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Keywords:

  • biodiversity;
  • yeasts;
  • Patagonia;
  • psychrophiles;
  • glacial meltwater environment

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

The occurrence of culturable yeasts in glacial meltwater from the Frías, Castaño Overo and Río Manso glaciers, located on Mount Tronador in the Nahuel Huapi National Park (Northwestern Patagonia, Argentina) is presented. Subsurface water samples were filtered for colony counting and yeast isolation. The total yeast count ranged between 6 and 360 CFU L–1. Physiologic and molecular methods were employed to identify 86 yeast isolates. In agreement with yeast diversity data from studies for Antarctic and Alpine glaciers, the genera Cryptococcus, Leucosporidiella, Dioszegia, Rhodotorula, Rhodosporidium, Mrakia, Sporobolomyces, Udeniomyces and Candida were found. Cryptococcus and Leucosporidiella accounted for 50% and 20% of the total number of strains, respectively. Among 21 identified yeast species, Cryptococcus sp. 1 and Leucosporidiella fragaria were the most frequent. The typically psychrophilic Mrakia yeast strain and three new yeast species, yet to be described, were also isolated. All yeast strains were able to grow at 5, 10, and 15°C. Among yeast strains expressing extracellular enzymatic activity, higher proteolytic and lipolytic activities were obtained at 4°C than at 20°C.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Extremophilic microorganisms, capable of developing in extreme environments, attract considerable attention due to their importance in biogeochemical nutrient cycling, their ecological role, and their biotechnological potential (Skidmore et al., 2000; Deming, 2002; Foght et al., 2004; Raspor & Zupan, 2005). Extremely cold environments (i.e. ice, snow, and sea ice) can be successfully colonized by a group of extremophilic microorganisms known as psychrophiles (Deming, 2002; Feller & Gerday, 2003). This group of microorganisms is currently defined by an optimum growth temperature of 15°C, a maximum growth temperature below 20°C, and a minimum growth temperature equal to or below 0°C (Morita, 1975). They can use complex carbon biopolymers as energy sources, because they are able to synthesize extracellular enzymes that are active at low temperatures (cold-adapted enzymes). Thus, psychrophilic yeasts play an essential role in nutrient cycling and biomass production processes in cold ecosystems (Margesin et al., 2002).

Psychrophilic yeasts have been isolated from marine waters, Arctic and Alpine glaciers, and Antarctic ecosystems, and their occurrence and abundance in these environments have been described (Vishniac, 1999; Díaz & Fell, 2000; Bergauer et al., 2005; Buzzini et al., 2005). However, these studies do not systematically describe fungal biodiversity, and in some cases are focused on contaminated areas rather than on pristine environments.

The Nahuel Huapi National Park (NHNP) in northwestern Patagonia (Argentina) has a cold to temperate climate, and includes vast areas with little or no human influence. Studies on the occurrence of yeasts in terrestrial and aquatic environments in the NHNP have allowed the characterization of native yeast communities and the description of new species (Brizzio & van Broock, 1998; Libkind et al., 2003, 2005a). However, the studies concerning aquatic environments have focused mainly on pigmented mesophilic yeasts (Libkind et al., 2003). The diversity of psychrophilic yeasts from Mount Tronador glacial meltwater remains so far undescribed. This work represents the first study on the isolation, identification and characterization of culturable yeasts in this particular environment.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Area description and sampling

Mount Tronador is an old, extinct volcanic cone located at 71°50′W, 41°10′S (3554 m above sea level) in the NHNP. Four of its 10 glaciers are located in Argentina, and the remaining six are in Chile. In Argentina, three of the glaciers, named Río Manso, Castaño Overo, and Alerce, belong to the high Manso river basin, which flows into the Mascardi Lake. This lake then drains through Chile into the Pacific Ocean by means of the Manso River. The fourth Argentine glacier, the Frías, flows into the Nahuel Huapi Lake (Argentina), which has an Atlantic drainage, through several lakes and rivers (Rabassa et al., 1978).

Water samples were collected from meltwater rivers corresponding to the glaciers Río Manso (two sites), Castaño Overo (one site) and Frías glaciers (three sites), during the late summer of 2004 (Fig. 1; Table 1). Three independent samples were obtained from each sampling site, and samples were stored at 4°C until processing. Water temperature and pH, air temperature and GPS positioning were recorded in situ.

image

Figure 1.  Location of Mount Tronador and study area. Ventisquero Negro lagoon (1), Garganta del Diablo waterfall (2), Castaño Overo river (3), Frías river (4–6).

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Table 1.   Location, physicochemical characteristics of sampling sites and viable yeast counts
GlaciersSampling sitesLocationTemperature (°C)pHYeasts viable counts (CFU L–1)
AirWaterMean ± SD*
  • *

    Mean values of three replicates and SD.

Río Manso1. Ventisquero Negro lagoon41°13′S 71°47′W2256.76 ± 12
2. Garganta del Diablo waterfall41°11′S 71°5′W2256.723 ± 40
Castaño Overo3. Castaño Overo river41°12′S 71°46′W2267.026 ± 43
4. Frías river41°07′S 71°46′W1056.4316 ± 407
Frías5. Frías river41°06′S 71°48′W1377.027 ± 46
6. Frías river41°02′S 71°47′W14137.0182 ± 118

Yeast isolation and quantitative analysis

Variable volumes of water (100–300 mL) were filtered through Millipore nitrocellulose membranes (0.45-μm pore size, 47-mm diameter), using a sterilized Nalgene device. The filters were placed on MYP agar (malt extract 7 g L–1; yeast extract 0.5 g L–1; soytone 2.5 g L–1; agar 15 g L–1; pH 4) containing chloramphenicol 100 mg L–1 in petridishes and incubated at 4–15°C for up to 1 month. Plates were periodically examined, and all emerging yeast colonies were transferred to MYP agar without antibiotics and purified. The isolates were stored in MYP or PD (glucose 20 g L–1; agar 15 g L–1; potato infusion 23% v/v) agar slants and kept at 4°C. Yeast colonies were recorded under a stereoscopic microscope OLYMPUS SZX9 (Tokyo, Japan) with × 60 magnification. Yeast colony forming units (CFUs) were registered for quantitative analysis of yeast occurrence. Yeast cell counts from three replicates were use to calculate means and SDs.

Yeast identification

Yeast identification was achieved by a polyphasic approach combining conventional and molecular techniques. Identification up to the genus level was performed according to morphologic characteristics and physiologic tests according to Yarrow (1998). Within each genus, strains with identical morphologic and physiologic characteristics were grouped together, and these groups were then subjected to PCR fingerprinting, employing the mini/microsatellite-primed PCR technique (MSP-PCR). Strains with identical DNA-banding patterns were grouped, and considered to belong to the same species (Sampaio et al., 2001; Gadanho & Sampaio, 2002). In some cases, DNA profiles of yeast isolates were compared to those of type or reference strains (Libkind et al., 2003). A representative strain of each MSP-PCR group was subjected to sequence analysis of the rRNA gene D1/D2 region. Physiologically distinct strains with unique MSP-PCR banding patterns were also selected for direct identification by rRNA gene D1/D2 region sequence analysis and compared with those of all fungal species available at the international Nucleotide Sequence Collaboration Databases (http://www.ncbi.nlm.nih.gov/BLAST/) using the Basic Local Alignment Search Tool (blast).

DNA extraction

Total DNA extraction was performed according to Libkind et al. (2003) with modifications. Two loopfuls of cultures grown on MYP agar were suspended in Eppendorf tubes containing 500 μL of lysing buffer [20 mmol Tris L–1; 250 mmol NaCl L–1; 50 mmol EDTA L–1; 0.3% (w/v) sodium dodecyl sulfate; pH 8] and 200 μL of 425–600 μm glass beads. After being vortexed for 3 min, the tubes were incubated for 1 h at 65°C. After being vortexed for another 3 min, the suspensions were centrifuged for 30 min at 5200 g. Finally, the collected supernatants were diluted 1 : 750, and 5 μL was applied for PCR studies. The leftover supernatants were immediately placed at −20°C.

MSP-PCR fingerprinting

The synthetic oligonucleotide (GAC)5 and the core sequence of the phage M13 (GAGGGTGGCGGTTCT) were used in minisatellite and microsatellite primed PCR experiments, respectively. PCR reactions were performed in 25-μL reaction volumes containing 12.5 μL of PCR Master Mix 2 × (Promega), 2.0 μL of 0.5 μM MgCl2, 4 μL of 5 μM primer, and 5 μL of genomic DNA. DNA amplification was performed in a Mini Cycler My Research; the PCR technical steps and negative controls were performed according to Libkind et al. (2003). Amplified DNA fragments were separated by electrophoresis in 1.4% (w/v) agarose gels (Gibco-BRL) in 0.5 × TBE (Tris–borate–EDTA) buffer at 120 V for up to 40 min, and stained with ethidium bromide (2 mg mL–1). A molecular size marker was included according to Libkind et al. (2003). DNA banding patterns were acquired with a Polaroid 667 camera.

rRNA gene sequence analysis

Total DNA was extracted using the methods described above, and amplified using primers ITS5 (5′-CGC AGT AAA AGT CGT AAC AAG G) and LR6 (5′-CGC CAG TTC TGC TTA CC-3′) as described by Libkind et al. (2003). Sequencing of the D1/D2 region of the 26S ribosomal subunit was performed directly from purified PCR products by the Sequencing Service from STAB VIDA (Oeiras, Portugal).

Influence of temperature on growth

Two experiments were carried out in order to assess the influence of temperature on growth of the isolated strains. In order to determine minimum and maximum yeast growth temperatures, all 86 isolates were tested for the ability to grow at different temperatures (5, 10, 15, 20, 25, 30, and 35°C) on MYP agar plates. The plates were inoculated with a calibrated suspension (106 cells mL–1) of yeast cells grown for 24–48 h, and incubated at different temperatures. Growth was monitored visually on a daily basis for 1 week, except for the Petri dishes incubated at 5°C, which were monitored weekly for 1 month. Colony growth was assessed qualitatively. After determination of minimum and maximum growth temperatures, the optimum growth temperature was established for selected strains in a liquid culture. This assay included: two strains of the most frequently isolated species, Leucosporidiella fragaria CRUB 1201 and Cryptococcus sp. 1 CRUB 1226; two psychrophilic strains, Mrakia frigida CRUB 1253 and Mrakia sp. CRUB 1272; and the mesophilic strain Rhodotorula mucilaginosa CRUB 1282. Erlenmeyer flasks (250 mL) containing 50 ml of MYP broth (malt extract 7 g L–1; yeast extract 0.5 g L–1; soytone 2.5 g L–1) were inoculated with yeast cell suspensions (105 cells mL–1) and incubated at 5, 10, 15, 20, 25 and 30°C with periodic shaking. Experiments were performed in duplicate. Growth kinetics were measured photometrically at 660 nm with a Spectronics 20 Genesys Spectrophotometer at 24-h intervals. After 210 h of incubation, dry biomass was determined at 105°C (Libkind et al., 2004).

Extracellular enzymatic activity

A set of 78 strains, representative of all identified species, was tested for the ability of the strains to degrade starch, protein (casein), lipids (tributirin and Tween-80), pectin, cellulose and chitin, according to the procedures described by Buzzini & Martini (2002). Calibrated suspensions (106 cells mL–1) of yeast cells grown for 24–48 h were used to inoculate the surface of agar plates using a multipoint inoculation device. Enzymatic activity was checked after 21 days in petri dishes incubated at 20°C, and after 5 days in dishes incubated at 4°C. The halo/colony (h/c) ratio was used as a semiquantitative assessment of extracellular enzymatic activity. Data for both temperatures were compared using the Student's t-test.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Yeast occurrence

Yeast viable counts were below 3 CFU L–1 in seven of the 18 samples. High viable counts were obtained only in the Frías River site sample (780 CFU L–1) (data not shown). Mean yeast counts are listed in Table 1. The low yeast counts reported here are in agreement with data reported by Libkind et al. (2003) for ultraoligotrophic lakes and lagoons of glacial origin, in the NHNP. Buzzini et al. (2005) also found low counts (10–20 CFU L–1) for meltwater draining from glaciers in the Italian Alps. These results are probably related to the fact that meltwater rivers draining from glaciers are oligotrophic (Hagler & Ahearn 1987), i.e. characterized by low nutrient concentrations (Pedrozo et al., 1993).

As shown by the SD values (Table 1), yeast counts were quite variable at all sampling sites, as expected for highly dynamic systems such as running waters. Considering that microbial populations in glacial ecosystems are primarily associated with sediments (Foght et al., 2004), yeast count variability could also be due to uneven distribution of clays and suspended particles in the samples. Neutral or slightly acidic pH water values were registered at all sampling sites (Table 1), but no pH influence on yeast colony counts was apparent.

Yeast identification

In total, 86 yeast isolates were collected and classified into nine genera on the basis of morphologic and physiologic identification tests. The genera were: Cryptococcus, Leucosporidiella, Dioszegia, Rhodotorula, Rhodosporidium, Mrakia, Sporobolomyces, Udeniomyces, and Candida. Ninety-nine per cent of the isolates belonged to the phylum Basidiomycetes.

Strain characterization by MSP-PCR fingerprinting showed 27 distinct profiles, 11 of which were shared by at least two strains and formed groups of identical DNA banding patterns (Table 2; Fig. 2). Two of the MSP-PCR groups were identified as Rhodotorula mucilaginosa and Sporobolomyces ruberrimus by direct comparison with the MSP-PCR type strain profiles (Fig. 2d). Sequences obtained in this study were deposited in GenBank, and the accession numbers are shown in Table 3. As suggested by Fell et al. (2000), strains that differed from the closest related type strain by two or fewer nucleotides in the D1/D2 region were considered to be the same species. The 26S rRNA gene D1/D2 region sequence analysis of representative strains from the nine remaining MSP-PCR groups allowed them to be assigned to the following species: Dioszegia crocea (two MSP-PCR patterns), Leucosporidiella fragaria (two MSP-PCR patterns), L. creatinivora, Rhodotorula colostri, Cryptococcus festucosus, and two novel species of Cryptococcus, henceforth referred to as Cryptococcus sp. 1 and Cryptococcus sp. 2 (Table 2). Leucosporidiella fragaria strains with different MSP-PCR patterns had identical D1/D2 sequences (Fig. 3b), whereas D. crocea strains with different MSP-PCR patterns differed by two nucleotide substitutions (Fig. 3c). Novel species Cryptococcus sp. 1 showed three nucleotide substitutions compared to Filobasidium globisporum; however, the comparison with equivalent sequences from GenBank showed that it is identical to the species Cr. ‘stepposus’, which has not yet been validly described (Fig. 3c). Cryptococcus sp. 2 species, on the other hand, differed substantially (44 substitutions) from the closest known species, Cr. fragicola (Fig. 3c), and its formal description is in progress.

Table 2.   Yeast species isolated from meltwater of three glaciers on Mount Tronador (NHNP)
IdentificationOrigin of the strains*Total number of strainsMSP-PCRblast results
123456
  • *

    Sampling sites: Ventisquero Negro lagoon (1), Garganta del Diablo waterfall (2), Castaño Overo river (3), Frías river (4–6).

  • 1 and 2 indicate the number of different MSP-PCR profiles displayed by each species. V, variable MSP-PCR profiles; ND, not determined.

  • Percentage of similarity with the sequence of the closest type strain (number of nucleotide differences).

  • §

    Compared with Filobasidium globisporum CBS 7642T.

  • Compared with Cryptococcus fragicola JCM 1530T.

  • Compared with Mrakia frigida CBS 5270T.

Cryptococcus sp. 1  122  23199% (3)§
Leucosporidiella fragaria   15  15299% (2)
Cryptococcus af. laurentii14 13211V
Dioszegia crocea   6  6299% (0.2)
Rhodotorula colostri4  1  5199% (2)
Sporobolomyces ruberrimus1  3  41
Cryptococcus sp. 2   3  3192% (44)
Rhodotorula mucilaginosa111   31
Cryptococcus festucosus1 1   2199% (2)
Leucosporidiella creatinivora    2 2199% (1)
Mrakia frigida 2    2ND100%
Dioszegia fristingensis   1  11100%
Cryptococcus macerans1     1199% (1)
Rhodosporidium kratochvilovae1     11100%
Rhodotorula laryngis1     11100%
Dioszegia hungarica   1  11100%
Mrakia sp. 1    1ND98% (8)
Cryptococcus cylindricus   1  1ND99% (1)
Cryptococcus adeliensis1     1ND100%
Udeniomyces pannonicus   1  1ND100%
Candida famata 1    1ND100%
image

Figure 2.  MSP-PCR fingerprintings obtained with the M13 primer. (a) Leucosporidiella fragaria, (b) Cryptococcus sp. 1, (c) Dioszegia crocea, (d) Rhodotorula mucilaginosa (left) and Sporobolomyces ruberrimus (right) identified with MSP-PCR. PYCC, Portuguese Yeast Culture Collection; CBS, Centraalbureau voor Schimmelcultures;T, type strain; M, molecular size marker (λ DNA cleaved with HindIII, and ΦX174 DNA cleaved with HaeIII).

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Table 3.   Yeast isolates subjected to 26S rRNA gene D1/D2 fragment sequencing
SpeciesStrain numberGeneBank accession no.
Candida famataCRUB 1270DQ513292
Cryptococcus cylindricusCRUB 1255DQ513289
Cryptococcus festucosusCRUB 1301DQ513283
Cryptococcus maceransCRUB 1178DQ513290
Cryptococcus sp. 1CRUB 1232DQ513273
Cryptococcus sp. 1CRUB 1240DQ513274
Cryptococcus sp. 2CRUB 1230DQ513279
Crytococcus adeliensisCRUB 1213DQ513272
Dioszegia croceaCRUB 1274DQ513280
Dioszegia croceaCRUB 1276DQ513275
Dioszegia croceaCRUB 1279DQ513276
Dioszegia fristingensisCRUB 1275DQ513281
Dioszegia hungaricaCRUB 1278DQ513277
Leucosporidiella creatinivoraCRUB 1214DQ513282
Leucosporidiella fragariaCRUB 1205DQ513269
Leucosporidiella fragariaCRUB 1211DQ513270
Leucosporidiella fragariaCRUB 1201DQ513271
Mrakia frigidaCRUB 1253DQ513285
Mrakia frigidaCRUB 1304DQ513284
Mrakia sp.CRUB 1272DQ513286
Rhodotorula colostriCRUB 1180DQ513291
Rhodosporidium kratochvilovaeCRUB 1179DQ513278
Rhodotorula laryngisCRUB 1183DQ513288
Udeniomyces pannonicusCRUB 1223DQ513287
image

Figure 3.  Phylogenetic placement of basidiomycetous Patagonian yeast isolates obtained by neighbor joining (distance K2P method) of the D1/D2 domains of the 26S rRNA gene. (a) Cystofilobasidiales Order, Hymenomycetes Class (rooted with Phaffia rhodozyma); (b) Leucosporidiales and Microbotryales Orders, Urediniomycetes Class (rooted with Rhodotorula nothofagi); (c) Tremellales and Filobasidiales Orders, Hymenomycetes Class (rooted with Bullera arundinariae) indicate a not yet valid species name. The scale indicates the number of substitutions accumulated every 100 nucleotides. Bootstrap values higher than 50% are shown (1000 replicates).

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Sixteen strains showed a unique MSP-PCR fingerprinting pattern; the 26S rRNA gene D1/D2 sequence analysis allowed the identification of five of them as D. fristingensis, D. hungarica, Cr. macerans, Rhodotorula laryngis, and Rhodosporidium kratochvilovae (Table 3). The remaining 11 strains were grouped together on the basis of identical results for 42 physiologic and morphologic tests, which suggested that they were closely related to Cr. laurentii, even though they had heterogeneous MSP-PCR profiles. The D1/D2 sequences of the 26S rRNA gene and internal transcribed spacer (ITS) regions have high intraspecific heterogeneity (Sugita et al., 2000; Takashima et al., 2003), which could explain the highly heterogenic profiles observed in this survey. The difficulty of accurately identifying strains of this species has been addressed before (Sugita et al., 2000; Takashima et al., 2003), so despite the fact that for the purpose of this article these 11 strains were designated ‘Cryptococcus af. laurentii’, the possibility that different species have been grouped under this name cannot be ruled out.

Finally, seven single strains were subjected directly to rRNA gene sequence analysis, and according to the blast-based identification results, they were assigned to M. frigida (two strains), Cr. adeliensis, Cr. cylindricus, Udeniomyces pannonicus, Candida famata and Mrakia sp. 1 (Table 3). The last of these, a novel species, differed from M. frigida by eight nucleotide positions (Fig. 3a), and its formal description is in progress.

Yeast diversity

As mentioned above, of the nine identified genera, the most frequently isolated was Cryptococcus (50% of total strains), whereas Leucosporidiella (formerly Rhodotorula, Sampaio et al., 2003) and Rhodotorula accounted for 20% and 9% of the total strains, respectively. In total, 21 species were presented in this work. Three of these were new species, two of which belonged to the genus Cryptococcus and one of which belonged to the genus Mrakia. Cryptococcus sp. 1 accounted for the highest number of isolates (23 strains), whereas Cryptococcus sp. 2 and Mrakia sp. were much less frequent (Table 2).

When the yeast diversity of Mount Tronador glacial meltwater was compared with that of other cold environments (Table 4), a notable resemblance to Antarctic environments was observed. Eight of the 21 species found in this study are present in Antarctic habitats. A higher percentage (>50%, 10 species) of common species was observed between glacial meltwater and other aquatic environments of the NHNP. In contrast, only two species were shared with Alpine glaciers. However, any comparison, in the case of Alpine glaciers, is limited due to the lack of yeast diversity studies based on molecular identification of isolates in such environments.

Table 4.   Comparison of yeasts isolated from glacial meltwater rivers from Mount Tronador with those isolated from other cold environments
Species from Mount Tronador glacial meltwaterAlpine glaciers*Antarctic environmentsNahuel Huapi National Park
Ultraoligotrophic aquatic environmentsTerrestrial environments§
Cryptococcus af. laurentii+++
Cryptococcus macerans+++
Rhodotorula mucilaginosa+++
Rhodotorula colostri++
Rhodosporidium kratochvilovae++
Rhodotorula laryngis++
Dioszegia hungarica++
Leucosporidiella creatinivora++
Mrakia frigida++
Candida famata++
Cryptococcus adeliensis+
Dioszegia fristingensis+
Sporobolomyces ruberrimus+
Leucosporidiella fragaria
Dioszegia crocea
Cryptococcus festucosus
Cryptococcus cylindricus
Udeniomyces pannonicus

This is the first report on the occurrence of the Mrakia genus in the NHNP, as well as on the occurrence of several other species (L. fragaria, D. crocea, Cr. festucosus, Cr. cylindricus and U. pannonicus) in glacial environments (Table 4).

Rhodotorula fragaria (Torulopsis fragaria) (Fell & Statzell-Tallman, 1998a) was isolated from fresh strawberries (35 strains) and blackcurrants (five strains) in the UK in 1971, and it was later named L. fragaria (Sampaio et al., 2003). According to temperature growth range experiments (see below), L. fragaria strains were able to grow well at 5°C, and their maximum growth temperature was 25°C under the assayed conditions. With regard to extracellular enzymatic activity tests, most L. fragaria strains showed extracellular proteolytic and lipolytic activities at 4 and 20°C. The intensity of degradation was higher at 4°C than at 20°C for both activities. The data reported here indicate that strains of L. fragaria are cold-adapted. With regard to Dioszegia spp., it has been suggested that members of this genus (D. aurantiaca, D. statzelliae, D. fristingensis and D. crocea) are present either on plant surfaces or in soil, and that some of them may be restricted to areas with cold climates (Inácio et al., 2005). The presence of the last two species in Patagonian glacial meltwater is in agreement with the suggested restricted distribution. These yeast species may appear in meltwater rivers as a consequence of run-off from the surrounding terrestrial environments.

Cryptococcus festucosus was first isolated from leaves in the Moscow region, Russia (Golubev et al., 2004). The type strain of Cr. cylindricus was isolated from lichen in the Arrábida Natural Park (Portugal) (Fonseca et al., 2000); a psychrophilic strain of this species was also isolated from soil in Japan (Nakagawa et al., 2004). This is the first report of the presence of Cr. cylindricus and Cr. festucosus in cold aquatic environments (Table 4). Two additional strains of the latter species have recently been isolated from Patagonian rivers (unpublished data).

Udeniomyces pannonicus was isolated for the first time from leaves in Hungary (Niwata et al., 2002). This is a ballistosporous genus usually associated with leaf surfaces (Nakase, 2000); other Udeniomyces species have been isolated from plants in Thailand, Japan, New Zealand, Tasmania and Australia, and from frozen fish in Japan (Niwata et al., 2002).

Likewise, the three species Cr. festucosus, Cr. cylindricus and U. pannonicus seemed to be primarily associated with soil and plants in temperate or cold climates. As was the case for Dioszegia spp., run-off from terrestrial environments could be responsible for their presence in meltwater aquatic environments in Patagonia.

Mrakia, a typically psychrophilic genus, has a restricted distribution in Antarctica, and Arctic and Alpine glaciers (Fell & Statzell-Tallman, 1998b; Margesin et al., 2005). Mrakia genus sexual homothallic reproduction by means of teliospores is a distinctive characteristic, which may influence its ability to survive in cold environments (Margesin et al., 2005). Considering data from other glacial meltwater environments, the occurrence of the psychrophilic Mrakia species in glacial meltwater in Patagonia is not surprising. However, the present work is the first reference to the genus in this type of environment in South America.

Effect of temperature on yeast growth

All isolated yeast strains were able to grow in MYP agar at 5, 10 and 15°C. Three psychrophilic Mrakia strains (two M. frigida and Mrakia sp. CRUB 1272) did not grow at 20°C, whereas six strains of D. crocea formed colonies at 20°C but not at 25°C. The 77 remaining strains (90%) grew at 25°C. Cryptococcus adeliensis CRUB 1213, C. famata CRUB 1270 and three strains of S. ruberrimus grew at 30°C. Finally, three strains of Rhodotorula mucilaginosa formed colonies in the same medium at 35°C.

The results for optimum growth temperatures of selected strains are shown in Fig. 4. Optimum growth temperatures were as follows: 15°C (c. 1.7 g dry biomass L–1) for psychrophilic Mrakia species, 20°C (3.1 g L–1) for Cryptococcus sp. 1, and between 20 and 25°C (1.7–1.8 g L–1) for L. fragaria. Rhodotorula mucilaginosa was the only species able to grow at 30°C, and probably has an even higher optimum growth temperature.

image

Figure 4.  Growth temperature profiles of selected yeast strains. Bars indicate the SD of mean values. These results were obtained after 216 h of incubation.

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Water temperature measured during sample collection ranged generally between 4.5 and 5.5°C (Table 1). As expected for yeasts isolated from such cold water, all strains grew well at similar temperatures in laboratory conditions. These results confirm that all strains isolated are adapted to the cold. Lower growth temperature limits are difficult to determine, because compounds required to prevent media from freezing are generally toxic (Vishniac, 2005). The psychrophilic nature of Mrakia species (including the new Mrakia species) has been confirmed experimentally (Fell & Statzell-Tallman, 1998b). Leucosporidiella fragaria and Cryptococcus sp. 1, which were the most frequently isolated species, had higher optimum growth temperatures than Mrakia species, but much lower optimum growth temperatures than mesophilic R. mucilaginosa.

Extracellular enzymatic activity

Ninety-five per cent of the 78 tested strains showed at least one extracellular enzyme activity at either 4°C or at 20°C. Lipolysis (hydrolysis of Tween-80 and/or tributirin) was the most widely expressed extracellular enzyme activity. Almost all strains showed the ability to hydrolyze at least one of the two lipophilic substrates, regardless of the temperature at which it was tested. Amylolytic, proteolytic and pectinolytic activities were less frequent, and were expressed in only 15–20% of the strains. None of the strains was able to hydrolyze chitin or cellulose at either of the assessed temperatures.

Differences in the expression of extracellular enzymatic activities at 4 and 20°C were investigated to determine the number of positive strains and/or the intensity of substrate degradation (Fig. 5). Four of five tested activities showed a higher number of positive strains at 4°C than at 20°C. The biggest differences were observed for lipolytic activity; 64 strains were able to hydrolyze Tween-80 at 4°C, whereas only nine strains retained this activity at 20°C. Pectinolytic activity was the only exception, with four and 11 positive strains at 4 and 20°C, respectively. With regard to the intensity of each activity, assessed by the h/c ratio, significantly higher activities of proteolysis (t=6.4907; P=0.00001) and hydrolysis of Tween-80 (t=4.6146; P=0.00015) were observed at 4°C than at 20°C. The remaining activities showed no significant intensity differences between the two temperatures.

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Figure 5.  Extracellular enzymatic activity of yeast strains from glacial meltwater rivers from the NHNP. Bars indicate the SD of mean values. N indicates the number of positive strains (out of 78 strains) for the corresponding enzymatic activity. aSignificantly different activity at 4 and 20°C (P<0.001).

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Sporobolomyces ruberrimus CRUB 1182 had the highest amylolytic activity at both tested temperatures (h/c=7.5 at 4°C, and h/c=5 at 20°C). Of the 15 positive strains, L. fragaria CRUB 1200 showed the highest proteolytic activity at 4°C (h/c=6). The highest lipolytic activity found at 4°C on tributirin agar corresponded to Cr. macerans CRUB 1290 (h/c=5), whereas for the same activity on Tween-80, S. ruberrimus CRUB 1287 had an h/c ratio of 4.5. Very high pectinolytic activity was observed at 4°C for Cryptococcus sp. 1 CRUB 1247 (h/c=8), and at 20°C for L. fragaria CRUB 1218 (h/c=7).

Final remarks

This report is the first on cold-adapted yeasts isolated from glacial meltwater in Patagonia, and reveals an interesting diversity of such yeasts, including three novel species. Vishniac (1996) suggested that for a species to be considered as indigenous, certain requirements must be met. Evidence must be presented that its occurrence is unique, i.e. that it is a new species. Additionally, it must be adapted to the environmental conditions, and it must be isolated frequently in the sampled area, so as to rule out possible immigration. Our results show the presence of at least three new species with low growth temperature profiles and important extracellular enzymatic activities at low temperatures. Moreover, cold-adapted species such as L. fragaria and Dioszegia spp. were isolated with high frequency. Leucosporidiella creatinivora, Cr. cylindricus, Cr. festucosus, M. frigida and U. pannonicus were less frequently isolated species, but are probably also autochthonous for these cold habitats, considering evidence from other publications and our own ecophysiologic observations. Consequently, a considerable fraction of the yeasts isolated from the glacial meltwater rivers of Mount Tronador (NHNP) might represent the indigenous microbial community of this extremely cold environment.

A significant proportion of the yeast isolates were able to hydrolyze natural compounds such as lipids, starch, protein and pectin at low temperatures, which, as discussed above, represents further evidence for the metabolic adaptation of these strains to cold environments. The above data suggest a significant ecological role, as organic matter decomposers and nutrient cyclers, of yeasts occurring in these unpolluted cold habitats. However, some organic compounds, such as cellulose and chitin, could not be degraded by any isolate, in accordance with results for yeast strains collected from Alpine glaciers (Buzzini et al., 2005). Possibly, our native yeasts may not be directly involved in the decomposition of these complex molecules. However, Margesin & Schinner (1997) and Bergauer et al. (2005) reported the ability of certain yeasts isolated from contaminated Alpine glacial environments to degrade hydrocarbon pollutants at low temperatures.

Several yeast species with distinctive properties reported in this study (including novel ones) could be used for further metabolic, ecological and biotechnological studies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

This work was partially funded by the Universidad Nacional del Comahue (Project B121) and CONICET (Project PIP2784) grants to M. van Broock, and SECYT (Project PICT 22200). Bilateral cooperation between Argentina and Italy was supported by a SECYT-MAE cooperation agreement (IT/PA03-BI/087). V. de García and D. Libkind were supported by CONICET PhD fellowships. We would like to thank the authorities of Parques Nacionales (Argentina) for providing permission for water sample collection within the NHNP. Our special thanks go to Celia Tognetti for critical review of the manuscript and Amalia de Negri for map design.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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