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. albus KCTC 9186T and N. luteus KCTC 9575T in that metric micro-well method (Ezaki et al. 1989), incor- it contained a greater proportion of iso-C16:0 iso-C15:0 and porating the modifications described by He et al. C17:1x6c and smaller proportions of C16:0,C17:0 and (2005). C17:1x8c. The chemotaxonomic characteristics were consistent with the affiliation of the strain to the genus Nucleotide sequence accession number Nocardioides (Yoon et al. 1997, 2006, 2010; Lee et al. 2012; Liu et al. 2012). The 16S rRNA gene sequence of strain KLBMP 1050T The nearly complete 16S rRNA gene sequence determined in this study has been deposited in (1449 bp) was obtained. Comparative analyses of the GenBank under the accession number HM153774. 16S rRNA gene sequence revealed that strain KLBMP 1050T belonged to the genus Nocardioides. Sequence similarity calculations indicated that the closest rela- Results and discussion tives of strain KLBMP 1050T were N. albus KCTC 9186T (99.38 %) and N. luteus KCTC 9575T Strain KLBMP 1050T was found to be Gram-positive (99.03 %). Levels of 16S rRNA gene sequence sim- and non-motile. Cells are rods (about 1.0–1.8 mm ilarity between strain KLBMP 1050T and the type long and 0.6–0.8 mm wide). The hyphae of the aerial strains of other recognized members of the genus mycelium were observed to fragment into rod-like Nocardioides were below 97.0 %. It is evident from elements (Supplementary Fig. S1). Growth of strain Fig. 1 that strain KLBMP 1050T formed a distinct 16S KLBMP 1050T was observed at temperatures in the rRNA gene subclade with N. albus KCTC 9186T and N. range 10–32 °C, the optimum being 25–28 °C. luteus KCTC 9575T at a high bootstrap value (100 % in Growth occurred at pH 6–11, the optimum pH being the neighbour-joining tree). This relationship was also 7–8. The strain required 0–12 % (w/v) NaCl for supported in the maximum-parsimony and maximum- growth, the optimum concentration being 3 %. Good likelihood trees (Supplementary Figs. S3, S4). 123 356 Antonie van Leeuwenhoek (2012) 102:353–360

Table 1 Characteristics Characteristics 1 2 3 4 that distinguish strain T KLBMP 1050 from its Colony colour on ISP 4 medium White White Yellow ND closest phylogenetic neighbours Growth on TSA medium Poor Good Good ND Growth at/with pH 11.0 ?-?- 37 °C -??? NaCl tolerance (%) 0–12 % 0–11 % 0–13 % 0.5–8 % Nitrate reduction ?--- Growth on sole carbon source Inositol W -?- Lactose -??- Ribose ?--- Growth on sole nitrogen source L-Lysine --?- L-Proline -??ND L-Tyrosine ?--- L-Valine -??ND Acid production from D-Arabinose ?-WND D-Fructose --?ND D-Galactose -??ND Glucose W ??- D-Maltose -?WND L-Rhamnose ?--ND Ribose ?--ND Decomposition of Chitin ?--ND

T Esculin ?--? Strains: 1, KLBMP 1050 ; 2, N.albus KCTC 9186T;3, Xylan --?ND N. luteus KCTC 9575T;4, API ZYM test results Nocardioides marinus Esterase (C4) ??-W CL-DD14T (data from Choi et al. 2007). Data were Lipase (C14) ?--- obtained from this study Cystine arylamidase --?- under identical growth Naphthol-AS-BI-phosphohydrolase ??-- conditions, except those as b-Galactosidase ?--- labeled. ?, Positive, utilized; -, negative, not a-Glucosidase ??-? utilized b-Glucosidase ?--- W weakly positive, ND no a-Mannosidase --?- data available N-Acetyl-b-glucosaminidase ?--- a Data from Yoon et al. DNA G?C content (mol%) 70.1 67a 68a 73 (2006)

Furthermore, the DNA–DNA hybridization analysis are 97 % 16S rRNA gene sequence similarity (mean value of five experiments) of strain KLBMP (Stackenbrandt and Goebel 1994) and 70 % DNA– 1050T to its closest relatives revealed 37.5 ± 3.5 % DNA relatedness (Wayne et al. 1987), it can be DNA–DNA relatedness to N. albus KCTC 9186T and concluded that isolate KLBMP 1050T belongs to a new 33 ± 2.3 % to N. luteus KCTC 9575T. Since the Nocardioides species. The G?C content of strain threshold values for the delineation of genomic species KLBMP 1050T was 70.1 mol%. 123 Antonie van Leeuwenhoek (2012) 102:353–360 357

Table 2 Fatty acid profiles Fatty acid KLBMP 1050T Nocardioides Nocardioides (%) of strain KLBMP albus KCTC luteus KCTC 1050T and its nearest 9186T 9575T neighbours Saturated

C14:0 – 0.84 0.74

C16:0 4.05 8.85 8.05

C17:0 2.83 4.83 10.68

C18:0 2.32 3.47 3.51 Unsaturated

C15:1x6c

C17:1x8c 5.84 7.60 13.60

C17:1x6c 8.77 6.54 7.31

C18:1x9c 11.20 15.02 8.51 Branched

iso-C14:0 1.92 1.27 0.79

iso-C15:0 5.83 3.12 3.80

iso-C16:1 G 1.43 – –

iso-C16:0 23.34 22.61 18.27

iso-C17:0 4.16 1.40 1.69

anteiso-C17:0 0.73 1.04 0.99

iso-C18:0 0.55 – –

iso-C18:1 H 1.23 1.37 –

iso-C19:1 I 1.54 0.75 – 10-Methyl

All the data are from this C17:0 2.40 1.77 3.83 study. All strains were C18:0 TBSA 7.37 5.06 5.24 grown on ISP 2 medium for Hydroxy 5 days at 28 °C. Fatty acids amounting to \0.5 % in all C16:0 2-OH – 0.76 0.61 species are not shown. C17:0 2-OH – 0.50 0.53 Values are percentages of Summed featurea total fatty acids; -, not Sum In Feature 3 5.03 5.04 4.98 detected Sum In Feature 4 1.71 2.61 0.60 TBSA tuberculostearic acid Sum In Feature 6 – – 0.55 a Summed features represent groups of two or Sum In Feature 7 0.59 – – three fatty acids that could Sum In Feature 8 0.88 1.16 0.82 not be separated by GLC Sum In Feature 9 2.48 1.31 1.05 with the MIDI system

In this study, the phenotypic features (NaCl require- of the the phenotypic, genotypic and phylogenetic data ment, temperature and pH ranges for growth, nitrate above, this isolate represents a novel species within the reductase activity, carbon and nitrogen utilization pattern genus Nocardioides, for which the name Nocardioides and API ZYM test results) clearly differentiated strain panzhihuaensis sp. nov. is proposed. KLBMP 1050T from its nearest neighbours N. albus KCTC 9186T and N. luteus KCTC 9575T.Furthermore, Description of N. panzhihuaensis sp. nov. the different G?C content, differences of fatty acid compositions and low level of DNA–DNA relatedness N. panzhihuaensis (pan.zhihua.en’sis. N.L.masc.adj. also supported the conclusion that strain KLBMP 1050T panzhihuaensis pertaining to Panzhihua city, from should be considered as a new species. Thus, on the basis where the strain was isolated). 123 358 Antonie van Leeuwenhoek (2012) 102:353–360

82* Nocardioides nitrophenolicus NSP 41T (AF005024) 59* Nocardioides kongjuensis A2 -4T (DQ218275) 97* Nocardioides caeni MN8T (FJ423551) Nocardioides aromaticivorans H-1T(AB087721) Nocardioides simplex KCTC 9106T (AF005009) 97* 61* Nocardioides ginsengisoli Gsoil 1124T (AB245396) Nocardioides humi DC Y2 4T (EF623863) 72* Nocardioides ultiminferus RP-B26T (FM997998) 84 99* Nocardioides maradoensis RP-B30T (FM998000) Nocardioides aestuarii JC2056T (AY423719) 61 Nocardioides tritolerans MSL-14T(EF466107) 82* KLBMP 1050T (HM153774 ) 100* Nocardioides albus KCTC 9186T (AF004988) Nocardioides luteus KCTC 9575T (AF005007) Nocardioides marinus CL-DD14T (DQ401093) Nocardioides panacihumi Gsoil 616T (AB271053) 56 84* Nocardioides terrae VA15T (FJ423762) 99* Nocardioides terrigena DS-17T (EF363712) Nocardioides sediminis MSL-01T (EF466110) Nocardioides furvisabuli SBS-26T (DQ411542) 87* Nocardioides ganghwensis JC2055T (AY423718) 100* Nocardioides oleivorans DSM 16090T (AJ698724) 98* Nocardioides exalbidusRC825T (AB273624) 73* Nocardioides hwasunensis HFW-21T (AM295258) Nocardioides fonticola NAA-13T (EF626689) Nocardioides caricicola YC6903T (FJ750845) Nocardioides hankookensis DS-30T (EF555584) 66 Nocardioides pyridinolyticus OS4T (U61298) 99 87 Nocardioides aquiterrae GW-9 T (AF529063) 94* Nocardioides aquaticus EL-17KT (X94145) Nocardioides koreensis MSL-09T (EF466115) Nocardioides kribbensis KSL-2T (AY835924) Nocardioides dokdonensis FR1436T (EF633986) Nocardioides marinisabuli SBS-12T (AM422448) 97 Nocardioides basaltis J112T (EU143365) 100 87* Nocardioides salarius CL-Z59T(DQ401092) 100* Nocardioides halotolerans MSL 23T (EF466122) Nocardioidesdilutus MSL-11T (EF466121) Nocardioides alkalitolerans KS L-1T (AY633969) 50 Nocardioides dubius KS L-1 04T (AY928902) 59 Nocardioides mesophilus MSL-22T (EF466117) 78 Nocardioides jensenii DSM 20641T (Z78210) 94* 78* Nocardioides daedukensis MDN22T(FJ842646) Terrabacter tumescens KCTC 9133T (AF005023)

0.01

Fig. 1 Neighbour-joining tree based on almost complete 16 C50 % are shown. GenBank accession numbers are given in rRNA gene sequences showing the position of strain KLBMP parentheses. Asterisks indicate that the corresponding nodes 1050T amongst its phylogenetic neighbours. Terrabacter tumes- were also recovered in the maximum-parsimony and maximum- cens KCTC 9133T was used as an outgroup. Numbers at the likelihood trees. Bar 0.01 substitutions per site nodes indicate the levels of bootstrap support (%); only values

Cells are aerobic, Gram-positive, non-motile, irreg- and L-serine as sole nitrogen sources. Acid is produced ular short rods. Colonies are white on the media tested. from D-arabinose, cellobiose, D-glucose, D-ribose, White aerial mycelium is observed. No soluble L-rhamnose and D-xylose. In the API ZYM sys- pigments are produced. Growth occurs at 10–32 °C, tem, alkaline phosphatase, esterase (C4), esterase pH 6–11 and in the presence of 0–12 % (w/v) NaCl. lipase (C8), lipase (C14), leucine arylamidase, Uses D-arabinose, cellobiose, D-fructose, D-galactose, valine arylamidase, naphthol-AS-BI-phosphohydrolase, inositol, D-maltose, mannose, D-ribose, L-rhamnose, b-galactosidase, a-glucosidase, b-glucosidase and trehalose and D-xylose as sole carbon and energy N-acetyl-b-glucosaminidase are positive but cystine sources. Uses L-glutamic acid, L-histidine, L-tyrosine arylamidase, trypsin, acid phosphatase, a-galactosidase, 123 Antonie van Leeuwenhoek (2012) 102:353–360 359 a-mannosidase and b-glucuronidase are negative. Fitch WM (1971) Toward defining the course of evolution: Cell-wall peptidoglycan contains LL-DAP as the minimum change for a specific tree topology. Syst Zool 20:406–416 diagnostic amino acid. The predominant menaquinone Gordon RE, Barnett DA, Handerhan JE, Pang CH-N (1974) is MK-8(H4) and phospholipids are diphosphatidyl- Nocardia coeliaca, Nocardia autotrophica, and the no- glycerol, phosphatidylglycerol and phosphatidylcho- cardin strains. Int J Syst Bacteriol 24:54–63 line. The major cellular fatty acids are iso-C , Hasegawa T, Takizawa M, Tanida S (1983) A rapid analysis for 16:0 chemical grouping of aerobic actinomycetes. J Gen Appl C18:1x9c and C17:1x6c. The DNA G?C content of the Microbiol 29:319–322 type strain is 70.1 mol%. He L, Li W, Huang Y, Wang L, Liu ZH (2005) Streptomyces The type strain, KLBMP 1050T (= KCTC 19888T jietaisiensis sp. nov., isolated from soil in northern China. = NBRC 108680T) was isolated from surface-steril- Int J Syst Evol Microbiol 55:939–944 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon ized stems of J. curcas L. collected from the city of YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing Panzhihua, Sichuan Province, south-west China. EzTaxon-e: a prokaryotic 16S rRNA gene sequence data- base with phylotypes that represent uncultured species. Int Acknowledgments The authors are grateful to KCTC for J Syst Evol Microbiol 62:716–721 kindly providing the type strain N. albus KCTC 9186T and Prof. Kimura M (1980) A simple method for estimating evolutionary Iain C. Sutcliffe for his valuable comments on the manuscript. rates of base substitutions through comparative studies of This research was partially supported by National Natural nucleotide sequence. 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