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Nocardioides Panzhihuaensis Sp. Nov., a Novel Endophytic Actinomycete Isolated from Medicinal Plant Jatropha Curcas L

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Nocardioides Panzhihuaensis Sp. Nov., a Novel Endophytic Actinomycete Isolated from Medicinal Plant Jatropha Curcas L Antonie van Leeuwenhoek (2012) 102:353–360 DOI 10.1007/s10482-012-9745-8 ORIGINAL PAPER Nocardioides panzhihuaensis sp. nov., a novel endophytic actinomycete isolated from medicinal plant Jatropha curcas L. Sheng Qin • Bo Yuan • Yue-Ji Zhang • Guang-Kai Bian • Tomohiko Tamura • Bing-Zhi Sun • Wen-Jun Li • Ji-Hong Jiang Received: 19 March 2012 / Accepted: 22 April 2012 / Published online: 3 May 2012 Ó Springer Science+Business Media B.V. 2012 Abstract A novel Gram-positive, aerobic, rod- similarity to Nocardioides albus KCTC 9186T shaped and mycelia-producing bacterial strain, desig- (99.38 %) and Nocardioides luteus KCTC 9575T nated KLBMP 1050T, was isolated from the stem of (99.03 %). However, the DNA–DNA relatedness of the oil-seed plant Jatropha curcas L. collected from isolate KLBMP 1050T to these two type strains were Sichuan Province, south-west China. Phylogenetic 37.5 ± 3.5 and 33 ± 2.3 %, respectively. Strain analysis based on the 16S rRNA gene sequence KLBMP 1050T grew at the pH range 6–11, temper- revealed that the isolate KLBMP 1050T belonged to ature range 10–32 °C and with 0–12 % NaCl. The the genus Nocardioides, with the highest sequence physiological properties of strain KLBMP 1050T differ from those of N. albus KCTC 9186T and N. luteus KCTC 9575T. The cell-wall peptidoglycan Sheng Qin and Bo Yuan contributed equally to this study contained LL-diaminopimelic acid and MK-8(H4) was the major respiratory quinone. The predominant Electronic supplementary material The online version of cellular fatty acid of strain KLBMP 1050T was iso- this article (doi:10.1007/s10482-012-9745-8) contains supplementary material, which is available to authorized users. C16:0 (23.3 %). The total DNA G?C content was 70.1 mol%. On the basis of the phenotypic, chemo- S. Qin Á B. Yuan Á Y.-J. Zhang Á G.-K. Bian Á taxonomic and phylogenetic data, strain KLBMP & B.-Z. Sun Á J.-H. Jiang ( ) 1050T represents a novel species of the genus Nocar- The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, School of Life Science, Jiangsu dioides, for which the name Nocardioides panzhihua- Normal University, Xuzhou 221116, Jiangsu, ensis sp. nov. is proposed. The type strain is KLBMP People’s Republic of China 1050T (= KCTC 19888T = NBRC 108680T). e-mail: [email protected] T. Tamura Keywords Nocardioides panzhihuaensis sp. nov. Á NITE Biological Resource Center (NBRC), National Endophytic Á 16S rRNA gene Á Polyphasic taxonomy Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan W.-J. Li (&) Introduction The Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education and Laboratory for The genus Nocardioides was proposed by Prauser Conservation and Utilization of Bio-Resources, Yunnan (1976) and currently contains 56 species with validly Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China published names. Members of this genus have been e-mail: [email protected] isolated from various environments including the 123 354 Antonie van Leeuwenhoek (2012) 102:353–360 recently described species Nocardioides tritolerans tryptone soya agar (TSA) (Bowman et al. 1996), R2A (Dastager et al. 2008), Nocardioides sediminis agar, Czapek’s agar and nutrient agar medium plates (Dastager et al. 2009), Nocardioides daedukensis (Waksman 1967) for 14 days at 28 °C. Carbon source (Yoon et al. 2010), Nocardioides alpinus (Zhang utilization was tested by using ISP 9 medium (Shirling et al. 2012), Nocardioides ginsengagri (Lee et al. and Gottlieb 1966) supplemented with 1 % (final 2012), Nocardioides szechwanensis and Nocardioides concentration) carbon sources. The utilization of amino psychrotolerans (Liu et al. 2012). At present, there is acids as sole nitrogen sources was tested as described by only one species, Nocardioides caricicola, that has Williams et al. (1983). Production of acid and other been isolated from the endophytic environment (Song physiological and biochemical characteristics were et al. 2011). During the course of investigating tested by using the well established procedures (Gordon endophytic actinomycetes associated with the oil-seed et al. 1974). All the above tests were carried out at 28 °C plant Jatropha curcas L., a Nocardioides-like endo- and incubated for 14 days. Enzyme activities were phytic strain, KLBMP 1050T, was isolated. Here, the determined by using API ZYM test strip at 28 °C polyphasic characterization of this strain indicated that according to the manufacturer’s instructions (bio- it represents a novel species of the genus Nocardioides. Merieux). Growth at different temperatures (4, 10, 20, 25, 28, 30, 32, 37, 40, 45, 50 °C), different NaCl concentrations (0–15 %, at intervals of 1 %) was tested Materials and methods on ISP 2 agar at 28 °C for 14 days. The pH range for growth was determined in ISP 2 broth that was adjusted Isolation and maintenance of isolate to various pH values (pH 5.0–12.0 at intervals of 0.5 pH unit) using 1 M NaOH and 1 N HCl solutions. Strain KLBMP 1050T was isolated from the healthy stems of a medicinal plant J. curcas L. collected from Chemotaxonomy Sichuan Province, south-west China, in 2010. For the isolation of endophytes, the samples were firstly Biomass used for chemotaxonomic analyses was surface sterilized following the procedure of Qin obtained from cultures grown in ISP 2 broth on a rotary et al. (2008). After that, the surface sterilized samples shaker (about 150 rpm) for 5 days at 28 °C. The were aseptically crumbled into smaller fragments diaminopimelic acid of the cell-wall peptidoglycan using a commercial Joyoung blender, spread onto was determined as described by Hasegawa et al. (1983). sodium propionate agar (Qin et al. 2009) and incu- Isoprenoid quinones were isolated according to the bated at 28 °C for 2–6 weeks. Strain KLBMP 1050T method of Collins et al. (1977) and analysed by HPLC as was obtained after incubation for two weeks. The described by Kroppenstedt (1985). Polar lipids were purified strain was maintained on yeast extract–malt extracted and identified by two-dimensional TLC extract agar (ISP 2) (Shirling and Gottlieb 1966) and according to the method described by Minnikin et al. as a glycerol suspension (20 %, w/v) at -80 °C. (1984). For analysis of cellular fatty acid composition, the cell mass of strains KLBMP 1050T and two Phenotypic characterization reference strains N. albus KCTC 9186T and N. luteus KCTC 9575T were harvested from ISP 2 plates after Gram staining was performed as described by Smibert incubation at 28 °C for 5 days. Fatty acids were and Krieg (1994). Cell morphology was observed using analysed using the standard MIDI (Microbial Identifi- a light microscopy (SA3300-PL) and a scanning cation, Sherlock version 6.0) procedure (Sasser 1990) electron microscope (Hitachi; S-3400N) and the pres- and the gas chromatograph Agilent GC 6850. The ence of flagella was investigated using a transmission resulting profiles were identified using the database electron microscope (Hitachi; H-7650) with cells grown library TSBA6 version 6.0. for 7 days at 28 °C on ISP 2 medium. Cultural characteristics of strain KLBMP 1050T were deter- Phylogenetic analyses mined using ISP 2, oatmeal agar (ISP 3), inorganic salts- starch agar (ISP 4), glycerol-asparagine agar (ISP 5) Genomic DNA extraction, PCR-mediated amplifica- (Shirling and Gottlieb 1966), potato-dextrose agar, tion of the 16S rRNA gene and sequencing of the PCR 123 Antonie van Leeuwenhoek (2012) 102:353–360 355 products were carried out as described by Li et al. growth was observed on all the tested media except for (2007). The almost complete 16S rRNA gene poor growth on TSA agar. Cells were positive for sequence was multiply aligned with selected oxidase activity, nitrate reductase, milk peptonization sequences obtained from the GenBank/EMBL/DDBJ and coagulation but were negative for urease and H2S databases by using the CLUSTAL W programme production. Cells were positive for the decomposition version 1.81 (Thompson et al. 1997). The identifica- of casein, chitin, esculin and Tween 80 but were tion of phylogenetic neighbours and the calculation of negative for the decomposition of xylan, adenine and pairwise 16S rRNA gene sequence identities were cellulose. The other results of physiological and achieved using the EzTaxon-e database (Kim et al. biochemical tests were given in the species description 2012). Phylogenetic and molecular evolutionary anal- and in Table 1. yses were conducted using MEGA version 5.0 The diagnostic diamino acid present in strain KLBMP T (Tamura et al. 2011). The evolutionary distances were 1050 was LL-2,6-diaminopimelic acid, which is charac- computed using the Kimura two-parameter method teristic of wall chemotype I sensu Lechevalier and (Kimura 1980). Phylogenetic relationships were ana- Lechevalier (1970). The predominant menaquinone was lyzed by using neighbour-joining (Saitou and Nei MK-8(H4) and the polar lipids were diphosphatidylglyc- 1987), maximum-parsimony (Fitch 1971) and maxi- erol, phosphatidylglycerol and phosphatidylcholine mum-likelihood (Felsenstein 1981) methods. The (Supplementary Fig. S2). The fatty acid profile (i.e. fatty topologies of the phylogenetic trees were evaluated acids representing [5 % of the total) comprised the by the bootstrap resampling method of Felsenstein following: iso-C16:0 (23.3 %), C18:1x9c (11.2 %), (1985) with 1,000 replicates. C17:1x6c (8.8 %), C17:1x8c (5.8 %), iso-C15:0 (5.8 %), 10-methyl C18:0 tuberculostearic acid (7.4 %) and DNA relatedness studies summed feature 3 (C16:1x7c and/or iso-C15:0 2-OH) (5.0 %). A detailed fatty acid profile comparison with its The G?C content of the DNA was determined by the nearest neighbour species is given in Table 2. The fatty HPLC method (Mesbah et al. 1989). DNA–DNA acid profile of strain KLBMP 1050T differed from that of hybridization was carried out according to the fluoro- N.
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