Study on biodiversity of yeasts isolated in samples

Study on biodiversity of yeasts isolated in samples, collected in Phongnha- Kebang National Park Dao Thi Luong*, Phan Thi Thu Mai, Tran Thi Le Quyen, Duong Van Hop Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, 144 Xuan Thuy, Hanoi Abstract From 53 samples (13 of leaves, 18 of litters and 22 of soils), which were collected in Phongnha- Kebang National Park, 57 yeast strains were isolated Those yeast strains were identified based on the morphological characteristics and the sequence analysis of D1/D2 domain of 26S rDNA They belonged to 13 genera and 26 species, of which were suspected as new species A mong 13 genera, Basidiomycetous yeasts were dominant from isolated samples Thirty-four strains out of 57 were mainly grouped into genera of Rhodotorula, Sporobolomyces, Candida and 11 species The other 18 strains belonged to the genera of Cryptococcus, Hannaella, Rhodosporidium, Pseudozyma and Trichosporon and 10 species The rest were identified as Jaminaea, Pichia, Quambalaria, Sporisorium, Saccharomycopsis and Trichosporon (5 strains, species) Keywords: Biodiversity of yeast from Phongnha- Kebang National Park Introduction Phongnha- Kebang National Park was first nominated as a UNESCO World Heritage Site in 1998 The reason given for the nomination was that this natural reservation was satisfied criteria of biodiversity, unique beauty and geo-diversity Phongnha - Kebang National Park is located in Quangbinh province with the distances of about 500 km south of the Hanoi capital The climate * Corresponding author Tel.: +84-904302964 E-mail: luongdaothi@yahoo.com in this national park is tropical (hot, and humid) The annual mean temperature is 23 to 25 °C Annual rainfall is 2,000 mm to 2,500 mm Average annual relative humidity is 84% The park is part of the Annamites eco-region By far the largest vegetation type is tropical dense moist evergreen forest on limestone under 800 m above sea level According to the results of initial surveys, the primary tropical forest in Phongnha-Kebang has 140 families, 427 genera, and 751 species of vascular plants The forest is also home to 98 families, 256 genera and 381 species of vertebrates, of the 59 recorded reptile and amphibian species The 72 fish species include species endemic to the area The park is home of over 200 bird species and 259 butterfly species of 11 families However, different from plants, invertebrates and animals, microorganisms living (eg yeasts) in the national environment such as forests were not studied until recent years The studies on the diversity of yeasts in the tropical forest have just started and further extensive studies are required This report discusses about yeasts found in Phongnha-Kebang National Park Materials and methods Yeast strains Yeast strains were isolated from plant leaves, litters and soils collected in Phongnha- Kebang National Park, Quangbinh province, using the direct streaking technique method on YM agar Morphological characteristics based on methods described by Yarrow (1998) [7] Sequencing and phylogenetic analysis The partial sequence of D1/D2 domain of 26S rDNA, was determined after PCR amplification of the DNA Both strands of the PCR products were sequenced directly (Kurtzman and Robnett, 1997) [2] Generated sequences were aligned with reference sequences of related species by using the CLUSTAL X computer program (Thompson et al., 1997) [6] Reference sequences used for the phylogenetic study were obtained from the database The phylogenetic tree was constructed from the evolutionary distance data according to Kimura (1980) using the neighbor-joining method (Saitou and Nei, 1987) [1, 5] Sites where gaps existed in any sequences were excluded Bootstrap analyses were performed from 1000 random repetitions Results and discussion Fifty-three samples were collected in Phongnha- Kebang National Park Yeasts were isolated from these samples at 30 oC by the direct streaking technique method on YM agar The colonies, the cells and ballistoconidia were observed and selected Fifty-seven strains of yeasts were isolated from 20 samples out of 53 collected The frequency of isolation was high when samples collected from the leaves, and very low from the litters (Table 1) These results clearly suggested that yeasts associated with plants in PhongnhaKebang National Park are rich in biodiversity and numerous strains live in these substrates Table The samples for the isolation of yeasts Sources No of samples No of strains isolated Frequency of isolation (%) Leaves 13 41 71.9 Litters 18 5.3 Soils 22 13 22.8 Total 53 57 100 The sequences of D1/D2 domain of 26 rDNA of 57 yeast strains were determined Ten strains belonged to ascomycetous yeasts and the remaining 47 strains belonged to basidiomycetous yeasts These yeasts were tentatively identified on the basis of the D1/D2 sequences by consulting a guideline of Kurzman and Robnett (1998), namely, strains having a nucleotide difference of zero to one are conspecific, those having a nucleotide difference of two to three are most conspecific or sister species of earlier described and those having a nucleotide difference of four or more are possible different species [3] On the basis of the above mentioned description, 57 isolates were identified as 26 species in 13 genera (Table 2) Ascomycetous yeasts included genera Candida, Pichia and Saccharomycopsis and occupid 17.5% of isolates Ten strains of ascomycetous yeasts were found to represent species (5 known species and undescribed species) Basidiomycetous yeast strains were dominant from isolated samples (82.5%), included 10 genera Cryptococcus, Jaminaea, Hannaella, Pseudozyma, Rhodosporidium, Rhodotorula, Quambalaria, Sporidiobolus, Sporobolomyces and Trichosporon They were classified into 20 species, 16 known species (43 strains), undescribed species (4 strains) (Fig – 4) Among 13 genera found in this study, Rhodotorula genus was the dominant (28.1% strains was found in 13 out of 20 samples), followed by Sporobolomyces genus (17.5% from samples of plant), Candida (14% from samples of soil and plants), Cryptococcus (8.8% from samples of soil) and Rhodosporidium (7.0% from samples of litter, soil and plants), Hannaella (5.3% from samples of plant), Pseudozyma (5.3% from samples of plant), Trichosporon (5.3% from plants) The remaining genera occupied 1.8 - 3.5% of isolates (Table 2) 0.05 0.05 100 Candida diversa_AB436390 100 Candida diversa_ AB436390 100 S16.4 S16.4100 Saturnispora saitoi_EF550218 100 Saturnispora saitoi_ EF550218 Saturnispora ahearnii_EF550217 Saturnispora ahearnii_ EF550217 Saturnispora besseyi_EF550216 100 besseyi_ EF550216 100 Saturnispora Candida_rugosa_ EF375701 Candida_rugosa_ EF375701 100 catenulata_ Candida catenulata_ AF374607 100 Candida AF37460 100 99 99 100 100 PL1.4 PL1.4 91 PL1.2 PL1.2 70 P05.3B 70 Candida tropicalis_FJ665624 Candida FJ665624 100tropicalis_ 100 Candida albicans_DQ318808 Candida albicans_ DQ318808 100 Candida oleophila_AF178051 100 Candida oleophila_ AF178051 100 Candida 100 Candida sojae_ U71070sojae_U71070 Candida metapsilosis_ AJ508577 Candida metapsilosis_ AJ508577 84 Candida Candida orthopsilosis_ AJ508576 orthopsilosis_ AJ508576 100 100 Candida parapsilosis_FJ432627 Candida 89 parapsilosis_FJ432627 89 100 PL5.1 100 PL5.1 100 tenuis_ Candida tenuis_AY964682 100 98 Candida AY964682 Candida trypodendroni_ AF017240 Candida trypodendroni_ AF017240 PL2.8 60 PL2.8 60 Candida fukuyamaensis_ U62311 Candida fukuyamaensis_ U62311 100 100 PL4.5 PL4.5 98 PL4.7 98 PL4.7 90 Pichia guilliermondii_ 90 Pichia guilliermondii_ EU182216 EU182216 Candida fermentati_AY894826 82 Candida 82 fermentati_AY894826 96 Pichia caribbica_EU177571 96 Pichia caribbica_EU177571 Saccharomycopsis U40099 97 Saccharomycopsis selenospora_selenospora_ U40099 97 Saccharomycopsis malanga_U40135 Saccharomycopsis malanga_U40135 100Saccharomycopsis_capsularis_ 100 Saccharomycopsis_capsularis_ AB196493 AB196493 56 56 Saccharomycopsis crataegensis_ U40079 Saccharomycopsis crataegensis_ U40079 100 100 Saccharomycopsis fibuligera_AB196495 Saccharomycopsis fibuligera_AB196495 PL3.3 PL3.3 59 67 59 Pichia Pichia ciferrii_ U74587ciferrii_U74587 100 silvicultrix_U69879 100 Candida silvicultrix_U69879 Candida Pichia subpelliculosa_ AB375306 Pichia subpelliculosa_ AB375306 PL4.6 PL4.6 90 90 Pichia anomala_EU753716 Pichia anomala_ EU753716 Saccharomyces cerevisiae_FJ770557 Saccharomyces cerevisiae_FJ770557 P05.3B91 84 98 67 Fig Phylogenetic tree constructed from neighbour-joining analysis of the sequences of 26S rDNA D1/D2 domain depicting the relationships of isolates belonged to ascomycetous yeasts with closely related taxa Ustilago alcornii_AY740165 Ustilago triodiae_AY740126 Ustilago echinata_AY740144 83 PL4.4 Ustilago esculenta_AB211926 51 67 57 0.005 52 Pseudozyma parantarctica_ AB089357 PL2.5 Pseudozyma shanxiensis_DQ00895 Pseudozyma prolifica_AM160639 Pseudozyma hubeiensis_DQ008953 100 P05.2 Sporisorium chrysopogonis_ AY740131 52 83 Fig Phylogenetic tree constructed from neighbour-joining analysis of the sequences of 26S rDNA D1/D2 domain depicting the relationships of isolates belonged to basidiomycetous yeasts with closely related taxa (Pseudozyma genus) 0.02 100 100 100 100 100 100coremiiforme Trichosporon coremiiforme_ AF189863 Trichosporon _ AF189863 Trichosporon faecale_AF105395 Trichosporon faecale_AF105395 100 89 69 88 64 84 75 81 100 99 0.02 Trichosporon moniliiforme_ AY953964 Trichosporon moniliiforme _ AY953964 100 Trichosporon Trichosporon mucoides _AF335988 mucoides_AF335988 Trichosporon Trichosporon dermatis _AY143555 dermatis_AY143555 100 100 PL4.2 PL4.2 Trichosporon Trichosporon asteroides _ AF075513 asteroides_ AF075513 89 PL1.1 PL1.1 69 88 Trichosporon Trichosporon japonicum _ AF308657 japonicum_ AF308657 Trichosporon asahii_AB363786 Trichosporon asahii 64 _AB363786 84 PL1.3 PL1.3 S09.3 S09.3 S09.5 S09.5 75 S13.3 S13.3 81 100 S02 S02 99 S13.2 S13.2 Cryptococcus Cryptococcus podzolicus _ EF068196podzolicus_ EF068196 Bullera ninhbinhensis_AB261011 Bullera ninhbinhensis_AB261011 P0321 P0321 84 67 84 Hannaella luteolus_AM160633 Hannaella luteolus_AM160633 100 100 P02.1B P02.1B 60 60 Hannaella kunmingensis_AB109558 Hannaella kunmingensis _AB109558 Hannaella zeae_AJ965480 Hannaella zeae_AJ965480 Hannaella sinensis_FJ743606 Hannaella 55 sinensis_FJ743606 55 100 100 P05,2B P05,2B Tremella encephala_AF189867 Tremella encephala_AF189867 67 67 67 Fig Phylogenetic tree constructed from neighbour-joining analysis of the sequences of 26S rDNA D1/D2 domain depicting the relationships of isolates belonged to basidiomycetous yeasts with closely related taxa (Cryptococcus, Hannaella, Trichosporon genera) 0.05 75 75 Sporobolomyces alborubescens_ AF207886 Rhodotorula mucilaginosa_FJ515267 100 P03.1B, P02.223, P03.222, P04.13, P02.1, P05.1, P03.11 Sporobolomyces alborubescens _ AF207886 100 S14, S15, S16, S17, S18, P06.1B, P06, P01B, P02.3 Rhodotorula FJ515267 P06.3B,mucilaginosa_ P05.1B, P04.1B, P06.2B, P05.3, P03.12 100 P03.1B, P02.223, P03.222, P04.13, P02.1, P05.1, P03.11 Rhodotorula dairenensis_ AY033551 90 100 S14, S15, S16, S17, S18, P06.1B, P06, P01B, P02.3 100 Rhodosporidium paludigenum_ AF514863 P06.3B, P05.1B, P04.1B, P06.2B, P05.3, P03.12 L2.1, L1.4, S13 Rhodotorula dairenensis_ AY033551 90 95 PL5.2 100 Rhodosporidium paludigenum_ AF514863 66 Rhodosporidium toruloides_ DQ832191 L2.1, L1.4, S13 95 PL5.2 Rhodosporidium diobovatum_ AB217490 57 66 Rhodotorulatoruloides araucariae _ AF070427 Rhodosporidium _ DQ832191 98 Rhodosporidium diobovatum _ AB217490 Rhodosporidium kratochvilovae _ EU373462 57 Rhodotorula araucariae _ AF070427 Rhodotorula glutinis_FJ345357 100 98 99 100 100 100 98 Sporobolomyces nylandii_ AB279629 P04.12 81 Sporobolomyces odoratus_ AF387126 P03.22B P04.12 Sporobolomyces poonsookiae_ AB217490 P03.22B Sporidiobolus ruineniae_AB217490 EU547494 Sporobolomyces poonsookiae_ 51 81 68 68 100 Rhodosporidium kratochvilovae_ EU373462 Sporobolomyces nylandii_ AB279629 Rhodotorula glutinis_FJ345357 Sporobolomyces odoratus_ AF387126 99 51 90 90 100 0.05 P054B 100 Sporidiobolus ruineniae_ EU547494 100 P054B Sporobolomyces carnicolor_ AY070008 100 Sporobolomyces Sporobolomyces carnicolor_ jilinensis_ AY070008 AY364838 61 Sporobolomyces blumeae _ AB279628 Sporobolomyces jilinensis_ AY364838 61 100Sporobolomyces blumeae_ AB279628 P04.2B 100 P04.2B 100 Cystobasidium fimetarium_ AY512843 100 Cystobasidium fimetarium_ AY512843 PL3.1, L1.7 PL3.1,Rhodotorula L1.7 58 nymphaeae_AB055194 58 Rhodotorula nymphaeae_AB055194 Rhodotorula minuta_EU583491 100 Rhodotorula minuta_EU583491 100 100 PL2.3 PL2.3 Quambalaria cyanescens_ DQ823442 Quambalaria cyanescens_ DQ823442 pitereka_ DQ823439 100100 Quambalaria Quambalaria pitereka_ DQ823439 100 100 S21 S21 Jaminaea angkoriensis _EU587489 Jaminaea angkoriensis _EU587489 100100 Pseudozyma hubeiensis_ DQ008953 Pseudozyma hubeiensis_ DQ008953 98 Fig Phylogenetic tree constructed from neighbour-joining analysis of the sequences of 26S rDNA D1/D2 domain depicting the relationships of isolates belonged to basidiomycetous yeasts with closely related taxa (Jaminaea, Quambalaria, Rhodosporidium Rhodotorula, Sporidiobolus, Sporobolomyces genera) Table The yeasts found in samples collected in National Phongnha- Kebang Park No Genus Candida Species No of strain isolated Candida catenulata Candida diversa Candida parapsilosis Candida sp Total number of strains in the genus 8(6b) 4(1a) Cryptococcus Cryptococcus podzolicus 5(1 b) Jaminaea Jaminaea angkoriensis 1(1 b) Hannaella luteolus Hannaella sinensis Hannaella Hannaella sp 1(1 a) 3(3 b) Pichia Pichia anomala 1(1 b) Pseudozyma Pseudozyma hubeiensis 3(3 b) Pseudozyma sp 2(2 a) Rhodosporidium Rhodosporidium paludigenum 4(1 b) Rhodotorula Rhodotorula mucilaginosa 14 16(2 b) Rhodotorula sp Quambalaria sp 1(1 a) 1(1 b) Quambalaria 10 Saccharomycopsis Saccharomycopsis fibuligera 1(1 b) 11 Sporidiobolus Sporidiobolus ruineniae 1(1 b) Sporobolomyces Sporobolomyces alborubescens Sporobolomyces blumeae Sporobolomyces odoratus Trichosporon sp 3(2 b) 57(5 a) 57(26 b) 12 13 Trichosporon Total 10(3 b) a The number of suspected new species (less than 99% homology in their D1/D2 rDNA to the nearest strains in BLAST search) b The number of species in the genus Twenty one of twenty six isolates belonged to known species Rhodotorula mucilaginosa most frequently isolated (24.6%), was found in 11 out of 20 samples, followed by Sporobolomyces alborubescens (12.3% from samples), Cryptococcus podzolicus (8.8% from samples) and Rhodosporidium paludigenum (7.0% from samples) The other 17 species occupied 1.8 – 3.5% of isolates and the remaining species (8.8%) were as undescribed species Some pictures of yeast strains were showed in Fig and In the previous studies, Dao Thi Luong and co-workers (2008) isolated 71 yeast strains from 17 of 20 leaf samples, which were collected in dioxin polluted area in Danang military airport Fifty yeast strains were identified based on the sequence analysis of D1/D2 domain of 26S rDNA They belonged to genera and 23 species, of which 10 species were suspected as new ones The differences from this study were that Pseudozyma and Cryptococcus (29/50 strains, species) were mainly grouped and ballistoconidium-forming yeasts were not found [4] Fig Colonies and vegetative cells of Rhodotorula mucilaginosa P021 grown in YM broth for days at 25 ˚C Fig Ballistoconidia of Sporobolomyces blumeae P042B produced on corn meal agar after days at 25 ˚C From the above results, yeasts found in the natural environment are rich in biodiversity and many undescribed species have been found Therefore, study of yeast in the natural environment in Vietnam should be done for the progress of yeast systematics and for the utilization of useful functions of yeasts for the welfare of humans Conclusion - Fifty-seven yeast strains were isolated from 53 samples which were collected in PhongnhaKebang National Park - They belonged to 13 genera and 26 species, of which were suspected as new species Acknowledgements This study was supported partly by special coordination funds of “Conserve and maintain microbial resources” - the independent project of Misnistry of Sceince and TechnologyVietnam Reference [1] Kimura M (1980), “A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences”, J Mol Evol., 16 pp 111 – 120 [2] Kurtzman CP, Robnett CJ (1997), “Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene”, J Clin Microbiol., 35 pp 1216 – 1223 [3] Kurtzman CP, Robnet CJ (1998), “Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences”, Antonie van Leeuwenhoek, 67 pp 151 – 171 [4] Luong Dao Thi, Quyen Tran Thi Le, Hop Duong Van (2008), “Study on biodiversity of yeasts isolated in leaves, collected in dioxin polluted area in Da Nang military airport”, Journal of Genetics and Applications, Special Issue: Biotechnology, pp – [5] Saitou N, Nei M (1987) “The neighbor-joining method: a new method for reconstructing phylogenetic trees”, Mol Biol Evol pp 406 – 425 [6] Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997), “The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tool”, Nucleic Acids Res 25 pp 4876 – 4882 [7] Yarrow D (1998) Methods for the isolation, maintenance and identification of yeasts The Yeasts, a Taxonomic Study, 4th edn (Kurtzman CP & Fell JW, eds), pp 77-100 Elsevier, Amsterdam  ... yeasts associated with plants in PhongnhaKebang National Park are rich in biodiversity and numerous strains live in these substrates Table The samples for the isolation of yeasts Sources No of. .. strains were determined Ten strains belonged to ascomycetous yeasts and the remaining 47 strains belonged to basidiomycetous yeasts These yeasts were tentatively identified on the basis of the D1/D2... for the progress of yeast systematics and for the utilization of useful functions of yeasts for the welfare of humans Conclusion - Fifty-seven yeast strains were isolated from 53 samples which were