mycological research 110 (2006) 713–724 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/mycres Hypersaline conditions induce changes in cell-wall melanization and colony structure in a halophilic and a xerophilic black yeast species of the genus Trimmatostroma Tina KOGEJa,*, Anna A. GORBUSHINAb, Nina GUNDE-CIMERMANa a University of Ljubljana, Biotech. Faculty, Dept. of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia Geomicrobiology, ICBM, Carl von Ossietzky Universität Oldenburg, POB 2503, D-26111 Oldenburg, Germany b article info abstract Article history: Melanized yeast-like meristematic fungi are characteristic inhabitants of highly stressed Received 1 July 2005 environments and are rare eukaryotic extremophiles. Therefore, they are attractive organ- Received in revised form isms for studies of adaptations. In this study we compared two meristematic species of the 21 December 2005 genus Trimmatostroma on media of differing water potentials isolated from distinct water- Accepted 25 January 2006 stressed environments: T. salinum from the hypersaline water of a solar saltern, and T. abie- Published online 12 June 2006 tis from a marble monument in Crimea. The morphology and melanization of both isolates Corresponding Editor: in response to sodium chloride-induced water stress were investigated by means of light Nicholas P. Money and electron microscopy. We describe and compare the colony form and structure, ultrastructure, and degree of cell-wall melanization of both species in reaction to salinity and Keywords: to inhibited melanin synthesis. The halophilic T. salinum responded to changed salinity Fungal physiology conditions on the level of individual cell ultrastructure and degree of cell wall melaniza- Hyphomycetes tion, whereas the xerophilic rock-inhabiting T. abietis responded with modification of its Marine fungi colony structure. Surprisingly, both the halophilic and the xerophilic Trimmatostroma spe- Melanin cies were able to adapt to hypersaline growth conditions, although their growth patterns Salinity show distinct adaptation of each species to their natural ecological niches. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Eukaryotic halophilic microorganisms are poorly investigated and little is known about their adaptation to growth at extremely hypersaline conditions (Petrovič et al. 2002). As black yeast-like meristematic fungi were frequently isolated from solar salterns around the world (Butinar et al. 2005), they represent highly appropriate organisms for studying the mechanisms of salt tolerance in eukaryotes (Petrovič et al. 1999; Petrovič et al. 2002; Turk & Plemenitaš 2002). One of the isolated halophilic species is from the black yeast genus Trimmatostroma (Dothideales), which contains 34 species (www.indexfungorum.org/names), primarily inhabiting dead or living plant material (Ellis 1971; Ellis & Ellis 1985), but also extreme habitats such as salt marshes (Abdel-Fattah et al. 1977), hypersaline water (Gunde-Cimerman et al. 2000; Gunde-Cimerman et al. 2004), desert soil (Abdel-Hafez 1982), arid and uv-stressed atmosphere-exposed inorganic and organic surfaces (Butin et al. 1996; Krumbein et al. 1996). We compared sodium chloride-induced morphological adaptations in two extremophilic Trimmatostroma species from distinct water-stressed environments. Halophilic T. salinum * Corresponding author. E-mail address: tina.kogej@bf.uni-lj.si. 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.01.014 714 (Zalar et al. 1999), which is a constitutively melanized fungus, was selected as it is the most melanized representative of halophilic black yeasts from the salterns. It has been repeatedly isolated from hypersaline water at the highest salinity level throughout the season of salt production (Gunde-Cimerman et al. 2000; Butinar et al. 2005). It was also isolated from microbial mats and wood, immersed in brine, and is able to decompose wood at hypersaline conditions (Zalar et al. 2005). Thus, it has an important, previously unrecognised ecological role in this environment. Xerophilic T. abietis is found on marble and rock surfaces, and similar air-exposed environments (Krumbein et al. 1996; Gorbushina et al. 1996). It was selected for comparison due to its ability to withstand reduced water activity, which is a consequence of low humidity and not increased sodium chloride. Both species undergo similar meristematic conidiogenesis, and have a similar, highly adapted morphological ecotype and the characteristic dark pigment, generally referred to as melanin (Wheeler & Greenblatt 1988; Giraud et al. 1995; Gorbushina & Krumbein 2000; Jacobson 2000; Petrovič et al. 2002), consistently present in their cell walls (Romero-Martı́nez et al. 2000). In order to investigate melanin distribution in the cells at different sodium chloride concentrations, both species were grown on normal media and on media amended with melanin-synthesis inhibitor. We examined them at the colonial and cellular level by light and TEM. In this study, we exposed the two black-yeast species to moderate and extremely saline conditions and identified their morphological adaptations by comparison. Materials and methods Strains The halophilic Trimmatostroma salinum MZKI B-734 was isolated from hypersaline water of a crystallization pond in the Adriatic solar saltern Sečovlje in Slovenia (Zalar et al. 1999); it is deposited in the Culture Collection of the National Institute of Chemistry, Ljubljana (MZKI), Slovenia. The xerophilic T. abietis A18 was isolated from the marble monuments in Chersonesos, Crimea, Ukraine (Gorbushina et al. 1996) and identified at the Centraalbureau voor Schimmelcultures (CBS, Utrecht); it is maintained in the culture collection of the Geomicrobiology group, Carl von Ossietzky University, Oldenburg, Germany. Media and growth conditions Fungal cultures were maintained on potato-dextrose slant agar (PDA) (Anon 1995) and stored at 4
C. They were grown on solid malt-extract medium (MEA), which consisted of 20 g l1 malt extract, 0.1 g l1 Bacto-peptone, 20 g l1 glucose, 20 g l1 agar with different concentrations of sodium chloride. For growth curve determination, fungal cultures were grown on solid MEA medium with sodium chloride [0–30 % (w/v) in steps of 2 %]. The diameter of the growing colonies was measured every 2 d for four months, and a mean value of two measured lines, perpendicular to each other, was taken. The increase in colony radius was plotted against time and the T. Kogej et al. linear regression was calculated in order to estimate growth rate (mm d1) under each set of salinity conditions for each species. Three to four replicate plates per fungal strain and per salinity were used in each experiment. For histological and ultrastructural studies, the fungi were grown on solid MEA media at minimal, optimal and maximal salinities. These were without sodium chloride, and with 4 % (0.68 M), 10 % (1.71 M), and 20 % (w/v) (3.42 M) sodium chloride for Trimmatostroma salinum, and without sodium chloride, and with 6 % (1.03 M), and 10 % (1.71 M) sodium chloride for T. abietis, respectively. A dihydroxynaphthalene (DHN)-melanin inhibitor tricyclazole [5-methyl-1,2,4-triazolo(3,4,b)-benzothiazole]; (Ely Lilly, Indianapolis) was used in control media to prevent melanin biosynthesis. Tricyclazole was dissolved in ethanol and added to the media after sterilization to obtain the final concentration of 30 mg ml1 and 1 %, respectively. Preculturing on the same MEA media as in the experiment was used to adapt the fungi to the media and growth conditions used. The inoculi from pre-cultures were prepared as cell suspensions in sterile water of the same salinity and drop-inoculated onto a piece of cellophane for TEM studies or onto a nitrocellulose filter for histological analyses. Both were overlaid on the surface of solid MEA media. The plates were incubated at 22
C for 10 d in the dark. After 9 d of growth, colonies were observed and photographed with a dissecting microscope Zeiss DR (Zeiss, Jena) combined with a camera M35 using a Kodak Ektachrome 64T film. TEM Ten day-old colonies were fixed in 2.5 % glutaraldehyde and 4 % formaldehyde in 0.1 M phosphate buffer at pH 7.2 (Brisson et al. 1996) for 2 h at room temperature and post-fixed in 1 % osmium tetroxide for 1 h at 4
C. Dehydration was performed in a series of graded ethanol solutions: 30 % (v/v) (15 min), 50 % (2  15 min), 70 % (2  15 min), 90 % (2  15 min), absolute ethanol (2  15 min, 1  30 min). Spurr’s resin (Spurr 1969) was used for embedding. Sections were cut using an ultramicrotome Ultracut (Reichert, Vienna) and contrasted with uranyl acetate and lead citrate (Reynolds 1963). The sections were examined with the transmission electron microscope Philips CM 100 (80 kV; digital camera Gatan Bioscan Camera 792) and with Zeiss EM 109 (50 kV; TFP camera, film Ilford). The experiment was repeated twice. Embedding in 2 % agarose was used to preserve the structure of the softer extracellular polymeric substances (EPS)-rich colonies of T. salinum grown on 4 % sodium chloride with tricyclazole, and on 10 % sodium chloride with tricyclazole, and T. abietis grown on all media and salinities (0, 6, 10 %, with and without tricyclazole). Histology and light microscopy Light-microscopy preparations of fungi were mounted in lactic acid, glycerol and water to observe cell morphology, and negatively stained with India ink to observe the presence of EPS. For histology, 10 d-old colonies were fixed in the same way as for TEM but without post-fixation. Several drops of 15 % (v/v) ethanol were added to the samples in the fixative solution to overcome the hydrophobicity of the fungal colonies. Salinity-induced changes in Trimmatostroma spp. Results A Growth (mm day-1) Dehydration was performed in a graded ethanol series as for TEM. Technovit 7100 resin was used for embedding according to the instructions of the manufacturer (Kulzer Heraeus HistoTechnik, Wehrheim/Taunus, Germany). Sections were cut using a Reichert microtome and stained with PAS (Whitlatch & Johnson 1974) and toluidine blue. The samples were observed with a light microscope Zeiss Axioskop 2 and photographed with an Olympus C-3030Zoom digital camera. 715 Growth curves 2 1.5 1 0.5 0 0 Colony structure of Trimmatostroma salinum In Trimmatostroma salinum, the development of the colony began with the growth of hyaline vegetative hyphae. The hyphae on the growing edge of the colony were colourless, becoming dark olive green towards the centre of the colony. They swelled, rounded, and developed transverse and longitudinal septa and underwent a meristematic conversion to release multicellular round and strongly melanized meristematic conidia by disarticulation, which were concentrated in the heaped cauliflower-like centre of the colony (Fig 2A). Submerged thick melanized substrate hyphae formed in older colonies at all salinities except at 20 % sodium chloride. The colonies were at first soft, but were dry and crumbling at all salinities after one month. The growth at different sodium chloride concentrations affected their size, colour and prevalent cell types (see Fig 2C, E, G and Table 1 for description). The hyaline hyphal growth on the colony margin was most pronounced at optimal growth salinities (4 and 10 %), and more restricted on non-saline medium. The colonies on 20 % sodium chloride had a reduced area of contact with the medium and pronounced growth away from the substrate. The addition of tricyclazole to the media caused reddish – brown pigmentation of the colonies and of the media, and less expressed hyphal growth on the colony margin. Histology showed the presence of obovate individual cells on the media without sodium chloride and with 4 % and 10 % sodium chloride (Fig 2B, D, F), and irregular multicellular clusters in abundant light-brown slime at 20 % sodium chloride (Fig 2H). Colony structure of Trimmatostroma abietis The colonies of Trimmatostroma abietis on non-saline MEA were initially yellowish brown with hyaline peripheral hyphae, 5 10 15 20 25 30 25 30 NaCl in the medium (%) B 60 Colony diameter (mm) Halophilic Trimmatostroma salinum could grow at up to 26 % sodium chloride with optimal growth at 2–6 % sodium chloride, whereas, surprisingly, xerophilic T. abietis could grow at up to 16 % sodium chloride with an optimum at 2 % (Fig 1A). Growth of both species was slower and more restricted on the medium without added sodium chloride. Maximum colony diameter at all salinities was obtained after 55 d for T. salinum and after 119 d for T. abietis (Fig 1B). Their maximum colony diameters were comparable at salinities up to 10 % sodium chloride. 50 40 30 20 10 0 0 5 10 15 20 NaCl in the medium (%) Fig 1 – Growth rates (A) and final colony diameter (B) of halophilic Trimmatostroma salinum (A) and of halotolerant T. abietis (:) on saline MEA media. For growth curve determination, the diameter of fungal cultures grown on solid MEA medium with sodium chloride was measured every 2 d for four months, and a mean of two lines perpendicular to each other was taken. Three to four replicate plates of each fungal strain and salinity were used in each experiment. (A) Final colony diameter was obtained after 55 d for T. salinum and after 119 d for T. abietis. (B) The temporal increase in radius was plotted against time and the linear regression was calculated in order to estimate the growth in millimetre per day under each set of salinity conditions for each species. which became olivaceous–green with age. Oblong two-to four-celled transversely septate melanized conidia (Fig 3A) formed in chains from hyphae in the centre of the colony. Young conidia were uniformly yellowish brown, older conidia had a ring or patches of cell-wall fragments around central part of the cells and hyaline cell wall at the apex. T. abietis colonies were soft, velvety on the edge, heaped and cauliflower-like at the centre. They were covered by aerial hyphae, which were more pronounced in older colonies and at higher salinities. The growth at different sodium chloride concentrations influenced the size of the colony, its colour and structure (see Fig 3A, C, E and Table 1 for description). T. abietis formed a layered colony of tightly interwoven hyphae on the saline MEA medium 716 T. Kogej et al. Fig 2 – Histological sections of Trimmatostroma salinum colonies grown on MEA media with: (A) no sodium chloride; (B) no sodium chloride with tricyclazole; (C) 4 % sodium chloride; (D) 4 % sodium chloride with tricyclazole; (E) 10 % sodium chloride; (F) 10 % sodium chloride with tricyclazole; (G) 20 % sodium chloride; (H) 20 % sodium chloride with tricyclazole. All sections were unstained. The scale bars represent 50 mm. B, bottom of the colony; T, top of the colony. (Fig 3C, E). The basal layer in the immediate vicinity of the sodium chloride-containing medium was darker, compact and relatively thin, while the middle layer was composed of more loose conidia and hyphae. The addition of tricyclazole to the media caused reddish brown pigmentation of the colonies at all salinities and diffused reddish pigmentation in non-saline MEA, reduced hyphal growth on the colony margin, and more aerial hyphae. The colony on non-saline tricyclazole-amended MEA formed a mat-like structure of tightly interwoven hyphae, and the layered colony structure was more evident at higher salinities (Fig 3B, D, F). Less conidia were produced at higher salinities as the hyphae did not disarticulate. TEM of Trimmatostroma salinum cells It has been reported that addition of non-toxic concentrations of the DHN-melanin biosynthesis inhibitor tricyclazole to the T. salinum MEA salinity No NaCl Colony colour Dark brown to yellowish/olivaceous green Filamentous, heaped, wrinkled, dark reverse Colony description 4 % NaCl 10 % NaCl 20 % NaCl Darker olivaceous green to black Yellowish green Dark greenish brown Filamentous, heaped, dark reverse Filamentous, heaped, octopus-like, dark reverse Dark, long, very thick tree-like branched substrate hyphae with mass of meristematic conidia and short hyaline apex Short yellowish Small, pyramid-like, restricted growth, dark reverse Smooth, very restricted short substrate hyphae Colony margin Sparse, short, very dense peripheral dark hyphae with hyaline apex Very dense, longer and branched peripheral dark hyphae with hyaline apex Aerial hyphae Short yellow Short yellowish Conidiation Formation of meristematic conidia by transverse septation, swelling, and longitudinal and disarticulation of hyphae Disarticulated meristematic multicellular clumps of round 1- few-celled conidia with melanized cell walls and abundant slime around the cells; endoconidiation in some of the cells (see Fig 2A) Formation of meristematic conidia by transverse septation, swelling, and longitudinal and disarticulation of hyphae Abundant and loose melanized meristematic clumps, composed of 2- to 3-celled endoconidia, rare multicellular clumps, loose aerial mycelium of melanized moniliform hyphae, no slime; conidia round, strongly melanized with thick cell walls, 1- to 2-celled; cell wall is peeling off, breaking (see Fig 2C) Histology T. abietis Formation of meristematic conidia by transverse septation, swelling, and longitudinal and disarticulation of hyphae Short colourless Formation of meristematic conidia by transverse septation, swelling, and longitudinal and disarticulation of hyphae Loose mass of Disarticulating separate disarticulated meristematic cells, some meristematic conidia, internally divided as in 1- to multicellular, endoconidiation; conidia round, prevailing 1-celled, round with thick melanized cell walls, rarely 2-celled, strongly brittle and melanized with thick cell disintegrating crust, wall, in some cells peeling off, no penetration frequent detached cell wall fragments into the filter, colony (see Fig 2G) compaction most expressed (see Fig 2E) no NaCl 6 % NaCl 10 % NaCl Yellowish brown to dark olivaceous green Filamentous, heaped, slimy, dark reverse Dark green Grey green Filamentous, heaped, dark reverse Short and thin hyaline hyphae Short and thin hyaline hyphae Small, hedgehog-like, restricted growth, dark reverse Sparse short light brown hyphae with hyaline apex Yellow Pronounced yellow Formation of conidia in chains by meristematic conversion and disarticulation of hyphae Formation of conidia in chains by meristematic conversion and disarticulation of hyphae Salinity-induced changes in Trimmatostroma spp. Table 1 – Colony characteristics of Trimmatostroma salinum and T. abietis grown at different sodium chloride (NaCl) concentrations Dense colourless Formation of conidia in chains by meristematic conversion and disarticulation of hyphae, disarticulation incomplete Single very compact Basal layer of closely Individual oblong thin layer of dense, interwoven tissue-like 2- to 4-celled tissue-like interwoven and lightly melanized melanized conidia hyphae, covered hyphae, covered by a and loosely organized thick layer of melanized with abundant aerial meristematic hyphae hyphae; scarce embedded in abundant meristematic oblong multicellular oblong 2- to 4-celled conidia colonial slime conidia, peeling off in loose chains, some (see Fig 3A) of the cell wall on interstitial slime conidia (see Fig 3E) between the cells (see Fig 3C) 717 718 T. Kogej et al. Fig 3 – Histological sections of Trimmatostroma abietis colonies grown on MEA media with: (A) no sodium chloride; (B) no sodium chloride with tricyclazole; (C) 6 % sodium chloride; (D) 6 % sodium chloride with tricyclazole; (E) 10 % sodium chloride; (F) 10 % sodium chloride with tricyclazole. All sections were stained with PAS. The scale bars represent 50 mm. B, bottom of the colony; T, top of the colony. culture medium eliminates or greatly diminishes the appearance of distinct electron-dense granules (Bell & Wheeler 1986). We used tricyclazole for inhibition of melanin biosynthesis in Trimmatostroma salinum and in T. abietis and compared the ultrastructure of melanized and tricyclazole-inhibited cells to identify the position of melanin. Melanin was observed as electron-dense (dark) granules in or on the electron translucent (light-coloured) cell walls, whereas electron-dense granules in the cell walls of cells with blocked melanin biosynthesis either disappeared or formed a less distinct electron-dense layer on the outer side of the cell wall, and were smaller and lighter in colour (compare left and right columns of Figs 4–5). On the medium without salt, melanin was deposited in large granules in the outer cell-wall layer and as a layer on the surface of the outer cell-wall stratum of the cell wall of T. salinum meristematic clumps and endoconidia (Fig 4A). On the medium containing 4 % sodium chloride, melanin granules were dense and coalesced to form a firm, thick, shield-like layer on the outer side of the cell wall (Fig 4C). On the medium containing 10 % sodium chloride, the thick and dense melanin layer was fragmentary, with separate melanin granules in the cell wall and extracellularly (Fig 4E). On the medium containing 20 % sodium chloride, the melanin layer on the outer side of the cell wall was very compact, dense and uniform, and no separate granules were observed (Fig 4G). The size of melanin granules decreased with increasing salinity (Table 2). On the tricyclazole-amended media, no melanin granules were visible in the cell wall, but a thin electron-dense layer was present on the outer side of the cell at 0, 4 and 10 % sodium chloride. At 20 % sodium chloride, pronounced EPS around the cells was electron dense. Also, no meristematic multicellular clusters were formed, but mostly one- to twocelled spherical, loose conidia were produced (Fig 4B, D, F). The exception was the medium with 20 % sodium chloride, where the prevalent cell type were irregular multicellular clusters (Fig 4H). Salinity-induced changes in Trimmatostroma spp. 719 Fig 4 – TEM micrographs of thin sections of Trimmatostroma salinum cells grown on MEA media with: (A) no sodium chloride; (B) no sodium chloride with tricyclazole; (C) 4 % sodium chloride; (D) 4 % sodium chloride with tricyclazole; (E) 10 % sodium chloride; (F) 10 % sodium chloride with tricyclazole; (G) 20 % sodium chloride; (H) 20 % sodium chloride with tricyclazole. Abbreviations: CW – cell wall, EPS – extracellular polymeric substances, M – melanin, V – vacuoles. Arrow – cell wall indentations. The cell types presented here were observed in at least 10 cells. 720 Table 2 – Ultrastructural characteristics of Trimmatostroma salinum and T. abietis grown at different sodium chloride concentrations T. salinum MEA salinity No NaCl 4 % NaCl 0.5–0.8 mm 0.8–1 mm Two distinct Irregular invaginations of plasma membrane T. abietis 10 % NaCl 20 % NaCl no NaCl 0.8 mm, Uneven thickness Extremely stratified 0.5 mm Two distinct 0.8–1 mm, Uneven thickness Two - - þ Melanization pattern Numerous separate granules in the cell wall coalescing into a layer on the cell surface Firm, thick, shield-like melanin layer, numerous small granules in the septa Location of melanin In the outer cell-wall layer and continually to the surface of the outer cell-wall stratum, in septa þ Visible only when some melanin granules were attached No separate electron-dense granules; less distinct, thinner electron-dense layer on the outer side of the cell wall; cell-wall thickness unchanged In the outer cell-wall layer and on its surface, in septa Thick and dense, but fragmentary layer, separate melanin granules on its inner side and around the cells, few in the septa In the outer cell-wall layer and on its surface, in septa Appo. cell-wall thickness Number of cell-wall layers Extracelullar polymeric substances (EPS) Ultrastructure of tricyclazole-inhibited cells 6 % NaCl 10 % NaCl Uneven thickness Two w1 mm, Uneven thickness Two, stratified þ - - þ Very compact, dense, and uniform melanin layer Separate melanin granules in the cell wall and in EPS, few small melanin granules in the septa Separate melanin granules in the cell wall and in EPS, few small melanin granules in the septa In the outer cell-wall layer and on its surface In the outer cell-wall layer and on its surface, in EPS, in septa Separate melanin granules, in some cells coalescing into a dense melanin layer, detached from the cell wall In the outer cell-wall layer and on its surface, in EPS Two In the outer cell-wall layer and on its surface, in EPS, in septa - þ Dark-stained EPS with clearly visible outer borderline in some cells þ Wide, fibrous þ Fibrous þ Well-defined with a visible borderline No separate electron-dense granules, thin granular electron-dense layer on the outer side of the layered, light-coloured cell wall; plasma membrane invaginations (Fig 4D, arrow) No separate electron-dense granules; outer cell-wall layer thin, patchy, cracked, composed of small and dispersed electron-dense granules, in some cells composed of two to three layers No separate electron-dense granules, uneven and stratified cell wall; pronounced electron-dense EPS around cell clusters; plasma membrane invaginations (Fig 4H, arrow) Separate very small electron-dense granules in the pronounced EPS; electron-translucent, layered thicker cell wall (w1 mm or more); plasma membrane invaginations (Fig 5B, arrow) Separate very small electron-dense granules in the pronounced EPS Separate very small electron-dense granules in the pronounced EPS; electron-translucent, layered thicker cell wall (w1 mm or more); numerous plasma membrane invaginations (Fig 5F, arrow) T. Kogej et al. - Salinity-induced changes in Trimmatostroma spp. 721 Fig 5 – TEM micrographs of thin sections of Trimmatostroma abietis cells grown on MEA media with: (A) no NaCl; (B) no NaCl with tricyclazole; (C) 6 % NaCl; (D) 6 % NaCl with tricyclazole; (E) 10 % NaCl; (F) 10 % NaCl with tricyclazole. Abbreviations: CW – cell wall, EPS – extracellular polymeric substances, M – melanin, V – vacuoles. Arrow – cell wall indentations. The cell types presented here were observed in at least 10 cells. TEM of Trimmatostroma abietis cells On the medium without salt and at 6 % sodium chloride, separate large melanin granules were present in the outer cell-wall layer and extracellularly in the fibrous material (EPS; Fig 5A). On the media containing 6 % sodium chloride, an additional dense melanin layer was present, which detached from the cell wall (Fig 5C). On the medium containing 10 % sodium chloride, smaller separate melanin granules were located mostly in the EPS, and were not forming any evident layer (Fig 5E). The size of melanin granules decreased only slightly with increasing salinity. On tricyclazole-amended media, no melanin granules were visible in the thick cell wall, while the presence of smaller electron-dense granules in the extracellular matrix was very characteristic (e.g. Fig 5D). The cytoplasm was dark and membranous invaginations were observed (Fig 5B, D, F). Discussion Eukaryotic microorganisms termed ‘‘black yeasts’’ (de Hoog & Hermanides-Nijhof 1977), microcolonial fungi (Staley et al. 722 1982), or meristematic ascomycetes (Sterflinger et al. 1999), are remarkably successful in adapting to extreme environments (Staley et al. 1982; Gorbushina et al. 1993; Nienow & Friedmann 1993; Wollenzien et al. 1995; Gunde-Cimerman et al. 2000; Gunde-Cimerman et al. 2004), as confirmed by the growth experiments with Trimmatostroma salinum and T. abietis in this study. Initially, both species were grown in vitro at increasing salinities to determine their growth rates and maximum colony size. The results confirmed the halophilic nature of T. salinum, but also showed a remarkable degree of adaptive response to sodium chloride-induced water stress in the rock-inhabiting T. abietis (Fig 1B). Although both strains originate from ecologically very different extreme niches, atmospheric desiccation and hypersalinity both impose a characteristic stress of low water potential. Black yeasts’s distinctive features are polymorphism, meristematic growth, endoconidiation or sarcinic conidiogenesis, frequently muriform cells, which develop by conversion from undifferentiated hyphae, and thick, melanized cell walls (de Hoog 1993; Zalar et al. 1999). This morphological ecotype is important for their growth and survival in various extreme environments (Staley et al. 1982; Gorbushina et al. 1993; Nienow & Friedmann 1993; Wollenzien et al. 1995; Gunde-Cimerman et al. 2000; Gunde-Cimerman et al. 2004). Meristematic growth, a common feature of both species, is an adaptation to stressed conditions, since it ensures an optimal surface:volume ratio of the colony (Wollenzien et al. 1995). The structure of the colony depends on the cell types present and their development. Histological analyses of the colonies of T. salinum showed that the prevailing cell types were round multicellular meristematic clusters, which developed from hyphae by transverse and longitudinal septation followed by disarticulation (Fig 2). At the highest salinity T. salinum produced separate, strongly melanized multicellular clusters and one- to two-celled conidia (Fig 2). In contrast, T. abietis colonies consisted of clumps of loosely organized oblong conidia on medium without sodium chloride, but at increasing salinity structurally different layers of the colony formed from tightly interwoven hyphae (Fig 3). Such basal layers could protect the cells in the upper layer of the colony from direct contact with the hyperosmotic environment by creating a salt gradient from the medium to the upper colony layers. The thicker layer observed at higher salinity could support this assumption. The ultrastructural analyses showed that the cell walls of T. salinum were the thickest within the optimal salinity range (Fig 4), whereas in T. abietis, the thickness of the cell wall increased on saline media and with the inhibition of melanin synthesis (Fig 5). The thickness of the cell walls of both species was between 0.5–1 mm, similar to the cell-wall thickness of Dendryphiella salina, a marine hyphomycete (Clipson et al. 1989). Such an exceptionally developed thick cell wall might be part of the mechanism enabling growth of fungi in saline (Clipson et al. 1989) and hypersaline environments. Cells of both species grown on the media with 10 % or more sodium chloride had numerous cell-wall and plasma-membrane invaginations, causing uneven cell-wall thickness (see arrows on Fig 4E, G, 5E), and thus were probably experiencing difficulties to cope with this stress, as observed in Aspergillus repens grown on medium with 2 M NaCl (or 11.7 % w/v) (Kelavkar et al. 1993). T. Kogej et al. T. salinum synthesizes DHN melanin under non-saline and hypersaline growth conditions (Kogej et al. 2003). In both investigated Trimmatostroma spp., TEM micrographs showed melanin as typical electron-dense granules in or on the cell walls and in septa, as indicated from TEM studies of other DHN-melanized fungi (Wheeler et al. 1978; Wheeler & Bell 1988; Butler & Day 1998; Carzaniga et al. 2002; Langfelder et al. 2003; Nosanchuk & Casadevall 2003). In halophilic T. salinum, the melanin granules were part of the outer cell-wall layer. They were separate at low salinities, and became coalesced at higher salinities, forming an electron-dense cellwall layer around individual cells (Fig 4). In halotolerant T. abietis, the melanin granules were less organized and less bound to the cell wall at all salinities (Fig 5) as in T. salinum, and a considerable portion was deposited in the outer polysaccharide matrix layer, which was relatively easily separated from the cell. We used tricyclazole, an inhibitor of reductase enzymes in the DHN-melanin biosynthesis pathway, for inhibition of melanin synthesis in both fungi to diminish the appearance of the distinct electron-dense granules (Bell & Wheeler 1986). The cell walls of tricyclazole-inhibited T. salinum contained a thin electron-dense layer in the outer cellwall layer, whereas in T. abietis the electron-dense granular material was located in the EPS outside of the cell wall, which possibly indicates different location of melanin in the cell walls. It has been reported that melanin in the cell walls of fungi can be structured in many ways. In spores and hyphae, it can appear as a heavily pigmented outer cell-wall layer or a layer on the outside of the wall, as scattered or bar-organized granules in the wall or as granules sloughing off the surface (Durell 1964; Ellis & Griffiths 1974). In Cladosporium cladosporioides, taxonomically related to Trimmatostroma spp., and in other dematiaceous fungi, the conidial cell wall comprised an electron-translucent inner layer and an electron-dense outer layer (Latgé et al. 1988). No previous reports exist on how high sodium chloride concentrations affect melanization. Melanins formed through the DHN pathway are of particular interest, as they are known to protect fungi against a number of environmental factors (Zhdanova & Pokhodenko 1974; Taylor et al. 1987; RomeroMartı́nez et al. 2000; Gunde-Cimerman et al. 2004; Kogej et al. 2004). Melanin plays an important role in fungal protection against different types of stress (Bell & Wheeler 1986), thus it might play a role during growth at hypersaline conditions as well. Generally, tricyclazole is reported as having no other apparent effect on treated cells except for inhibition of melanization (Bell & Wheeler 1986), but we observed morphological modifications in both species with inhibited melanin synthesis. The addition of tricyclazole in very low amounts such as 2 mg ml1 (data not shown) as well as at 30 mg ml1 caused cell separation in T. salinum on the medium without salt (Figs 2B, 4B), and a complete disintegration of meristematic cell clusters on the saline media (Fig 2D, F). One could speculate that melanin is an integral component of the cell walls of T. salinum and as such might have an important role in morphological development and cell cluster formation. In T. abietis, the addition of 30 mg ml1 tricyclazole induced a layered colony structure on the medium without sodium chloride (Fig 3B), and an increased thickness of a tissue-like basal layer Salinity-induced changes in Trimmatostroma spp. on saline media (Figs 3D, F). Here, melanization of the cells in the basal layer might be important for the protection of the upper colony layers from direct contact with salt, so in the absence of melanin a thicker layer would develop to isolate the upper cells from the high sodium chloride concentration. In conclusion, the combination of histological and ultrastructural methods has demonstrated morphological adaptation on the cellular and on the colonial level to increased salinity in these two extremophilic Trimmatostroma spp. The adaptive responses in halophilic T. salinum to increased sodium chloride concentrations were more expressed on the cellular level. Each cell was protected by a thick cell wall with a distinct melanin layer, which was more compact and dense at higher salinities. Conversely the xerophilic rockinhabiting T. abietis reacted to sodium chloride-induced water stress at colony level by expressed pseudotissue development and a layered distribution of cell types inside the colonies. In T. abietis, the melanization of the NaCl-exposed basal layer of the colony was more pronounced, whereas melanization of the cells was not much affected by a change in salinity on the contrary, in T. Salinum grown on saline media, the melanin granules coalesced to form a compact layer around individual loosely arranged cells . Melanin inhibition disrupted this type of fungal growth, provoking ultrastructural changes, and a generally stressed response. It is known that the cell-wall melanization in phytopathogenic fungi conveys increased mechanical strength (Frederick et al. 1999; Butler et al. 2001), maintains high internal solute concentration, and thus the necessary internal hydrostatic pressure, particularly in plant-invading fungal structures (Howard & Valent 1996). Our study indicates that the structure of melanin cell-wall layers is affected by a change in sodium chloride concentration in the halophilic T. salinum, but not in the xerophilic T. abietis. Based on these results and on the observation of a similar formation of a melanin layer at saline growth conditions in other halophilic black yeasts (our unpublished results), we propose that melanization of the cell wall is one of the mechanisms enabling growth of halophilic and halotolerant black yeasts at hypersaline conditions. A heavily melanized cell wall probably provides protection against water loss and leakage of intracellular compatible solutes, but this needs to be studied further. The comparison of both black yeast species thus contributes to the understanding of eukaryotic adaptations to life in extreme environments. Acknowledgments The research was supported by the Ministry of Education, Science, and Sports of the Republic of Slovenia and by International Bureau of BMBF. Financial assistance to A.A.G. was additionally provided by a DFG project Go 897/2-1 and 897/2-2. 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