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 phyloge- netic 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 acti- nomycetes 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.