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Int J Syst Evol Microbiol 56 (2006), 2193-2197; DOI  10.1099/ijs.0.64385-0
© 2006 International Union of Microbiological Societies

Pseudonocardia oroxyli sp. nov., a novel actinomycete isolated from surface-sterilized Oroxylum indicum root

Qiang Gu1,2, Hongli Luo1, Wen Zheng1, Zhiheng Liu1 and Ying Huang1

1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China
2 Department of Biology, Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China

Correspondence
Ying Huang
huangy{at}im.ac.cn


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A high-G+C-content, Gram-positive bacterium, strain D10T, was isolated from the root of Oroxylum indicum, a Chinese medicinal plant. Based on 16S rRNA gene sequence analysis, strain D10T was a member of the genus Pseudonocardia and was most closely related, albeit loosely, to Pseudonocardia halophobica. Morphological and chemotaxonomic characteristics support the affiliation of strain D10T to the genus Pseudonocardia. Results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain D10T from related Pseudonocardia species. Strain D10T (=CGMCC 4.3143T=DSM 44984T) therefore represents a novel species, for which the name Pseudonocardia oroxyli sp. nov. is proposed.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain D10T is DQ343154.

A neighbour-joining tree based on 16S rRNA gene sequences of type strains of Pseudonocardia species and SEMs of strain D10T grown on ISP medium 2 agar are available as supplementary material in IJSEM Online.


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Pseudonocardia, initially proposed by Henssen (1957)Go, is the type genus of the family Pseudonocardiaceae (Embley et al., 1988Go; Warwick et al., 1994Go), which belongs to the suborder Pseudonocardineae (Stackebrandt et al., 1997Go). The genus currently comprises 26 species, the majority of which have been described by polyphasic taxonomic approaches (Warwick et al., 1994Go; Reichert et al., 1998Go; Huang et al., 2002Go). Most of these micro-organisms were isolated from soil samples, although they have also been isolated from plant samples, such as root nodules and tree-bark compost (Evtushenko et al., 1989Go; Reichert et al., 1998Go). In this paper, a novel Pseudonocardia species is proposed for a strain that was isolated from the surface-sterilized root of a higher plant.

Strain D10T was isolated from the root elongation zone of Oroxylum indicum, a traditional Chinese medicinal plant, which was collected in the rainforest around Liusha River, southwest of Jinghong City, Yunnan Province, China. After being surface-sterilized by the procedure established by Coombs & Franco (2003)Go, root samples were sliced into pieces with a sterile blade followed by plating on BL-2 agar plates (glucose, 5 g; soluble starch, 5 g; acid hydrolysates of casein, 2 g; yeast extract, 1 g; NaCl, 5 g; CaCO3, 5 g; agar, 15 g; distilled water, 1 l; supplemented with 100 µg penicillin ml–1), which were incubated at 27 °C for 2–4 weeks. The organism, seen around the root sample under a light microscope, was transferred onto fresh yeast extract/malt extract (ISP medium 2) agar (Shirling & Gottlieb, 1966Go) and well maintained.

Genomic DNA extraction and PCR amplification of the 16S rRNA gene from strain D10T were performed using an established method (Chun & Goodfellow, 1995Go). The PCR product was purified and sequenced directly using a Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) and universal primers 27F and 1492R. Sequence gel electrophoresis was carried out and the nucleotide sequences were obtained automatically using an Applied Biosystems DNA sequencer (model 3730XL) and software provided by the manufacturer. Preliminary phylogenetic analysis based on the results of the search program BLAST showed that strain D10T is closely related to members of the genus Pseudonocardia. Alignment of 16S rRNA gene sequences and construction of neighbour-joining (Saitou & Nei, 1987Go) and maximum-parsimony (Kluge & Farris, 1969Go) trees were carried out using the software package MEGA (Molecular Evolutionary Genetics Analysis) version 3.1 (Kumar et al., 2004Go). Pairwise distances for the neighbour-joining algorithm were calculated with the Kimura two-parameter model (Kimura, 1980Go) and close-neighbour-interchange (search level=2, random additions=100) was applied in maximum-parsimony analysis. Bootstrap values were based on 1000 replicates. A neighbour-joining tree (see Supplementary Fig. S1 in IJSEM Online) containing available 16S rRNA gene sequences of type strains of Pseudonocardia species confirmed placement of strain D10T in the genus Pseudonocardia. It is evident from Fig. 1Go that strain D10T is closely related to Pseudonocardia halophobica IMSNU 21327T (16S rRNA gene sequence similarity of 97.8 %), Pseudonocardia hydrocarbonoxydans IMSNU 22140T (97.5 %), Pseudonocardia sulfidoxydans DSM 44248T (97.5 %), Pseudonocardia benzenivorans B5T (97.3 %) and Pseudonocardia dioxanivorans CB1190T (97.2 %). The 16S rRNA gene sequence similarities between strain D10T and other Pseudonocardia species are below 97 %. The relationship between strain D10T and its closest neighbour, P. halophobica, is supported by both neighbour-joining and maximum-parsimony algorithms with bootstrap values of 82 and 69 %, respectively (Fig. 1Go).


Figure 1
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  		Fig. 1. Phylogenetic analyses based on 16S rRNA gene sequences (accession nos are given in parentheses), displaying relationships between strain D10T and related representatives of Pseudonocardia species. Neighbour-joining and maximum-parsimony trees were both performed using the software package MEGA (Molecular Evolutionary Genetics Analysis) version 3.1 (Kumar et al., 2004Go). Bootstrap values based on 1000 replicates are listed as percentages; those inferred from the neighbour-joining method are above the branches and those from the maximum-parsimony algorithm are below. Bar, 0.01 substitutions per nucleotide position.

 
Morphological characteristics of isolate D10T were examined by light (Axioskop 20; Zeiss) and scanning electron (FEI QUANTA) microscopy of 14-day-old cultures grown on ISP medium 2 agar. The organism showed morphology that was typical of the genus Pseudonocardia in that both substrate and aerial mycelium fragmented into rod-shaped, smooth elements and swollen segments were formed at the tip of mycelium (see Supplementary Fig. S2 in IJSEM Online). The physiological characteristics of strain D10T and its closest neighbours were tested together using well-established procedures, i.e. utilization of sole carbon sources for energy and growth was assessed as described by Yassin et al. (1995)Go, acid production from carbohydrates was determined using media and methods described by Gordon et al. (1974)Go, and biochemical and biodegradation tests were performed according to the method of Gordon & Mihm (1957)Go. The results of these tests enabled strain D10T to be distinguished easily from related Pseudonocardia species (Table 1Go).


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  		Table 1. Physiological characteristics of the type strains of some Pseudonocardia species

Strains: 1, D10T; 2, P. halophobica IMSNU 21327T; 3, P. sulfidoxydans DSM 44248T; 4, P. hydrocarbonoxydans IMSNU 22140T; 5, P. benzenivorans B5T; 6, P. dioxanivorans CB1190T. +, Positive; –, negative; (+), weakly positive. All strains are positive for acid production from D-galactose and D-glucose, but negative for acid production from D-cellobiose, methyl {alpha}-D-glucoside, D-lactose and D-raffinose. All strains are positive for assimilation of D-fructose, D-glucose, glycerol, malate, D-mannose, succinate and D-trehalose, but negative for assimilation of nicotinamide and D-sorbose. Assimilation of D-glucose, glycerol, D-mannose and D-trehalose by P. sulfidoxydans, P. hydrocarbonoxydans and P. dioxanivorans was tested by Mahendra & Alvarez-Cohen (2005)Go using a different method; congruent results were obtained.

 
Standard methods were used for extraction and analysis of the isomers of diaminopimelic acid (Hasegawa et al., 1983Go), whole-cell sugars (Lechevalier & Lechevalier, 1980Go), menaquinones (Collins, 1985Go; Wu et al., 1989Go), polar lipids (Lechevalier et al., 1977Go; Minnikin et al., 1984Go) and fatty acids (Sasser, 1990Go; Kämpfer & Kroppenstedt, 1996Go). Mycolic acids were detected by the acid methanolysis procedure (Minnikin et al., 1975Go). Strain D10T produced whole-organism hydrolysates rich in meso-diaminopimelic acid, arabinose and galactose (wall chemotype IV sensu Lechevalier & Lechevalier, 1970Go) and contained MK-8 (H4) as its predominant menaquinone. Major polar lipids were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylinositol, two phospholipids of unknown structure containing glucosamine and two glycolipids. The fatty acid profile of strain D10T was composed of C16 : 0 iso (45.1 %), C16 : 0 10-methyl (11.1 %), C17 : 1{omega}6c (7.4 %), C16 : 1 iso H (7.1 %), C18 : 1{omega}9c (5.1 %), C15 : 0 iso (4.4 %), C16 : 1{omega}7c (4.3 %), C14 : 0 iso (3.5 %) and C16 : 0 (3.0 %), but it lacked mycolic acids. The G+C content of genomic DNA of strain D10T (70.6 mol%) was determined using the thermal denaturation method (Marmur & Doty, 1962Go) with Escherichia coli K-12 as a control. All data supported affiliation of strain D10T to the genus Pseudonocardia (Reichert et al., 1998Go; Huang et al., 2002Go).

Genomic hybridization experiments between strain D10T and its closest phylogenetic neighbour, P. halophobica IMSNU 21327T, were carried out using the method described by He et al. (2005)Go, with the result that they shared low DNA–DNA relatedness of 35 %. Moreover, strain D10T could be easily differentiated from the type strain of Pseudonocardia spinosa, whose 16S rRNA gene sequence is not available, in that the latter produces spiny spores, grows extremely slowly and lacks septa in hyphae (Henssen & Schäfer, 1971Go).

The combined genotypic and phenotypic data show that strain D10T should be classified as a representative of a novel species within the genus Pseudonocardia. The name proposed for this novel species is Pseudonocardia oroxyli sp. nov.

Description of Pseudonocardia oroxyli sp. nov.
Pseudonocardia oroxyli (o.ro.xy'li. N.L. gen. n. oroxyli of the plant genus Oroxylum).

Aerobic, Gram-positive actinomycete that forms cinnamon-buff substrate mycelium and pale pinkish cinnamon aerial mycelium. Both types of mycelium fragment into rod-shaped smooth elements. Swellings are produced at the tip of hyphae. No pigment is produced. Good growth occurs after 3 days incubation on ISP medium 2 agar at 25–30 °C. Decomposition of urea and production of H2S are negative. Main polar lipids are phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylinositol, two phospholipids of unknown structure containing glucosamine and two glycolipids. The predominant fatty acid is C16 : 0 iso (45.1 %). Physiological properties, including acid production, carbon source utilization, biodegradation and growth in the presence of sodium chloride, are indicated in Table 1Go.

The type strain is D10T (=CGMCC 4.3143T=DSM 44984T), isolated from a surface-sterilized root of Oroxylum indicum collected in the rainforest around Liusha River, southwest of Jinghong City, Yunnan Province, China. The G+C content of genomic DNA of strain D10T is 70.6 mol%.


   ACKNOWLEDGEMENTS
 
This work was supported by the Knowledge Innovation Project of the Chinese Academy of Sciences and by a grant from Hisun Pharmaceutical Company. The authors are grateful to Professor R. M. Kroppenstedt (DSMZ) for providing some type strains of Pseudonocardia, and to Professors Cheng-Lin Jiang and Li-Hua Xu (Yunnan University) for their help in plant sample collection.


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