Pseudonocardia sichuanensis sp. nov., a novel endophytic ...

Antonie van Leeuwenhoek (2011) 99:395–401 DOI 10.1007/s10482-010-9504-7

ORIGINAL PAPER

Pseudonocardia sichuanensis sp. nov., a novel endophytic actinomycete isolated from the root of Jatropha curcas L. Sheng Qin • Ke Xing • Shi-Min Fei • Qiang Lin • Xiu-Min Chen • Cheng-Liang Cao • Yong Sun • Yun Wang • Wen-Jun Li • Ji-Hong Jiang

Received: 21 July 2010 / Accepted: 31 August 2010 / Published online: 9 September 2010 Ó Springer Science+Business Media B.V. 2010

Abstract A novel isolate, designated strain KLBMP 1115T was isolated from the surface-sterilized root of oil-seed plant Jatropha curcas L. collected from Sichuan Province, south-west China. Characterization of the isolate was based on a polyphasic approach. 16S rRNA gene sequence analysis indicated that strain KLBMP 1115T belongs to the phylogenetic cluster of the genus Pseudonocardia and was most closely related to Pseudonocardia adelaidensis EUM 221T (98.9%) and Pseudonocardia zijingensis DSM 44774T

S. Qin K. Xing C.-L. Cao Y. Sun J.-H. Jiang (&) The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, School of Life Science, Xuzhou Normal University, Xuzhou 221116, Jiangsu, People’s Republic of China e-mail: [email protected] W.-J. Li (&) The Key Laboratory for Microbial Resources of the Ministry of Education and Laboratory for Conservation and Utilization of Bio-resources, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, Yunnan, People’s Republic of China e-mail: [email protected] S.-M. Fei Q. Lin X.-M. Chen Sichuan Academy of Forestry, Chengdu 610081, Sichuan, People’s Republic of China Y. Wang Xinjiang Institute of Microbiology, Xinjiang Academy of Agricultural Science, Urumqi 830091, Xinjiang, People’s Republic of China

(98.6%), whereas the DNA–DNA relatedness values between strain KLBMP 1115T and the two type strains were 47.3 and 39.7%, respectively. Levels of lower similarities to the type strains of other recognized Pseudonocardia species ranged from 94.4 to 98.4%. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The predominant respiratory quinone was MK-8(H4). The major fatty acids of strain KLBMP 1115T was iso-C16: 0. The chemotaxonomic properties of strain KLBMP 1115T were consistent with those shared by members of the genus Pseudonocardia. On the basis of the phenotypic features and the DNA–DNA hybridization data, strain KLBMP 1115T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia sichuanensis sp. nov. is proposed. The type strain is KLBMP 1115T (=KCTC 19781T = CCTCC AA 2010002T). Keywords Pseudonocardia sichuanensis sp. nov. Polyphasic taxonomy 16S rRNA

Introduction Endophytic actinobacteria is a research hotspot in recent years. Increasing studies have revealed a large richness of endophytic actinobacterial species and their diverse functions (Araujo et al. 2002; Coombs and Franco 2003; Ryan et al. 2008; Qin et al. 2009a).

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These actinobacteria are relatively unstudied and potential sources of novel natural products for exploitation in medicine, agriculture and industry (Strobel et al. 2004). The genus Pseudonocardia was firstly proposed by Henssen (1957) to accommodate nocardioform actinomycetes that have a type IV cell wall and lack mycolic acids. Its description was later emended by McVeigh et al. (1994), Warwick et al. (1994), Reichert et al. (1998), Huang et al. (2002) and Park et al. (2008). At the time of writing, there are 36 recognized species belonging to the genus Pseudonocardia, including the recently described organisms P. oroxyli (Gu et al. 2006), P. endophytica (Chen et al. 2009), P. acaciae (Duangmal et al. 2009) and P. tropica (Qin et al. 2009b) that were all isolated from the endophytic environments of surface sterilized plants. During a study of the diversity and function of endophytic actinobacteria from oil-seed plant Jatropha curcas L., an actinomycete strain, designated KLBMP 1115T was isolated that exhibited the typical morphological characteristics of the genus Pseudonocardia. Further study of this strain, using a polyphasic taxonomic approach, confirmed its position as a representative of a novel species within the genus Pseudonocardia, for which the name Pseudonocardia sichuanensis sp. nov. is proposed.

Antonie van Leeuwenhoek (2011) 99:395–401

glycerol suspension (20%, v/v) at -20°C. Strain KLBMP 1115T was deposited in the Collection Center of Typical Cultures, China (CCTCC) as strain CCTCC AA 2010002T and in the Korean Collection for Type Cultures (KCTC) as strain KCTC 19781T. Phenotypic characterization Cultural characteristics of the organism were observed on ISP 2, oatmeal agar (ISP 3), inorganic salts-starch agar (ISP 4), glycerol-asparagine agar (ISP 5) (Shirling and Gottlieb 1966), as well as potato-dextrose agar, Czapek’s agar and nutrient agar (Waksman 1967) for 14 days at 28°C. The colony colour was determined with the ISCC–NBS colour charts (Kelly 1964). The morphology of a 3 weeks old ISP 2 agar culture (growth at 28°C) was observed using light microscopy (SA3300-PL) and scanning electron microscopy (Hitachi; S-3400N). The pH range (pH 4.0–11.0 at intervals of 0.5 pH units, at 28°C), growth temperature (0, 4, 10, 15, 20, 28, 37, 40, 45 and 55°C), NaCl tolerance (0–20%, w/v) (at intervals of 0.5%, 28°C) were checked using ISP 2 medium. Utilization of sole carbon sources for energy and growth, and decomposition of test substances was carried out according to Kurup and Schmitt (1973) and Gordon et al. (1974). Two type strains, P. adelaidensis EUM 221T and P. zijingensis DSM 44774T were tested together under the same conditions.

Materials and methods Chemotaxonomy Isolation and maintenance of organism Strain KLBMP 1115T was isolated from the healthy root of a oil-seed plant Jatropha curcas L., collected from the city of Panzhihua, Sichuan Province, southwest China. The root samples were air dried at room temperature after thoroughly washed under tap water, and surface sterilized according to the five-step sterilization procedure (Qin et al. 2008). Then the surface sterilized roots were aseptically crumbled into smaller fragments by commercial blender (Joyoung, XC-001) and plated on tap water-yeast extract agar (TWYE, Crawford et al. 1993), incubated at 28°C for 2–8 weeks. This strain was picked from the TWYE plate after incubation for 21 days. The purified culture was maintained on yeast extractmalt extract agar (ISP 2 medium) at 4°C and as a

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The G?C content of the DNA was determined by the method of Mesbah et al. (1989). For cellular fatty acid analysis, strain KLBMP 1115T was grown in TSB at 150 rpm for 10 days at 28°C. Whole cell fatty acids were extracted from 50 mg wet cells, saponified, methylated and fatty acid methyl esters were analysed using the Microbial Identification system (MIDI, TSBA library version 3.9; method: TSBA6) (Sasser 1990). Amino acids and sugars of whole-cell hydrolysates were analyzed by TLC as described previously (Staneck and Roberts 1974). Polar lipids were extracted, examined by using two-dimensional TLC and identified by the method of Minnikin et al. (1979). Menaquinones were extracted and purified as described by Collins et al. (1977) and analysed by HPLC (Groth et al. 1997).


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Molecular analysis Genomic DNA preparation, PCR amplification and sequencing of the 16S rRNA gene were carried out using procedures described by Li et al. (2007). The obtained sequence was compared with 16S rRNA reference gene sequences retrieved from the GenBank and EMBL databases by BLAST search. Multiple alignments of the sequence were carried out using the CLUSTAL_X (Thompson et al. 1997) software. Evolutionary distances were calculated using the Kimura two-parameter model (Kimura 1980). The phylogenetic tree was constructed by using the neighbour-joining (Saitou and Nei 1987) and maximum-parsimony methods (Kluge and Farris 1969) in the MEGA 4.0 program (Tamura et al. 2007). The confidence values for branches of the phylogenetic tree were determined using bootstrap analyses (Felsenstein 1985) based on 1,000 resamplings. Levels of DNA–DNA relatedness were determined according to the fluorometric micro-well method (Ezaki et al. 1989; He et al. 2005) and the hybridizations were carried out with three replications. Nucleotide sequence accession number The 16S rRNA gene sequence of strain KLBMP 1115T determined in this study has been deposited in GenBank under the accession number HM153789.

Results and discussion Strain KLBMP 1115T showed typical characteristics of the genus Pseudonocardia (Warwick et al. 1994; Huang et al. 2002). Morphological observation of the 2 weeks old culture of strain KLBMP 1115T revealed that both aerial and vegetative hyphae were abundant, well developed and fragmented. Chains of rodshaped spores with smooth surface were produced on ISP 2 medium agar (Fig. 1). Strain KLBMP 1115T showed good growth on ISP 2, 3 and 5, Czapek’s and PDA media, moderate growth on ISP 4 medium and poor growth on nutrient agar. Yellow–white aerial mycelium was produced on ISP 2 medium, and white aerial mycelia was produced on ISP 3, ISP 4, ISP 5, Czapek’s agar and Potato dextrose agar media, but no aerial mycelium was formed on nutrient agar. The

Fig. 1 Scanning electron micrograph of strain KLBMP 1115T grown on ISP 2 medium for 21 days at 28°C. Bar, 10 lm

substrate mycelium ranges from yellowish (on Potato dextrose agar)/yellow–white (on ISP 5 agar) to white (on ISP3, ISP4 and Czapek’s agar media) or yellow (on ISP 2 and Nutrient agar). No diffusible pigment was detected on any of the tested media. Growth of strain KLBMP 1115T occurred in the pH range 6.0–8.0 and 0–3% NaCl (w/v), with optimum growth at pH 7.0 and 0% NaCl (w/v). The temperature range for growth was 15–45°C, with the optimum temperature being 28°C. The physiological and biochemical properties are given in the species description and Table 1. The cell wall diamino acid in the peptidoglycan layer of strain KLBMP 1115T was meso-diaminopimelic acid and the whole-cell sugars were arabinose and galactose. Polar lipids of this novel strain comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol (phospholipid III; Lechevalier et al. 1977). The predominant menaquinone was MK-8(H4). The major fatty acids were iso-C16:0 (31.77%), anteiso-C17:0 (13.05%), iso-C17:1x9c/C16:0 10 methyl (12.22%), C16:0 (10.68%), iso-C15:0 (5.88%) and iso-C17:0 (5.23%), with C17:0 10-methyl (3.39%), anteiso-C15:0 (2.70%), iso-C14:0 (2.35%), iso-C16:1 H (1.59%), C16:1 x7c/C16:1x6c (1.64%), C17:1 x6c (1.47%), C14:0 (1.11%) and C17:0 (1.07%) present as minor components; iso-C12:0, iso-C13:0, anteiso-C13:0, iso-C14:03H, C17:1x8c and iso-C18:0 were detected at a level of \1%. The DNA G?C content of strain KLBMP

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Table 1 Characteristics that distinguish strain KLBMP 1115T from its closest phylogenetic neighbours Characteristics

1

2

3

Growth on ISP 3 medium

Good

Poor

Moderate

Growth at/with pH 10.0

-

?

w

45°C

w

w

?

NaCl (5%)

-

?

?

Arabinose

w

-

-

Cellobiose

-

?

-

Fructose

-

?

-

Inositol

-

?

?

Lactose

?

?

-

Maltose

?

-

?

Mannitol

-

-

?

Mannose

?

?

-

Rhamnose Ribose

? w

?

? w

Sorbitol

-

?

-

Trehalose

?

-

?

Xylitol

-

?

-

Xylose

?

-

?

Acid production from raffinose

?

-

-

Decomposition of adenine

-

-

?

Hydrolysis of starch

?

-

?

Catalase activity

-

?

?

Reduction of nitrate

-

-

?

DNA G?C content (mol%)

69.8

78.9*

70.9*

Growth on sole carbon source

T

Strains: 1, P. sichuanensis KLBMP 1115 ; 2, P. adelaidensis EUM 221T; 3, P. zijingensis DSM 44774T Note: Data obtained during this study were carried out under identical growth conditions ?, Positive, utilized; -, negative, not utilized; w, weakly positive *The DNA G?C content (mol%) of strains P. adelaidensis EUM 221T and P. zijingensis DSM 44774T indicated with asterisks were from Kaewkla and Franco (2010) and Huang et al. (2002)

1115T was 69.8 mol%, which falls within the range (68–79 mol%) described for the genus Pseudonocardia. The results of chemical analysis indicated that the organism has chemotaxonomic markers typical of the genus Pseudonocardia. The almost complete 16S rRNA gene sequence of strain KLBMP 1115T (1,497 bp) was determined in this study. Phylogenetic analysis based on 16S rRNA

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gene sequences revealed that strain KLBMP 1115T belonged to the genus Pseudonocardia; levels of similarity between strain KLBMP 1115T and the type strains of recognized Pseudonocardia species ranged from 94.4 to 98.9%. Strain KLBMP 1115T was related most closely to the type strain of P. adelaidensis EUM 221T (98.9%, 16S rRNA gene sequence similarity) and P. zijingensis DSM 44774T (98.6%). Phylogenetic trees constructed with the neighbour-joining and maximum-parsimony methods also indicated that strain KLBMP 1115T was a member of the genus Pseudonocardia and it formed a distinct subclade with two unvalidly published species ‘Pseudonocardia sp. YIM 63158’ and ‘Pseudonocardia sp. HBUM79244’ within the P. adelaidensis EUM 221T-P. zijingensis DSM 44774T-P. petroleophila ATCC 15777T cluster with a high bootstrap value (Fig. 2), which indicated that the novel strain should not be assigned to any of the recognized Pseudonocardia species. It has been shown that some Pseudonocardia species have high 16S rRNA gene sequence similarities, but have low DNA–DNA relatedness values below the 70% cut-off point (Prabahar et al. 2004; Ka¨mpfer et al. 2006; Park et al. 2008). For example, the type strains P. adelaidensis EUM 221T and P. zijingensis DSM 44774T share a 16S rRNA gene sequence similarity of 98.7%, but have a DNA–DNA relatedness of 60% (Kaewkla and Franco 2010). Recently, Stackebrandt and Ebers (2006) recommended an increase of about 2% (from 97 to 98.7–99%) to the 16S rRNA gene sequence similarity threshold used to determine the uniqueness of a new isolate provided that these data are supported by clear phenotypic difference. To establish the precise taxonomic position of strain KLBMP 1115T, DNA–DNA relatedness experiments were performed between the novel isolate and the type strains of P. adelaidensis EUM 221T and P. zijingensis DSM 44774T, and the levels of DNA–DNA relatedness between them were 47.3% (SD 2.6%) and 39.7% (SD 1.8%), respectively, which are well below the 70% cut-off point for recognition of genomic species (Stackebrandt and Goebel 1994), suggesting strongly that strain KLBMP 1115T represents a novel species of the genus Pseudonocardia. The morphological and chemotaxonomic characteristics determined for strain KLBMP 1115T were in accordance with those of members of the genus Pseudonocardia. However, strain KLBMP 1115T could be differentiated clearly from P. adelaidensis


399

P. sichuanensis KLBMP 1115T (HM153789) ‘Pseudonocardia sp’. HBUM79244 (EU119255) 71* ‘Pseudonocardia sp’. YIM 63158 (FJ817377) P. adelaidensis EUM 221T (FJ805427) 84* 61 P. zijingensis 6330T (AF325725) 45 P. petroleophila ATCC 15777T (X80596) P. xinjiangensis AS 4.1538T (AF325728) P. aurantiaca AS 4.1537T (AF325727) 44 T 100* P. alaniniphila YIM 16303 (EU722519) P. yunnanensis IFO 15681T (D85472) P. saturnea IMSNU 20052T (AJ252829) P. mongoliensis MN08-A0270T (AB521671) P. chloroethenivorans SL-1T (AF454510) 80* P. thermophila IMSNU 20112T (AJ252830) 98* 100* P. khuvsgulensis MN08-A0297T (AB521672) P. rhizophila YIM 67013T (GU322368) P. asaccharolytica DSM 44247T (Y08536) P. spinosispora LM 141T (AJ249206) P. eucalypti EUM 374T (FJ805426) 64 P. acaciae GMKU095T (EU921261)T P. artemisiae YIM 63587T (GU227146) P. oroxyli D10T (DQ343154) 84* P. ailaonensis YIM45505T (DQ344632) P. halophobica DSM 43089T (AJ252827) P. tetrahydrofuranoxydans K1T (AJ249200) P. hydrocarbonoxydans IMSNU 22140T (AJ252826) 93 90* P. sulfidoxydans DSM 44248T (Y08537) T 100* P. benzenivorans B5 (AJ556156) P. dioxanivorans CB1190T (AY340622) 90 P. carboxydivorans Y8T (EF114314) 96* P. antarctica DVS 5a1T (AJ576010) 99* P. alni DSM 44104T (Y08535) P. tropica YIM 61452T (GQ906587) P. parietis 04-St-002T (FM863703) P. endophytica YIM 56035T (DQ887489) 99* P. ammonioxydans H9T (AY500143) 68 P. kongjuensis LM 157T (AJ252833) 67 T 100* P. autotrophica IMSNU 20050 (AJ252824) P. compacta IMSNU 20111T (AJ252825) Streptoalloteichus hindustanus NBRC 15115T (D85497) 90* 100*

44*

78 92

0.005

Fig. 2 Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the positions of strain KLBMP 1115T and some related taxa. Numbers at the nodes are the bootstrap values based on 1,000 resamplings. Bootstrap values (expressed as percentages of 1,000 replications) of above 40%

are shown at branch points. Asterisks indicate the clades that were conserved when neighbour-joining and maximum-parsimony methods were used to construct phylogenetic trees. Bar, 0.005 inferred substitutions per site. Streptoalloteichus hindustanus NBRC 15115T (D85497) was used as an outgroup

EUM 221T and P. zijingensis DSM 44774T on the basis of combination of unique phenotypic characteristics, G?C contents (Table 1) and the low level of DNA– DNA relatedness, thus strain KLBMP 1115T was not affiliated with any recognized species of the genus Pseudonocardia. It is evident from the genotypic and phenotypic data that strain KLBMP 1115T occupies a new taxonomic position in the genus Pseudonocardia, for which the name P. sichuanensis sp. nov. is proposed.

Description of P. sichuanensis sp. nov P. sichuanensis (si.chu.a.nen’sis. N.L. masc. adj. sichuanensis pertaining to Sichuan, a province of south-west China). Cells are Gram-positive and aerobic, forms yellowish/yellow–white to white or yellow substrate mycelia and yellowish-white to white aerial mycelia on media tested. No diffusible pigment is produced. The rod-shaped spores occur in chains after 10 days

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of incubation on ISP 2 medium. Growth occurs between 15 and 45°C, pH 6.0 to 8.0, and 0–3% NaCl on ISP 2 agar. Acid is produced from mannose, raffinose, rhamnose and cellobiose. Uses L-Alanine, L-arginine, L-asparagine, L-lysine, L-proline, L-serine, L-threonine, L-tyrosine and L-valine as sole nitrogen sources, but negative for assimilation of L-glycine, L-glutamic acid and L-histidine. Positive for starch hydrolysis, but negative for nitrate reduction, milk peptonization and coagulation, gelatin liquefaction, growth in cellulose, melanin formation, H2S and urease production. The cell wall chemotype is type IV. Predominant menaquinone is MK-8(H4). The phospholipids are diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol (type PIII pattern). Major fatty acids are iso-C16: 0 (31.77%), anteiso-C17: 0 (13.05%), iso-C17: 1 x9c/C16: 0 10 methyl (12.22%), C16: 0 (10.68%), isoC15: 0 (5.88%) and iso-C17: 0 (5.23%). The DNA G?C content is 69.8 mol%. The type strain KLBMP 1115T (=KCTC 19781T = CCTCC AA 2010002T) was isolated from surfacesterilized root of Jatropha curcas L. collected from the city of Panzhihua, Sichuan Province, south-west China. Acknowledgments The authors are grateful to Prof. HansPeter Klenk (DSMZ, Germany) for kindly providing the type strain of P. adelaidensis EUM 221T. This research was partially supported by National Natural Science Foundation of China (Project no. 30872028, 31000005), the Major Fundamental Research Program of Natural Science Foundation of the Jiangsu Higher Education Institutions of China (08KJA350001), the program of the Demonstration and study of Standardization Seeding Technology of Jatropha (2007BAD50B0204) and Grants from Natural Science Foundation by Xuzhou Normal University (09XLR12, 09XLR19).

References Araujo WL, Marcon J, Maccheroni W Jr, Van Elsas JD, Van Vuurde JW, Azevedo JL (2002) Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl Environ Microbiol 68:4906–4914 Chen HH, Qin S, Li J, Zhang YQ, Xu LH, Jiang CL, Kim CJ, Li WJ (2009) Pseudonocardia endophytica sp. nov., isolated from the pharmaceutical plant Lobelia clavata. Int J Syst Evol Microbiol 59:559–563 Collins MD, Pirouz T, Goodfellow M, Minnikin DE (1977) Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100:221–230

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Antonie van Leeuwenhoek (2011) 99:395–401 Coombs JT, Franco CMM (2003) Isolation and identification of actinobacteria isolated from surface-sterilized wheat roots. Appl Environ Microbiol 69:5603–5608 Crawford DL, Lynch JM, Whipps JM, Ousley MA (1993) Isolation and characterization of actinomycete antagonists of a fungal root pathogen. Appl Environ Microbiol 59: 3899–3905 Duangmal K, Thamchaipenet A, Matsumoto A, Takahashi Y (2009) Pseudonocardia acaciae sp. nov., isolated from roots of Acacia auriculiformis A. Cunn. ex Benth. Int J Syst Evol Microbiol 59:1487–1491 Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid–deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39:224–229 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–789 Gordon RE, Barnett DA, Handerhan JE, Pang CH-N (1974) Nocardia coeliaca, Nocardia autotrophica, and the nocardin strains. Int J Syst Bacteriol 24:54–63 Groth I, Schumann P, Rainey FA, Martin K, Schuetze B, Augsten K (1997) Demetria terragena gen nov., sp. nov., a new genus of actinomycetes isolated from compost soil. Int J Syst Bacteriol 47:1129–1133 Gu Q, Luo H, Zheng W, Liu Z, Huang Y (2006) Pseudonocardia oroxyli sp. nov., a novel actinomycete isolated from surface-sterilized Oroxylum indicum root. Int J Syst Evol Microbiol 56:2193–2197 He L, Li W, Huang Y, Wang L, Liu ZH (2005) Streptomyces jietaisiensis sp. nov., isolated from soil in northern China. Int J Syst Evol Microbiol 55:939–944 Henssen A (1957) Beitra¨ge zur Morphologie und Systematic der thermophilen Actinomyceten. Arch Mikrobiol 26: 377–414 Huang Y, Wang L, Lu Z, Hong L, Liu Z, Tan GYA, Goodfellow M (2002) Proposal to combine the genera Actinobispora and Pseudonocardia in an emended genus Pseudonocardia, and description of Pseudonocardia zijingensis sp. nov. Int J Syst Evol Microbiol 52:977–982 Kaewkla O, Franco CMM (2010) Pseudonocardia adelaidensis sp. nov., an endophytic actinobacterium isolated from the surface-sterilized stem of a Grey Box tree. Int J Syst Evol Microbiol. doi:10.1099/ijs.0.019208-0 Ka¨mpfer P, Kohlweyer U, Thiemer B, Andreesen JR (2006) Pseudonocardia tetrahydrofuranoxydans sp. nov. Int J Syst Evol Microbiol 56:1535–1538 Kelly KL (1964) Color-name charts illustrated with centroid colors. Inter-Society Color Council-National Bureau of Standards, Chicago (Published in US) Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequence. J Mol Evol 16:111–120 Kluge AG, Farris FS (1969) Quantitative phyletics and the evolution of anurans. Syst Zool 18:1–32 Kurup PV, Schmitt JA (1973) Numerical taxonomy of Nocardia. Can J Microbiol 19:1035–1048 Lechevalier MP, De Bievre C, Lechevalier HA (1977) Chemotaxonomy of aerobic actinomycetes: phospholipid composition. Biochem Syst Ecol 5:249–260

Antonie van Leeuwenhoek (2011) 99:395–401 Li WJ, Xu P, Schumann P, Zhang YQ, Pukall R, Xu LH, Stackebrandt E, Jiang CL (2007) Georgenia ruanii sp. nov., a novel actinobacterium isolated from forest soil in Yunnan (China) and emended description of the genus Georgenia. Int J Syst Evol Microbiol 57:1424–1428 McVeigh HP, Munro J, Embley TM (1994) The phylogenetic position of Pseudoamycolata halophobica (Akimov et al. 1989) and a proposal to reclassify it as Pseudonocardia halophobica. Int J Syst Bacteriol 44:300–302 Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G?C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159–167 Minnikin DE, Collins MD, Goodfellow M (1979) Fatty acid and polar lipid composition in the classification of Cellulomonas, Oerskovia and related taxa. J Appl Bacteriol 47:87–95 Park SW, Park ST, Lee JE, Kim YM (2008) Pseudonocardia carboxydivorans sp. nov., a carbon monoxide-oxidizing actinomycete, and an emended description of the genus Pseudonocardia. Int J Syst Evol Microbiol 58:2475–2478 Prabahar V, Dube S, Reddy GSN, Shivaji S (2004) Pseudonocardia antarctica sp. nov., an actinomycetes from McMurdo Dry Valleys, Antarctica. Syst Appl Microbiol 27:66–71 Qin S, Wang HB, Chen HH, Zhang YQ, Jiang CL, Xu LH, Li WJ (2008) Glycomyces endophyticus sp. nov., an endophytic actinomycete isolated from the root of Carex baccans Nees. Int J Syst Evol Microbiol 58:2525–2528 Qin S, Li J, Chen HH, Zhao GZ, Zhu WY, Jiang CL, Xu LH, Li WJ (2009a) Isolation, diversity, and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rain forests in Xishuangbanna, China. Appl Environ Microbiol 75:6176–6186 Qin S, Zhu WY, Jiang JH, Klenk HP, Li J, Zhao GZ, Xu LH, Li WJ (2009b) Pseudonocardia tropica sp. nov., a novel endophytic actinomycete isolated from the stem of Maytenus austroyunnanensis. Int J Syst Evol Microbiol. doi:10.1099/ijs.0.020099-0 Reichert K, Lipski A, Pradella S, Stackebrandt E, Altendorf K (1998) Pseudonocardia asacccharolytica sp. nov. and Pseudonocardia sulfidoxydans sp. nov., two new dimethyl

401 disulfide-degrading actinomycetes and emended description of the genus Pseudonocardia. Int J Syst Bacteriol 48:441–449 Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent development and applications. FEMS Microbiol Lett 278:1–9 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic tree. Mol Biol Evol 4:406–425 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. MIDI Inc., Newwark, DE Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340 Stackebrandt E, Ebers J (2006) Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 33:152–155 Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA–DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849 Staneck JL, Roberts GD (1974) Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl Microbiol 28:226–231 Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from endophytic microorganisms. J Nat Prod 67: 257–268 Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599 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 tools. Nucleic Acids Res 25:4876–4882 Waksman SA (1967) The actinomycetes. A summary of current knowledge. Ronald Press, New York Warwick S, Bowen T, McVeigh HP, Embley TM (1994) A phylogenetic analysis of the family Pseudonocardiaceae and the genera Actinokineospora and Saccharothrix with 16S rRNA sequences and a proposal to combine the genera Amycolata and Pseudonocardia in an emended genus Pseudonocardia. Int J Syst Bacteriol 44:293–299

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