances between the genomes failed to reflect either the isolation habitat of the strains or their geographical origin. Strains from different parts of the world appeared to represent a relatively homogenous and widespread population. The lack of concordance between individual gene phylogenies and the decay of linkage disequilibrium indicated that W. mellicola is at least occasionally recombining. Two versions of a putative mating-type locus have been found in all sequenced genomes, each present in approximately half of the strains. W. mellicola thus appears to be capable of (sexual) recombination and shows no signs of allopatric speciation or specialization to specific habitats.

Population Genomics of an Obligately Halophilic Basidiomycete Wallemia ichthyophaga.

BACKGROUND: Wallemia ichthyophaga is a highly specialized basidiomycetous fungus. It is one of the most halophilic fungi ever described, only able to grow at low water activity. This specialization is thought to explain why it is only rarely isolated from nature. RESULTS: Genomes of 21 W. ichthyophaga strains were sequenced with PE150 reads on BGISEQ500 platform. The genomes shared high similarity with the reference genome of the species, they were all smaller than 10 Mbp and had a low number of predicted genes. Groups of strains isolated in the same location encompassed clones as well as very divergent strains. There was little concordance between phylogenies of predicted genes. Linkage disequilibrium of pairs of polymorphic loci decayed relatively quickly as a function of distance between the loci (LD decay distance 1270 bp). For the first time a putative mating-type locus was identified in the genomes of W. ichthyophaga. CONCLUSION: Based on the comparison of W. ichthyophaga genomes it appears that some phylogenetic lineages of the species can persist in the same location over at least several years. Apart from this, the differences between the strains do not reflect the isolation habitat or geographic location. Together with results supporting the existence of (sexual) recombination in W. ichthyophaga, the presented results indicate that strains of W. ichthyophaga can form a single recombining population even between different habitats and over large geographical distances.

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.

The Genus Wallemia-From Contamination of Food to Health Threat.

The fungal genus Wallemia of the order Wallemiales (Wallemiomycotina, Basidiomycota) comprises the most xerotolerant, xerophilic and also halophilic species worldwide. Wallemia spp. are found in various osmotically challenged environments, such as dry, salted, or highly sugared foods, dry feed, hypersaline waters of solar salterns, salt crystals, indoor and outdoor air, and agriculture aerosols. Recently, eight species were recognized for the genus Wallemia, among which four are commonly associated with foods: W. sebi, W. mellicola, W. muriae and W. ichthyophaga. To date, only strains of W. sebi, W. mellicola and W. muriae have been reported to be related to human health problems, as either allergological conditions (e.g., farmer's lung disease) or rare subcutaneous/cutaneous infections. Therefore, this allergological and infective potential, together with the toxins that the majority of Wallemia spp. produce even under saline conditions, defines these fungi as filamentous food-borne pathogenic fungi.

Chaophilic or chaotolerant fungi: a new category of extremophiles?

It is well known that few halophilic bacteria and archaea as well as certain fungi can grow at the highest concentrations of NaCl. However, data about possible life at extremely high concentrations of various others kosmotropic (stabilizing; like NaCl, KCl, and MgSO4) and chaotropic (destabilizing) salts (NaBr, MgCl2, and CaCl2) are scarce for prokaryotes and almost absent for the eukaryotic domain including fungi. Fungi from diverse (extreme) environments were tested for their ability to grow at the highest concentrations of kosmotropic and chaotropic salts ever recorded to support life. The majority of fungi showed preference for relatively high concentrations of kosmotropes. However, our study revealed the outstanding tolerance of several fungi to high concentrations of MgCl2 (up to 2.1 M) or CaCl2 (up to 2.0 M) without compensating kosmotropic salts. Few species, for instance Hortaea werneckii, Eurotium amstelodami, Eurotium chevalieri and Wallemia ichthyophaga, are able to thrive in media with the highest salinities of all salts (except for CaCl2 in the case of W. ichthyophaga). The upper concentration of MgCl2 to support fungal life in the absence of kosmotropes (2.1 M) is much higher than previously determined to be the upper limit for microbial growth (1.26 M). No fungal representatives showed exclusive preference for only chaotropic salts (being obligate chaophiles). Nevertheless, our study expands the knowledge of possible active life by a diverse set of fungi in biologically detrimental chaotropic environments.

Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective.

Molecular studies of salt tolerance of eukaryotic microorganisms have until recently been limited to the baker's yeast Saccharomyces cerevisiae and a few other moderately halotolerant yeast. Discovery of the extremely halotolerant and adaptable fungus Hortaea werneckii and the obligate halophile Wallemia ichthyophaga introduced two new model organisms into studies on the mechanisms of salt tolerance in eukaryotes. H. werneckii is unique in its adaptability to fluctuations in salt concentrations, as it can grow without NaCl as well as in the presence of up to 5 M NaCl. On the other hand, W. ichthyophaga requires at least 1.5 M NaCl for growth, but also grows in up to 5 M NaCl. Our studies have revealed the novel and intricate molecular mechanisms used by these fungi to combat high salt concentrations, which differ in many aspects between the extremely halotolerant H. werneckii and the halophilic W. ichthyophaga. Specifically, the high osmolarity glycerol signaling pathway that is important for sensing and responding to increased salt concentrations is here compared between H. werneckii and W. ichthyophaga. In both of these fungi, the key signaling components are conserved, but there are structural and regulation differences between these pathways in H. werneckii and W. ichthyophaga. We also address differences that have been revealed from analysis of their newly sequenced genomes. The most striking characteristics associated with H. werneckii are the large genetic redundancy, the expansion of genes encoding metal cation transporters, and a relatively recent whole genome duplication. In contrast, the genome of W. ichthyophaga is very compact, as only 4884 protein-coding genes are predicted, which cover almost three quarters of the sequence. Importantly, there has been a significant increase in their hydrophobins, cell-wall proteins that have multiple cellular functions.

Morphological responses to high sugar concentrations differ from adaptation to high salt concentrations in the xerophilic fungi Wallemia spp.

Fungi from the food-borne basidiomycetous genus Wallemia, which comprises Wallemia ichthyophaga, Wallemia muriae and Wallemia sebi, are among the most xerophilic organisms described. Their morphological adaptations to life at high NaCl concentrations are reflected in increased cell-wall thickness and size of cellular aggregates. The objectives of this study were to examine their growth and to define cell morphology and any ultrastructural cell-wall changes when these fungi are grown in low and high glucose and honey concentrations, as environmental osmolytes. We analysed their growth parameters and morphological characteristics by light microscopy and transmission and scanning electron microscopy. Wallemia ichthyophaga grew slowly in all of the sugar-based media, while W. muriae and W. sebi demonstrated better growth. Wallemia ichthyophaga adapted to the high glucose and honey concentrations with formation of larger cellular aggregates, while cell-wall thickness was increased only at the high glucose concentration. Wallemia muriae and W. sebi demonstrated particularly smaller sizes of hyphal aggregates at the high glucose concentration, and different and less explicit changes in cell-wall thickness. Adaptive responses show that the phylogenetically more distant W. ichthyophaga is better adapted to high salt conditions, whereas W. muriae and W. sebi cope better with a high sugar environment.

Adaptation of the glycerol-3-phosphate dehydrogenase Gpd1 to high salinities in the extremely halotolerant Hortaea werneckii and halophilic Wallemia ichthyophaga.

We report the first identification and characterisation of the glycerol-3-phosphate dehydrogenase (GPD) genes from extremely halophilic fungi. The black ascomycetous yeast Hortaea werneckii and the non-melanised basidiomycetous fungus Wallemia ichthyophaga inhabit similar hypersaline environments, yet they have two different strategies of haloadaptation through Gpd1-regulated glycerol synthesis. The extremely halotolerant H. werneckii codes for two salt-inducible GPD1 genes that show similar gene transcription regulation and have 98% amino-acid sequence identity between paralogues; however, they have distinct effects when expressed heterologously in Saccharomyces cerevisiae gpd mutants. Only the HwGpd1B isoform complements the function of Gpd in the gpd1 mutant, whereas none of the Gpd1 isoforms can rescue the salt sensitivity of the gpd1gpd2 double mutant. The obligate halophile W. ichthyophaga codes for only one GPD1 orthologue, the transcription of which is less affected by salt when compared to the H. werneckii homologues. Heterologous expression of WiGPD1 in S. cerevisiae recovers halotolerance of the gpd1 and gpd1gpd2 mutant strains, which is probably due to the overall high amino-acid similarity of the Gpd1 protein in W. ichthyophaga and S. cerevisiae. Phylogenetic analysis of amino-acid sequences reveals that the evolutionary origins of all of these three novel enzymes correspond to the phylogeny of the fungal species from which the genes were identified.