- Top of page
- Materials and methods
- Results and discussion
A total of 71 isolates were collected from lake sediment and soil surrounding lakes in the Skarvsnes area, Antarctica. Based on ITS region sequence similarity, these isolates were classified to 10 genera. Twenty-three isolates were categorized as ascomycetous fungi from five genera (Embellisia, Phoma, Geomyces, Tetracladium or Thelebolus) and 48 isolates were categorized as basidiomycetous fungi in five genera (Mrakia, Cryptococcus, Dioszegia, Rhodotorula or Leucosporidium). Thirty-five percent of culturable fungi were of the genus Mrakia. Eighteen isolates from eight genera were selected and tested for both antifreeze activity and capacity for growth under temperatures ranging from −1 to 25 °C. Rhodotorula sp. NHT-2 possessed a high degree of sequence homology with R. gracialis, while Leucosporidium sp. BSS-1 possessed a high degree of sequence homology with Leu. antarcticum (Glaciozyma antarctica), and these two isolates demonstrated antifreeze activity. All isolates examined were capable of growth at −1 °C. Mrakia spp., while capable of growth at −1 °C, did not demonstrate any antifreeze activity and exhibited only limited secretion of extracellular polysaccharides. Species of the genus Mrakia possessed high amounts of unsaturated fatty acids, suggesting that members of this genus have adapted to cold environments by increasing their membrane fluidity.
- Top of page
- Materials and methods
- Results and discussion
Cold environments cover a large portion of our planet, with many ecosystems permanently exposed to temperatures below 5 °C (Feller & Gerday, 2003). Since microbes adapted to cold environments are capable of growth at temperatures below 0 °C, it is expected that these microbes employ unique physiological tools, such as cold-adapted enzymes and antifreeze proteins (AFP), in order to survive (Buzzini et al., 2012). Antarctica is the southernmost landmass on Earth and has an area of c. 14 million km2, making it the fifth-largest continent in the world. Approximately 98% of Antarctica is covered by ice and snow and temperatures in coastal areas usually range from 5 to −35 °C. Temperatures on Antarctic plateaus are much more extreme, ranging from c. −25 °C in summer to c. −70 °C in winter (Ravindra & Chaturvedi, 2011). The Skarvsnes ice-free area is located along the central Soya coast, East Antarctica, and contains many small oligotrophic freshwater lakes that constitute the only unfrozen water in the area (Imura et al., 1999). The surface of these lakes is covered by 1–2 m of ice for 11 months of the year (Tanabe et al., 2008); however, these lakes do not freeze below a depth of 3 m.
To our knowledge, over 700 fungal species have been isolated and recorded from Antarctica (Bridge et al., 2010). Twelve ascomycetous and four basidiomycetous species were reported from around Syowa station, East Antarctica. (Soneda, 1961; Tsubaki, 1961a , b; Tsubaki & Asano, 1965). Despite previous research into the mycoflora of the Skarvesnes ice-free area, knowledge of fungal biodiversity in this area remains incomplete. The present study aimed to assess the diversity of fungi isolated from soil collected around lakes as well as lake sediments in the Skarvsnes ice-free area, and to determine any relevant adaptations to cold environments.
Results and discussion
- Top of page
- Materials and methods
- Results and discussion
Samples were collected from 16 sites in the Skarvsnes ice-free area (Fig. 1). A total of 71 isolates were obtained and classified to 10 genera based on sequences covering the ITS region (Figs 2 and 3). The average length of the ITS region reads was 518 bp, with a maximum length of 616 bp and minimum length of 400 bp. Twenty-three isolates were classified as ascomycetous fungi and assigned to five genera (Embellisia, Phoma, Geomyces, Tetracladium, Thelebolus). Forty-eight isolates were classified as basidiomycetes and assigned also to five genera [Mrakia, Cryptococcus, Dioszegia, Rhodotorula, Leucosporidium (Glaciozyma)]. Dominant isolates belonged to the genera Mrakia (35.2%), Cryptococcus (22.5%), Thelebolus (11.3%) and Tetracladium (9.9%). These genera were collected from soils around lakes and lake sediments. Genera Rhodotorula, Leucosporidium and Geomyces were also collected from both the soil around lakes and lake sediments. Fungi of the genera Phoma, Embelisia and Dioszegia were isolated from soil surrounding lakes. No fungal species were exclusively isolated from the lake sediments.
Species of the genus Embellisia have generally been isolated from higher plants, often as endophytes or pathogens, in areas of moderate temperature (Li & Nan, 2007). Brander et al. (2000) previously isolated an Embellisa sp. from moss in Victoria Land, Antarctica. In the present study, two isolates of Embellisia sp. were collected from surface soil surrounding Lake Mago-ike. The genus Phoma has been reported from various cold environments (Fletcher et al., 1985). In Antarctica, this genus was isolated from Bunger Hill (Barker, 1977), MacRobertson Land (Fletcher et al., 1985) and Victoria Land (Toshi et al., 2002). The majority of these samples were isolated from moss, which led researchers to believe that the genus preferred moss as a substrate; however, Gonçalves et al. (2012) reported that a Phoma sp. was collected from water in a King George Island lake. The present study identified four isolates of Phoma sp. that were collected from soil surrounding lakes Ohgi-ike and Naga-ike. Isolates of the genus Geomyces have been obtained numerous times from soil samples taken from Kay Land, Terra Nova, and Cape King, Antarctica (Frate & Caretta, 1990). In the present study, only two isolates of Geomyces sp. were isolated from the Skarvsnes ice-free area and eight isolates of Thelobolus sp. were isolated from both the soil around lakes and lake sediments in the Skarvsnes ice-free. Ruisi et al. (2007) determined that a Thelebolus sp. was one of the fungi most frequently isolated from the Antarctic continent. Thelebolus strains have been isolated from both littoral mats and under ice mats in Ace Lake, Lake Druzhby and Highway Lake (Vestfold Hills); Organic Lake, and Red Lake on Manning Island (Larsemann Hills); and Lake Fryxell and Lake Hoare (McMurdo Dry Valleys) (Göttlich et al., 2003). Seven isolates of Tetracladium sp. were isolated from soil and lake sediment taken from the Skarvsnes region. Tetracladium sp. had been previously isolated from soil taken from West Antarctica and plant matter taken from Admiralty Bay (Bridge & Newsham, 2009; Rosa et al., 2009). We obtained a total of 23 ascomycetous isolates from soil surrounding lakes and lake sediments. From lake sediments, we collected a total of six isolates from Tokkuri-ike, Kuwai-ike, Jizou-ike and Naga-ike. These isolates belonged to the genera Thelebolus spp. and Tetracladium spp.
These results indicate a low biodiversity of ascomycetous fungus in lake sediments at the Skarvsnes ice-free area, as compared with the soil surrounding the lakes.
Sixteen of the 71 isolates obtained belong to Cryptococcus; of those 16 isolates, 12 possessed high sequence similarity with Cryptococcus victoriae, three were close related to Cry. gastricus and one was related to Cry. friedmannii. Cryptococcus victoriae is a cosmopolite yeast and has been isolated from cold environments around the world. Cryptococcus victoriae, in Antarctica, has also been collected from soil samples taken from Victoria Land, Lichen Valley, Vestfold Hills and Davis Base (Montes et al., 1999; Thomas-Hall et al., 2002; Vaz et al., 2011). Two isolates of Rhodotorula spp., three isolates of Leucosporidium spp. (recently Leu. antarcticum was redefined as Glaciozyma antarctica; Turchetti et al., 2011) and two isolates of Dioszegia spp. were also collected during the present study. Species of these genera have been identified in South Victoria Land and various other cold temperature environments (Fell et al., 1969; Buzzini et al., 2012). Furthermore, a Leucospridium sp. (Glaciozyma sp.) has also been isolated from meltwater stream sediment, glacial meltwater stream sediment and sea water (Vishniac & Klingler, 1986; Klingler & Vishniac, 1988; Turchetti et al., 2011).
Di Menna (1966) reported that c. 24% of culturable yeasts from soil in Antarctica were Mrakia spp. Mrakia spp. have also been identified previously in various cold regions such as the Arctic, Siberia, the Alps, Patagonia and Antarctica (Morgesin et al., 2005; de Garcia et al., 2007, 2012; Pathan et al., 2010; Thomas-Hall et al., 2010; Singh & Singh, 2012; Tsuji et al., 2013). Mrakia spp. were the major mycoflora and some of the most adaptive fungi in the lake sediments at Skarvsnes ice-free area, East Antarctica.
Some fungi secrete extracellular AFP to prevent freezing of the cell when exposed to extreme cold conditions. Fungal AFPs have been found in snow mold fungi and have been identified as phytopathogens in both wheat and grass (Snider et al., 2000; Hoshino et al., 2003, 2009). Xiao et al. (2010a) tested 23 fungal species from Antarctica for antifreeze activity and determined antifreeze activity in only two species. Xiao et al. (2010b) also reported that 13 of a total of 145 eukaryotic microorganisms (fungi and stramenopile) isolates obtained in Antarctica demonstrated antifreeze activity. Nine of the 13 isolates that demonstrated antifreeze activity were basidiomycetous yeast, of which seven were Leu. cantarcticum (Glaciozyma antarctica) and two were Rhodotorula glacialis (Xiao et al., 2010b). In the present study, 18 isolates of eight genera were chosen from 71 total isolates and tested for antifreeze activity and capacity for growth at temperatures ranging from −1 to 25 °C. Rhodotorula sp. NHT-2 demonstrated high ITS region sequence homology with R. glacialis, and Leucosporidium sp. BSS-1 demonstrated high ITS region sequence homology with Leu. antarcticum (Glaciozyma antarctica) –only these two isolates demonstrated antifreeze activity. Results of the present study as well as those of Xiao et al. (2010a , b) indicate that Antarctic fungi rarely demonstrated antifreeze activity and that almost all fungi employed other strategies to survive in these extreme environments. Eighteen of the isolates tested in the present study were capable of growth at −1 °C. Leucosporidium sp BSS-1 was not capable of growth at 20 °C, although Phoma sp. NGK-4, Tetracladium spp. AGK-1 and JZS-2 and Thelebolus sp. EBN-3 were capable of growth at 25 °C (Table 1).
Table 1. Antifreeze activity, presence of EPS and growth at various culture temperatures of all isolates
|Isolates||Antifreeze activity||EPS||Culture temperature|
|−1 °C||4 °C||10 °C||20 °C||25 °C|
|Mrakia sp. AGK-2||−||w||+||+||+||w||−|
|Mrakia sp. EBN-1||−||w||+||+||+||w||−|
|Mrakia sp. KWS1-1||−||w||+||+||+||w||−|
|Mrakia sp. SK-4||−||w||+||+||+||w||−|
|Mrakia sp. SMS-2||−||w||+||+||+||w||−|
|Mrakia sp. TKS-1||−||w||+||+||+||w||−|
|Cryptococcus sp. OGA-1||−||w||+||+||+||+||−|
|Cryptococcus sp. NHU-1||−||w||+||+||+||+||−|
|Cryptococcus sp. MOA-2||−||w||+||+||+||+||−|
|Rhodotorula sp. ABH-3||−||w||+||+||+||+||−|
|Rhodotorula sp. NHT-2||+||w||+||+||+||+||−|
|Leucospridium sp. BSS-1||+||+||+||+||+||−||−|
|Dioszegia sp. ARJ-3||−||w||+||+||+||+||−|
|Phoma sp. NGK-4||−||w||+||+||+||+||+|
|Tetracladium sp. AGK-1||−||w||+||+||+||+||+|
|Tetracladium sp. JZS-2||−||w||+||+||+||+||+|
|Thelebolus sp. EBN-3||−||w||+||+||+||+||+|
|Thelebolus sp. TKS-1||−||w||+||+||+||+||+|
Robinson (2001) reported that physiological characteristics such as cryoprotectant sugars, polyols, fatty acids, AFPs and cold-active enzymes are advantageous to soil organisms for survival during the polar winters. Leucosporidium antarcticum (Glaciozyma antarcticum), a cryophilic yeast, (Hoshino & Matsumoto, 2012), has been known to form colonies within frost columns and secrete AFP under low temperature conditions (Lee et al., 2010). Moreover, the species has been shown to secrete both AFP and extracellular polysaccharides (EPS) when exposed to low temperatures (Fujiu 2010; Master's thesis, Graduate School of Science, Hokkaido University). Leucosporidium antarcticum (Glaciozyma antarcticum) are known to have the capacity to survive at low temperatures, but although species of this genus have not demonstrated antifreeze activity, and very few of them have been known to secrete EPS, Mrakia spp. have been identified as the dominant culturable fungi in lake sediments at the Skarvsnes ice-free area, East Antarctic, Pathan et al. (2010) determined that cold-adapted yeasts commonly contained high concentrations of unsaturated fatty acids, such as C18:1 and C18:2, and that these fatty acids were considered essential for survival at low temperatures. Results of fatty acid composition indicated that Mrakia spp. and Cry. victoriae had adapted to low temperature conditions by achieving high membrane fluidity (Turk et al., 2011; Singh et al., 2013). Moreover, Mrakia blollopis SK-4 has high concentrations of C18:2 and totals of C18:1 and C18:2, compared with those of M. blollopis CBS8921T (Table 2). It was therefore concluded that strain SK-4 was more highly adapted for survival in cold lake environments than the other Mrakia spp. analyzed.
Table 2. Fatty acid composition of Mrakia spp., Rhodotorula glacialis, Cryptococcus victoriae and Leucosporidium sp. Fatty acid analysis results for M. blollopis SK-4 and other species are shown. Fatty acid data were taken from Thomas-Hall et al. (2002, 2010), Rossi et al. (2009) and this study
|Fatty acids||M. blollopi s SK-4||M. blollopis CBS 8921T||M. frigida CBS5270T||M. robertii CBS8912T||R. glacialis DBVPG4785||Cry. victoriae CBS8920||Leu. sp. BSS-1|
|Palmitic acid (C16:0)||8||19||13||16||14.5||10||12|
|Palmitoleic acid (C16:1)||8||2||0||1||1.4||ND||3|
|Oleic acid (C18:1n9)||22||16||8||30||39.4||50||58|
|Linoleic acid (C18:2)||55||33||52||35||21.2||30||13|
|Linolenic acid (C18:3n3)||2||30||27||18||15.4||7||5|