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Rhodotorula pinicola sp. nov., a basidiomycetous yeast species isolated from xylem of pine twigs
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Rhodotorula pinicola sp. nov., a basidiomycetous yeast species isolated from xylem of pine twigs Jian-Hua Zhao, Feng-Yan Bai , Liang-Dong Guo, Jian-Hua Jia Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, Chinese Academy of Sciences, P.O. Box 2714, Beijing 100080, PR China Received 26 November 2001; received in revised form 27 March 2002 ; accepted 4 April 2002 First published online 25 April 2002
Abstract Three pink-colored yeast strains 3-1-3, 10-3-3 and 19-3-3 were isolated from xylem of surface-sterilized twigs of Pinus tabulaeformis collected from Dongling Mountain, Beijing, in different seasons. These strains were identified as Rhodotorula minuta (Saito) F.C. Harrison by conventional taxonomic characterization. However, molecular phylogenetic analysis based on internal transcribed spacer region (including 5.8S rDNA) and large-subunit rDNA D1/D2 domain sequences indicated that they represent a novel basidiomycetous yeast species, for which Rhodotorula pinicola is proposed (type strain: AS 2.2193T = CBS 9130T ). The new species was most closely related to Rhodotorula laryngis Reierso«l in the R. minuta complex. ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Endophytic fungi; Rhodotorula pinicola sp. nov.; Rhodotorula laryngis; Rhodotorula minuta
1. Introduction
2. Materials and methods
In a systematic and ecological survey on endophytic fungi of pine, three yeast strains were isolated from xylem parts of surface-sterilized pine twigs collected from Dongling Mountain, Beijing, in di¡erent seasons. These strains were identi¢ed as belonging to the basidiomycetous yeast genus Rhodotorula F.C. Harrison by conventional taxonomic investigation. Molecular phylogenetic analysis based on internal transcribed spacer (ITS) region (including 5.8S rDNA) and large-subunit (26S) rDNA D1/D2 domain sequences indicated that these three strains represent a novel yeast species with a close phylogenetic relationship to Rhodotorula laryngis Reierso«l, for which Rhodotorula pinicola is proposed.
2.1. Sampling and strain isolation One- to three-year-old twigs of the pine, Pinus tabulaeformis Carr. were collected from Dongling Mountain, Beijing, in the autumn (October, 2000), winter (February, 2001) and early summer (May, 2001), respectively. Needles were surface-sterilized and then cut into 5-mm length fragments. After needles were removed, twigs were cut into 5-mm length fragments, surface-sterilized, and then separated into parts of bark and xylem. Surface sterilization was performed by the method commonly employed in the studies of endophytes [1^4]. Sets of four to six fragments of needles, bark or xylem of twigs were evenly placed in 90-mm Petri dishes containing 2% malt extract agar supplemented with 1 mg ml31 streptomycin sulfate. Petri dishes were sealed, incubated at 25‡C for 2 months, and examined periodically. Fungal colonies were transferred to new malt agar plates or slants when developed from the plant material. 2.2. Phenotypic characterization
* Corresponding author. Tel. : +86 (10) 6262 6752 ; Fax : +86 (10) 6262 6752. E-mail address : [email protected] (F.-Y. Bai).
Morphological, physiological and biochemical characteristics were examined according to the standard methods
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employed in yeast taxonomy [5]. Extraction, puri¢cation and identi¢cation of ubiquinones were carried out according to Yamada and Kondo [6]. 2.3. DNA sequencing and molecular phylogenetic analysis Nuclear DNA was extracted by the method of Makimura et al. [7]. The DNA fragment covering the ITS region (including 5.8S rDNA) and 26S rDNA D1/D2 domain was ampli¢ed with a pair of primers, ITS1 (5P-TCC GTA GGT GAA CCT GCG G-3P) and NL4 (5P-GGT CCG TGT TTC AAG ACG G-3P). The polymerase chain reaction (PCR) reaction was performed for 36 cycles with denaturation at 94‡C for 1 min, annealing at 55‡C for 1 min and extension at 72‡C for 2 min. After puri¢cation, the PCR products were directly sequenced with the forward primers ITS1 and NL1 (5P-GCA TAT CAA TAA GCG GAG GAA AAG-3P) and the reverse primers ITS4 (5P-TCC TCC GCT TAT TGA TAT GC-3P) and NL4 using the ABI BigDye terminator cycle sequencing kit. Electrophoresis was done on an ABI PRISM 377 DNA sequencer. The ITS or D1/D2 sequences of the strains determined in this study and those of reference type strains obtained from GenBank (with accession numbers indicated in the trees) were aligned with the Clustal X program [8]. Phylogenetic trees were constructed from the evolutionary distance data calculated from Kimura’s two-parameter model [9] by using the neighbor-joining method [10]. Bootstrap analyses [11] were performed from 1000 random resamplings. Fig. 1. Phylogenetic trees depicting the relationships of strains 3-1-3, 103-3 and 19-3-3 with closely related species based on (A) ITS1-5.8S rDNA-ITS2 region and (B) 26S rDNA D1/D2 domain sequences. The phylograms were constructed by neighbor-joining analysis. Bootstrap percentages over 50% from 1000 bootstrap replicates are shown. The GenBank accession numbers of the sequences are given.
3. Results and discussion 3.1. Phenotypic investigation Three yeast strains 3-1-3, 10-3-3 and 19-3-3 were isolated from xylem of the surface-sterilized fragments of di¡erent twigs, which were collected at di¡erent times from three di¡erent trees of P. tabulaeformis. Strain 3-17 was isolated from a 1-year-old twig of a pine and strains 10-3-3 and 19-3-3 from the 3-year-old twigs of two other pines. The three strains form similar pink-colored colonies and have almost identical physiological and biochemical characters. Based on the conventional criteria examined (Table 1), these strains could be assigned to the basidiomycetous yeast genus Rhodotorula F.C. Harrison, and, furthermore, be identi¢ed as Rhodotorula minuta (Saito) F.C. Harrison according to the recent taxonomic system of the genus [12]. 3.2. Molecular phylogenetic analysis R. minuta is a heterogeneous species as shown by the remarkable variations in physiological properties, G+C
contents, as well as DNA^DNA homology values among the strains assigned to this species [12,13]. The recent molecular phylogenetic study based on 26S rDNA D1/D2 domain and ITS region sequence comparison indicated that, among the synonyms of R. minuta, R. laryngis Reierso«l, Rhodotorula pallida Lodder and Rhodotorula sloof¢ae Nova¤k and Vo«ro«s-Felkai represented three distinct species [14]. The sequences of the whole DNA fragments covering ITS1-5.8S rDNA-ITS2 regions and 26S rDNA D1/D2 domains of strains 3-1-3, 10-3-3 and 19-3-3 determined in this study were identical, indicating that the three strains represent a single species. Molecular phylogenetic analysis based on the ITS and D1/D2 sequences showed that these strains belong to the Erythrobasidium clade of the Urediniomycetes [14] and are most closely related to R. laryngis (Fig. 1). They di¡er from the type strain of this species in 6 (1.8%) and 7 (1.1%) nucleotides in the ITS and D1/D2
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regions, respectively. The three strains are also closely related with an Antarctic yeast CBS 8923 as shown in Fig. 1, they di¡er from the latter in eight nucleotides in both ITS and D1/D2 sequences. Previous studies on basidiomycetous yeasts have indicated that conspeci¢c strains usually have fewer than 1% nucleotide divergences in the ITS1 and 2 regions overall [15^17]. However, occasional cases have been found where strains with up to approximately 2% nucleotide di¡erences
161
in the ITS regions may be conspeci¢c as indicated by DNA^DNA relatedness [18,19]. On the basis of an extensive study on basidiomycetous yeasts, Fell et al. [14] showed that strains with two or more nucleotide divergences in the D1/D2 domain represented di¡erent species. Though the taxonomic status of strains 3-1-3, 10-3-3 and 19-3-3 could not be de¢nitely determined based only on the ITS sequences, the D1/D2 sequence comparison con¢rmed that the three strains represent a distinct species.
Table 1 Physiological characteristics of R. pinicola sp. nov. and closely related species Property
Assimilationb : Glucose Galactose L-Sorbose Sucrose Maltose Cellobiose Trehalose Lactose Melibiose Ra⁄nose Melezitose Inulin Soluble starch D-Xylose L-Arabinose D-Arabinose D-Ribose L-Rhamnose D-Glucosamine Methanol Ethanol Glycerol Erythritol Ribitol Galactitol D-Mannitol D-Glucitol Methyl K-D-glucoside Salicin DL-Lactic acid Succinic acid Citric acid Inositol Hexadecane Potassium nitrate Sodium nitrite EthylamineWHCl L-Lysine Cadaverine Additional growth test: Vitamin-free 30‡C 35‡C Starch formation Urease DBB reaction a b
R. laryngisa
R. pallida
R. minuta
R. sloo⁄ae
AS 2.2193T
AS 2.2239
AS 2.2233
CBS 2221T
CBS 320T
CBS 319T
CBS 5706T
+ 3 d + 3 + + d 3 3 + 3 3 + + + 3 3 3 3 + + 3 w 3 + 3 3 + 3 + 3 3 3 3 3 3 + 3
+ 3 3 + 3 d d d 3 3 + 3 3 + d + 3 3 3 3 + + 3 3 3 d 3 3 d 3 + 3 3 3 3 3 3 + 3
+ 3 3 + 3 d + w,d 3 3 + 3 3 + + + 3 3 3 3 + + 3 3 3 d w,d 3 + 3 d 3 3 3 3 3 3 + 3
+ d 3 + 3 + + 3 3 3 + 3 3 + + + 3 3 3 3 + + 3 + 3 + + 3 + d + 3 3
+ 3 w,d,3 3 3 3 + 3 3 3 3 3 3 + 3 3 + 3 3 3 + + 3 d 3 + w,d,+ 3 3 d + 3 3
+ w,d,+ + + 3 + + d 3 3 + 3 3 + + + + 3 3 3 + + 3 d 3 w,d,3 w,d,3 3 d 3 + 3 3
+ 3 d + 3 + + w,d,+ 3 3 + 3 3 3 + w,d,+ d 3 3 3 + + 3 d 3 w,d,3 w,d,+ 3 d d + 3 3
3 3 3 w,d,+ 3
3 3 3 w 3
3 3 3 + 3
3 3 3,+ 3 +
3 + 3 3 + +
3 + 3 3 + +
3 + 3 3 + +
3 + 3 3 + nd
3 + 3 3 + +
3 +
3 + 3 3 + +
R. pinicola
The characters of the CBS strains are from CBS online yeast database. +, positive; 3, negative; d, delayed; w, weakly positive ; nd, not determined.
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3 + +
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The heterogeneity of the genus Rhodotorula has been revealed by molecular phylogenetic analysis [14]. The type species of this genus, Rhodotorula glutinis, which was located in a di¡erent clade as shown by Fell et al. [14], is only distantly related to strains 3-1-3, 10-3-3 and 19-3-3 (Fig. 1). However, before the emendation of this genus and other related basidiomycetous yeast genera was made, the new species represented by the three strains studied can only be assigned to the genus Rhodotorula according to the present taxonomic system of basidiomycetous yeasts [20]. A new species name, R. pinicola, is therefore proposed. The isolates of the new basidiomycetous yeast species are very rare among the endophytic fungi that have been isolated from needles and twigs of P. tabulaeformis collected from Dongling Mountain, Beijing. A total of approximately 1000 plates inoculated with fragments of needles, bark or xylem of 1- to 3-year-old twigs from 30 di¡erent pine trees have been examined, only these three yeast colonies have been found, which grew out from xylem inoculated in three di¡erent plates. In these plates, no ¢lamentous fungi were found. Theoretically, the yeast cells of this species should also occur in the bark of the pine twigs. Probably because of the frequent occurrence of fast-growing ¢lamentous fungi in the bark, it was di⁄cult for the yeast cells to grow and form visible colonies. Basidiomycetous yeasts have been reported as rare endophytic fungi from palms [1], but they have not been identi¢ed at genus level. 3.3. Description of the new species 3.3.1. Latin diagnosis of R. pinicola Bai, Guo et Zhao sp. nov. In liquido malti post dies 3 ad 20‡C, cellulae vegetativae ovoideae, ellipsoideae vel elongatae, (3.5^4.7)U(3.5^7.6) Wm, singulae aut binae. Post unum mensem ad 20‡C, annulus et sedimentum formantur. In agaro malti post unum mensem ad 20‡C, colonia rosea, glabra, butyracea, margine glabra. In agaro farinae zeae pseudomycelium non formantur. Fermentatio nulla. Glucosum, saccharosum, cellobiosum (fortasse lente), trehalosum, lactosum (lente), melezitosum, D-xylosum, L-arabinosum, D-arabinosum, ethanolum, glycerolum, D-mannitolum (fortasse lente), salicinum et acidum succinicum assimilantur at non galactosum, L-sorbosum (vel lente), maltosum, melibiosum, ra⁄nosum, inulin, amylum solubile, D-ribosum, L-rhamnosum, D-glucosaminum, methanolum, erithritolum, ribitolum (vel exigue), galactitolum, D-glucitolum (vel exigue et lente), methyl K-D-glucosidum, acidum DL-lacticum, acidum citricum, inositolum nec hexadecanum. L-lysinum assimilantur at non kalium nitricum, natrum nitrosum, ethylaminum nec cadaverinum. Maxima temperatura crescentiae: 30^31‡C. Materia amyloidea iodophila non formantur. Urea ¢nditur. Vitaminae externae ad crescentiam necessaria sunt. Diazonium caeruleum B positivum. Ubiq-
Fig. 2. R. pinicola AS 2.2193T , vegetative cells grown on malt agar for 3 days at 20‡C. Bar represents 10 Wm.
uinonum majus : Q-10. Typus: isolatus ex xylema ramuli P. tabulaeformis, AS 2.2193T (originaliter ut 3-1-3T ) depositus in collectione China General Microbiological Culture Collection Center, Academia Sinica. 3.3.2. Description of R. pinicola Bai, Guo et Zhao sp. nov. Growth in malt extract : after 3 days at 20‡C, the cells are ovoid, ellipsoidal to elongate (3.5^4.7)U(3.5^7.6) Wm, single or in pairs (Fig. 2). A thin ring is formed. After 1 month at 20‡C, a ring and sediment are formed. Growth on malt extract agar : after 1 month at 20‡C, the streak culture is pink to orange-pink, smooth and glistening with an entire margin. Dalmau plate culture on corn meal agar: pseudohyphae are not formed. Fermentation is absent. Assimilation reactions and other physiological properties are given in Table 1. The major ubiquinone is Q-10. The type strain, 3-1-3T , was isolated from xylem of a surface-sterilized twig of P. tabulaeformis collected from Dongling Mountain, Beijing, in October, 2000. This strain has been deposited in the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, as AS 2.2193T , and in the Centraalbureau voor Schimmelcultures, The Netherlands, as CBS 9130T . Etymology : The speci¢c epithet pinicola refers to the genus name for pine (Pinus), the source of the type strain. Conventionally, it is not easy to distinguish the new species R. pinicola from the other taxa in the R. minuta complex as shown in Table 1. In this complex, only R. pallida can be clearly separated from the others by its inability to assimilate sucrose, cellobiose, melezitose and Larabinose (Table 1). R. pinicola is slightly di¡erent from its closest species R. laryngis in the assimilation reactions of galactose, lactose, ribitol and D-glucitol (Table 1). The new species di¡ers from R. minuta and R. sloo⁄ae in the as-
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similation reactions of ribose and (Table 1).
D-xylose,
respectively [10]
Acknowledgements
[11]
This study was supported by grants G2000046802 and 30070018 from the National Natural Science Foundation of China (NSFC).
[12]
References
[14]
[1] Fro«hlich, J., Hyde, K.D. and Petrini, O. (2000) Endophytic fungi associated with palms. Mycol. Res. 104, 1202^1212. [2] Guo, L.-D., Hyde, K.D. and Liew, E.C.Y. (2000) Identi¢cation of endophytic fungi from Livistona chinensis based on morphology and rDNA sequences. New Phytol. 147, 617^630. [3] Rodrigues, K.F. (1994) The foliar fungal endophytes of the Amazonian palm Euterpe oleracea. Mycologia 86, 376^385. [4] Taylor, J.E., Hyde, K.D. and Jones, E.B.G. (1999) Endophytic fungi associated with the temperate palm, Trachycarpus fortunei, within and outside its natural geographic range. New Phytol. 142, 335^346. [5] Yarrow, D. (1998) Methods for the isolation, maintenance and identi¢cation of yeasts. In: The Yeasts, a Taxonomic Study, 4th edn. (Kurtzman, C.P. and Fell, J.W., Eds.), pp. 77^100. Elsevier Science, Amsterdam. [6] Yamada, Y. and Kondo, K. (1973) Coenzyme Q system in the classi¢cation of the yeast genera Rhodotorula and Cryptococcus and the yeast like genera Sporobolomyces and Rhodosporidium. J. Gen. Appl. Microbiol. 19, 59^77. [7] Makimura, K., Murayama, Y.S. and Yamaguchi, H. (1994) Detection of a wide range of medically important fungal species by polymerase chain reaction (PCR). J. Med. Microbiol. 40, 358^364. [8] Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The Clustal X windows interface: £exible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 4876^4882. [9] Kimura, M. (1980) A simple method for estimating evolutionary rate
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of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111^120. Saitou, N. and Nei, M. (1987) The neighbor-joining method : a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406^425. Felsenstein, J. (1985) Con¢dence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783^791. Fell, J.W. and Statzell-Tallman, A. (1998) Rhodotorula F.C. Harrison. In: The Yeasts, a Taxonomic Study, 4th edn. (Kurtzman, C.P. and Fell, J.W., Eds.), pp. 800^827. Elsevier Science, Amsterdam. Hamamoto, M., Sugiyama, J. and Komagata, K. (1987) DNA^DNA reassociation studies of strains in the genera Rhodosporidium and Rhodotorula. J. Gen. Appl. Microbiol. 33, 57^63. Fell, J.W., Boekhout, T., Fonseca, A., Scorzetti, G. and Statzell-Tallman, A. (2000) Biodiversity and systematics of basidiomycetous yeasts as determined by large subunit rDNA D1/D2 domain sequence analysis. Int. J. Syst. Evol. Microbiol. 50, 1351^1371. Sugita, T., Can‹ete-Gibas, C.F., Takashima, M. and Nakase, T. (1999) Three new species of Bullera isolated from leaves in the Ogasawara Islands. Mycoscience 40, 491^501. Sugita, T., Nishikawa, A., Ikeda, R. and Shinoda, T. (1999) Identi¢cation of medically relevant Trichosporon species based on sequences of internal transcribed spacer regions and construction of a database for Trichosporon identi¢cation. J. Clin. Microbiol. 37, 1985^ 1993. Takashima, M. and Nakase, T. (2000) Four new species of the genus Sporobolomyces isolated from leaves in Thailand. Mycoscience 41, 357^369. Bai, F.-Y., Takashima, M. and Nakase, T. (2001) Description of Bullera kunmingensis sp. nov., and clari¢cation of the taxonomic status of Bullera sinensis and its synonyms based on molecular phylogenetic analysis. FEMS Yeast Res. 1, 103^109. Bai, F.-Y., Takashima, M. and Nakase, T. (2001) Phylogenetic analysis of strains originally assigned to Bullera variabilis: descriptions of Bullera pseudohuiaensis sp. nov., Bullera komagatae sp. nov. and Bullera pseudoschimicola sp. nov.. Int. J. Syst. Evol. Microbiol. 51, 2177^ 2187. Boekhout, T. (1998) Diagnostic descriptions of and key to presently accepted heterobasidiomycetous genera. In: The Yeasts, a Taxonomic Study, 4th edn. (Kurtzman, C.P. and Fell, J.W., Eds.), pp. 627^ 634. Elsevier Science, Amsterdam.
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Rhodotorula pinicola sp. nov., a basidiomycetous yeast species isolated from xylem of pine twigs Jian-Hua Zhao, Feng-Yan Bai , Liang-Dong Guo, Jian-Hua Jia Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, Chinese Academy of Sciences, P.O. Box 2714, Beijing 100080, PR China Received 26 November 2001; received in revised form 27 March 2002 ; accepted 4 April 2002 First published online 25 April 2002
Abstract Three pink-colored yeast strains 3-1-3, 10-3-3 and 19-3-3 were isolated from xylem of surface-sterilized twigs of Pinus tabulaeformis collected from Dongling Mountain, Beijing, in different seasons. These strains were identified as Rhodotorula minuta (Saito) F.C. Harrison by conventional taxonomic characterization. However, molecular phylogenetic analysis based on internal transcribed spacer region (including 5.8S rDNA) and large-subunit rDNA D1/D2 domain sequences indicated that they represent a novel basidiomycetous yeast species, for which Rhodotorula pinicola is proposed (type strain: AS 2.2193T = CBS 9130T ). The new species was most closely related to Rhodotorula laryngis Reierso«l in the R. minuta complex. ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Endophytic fungi; Rhodotorula pinicola sp. nov.; Rhodotorula laryngis; Rhodotorula minuta
1. Introduction
2. Materials and methods
In a systematic and ecological survey on endophytic fungi of pine, three yeast strains were isolated from xylem parts of surface-sterilized pine twigs collected from Dongling Mountain, Beijing, in di¡erent seasons. These strains were identi¢ed as belonging to the basidiomycetous yeast genus Rhodotorula F.C. Harrison by conventional taxonomic investigation. Molecular phylogenetic analysis based on internal transcribed spacer (ITS) region (including 5.8S rDNA) and large-subunit (26S) rDNA D1/D2 domain sequences indicated that these three strains represent a novel yeast species with a close phylogenetic relationship to Rhodotorula laryngis Reierso«l, for which Rhodotorula pinicola is proposed.
2.1. Sampling and strain isolation One- to three-year-old twigs of the pine, Pinus tabulaeformis Carr. were collected from Dongling Mountain, Beijing, in the autumn (October, 2000), winter (February, 2001) and early summer (May, 2001), respectively. Needles were surface-sterilized and then cut into 5-mm length fragments. After needles were removed, twigs were cut into 5-mm length fragments, surface-sterilized, and then separated into parts of bark and xylem. Surface sterilization was performed by the method commonly employed in the studies of endophytes [1^4]. Sets of four to six fragments of needles, bark or xylem of twigs were evenly placed in 90-mm Petri dishes containing 2% malt extract agar supplemented with 1 mg ml31 streptomycin sulfate. Petri dishes were sealed, incubated at 25‡C for 2 months, and examined periodically. Fungal colonies were transferred to new malt agar plates or slants when developed from the plant material. 2.2. Phenotypic characterization
* Corresponding author. Tel. : +86 (10) 6262 6752 ; Fax : +86 (10) 6262 6752. E-mail address : [email protected] (F.-Y. Bai).
Morphological, physiological and biochemical characteristics were examined according to the standard methods
1567-1356 / 02 / $22.00 ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 0 9 3 - 4
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employed in yeast taxonomy [5]. Extraction, puri¢cation and identi¢cation of ubiquinones were carried out according to Yamada and Kondo [6]. 2.3. DNA sequencing and molecular phylogenetic analysis Nuclear DNA was extracted by the method of Makimura et al. [7]. The DNA fragment covering the ITS region (including 5.8S rDNA) and 26S rDNA D1/D2 domain was ampli¢ed with a pair of primers, ITS1 (5P-TCC GTA GGT GAA CCT GCG G-3P) and NL4 (5P-GGT CCG TGT TTC AAG ACG G-3P). The polymerase chain reaction (PCR) reaction was performed for 36 cycles with denaturation at 94‡C for 1 min, annealing at 55‡C for 1 min and extension at 72‡C for 2 min. After puri¢cation, the PCR products were directly sequenced with the forward primers ITS1 and NL1 (5P-GCA TAT CAA TAA GCG GAG GAA AAG-3P) and the reverse primers ITS4 (5P-TCC TCC GCT TAT TGA TAT GC-3P) and NL4 using the ABI BigDye terminator cycle sequencing kit. Electrophoresis was done on an ABI PRISM 377 DNA sequencer. The ITS or D1/D2 sequences of the strains determined in this study and those of reference type strains obtained from GenBank (with accession numbers indicated in the trees) were aligned with the Clustal X program [8]. Phylogenetic trees were constructed from the evolutionary distance data calculated from Kimura’s two-parameter model [9] by using the neighbor-joining method [10]. Bootstrap analyses [11] were performed from 1000 random resamplings. Fig. 1. Phylogenetic trees depicting the relationships of strains 3-1-3, 103-3 and 19-3-3 with closely related species based on (A) ITS1-5.8S rDNA-ITS2 region and (B) 26S rDNA D1/D2 domain sequences. The phylograms were constructed by neighbor-joining analysis. Bootstrap percentages over 50% from 1000 bootstrap replicates are shown. The GenBank accession numbers of the sequences are given.
3. Results and discussion 3.1. Phenotypic investigation Three yeast strains 3-1-3, 10-3-3 and 19-3-3 were isolated from xylem of the surface-sterilized fragments of di¡erent twigs, which were collected at di¡erent times from three di¡erent trees of P. tabulaeformis. Strain 3-17 was isolated from a 1-year-old twig of a pine and strains 10-3-3 and 19-3-3 from the 3-year-old twigs of two other pines. The three strains form similar pink-colored colonies and have almost identical physiological and biochemical characters. Based on the conventional criteria examined (Table 1), these strains could be assigned to the basidiomycetous yeast genus Rhodotorula F.C. Harrison, and, furthermore, be identi¢ed as Rhodotorula minuta (Saito) F.C. Harrison according to the recent taxonomic system of the genus [12]. 3.2. Molecular phylogenetic analysis R. minuta is a heterogeneous species as shown by the remarkable variations in physiological properties, G+C
contents, as well as DNA^DNA homology values among the strains assigned to this species [12,13]. The recent molecular phylogenetic study based on 26S rDNA D1/D2 domain and ITS region sequence comparison indicated that, among the synonyms of R. minuta, R. laryngis Reierso«l, Rhodotorula pallida Lodder and Rhodotorula sloof¢ae Nova¤k and Vo«ro«s-Felkai represented three distinct species [14]. The sequences of the whole DNA fragments covering ITS1-5.8S rDNA-ITS2 regions and 26S rDNA D1/D2 domains of strains 3-1-3, 10-3-3 and 19-3-3 determined in this study were identical, indicating that the three strains represent a single species. Molecular phylogenetic analysis based on the ITS and D1/D2 sequences showed that these strains belong to the Erythrobasidium clade of the Urediniomycetes [14] and are most closely related to R. laryngis (Fig. 1). They di¡er from the type strain of this species in 6 (1.8%) and 7 (1.1%) nucleotides in the ITS and D1/D2
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regions, respectively. The three strains are also closely related with an Antarctic yeast CBS 8923 as shown in Fig. 1, they di¡er from the latter in eight nucleotides in both ITS and D1/D2 sequences. Previous studies on basidiomycetous yeasts have indicated that conspeci¢c strains usually have fewer than 1% nucleotide divergences in the ITS1 and 2 regions overall [15^17]. However, occasional cases have been found where strains with up to approximately 2% nucleotide di¡erences
161
in the ITS regions may be conspeci¢c as indicated by DNA^DNA relatedness [18,19]. On the basis of an extensive study on basidiomycetous yeasts, Fell et al. [14] showed that strains with two or more nucleotide divergences in the D1/D2 domain represented di¡erent species. Though the taxonomic status of strains 3-1-3, 10-3-3 and 19-3-3 could not be de¢nitely determined based only on the ITS sequences, the D1/D2 sequence comparison con¢rmed that the three strains represent a distinct species.
Table 1 Physiological characteristics of R. pinicola sp. nov. and closely related species Property
Assimilationb : Glucose Galactose L-Sorbose Sucrose Maltose Cellobiose Trehalose Lactose Melibiose Ra⁄nose Melezitose Inulin Soluble starch D-Xylose L-Arabinose D-Arabinose D-Ribose L-Rhamnose D-Glucosamine Methanol Ethanol Glycerol Erythritol Ribitol Galactitol D-Mannitol D-Glucitol Methyl K-D-glucoside Salicin DL-Lactic acid Succinic acid Citric acid Inositol Hexadecane Potassium nitrate Sodium nitrite EthylamineWHCl L-Lysine Cadaverine Additional growth test: Vitamin-free 30‡C 35‡C Starch formation Urease DBB reaction a b
R. laryngisa
R. pallida
R. minuta
R. sloo⁄ae
AS 2.2193T
AS 2.2239
AS 2.2233
CBS 2221T
CBS 320T
CBS 319T
CBS 5706T
+ 3 d + 3 + + d 3 3 + 3 3 + + + 3 3 3 3 + + 3 w 3 + 3 3 + 3 + 3 3 3 3 3 3 + 3
+ 3 3 + 3 d d d 3 3 + 3 3 + d + 3 3 3 3 + + 3 3 3 d 3 3 d 3 + 3 3 3 3 3 3 + 3
+ 3 3 + 3 d + w,d 3 3 + 3 3 + + + 3 3 3 3 + + 3 3 3 d w,d 3 + 3 d 3 3 3 3 3 3 + 3
+ d 3 + 3 + + 3 3 3 + 3 3 + + + 3 3 3 3 + + 3 + 3 + + 3 + d + 3 3
+ 3 w,d,3 3 3 3 + 3 3 3 3 3 3 + 3 3 + 3 3 3 + + 3 d 3 + w,d,+ 3 3 d + 3 3
+ w,d,+ + + 3 + + d 3 3 + 3 3 + + + + 3 3 3 + + 3 d 3 w,d,3 w,d,3 3 d 3 + 3 3
+ 3 d + 3 + + w,d,+ 3 3 + 3 3 3 + w,d,+ d 3 3 3 + + 3 d 3 w,d,3 w,d,+ 3 d d + 3 3
3 3 3 w,d,+ 3
3 3 3 w 3
3 3 3 + 3
3 3 3,+ 3 +
3 + 3 3 + +
3 + 3 3 + +
3 + 3 3 + +
3 + 3 3 + nd
3 + 3 3 + +
3 +
3 + 3 3 + +
R. pinicola
The characters of the CBS strains are from CBS online yeast database. +, positive; 3, negative; d, delayed; w, weakly positive ; nd, not determined.
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The heterogeneity of the genus Rhodotorula has been revealed by molecular phylogenetic analysis [14]. The type species of this genus, Rhodotorula glutinis, which was located in a di¡erent clade as shown by Fell et al. [14], is only distantly related to strains 3-1-3, 10-3-3 and 19-3-3 (Fig. 1). However, before the emendation of this genus and other related basidiomycetous yeast genera was made, the new species represented by the three strains studied can only be assigned to the genus Rhodotorula according to the present taxonomic system of basidiomycetous yeasts [20]. A new species name, R. pinicola, is therefore proposed. The isolates of the new basidiomycetous yeast species are very rare among the endophytic fungi that have been isolated from needles and twigs of P. tabulaeformis collected from Dongling Mountain, Beijing. A total of approximately 1000 plates inoculated with fragments of needles, bark or xylem of 1- to 3-year-old twigs from 30 di¡erent pine trees have been examined, only these three yeast colonies have been found, which grew out from xylem inoculated in three di¡erent plates. In these plates, no ¢lamentous fungi were found. Theoretically, the yeast cells of this species should also occur in the bark of the pine twigs. Probably because of the frequent occurrence of fast-growing ¢lamentous fungi in the bark, it was di⁄cult for the yeast cells to grow and form visible colonies. Basidiomycetous yeasts have been reported as rare endophytic fungi from palms [1], but they have not been identi¢ed at genus level. 3.3. Description of the new species 3.3.1. Latin diagnosis of R. pinicola Bai, Guo et Zhao sp. nov. In liquido malti post dies 3 ad 20‡C, cellulae vegetativae ovoideae, ellipsoideae vel elongatae, (3.5^4.7)U(3.5^7.6) Wm, singulae aut binae. Post unum mensem ad 20‡C, annulus et sedimentum formantur. In agaro malti post unum mensem ad 20‡C, colonia rosea, glabra, butyracea, margine glabra. In agaro farinae zeae pseudomycelium non formantur. Fermentatio nulla. Glucosum, saccharosum, cellobiosum (fortasse lente), trehalosum, lactosum (lente), melezitosum, D-xylosum, L-arabinosum, D-arabinosum, ethanolum, glycerolum, D-mannitolum (fortasse lente), salicinum et acidum succinicum assimilantur at non galactosum, L-sorbosum (vel lente), maltosum, melibiosum, ra⁄nosum, inulin, amylum solubile, D-ribosum, L-rhamnosum, D-glucosaminum, methanolum, erithritolum, ribitolum (vel exigue), galactitolum, D-glucitolum (vel exigue et lente), methyl K-D-glucosidum, acidum DL-lacticum, acidum citricum, inositolum nec hexadecanum. L-lysinum assimilantur at non kalium nitricum, natrum nitrosum, ethylaminum nec cadaverinum. Maxima temperatura crescentiae: 30^31‡C. Materia amyloidea iodophila non formantur. Urea ¢nditur. Vitaminae externae ad crescentiam necessaria sunt. Diazonium caeruleum B positivum. Ubiq-
Fig. 2. R. pinicola AS 2.2193T , vegetative cells grown on malt agar for 3 days at 20‡C. Bar represents 10 Wm.
uinonum majus : Q-10. Typus: isolatus ex xylema ramuli P. tabulaeformis, AS 2.2193T (originaliter ut 3-1-3T ) depositus in collectione China General Microbiological Culture Collection Center, Academia Sinica. 3.3.2. Description of R. pinicola Bai, Guo et Zhao sp. nov. Growth in malt extract : after 3 days at 20‡C, the cells are ovoid, ellipsoidal to elongate (3.5^4.7)U(3.5^7.6) Wm, single or in pairs (Fig. 2). A thin ring is formed. After 1 month at 20‡C, a ring and sediment are formed. Growth on malt extract agar : after 1 month at 20‡C, the streak culture is pink to orange-pink, smooth and glistening with an entire margin. Dalmau plate culture on corn meal agar: pseudohyphae are not formed. Fermentation is absent. Assimilation reactions and other physiological properties are given in Table 1. The major ubiquinone is Q-10. The type strain, 3-1-3T , was isolated from xylem of a surface-sterilized twig of P. tabulaeformis collected from Dongling Mountain, Beijing, in October, 2000. This strain has been deposited in the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, as AS 2.2193T , and in the Centraalbureau voor Schimmelcultures, The Netherlands, as CBS 9130T . Etymology : The speci¢c epithet pinicola refers to the genus name for pine (Pinus), the source of the type strain. Conventionally, it is not easy to distinguish the new species R. pinicola from the other taxa in the R. minuta complex as shown in Table 1. In this complex, only R. pallida can be clearly separated from the others by its inability to assimilate sucrose, cellobiose, melezitose and Larabinose (Table 1). R. pinicola is slightly di¡erent from its closest species R. laryngis in the assimilation reactions of galactose, lactose, ribitol and D-glucitol (Table 1). The new species di¡ers from R. minuta and R. sloo⁄ae in the as-
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similation reactions of ribose and (Table 1).
D-xylose,
respectively [10]
Acknowledgements
[11]
This study was supported by grants G2000046802 and 30070018 from the National Natural Science Foundation of China (NSFC).
[12]
References
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