Pseudonocardia Nantongensis Sp. Nov., a Novel Endophytic Actinomycete Isolated from the Coastal Halophyte Tamarix Chinensis Lour

Home , Pseudonocardia, Pseudonocardiaceae, Pseudonocardia alni

Antonie van Leeuwenhoek DOI 10.1007/s10482-012-9764-5

ORIGINAL PAPER

Pseudonocardia nantongensis sp. nov., a novel endophytic actinomycete isolated from the coastal halophyte Tamarix chinensis Lour

Ke Xing • Sheng Qin • Guang-Kai Bian • Yue-Ji Zhang • Wen-Di Zhang • Chuan-Chao Dai • Chang-Hong Liu • Wen-Jun Li • Ji-Hong Jiang

Received: 5 April 2012 / Accepted: 13 June 2012 Ó Springer Science+Business Media B.V. 2012

Abstract A novel isolate, designated strain KLBMP Pseudonocardia, being most closely related to Pseud- 1282T was isolated from the surface-sterilized leaves of onocardia kongjuensis LM 157T (98.33 %), Pseudono- a coastal halophyte Tamarix chinensis Lour., collected cardia autotrophica IMSNU 20050T (97.77 %), from Nantong, Jiangsu Province, east of China. Phylo- Pseudonocardia endophytica YIM 56035T (97.63 %), genetic analysis based on 16S rRNA gene sequences Pseudonocardia ammonioxydans H9 T (97.62 %) and revealed that this strain belongs to the genus Pseudonocardia compacta IMSNU 20111T (97.56 %); similarity to other type strains of the genus Pseudono- cardia was\97.5 %. Chemotaxonomic data conﬁrmed T Electronic supplementary material The online version the afﬁliation of strain KLBMP 1282 to the genus of this article (doi:10.1007/s10482-012-9764-5) contains Pseudonocardia. Strain KLBMP 1282T contained MK- supplementary material, which is available to authorized users. 8(H4) as the predominant ubiquinone and iso-C16:0 as K. Xing Á S. Qin (&) Á G.-K. Bian Á Y.-J. Zhang Á the major fatty acid. The polar lipids detected in strain T W.-D. Zhang Á J.-H. Jiang (&) KLBMP 1282 were diphosphatidylglycerol, phosphat- The Key Laboratory of Biotechnology for Medicinal Plant idylglycerol, phosphatidylcholine, phosphatidylmethyletha- of Jiangsu Province, School of Life Science, Jiangsu nolamine, phosphatidylethanolamine, phosphatidylinositol, Normal University, Xuzhou, Jiangsu 221116, People’s Republic of China phosphatidylinositol mannosides, one unknown phospho- e-mail: [email protected] lipid and four unknown glycolipids. The DNA G ? C T J.-H. Jiang content of strain KLBMP 1282 was 73.1 mol %. The e-mail: [email protected] results of DNA–DNA hybridizations and the phylogenetic analysis, together with the phenotypic and C.-C. Dai biochemical tests, allowed the differentiation of strain College of Life Sciences, Nanjing Normal University, T Nanjing 210046, China KLBMP 1282 from strains of other recognized Pseudonocardia species. Therefore, strain KLBMP C.-H. Liu 1282T represents a novel species of the genus Pseud- College of Life Sciences, Nanjing University, onocardia, for which the name Pseudonocardia nan- Nanjing 210093, China tongensis sp. nov. is proposed. The type strain is T T T W.-J. Li KLBMP 1282 (=KCTC 29053 = NBRC 108677 ). The Key Laboratory for Microbial Resources of the Ministry of Education and Laboratory for Conservation Keywords Pseudonocardia nantongensis sp. nov Á and Utilization of Bio-resources, Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan Coastal halophyte Á Endophytic Á Polyphasic 650091, People’s Republic of China taxonomy 123 Antonie van Leeuwenhoek

Introduction Materials and methods

The genus Pseudonocardia was firstly proposed by Isolation and maintenance of organism Henssen (1957) to accommodate nocardioform actinomycetes that have a type IV cell wall (contain meso- Strain KLBMP 1282T was isolated from the healthy diaminopimelic acid, with arabinose and galactose as leaves of a coastal halophyte Tamarix chinensis Lour., characteristic sugars) and lack mycolic acids. Mem- collected from the city of Nantong, Jiangsu Province, bers of the genus Pseudonocardia are composed of east of China. The samples were treated and surface vegetative and aerial mycelium with spore chains sterilized according to the five-step sterilization pro- produced by acropetal budding or fragmentation. The cedure (Qin et al. 2008). The samples were then major menaquinone is MK-8(H4)orMK-9and aseptically crumbled into smaller fragments using a phospholipids are either type PII or PIII pattern. The commercial blender (Joyoung, XC-001) and plated on DNA G ? C content for members of this genus ranges the selective isolation medium agar (starch 5 g, from 68 to 79 mol % (Lee et al. 2001). The description glucose 5 g, casein 2 g, yeast 1 g, CaCO3 2 g, agar of the genus was later emended by McVeigh et al. 15 g, 1 L distilled water, pH 7.0). The plates were (1994), Warwick et al. (1994), Reichert et al. (1998), incubated at 28 °C for 2–8 weeks and colonies were Huang et al. (2002), Park et al. (2008). At the time of obtained and subcultured on yeast extract-malt extract writing, the genus Pseudonocardia encompasses more agar (ISP 2 medium) at 4 °C and as glycerol suspen- than 40 species with validly published names, origi- sion (20 %, v/v) at -20 and -80 °C. Strain KLBMP nating from various environments (http://www.bacter 1282T was deposited in the NITE Biological Resource io.cict.fr/p/pseudonocardia.html). Interestingly, many Center (NBRC) as strain NBRC 108677T and in the new species of the genus Pseudonocardia were iso- Korean Collection for Type Cultures (KCTC) as strain lated from surface sterilized plants recently, such as KCTC 29053T. Pseudonocardia oroxyli (Gu et al. 2006), Pseudono- cardia endophytica (Chen et al. 2009), Pseudonocar- Phenotypic characterization dia acaciae (Duangmal et al. 2009), Pseudonocardia tropica (Qin et al. 2010), Pseudonocardia adelaiden- Cultural characteristics were observed on ISP 2, sis (Kaewkla and Franco 2010), Pseudonocardia oatmeal (ISP 3), inorganic salts-starch (ISP 4), glyc- kunmingensis (Zhao et al. 2011a), Pseudonocardia erol-asparagine (ISP 5) (Shirling and Gottlieb 1966), eucalypti (Kaewkla and Franco 2011), Pseudonocar- as well as potato-dextrose (PDA), Czapek’s and dia sichuanensis (Qin et al. 2011), Pseudonocardia nutrient agars (Waksman, 1967) for 14 days at artemisiae (Zhao et al. 2011b), Pseudonocardia ban- 28 °C. The colony colour was determined with the naensis (Zhao et al. 2011c) and Pseudonocardia ISCC–NBS colour charts (Kelly 1964). Cell morphol- serianimatus (Zhao et al. 2011d), indicating its high ogy was observed using light microscopy (SA3300- diversity within higher plants. PL) and scanning electron microscopy (Hitachi; During a study of the diversity of culturable S-3400N) using 4 weeks old nutrient agar medium endophytic actinobacteria associated with coastal culture (growth at 28 °C). Physiological tests of halophytes in eastern of China, an actinomycete growth at different temperatures (4, 10, 15, 20, 28, strain, designated KLBMP 1282T was isolated. The 32, 37, 45, 50 and 55 °C) and NaCl concentrations strain exhibited the typical morphological character- (0–20 %, w/v) (at intervals of 1 %, 28 °C) were istics of the genus Pseudonocardia. Here, we report examined by growing the novel strain on ISP 2 basal on the taxonomic characterization and classification medium. Growth at various pH values (4.0–11.0) were of strain KLBMP 1282T and our present study examined as described by Xu et al. (2005) by growing confirmed that it was a representative of a novel the strain in ISP 2 broth basal medium. Carbon source species within the genus Pseudonocardia, for which utilization tests and other phenotypic characteristics the name Pseudonocardia nantongensis sp. nov. is were carried out according to Kurup and Schmitt proposed. (1973) and Gordon et al. (1974). Nitrogen source

123 Antonie van Leeuwenhoek utilization was assessed according to Williams et al. 1981) algorithms. The phylogenetic consensus trees (1989). The type strains P. kongjuensis LM 157T, P. were reconstructed using MEGA version 5 (Tamura autotrophica IMSNU 20050T, P. endophytica YIM et al. 2011) for the neighbour-joining, maximum- 56035T, P. ammonioxydans H9T and P. compacta parsimony and maximum-likelihood methods. Boot- IMSNU 20111T were tested together under the same strap analysis was performed with 1,000 replications. conditions. DNA–DNA hybridization was performed using the microplate hybridization method (Ezaki et al. 1989;He Chemotaxonomy et al. 2005) and photobiotin-labelled DNA from strain KLBMP 1282T and P. kongjuensis LM 157T. DNA– For most chemotaxonomic analyses, cell mass was DNA relatedness was calculated as the mean of obtained from ISP 2 broth medium after cultivation for triplicate measurements. 10 days (at logarithmic growth phase) at 28 °C (150 rev min-1). Amino acids and sugars of whole- Nucleotide sequence accession number cell hydrolysates were analyzed by TLC as described previously (Staneck and Roberts 1974). For fatty acid The 16S rRNA gene sequence of strain KLBMP 1282T analysis, strain KLBMP 1282T and the reference strain determined in this study has been deposited in P. kongjuensis LM 157T were grown in nutrient broth GenBank under the accession number JQ819252. for 7 days at 28 °C. Extraction of fatty acid methyl esters, washing of extracts and GC analysis were performed according to the standard procedures of the Results and discussion Microbial Identification System (MIDI; Microbial ID) (Sasser 1990) using the MIDI Sherlock Version 6. 1, Cells of strain KLBMP 1282T were Gram-positive and MIDI database TSBA6. Polar lipids were extracted aerobic. Morphological observation of the 4 weeks according to the procedures described by Minnikin old culture of strain KLBMP 1282T revealed that both et al. (1979) and separated by two-dimensional TLC. aerial and vegetative hyphae were abundant, well Menaquinones were extracted and purified as developed and fragmented. The mycelia displayed described by Collins et al. (1977) and analysed by long spore chains, containing rod-shaped and smooth- HPLC (Groth et al. 1997). The G ? C content of the surfaced spores on NA medium agar (Supplementary DNA was determined by the method of Mesbah et al. Fig. S1). Strain KLBMP 1282T showed good growth (1989). on NA and PDA media, moderate growth on ISP 2, ISP 3 and ISP 5 media, and poor growth on ISP 4 and Molecular analysis Czapek’s agar media. White aerial mycelia were produced on tested media. The substrate mycelium Genomic DNA preparation, PCR amplification and varies from yellowish/yellow to orange-yellow. No sequencing of the 16S rRNA gene were carried out diffusible pigments were produced. Growth of strain using procedures described by Li et al. (2007). The KLBMP 1282T occurred in the pH range 6.0–9.0 and obtained sequence was compared with 16S rRNA 0–15 % NaCl (w/v), with optimum growth at pH 7.0 reference gene sequences retrieved from the Gen- and 3 % NaCl (w/v). The temperature range for Bank/EMBL/DDBJ databases by BLAST search. growth was 15–32 °C, with the optimum temperature Multiple alignments of the sequence were carried out being 28 °C. The detailed physiological and biochem- using the CLUSTAL_X (Thompson et al. 1997) ical properties are given in the species description and software. The identification of phylogenetic neigh- Table 1. Strain KLBMP 1282T can be distinguished bours and the calculation of pairwise 16S rRNA gene from its closest relatives by many phenotypic charac- sequence identities were achieved using the EzTaxon- teristics, including differences in utilization of sole e database (Kim et al. 2012). The phylogenetic carbon and nitrogen sources, degradation activity and relationship between the isolate and closely related different cultural characteristics on tested media. For strains was investigated using the neighbour-joining example, strain KLBMP 1282T can be distinguished (Saitou and Nei 1987), maximum-parsimony (Kluge from its closest relative P. kongjuensis LM 157T and Farris 1969) and maximum-likelihood (Felsenstein because it is in positive for nitrate reduction and 123 Antonie van Leeuwenhoek

Table 1 Characteristics Characteristic 1 2 3 4 5 6 that distinguish strain T KLBMP 1282 from its Assimilation of sole carbon sources closest phylogenetic neighbours D-arabinose w ??- ?- D-cellobiose - ??- -w Dextrin - ??? ?w Erythritol ? ??? ?- D-fructose w ??? ?- D-galactose w ??? ?w Inositol - ??? -- D-lactose - ??- ?? Maltose - ??? ?- D-rafﬁnose - ??- ?- D-rhamnose ? -?- ?- D-ribose ? ??? ?- D-sorbitol - ??? ?- Sucrose ? ??? ?- Trehalose w ??? ?- Xylose w ??? ?- Growth on sole nitrogen source L-alanine w ??- ?? L-histidine - ??? -- L-proline - ??? ?- Decomposition of Hypoxanthine ? ?-? -- Tyrosine - ?-? ?- Hydrolysis of Casein - ?-- ?- Strains: 1, P. nantongensis Gelatin - --- w - KLBMP 1282T; 2, P. kongjuensis LM 157T;3, Cellulose - ??- -- P. autotrophica IMSNU Nitrate reduction to nitrite ? --- ?? T 20050 ;4,P. endophytica Milk coagulation - ??- ?? YIM 56035T;5, P.ammonioxydans H9T;6, H2S production - ??- ?- P. compacta IMSNU Growth at 10 °C - ??- ?? T 20111 . All the data Growth at 37 °C - ??? ?- obtained during this study Growth on 7 % NaCl ? ??- ?- were carried out under identical growth conditions. Growth in pH 5.0 - ?-- -- ? positive, utilized, Growth on ISP 2 medium Moderate Good Good Good Good Good - negative, not utilized, Growth on ISP 4 medium Poor Good Good Moderate Good Good w weakly positive

negative for milk peptonization and coagulation and diaminopimelic acid as the diagnostic diamino acid,

H2S production, and its unable to grow at 10, 37 °C arabinose and galactose as the whole-cell sugars. Polar and in pH 5.0 and poor growth on ISP 4 medium. lipids of this novel strain comprised diphosphatidyl- The results of chemotaxonomic analyses were glycerol, phosphatidylglycerol, phosphatidylcholine, typical for the genus Pseudonocardia. Whole-cell phosphatidylmethylethanolamine, phosphatidyletha- hydrolysates of strain KLBMP 1282T contained meso- nolamine, phosphatidylinositol, phosphatidylinositol

123 Antonie van Leeuwenhoek

Table 2 Fatty acid profiles (%) of strain KLBMP 1282T and differentiated from its phylogenetically closest rela- the closely related type strain P. kongjuensis LM 157T tive P. kongjuensis LM 157T by some quantitative KLBMP P. kongjuensis differences of major component (iso-C16:0) and by the T T 1282 LM 157 presence of iso-C18:0 and absence of C17:0 and C x6c. The DNA G ? C content of strain KLBMP C 4.5 2.0 15:1 16:0 1282T was 73.1 mol %, which falls within the range C – 0.8 17:0 (68–79 mol %) described for the genus Pseudono- C 0.5 – 18:0 cardia. C x6c – 1.3 15:1 The almost complete 16S rRNA gene sequence C x8c 0.9 6.7 17:1 (1,443 nucleotide) of strain KLBMP 1282T was C18:1x9c 2.2 0.6 obtained and compared with sequences of known iso-C14:0 1.7 0.6 species on the EzTaxon-e database server (Kim et al. iso-C15:0 1.4 5.7 2012). The result revealed that strain KLBMP 1282T iso-C16:0 58.5 33.2 belonged to the genus Pseudonocardia. Sequence iso-C17:0 2.4 6.1 similarity calculation indicated that the closest rela- iso-C18:0 1.7 – tives of strain KLBMP 1282T were P. kongjuensis LM iso-C16:1 H 7.2 8.5 157T (98.33 %), P. autotrophica IMSNU 20050T T anteiso-C17:0 1.7 4.8 (97.77 %), P. endophytica YIM 56035 (97.63 %), T C17:0 10-methyl 1.9 7.2 P. ammonioxydans H9 (97.62 %) and P. compacta Sum In Feature 3 8.4 7.1 IMSNU 20111T (97.56 %), whereas the sequence Sum In Feature 5 0.7 – similarities between strain KLBMP 1282T and other Sum In Feature 9 3.4 12.2 members of the genus Pseudonocardia were below Summed features represent groups of two or three fatty acids 97.5 %. It is apparent from the neighbour-joining that cannot be separated by GC with the MIDI system. Summed phylogenetic tree (Fig. 1) that strain KLBMP 1282T features 3, 5, 9 comprised C16:1x7c/C16:1x6c, C18:2x6,9c and/ formed a distinct clade and this phylogenetic relation- or anteiso-C18:0, iso-C17:1x9c/C16:0 10-methyl, respectively ship was supported by a high bootstrap value of 67 %. All the data are from this study. All strains were grown in The phylogenetic relationship was also found and nutrient broth for 7 days at 28 °C. Fatty acids amounting to \0.5 % in all species are not shown. Values are percentages of supported in trees constructed with other maximum- total fatty acids; – not detected parsimony and maximum-likelihood tree-making algorithms (Supplementary Fig. S3–S4), which indicated that strain KLBMP 1282T should not be assigned mannosides, an unknown phospholipid and four to any of the recognized Pseudonocardia species. unknown glycolipids (phospholipid III; Lechevalier DNA–DNA hybridization between strain KLBMP et al. 1977) (Supplementary Fig. S2). The predominant 1282T and its closest phylogenetic neighbour P. T menaquinone was MK-8(H4) (93 %), minor compo- kongjuensis LM 157 was carried out by applying nents (\3 %) were also detected as MK-8, MK-8(H2) the fluorometric micro-well method. The determined and MK-9(H2). The major fatty acids were iso-C16:0 DNA–DNA relatedness value was 34.5 ± 3.6 %, (58.5 %), sum in feature 3(C16:1x7c/C16:1x6c) which is well below the 70 % cut-off point for (8.4 %), iso-C16:1H (7.2 %) and C16:0 (4.5 %), and recognition of genomic species (Stackebrandt and T sum in feature 9(iso-C17:1x9c/C16:0 10-methyl) Goebel 1994), suggesting that strain KLBMP 1282 (3.4 %), iso-C17:0 (2.4 %), C18:1x9c (2.2 %), C17:0 should be considered as a different genomic species of 10-methyl (1.9 %), anteiso-C17:0 (1.7 %), iso-C18:0 the genus Pseudonocardia. The DNA–DNA hybrid- (1.7 %), iso-C15:0 (1.4 %) present as minor compo- ization studies were not carried out between strain T nents; C17:1x8c (0.9 %), sum in feature 5(C18:2 KLBMP 1282 and other closest phylogenetic rela- x6,9c and/or anteiso-C18:0) (0.7 %) and C18:0 tives given the low level of 16S rRNA gene sequence (0.5 %) were detected at a level of\1 %. The cellular similarity (\98.0 %). It has been shown that some fatty acid profiles of strain KLBMP 1282T and its Pseudonocardia species have high 16S rRNA gene nearest neighbour P. kongjuensis LM 157T are sum- sequence similarities, but have low DNA–DNA relat- marized in Table 2. Strain KLBMP 1282T could be edness values below the 70 % cut-off point (Prabahar 123 Antonie van Leeuwenhoek

100* Pseudonocardia sichuanensis KLBMP 1115T (HM153789) 98* Pseudonocardia kunmingensis YIM 63158T (FJ817377) Pseudonocardia zijingensis 6330T (AF325725) 66* Pseudonocardia adelaidensis EUM 221T (FJ805427) Pseudonocardia aurantiaca DSM 44773T (FR749916) T 52 Pseudonocardia alaniniphila YIM 16303 (EU722519) 100* Pseudonocardia yunnanensis IFO 15681T (D85472) Pseudonocardia petroleophila ATCC 15777T (X80596) 99* Pseudonocardia serianimatus YIM 63233T (FJ817379) Pseudonocardia mongoliensis MN08-A0270T (AB521671) Pseudonocardia chloroethenivorans SL-1T (AF454510) Pseudonocardia thermophila IMSNU 20112T (AJ252830) T 99* Pseudonocardia khuvsgulensis MN08-A0297 (AB521672) 100* Pseudonocardia rhizophila YIM 67013T (GU322368) Pseudonocardia asaccharolytica DSM 44247T (Y08536) Pseudonocardia babensis VN05A0561T (AB514449) Pseudonocardia saturnea IMSNU 20052T (AJ252829) Pseudonocardia xinjiangensis AS 4.1538T (AF325728) Pseudonocardia bannaensis YIM 63101T(FJ817375) 100* Pseudonocardia artemisiae YIM 63587T (GU227146) 65* Pseudonocardia eucalypti EUM 374T (FJ805426) 57* Pseudonocardia acaciae GMKU095T (EU921261) Pseudonocardia spinosispora LM 141T (AJ249206) 100* Pseudonocardia autotrophica IMSNU 20050T (AJ252824) 75 Pseudonocardia compacta IMSNU 20111T (AJ252825) T 67* Pseudonocardia kongjuensis LM 157 (AJ252833) T 82* KLBMP 1282 (JQ819252) Pseudonocardia ammonioxydans H9T (AY500143) Pseudonocardia endophytica YIM 56035T (DQ887489) 96* Pseudonocardia parietis 04-St-002T (FM863703) Pseudonocardia nitrificans IFAM 379T (X55609) 44 94 Pseudonocardia carboxydivorans Y8T (EF114314) 60 Pseudonocardia antarctica DVS 5a1T (AJ576010) 95* Pseudonocardia alni DSM 44104T (Y08535) 95 Pseudonocardia tropica YIM 61452T (GQ906587) 84* Pseudonocardia ailaonensis YIM45505T (DQ344632) 65* Pseudonocardia oroxyli D10T (DQ343154) Pseudonocardia halophobica DSM 43089T (Y08534) Pseudonocardia tetrahydrofuranoxydans K1T (AJ249200) 90* Pseudonocardia hydrocarbonoxydans IMSNU 22140T (AJ252826) 99* Pseudonocardia sulfidoxydans DSM 44248T (Y08537) 73 Pseudonocardia benzenivorans B5T (AJ556156) 100* Pseudonocardia dioxanivorans CB1190T (CP002593) Kutzneria kofuensis NRRL B-24061T (AF114801)

0.01

Fig. 1 Neighbour-joining phylogenetic tree, based on 16S branches of the tree that were also recovered using maximum rRNA gene sequences, showing the positions of strain KLBMP parsimony and maximum-likelihood analyses. Kutzneria kofu- 1282T and some related taxa. Numbers at the nodes are the ensis NRRL B-24061T (AF114801) was used as an outgroup. bootstrap values based on 1,000 resamplings. Bootstrap values Bar 0.01 substitutions per site of above 40 % are shown at branch points. Asterisks indicate

123 Antonie van Leeuwenhoek

et al. 2004;Ka¨mpfer et al. 2006; Park et al. 2008). For liquefaction, cellulose hydrolysis and H2S production. example, the closest phylogenetic neighbour P. kon- Uses L-alanine and L-asparagine as sole nitrogen T gjuensis LM 157 showed 98.8–98.5 % 16S rRNA sources, but negative for assimilation of L-arginine, gene sequence similarities to P. autotrophica IMSNU L-lysine, L-proline, L-serine, L-threonine, L-tyrosine T T T 20050 and P. compacta IMSNU 20111 , but exhib- and L-histidine. The cell wall of strain KLBMP 1282 ited low levels of DNA relatedness to its phylogenetic contains meso-DAP. The whole-cell sugar pattern neighbours P. autotrophica (32 %) and P. compacta consists of arabinose and galactose. The cell wall (14 %) (Lee et al. 2001). Recently, Stackebrandt and chemotype is type IV. Predominant menaquinone is

Ebers (2006) recommended an increase of about 2 % MK-8(H4). The phospholipids are diphosphatidyl- (from 97 to 98.7–99 %) for the 16S rRNA gene glycerol (DPG), phosphatidylglycerol (PG), phospha- sequence similarity threshold used to determine the tidylcholine (PC), phosphatidylmethylethanolamine uniqueness of a new isolate provided that these data (PME), phosphatidylethanolamine (PE), phosphati- are supported by clear phenotypic differences. dylinositol (PI), phosphatidylinositol mannosides Together with the fact that our present species (PIM), an unknown phospholipid (PL) and four exhibiting more than 98.0 % 16S rRNA gene unknown glycolipids (GL). Major fatty acids are iso- sequence similarity to its closest phylogenetic neigh- C16:0 (58.5 %), sum in feature 3(C16:1x7c/C16:1x6c) bour, in future studies, highest values below 98.0 % (8.4 %) and iso-C16:1 H (7.2 %). The DNA G ? C can be considered to suggest the detection of a novel content is 73.1 mol %. species of the genus Pseudonocardia. The type strain KLBMP 1282T (= KCTC Strain KLBMP 1282T could be differentiated from 29053T = NBRC 108677T) was isolated from sur- P. kongjuensis LM 157T, P. autotrophica IMSNU face-sterilized leaves of a coastal halophyte Tamarix 20050T, P. endophytica YIM 56035T, P. ammonioxy- chinensis Lour., collected from the city of Nantong, dans H9T and P. compacta IMSNU 20111T by several Jiangsu Province, east of China. phenotypic characteristics, including its growth temperature range, growth in medium with added NaCl, Acknowledgments The authors are grateful to Prof. the pattern of assimilation of sole carbon and nitrogen Tomohiko Tamura (NITE Biological Resource Center, NBRC) for kindly providing the type strains and Prof. Martha E. Trujillo sources (Table 1). Phenotypic and genetic character- for his valuable comments on the manuscript. This research was istics and phylogenetic analysis demonstrate that partially supported by National Natural Science Foundation of strain KLBMP 1282T represents a novel species China (31000005, 31101502), the Program of Natural Science within the genus Pseudonocardia, for which the name Foundation of the Jiangsu Higher Education Institutions of China (10KJB180008, 11KJD210002), the Project Funded by P. nantongensis sp. nov. is proposed. the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Natural Science Foundation by Xuzhou City (No. XZZD1004). Description of P. nantongensis sp nov

Pseudonocardia nantongensis sp. nov. (nan.tong.en’- sis. N.L.masc.adj. nantongensis pertaining to Nantong References city, from where the strain was isolated). Chen HH, Qin S, Li J, Zhang YQ, Xu LH, Jiang CL, Kim CJ, Li Aerobic, Gram-positive actinomycete that forms WJ (2009) Pseudonocardia endophytica sp. nov., isolated extensively branched substrate mycelia and aerial from the pharmaceutical plant Lobelia clavata. Int J Syst mycelia which carry smooth-surfaced rod-shaped Evol Microbiol 59:559–563 spores. Forms yellowish/yellow or orange-yellow Collins MD, Pirouz T, Goodfellow M, Minnikin DE (1977) Distribution of menaquinones in actinomycetes and cory- substrate mycelia and white aerial mycelia on media nebacteria. J Gen Microbiol 100:221–230 tested. No diffusible pigments are produced. Growth Duangmal K, Thamchaipenet A, Matsumoto A, Takahashi Y occurs between 15 and 32 °C, pH 6.0–9.0, and 0–15 % (2009) Pseudonocardia acaciae sp. nov., isolated from NaCl on ISP 2 agar. Tween 20 and 40 are degraded, roots of Acacia auriculiformis A. Cunn. ex Benth. Int J Syst Evol Microbiol 59:1487–1491 but not Tween 80. Positive for starch hydrolysis, Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric nitrate reduction and urease production but negative deoxyribonucleic acid-deoxyribonucleic acid hybridiza- for milk peptonization and coagulation, gelatin tion in microdilution wells as an alternative to membrane 123 Antonie van Leeuwenhoek

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