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Pseudonocardia Nematodicida Sp. Nov., Isolated from Mangrove Sediment in Hainan, China

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Pseudonocardia Nematodicida Sp. Nov., Isolated from Mangrove Sediment in Hainan, China Antonie van Leeuwenhoek (2015) 108:571–577 DOI 10.1007/s10482-015-0512-5 ORIGINAL PAPER Pseudonocardia nematodicida sp. nov., isolated from mangrove sediment in Hainan, China Min Liu . Shan-shan Xing . Wei-dao Yuan . Hua Wei . Qian-guang Sun . Xiang-zhi Lin . Hui-qin Huang . Shi-xiang Bao Received: 19 March 2015 / Accepted: 17 June 2015 / Published online: 27 June 2015 Ó Springer International Publishing Switzerland 2015 Abstract Two aerobic, Gram-stain positive acti- phosphatidylmethylethanolamine, phosphatidylglyc- nobacterial strains with nematicidal activity, designated erol, diphosphatidylglycerol, phosphatidylcholine, HA11164T and HA12591, were isolated from mangrove phosphatidylinositol, five unidentified glycolipids and sediments in Hainan, China. Phylogenetic analysis four unidentified polar lipids. The predominant mena- based on the 16S rRNA gene sequences indicated that quinone of strain HA11164T was identified as MK-8 T strains HA11164 and HA12591 belong to the genus (H4), and the major fatty acids were identified as iso- Pseudonocardia and are closely related to Pseudono- C16:0,C17:1 x10, C16:0 and C16:1 x9. The G?Ccontent cardia carboxydivorans (with the similarities of 98.30 of strain HA11164T wasdeterminedtobe74.9mol%. and 98.24 %, respectively), Pseudonocardia alni (98.23 The DNA–DNA relatedness values between strains and 98.16 %, respectively) and Pseudonocardia antimi- HA11164T and P. alni, Pseudonocardia tropica, crobica (98.10 and 98.03 %, respectively). The major Pseudonocardia antarctica, P. carboxydivorans and polar lipids of the strain HA11164T, as a representative Pseudonocardia parietis were 58.3, 56.2, 50.0, 57.1 and strain of the two strains, were found to consist of 46.0 %, respectively. Based on the results of this polyphasic study, strains HA11164T and HA12591 are considered to represent a novel species of the genus Electronic supplementary material The online version of Pseudonocardia, for which the name Pseudonocardia this article (doi:10.1007/s10482-015-0512-5) contains supple- nematodicida sp. nov. is proposed. The type strain is mentary material, which is available to authorized users. HA11164T (=CGMCC 4.7118T = DSM 45940T). M. Liu Á S. Xing Á W. Yuan Á H. Wei Á Q. Sun Á H. Huang (&) Á S. Bao (&) Keywords Pseudonocardia nematodicida sp. nov. Á Key Laboratory of Biology and Genetic Resources of 16S rRNA gene Á Polyphasic taxonomy Tropical Crops of Ministry of Agriculture, Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, People’s Republic of China Introduction e-mail: [email protected] S. Bao The genus Pseudonocardia first proposed by Henssen e-mail: [email protected] (1957) for nocardioform actinomycetes that lack mycolic acids and have a type IV cell wall, reflecting X. Lin the presence of meso-diaminopimelic acid and the Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, sugars arabinose and galactose (Evtushenko et al. Xiamen 361005, Fujian, People’s Republic of China 1989; Prabahar et al. 2004; Qin et al. 2008; Park et al. 123 572 Antonie van Leeuwenhoek (2015) 108:571–577 2008; Scha¨fer et al. 2009; Qin et al. 2010), comprises Center (CGMCC), China, as strain CGMCC 4.7118T. 53 species at the time of writing (http://www.bacterio. Pseudonocardia tropica YIM 61452T, Pseudonocar- net/pseudonocardia.html). Bacterial species of this dia antarctica DVS 5a1T, Pseudonocardia alni DSM genus have been isolated from various environments, 44104T and Pseudonocardia parietis 04-St-002T were such as soil (Henssen et al. 1983; Lee et al. 2002; Park obtained from the DSMZ and Pseudonocardia car- et al. 2008; Qin et al. 2008; Ara et al. 2011), plant roots boxydivorans Y8T from the Korean Culture Center of (Duangmal et al. 2009; Kaewkla and Franco 2011; Micro-organisms (KCCM), and cultured under the Zhao et al. 2011), plant stem (Qin et al. 2010), coastal same conditions for comparative analysis. sediment (Liu et al. 2006), deep-sea sediment (Tian et al. 2013), industrial sludge (Mahendra and Alvarez- Morphological, physiological and biochemical Cohen 2005) and a gold mine cave (Lee et al. 2001). characterization Mangrove ecosystems, as highly productive wetlands distributed along estuaries, provide refuge, growing, Cultural characteristics, including the colour and breeding and feeding zones for many marine organ- growth of the aerial and substrate mycelia and the isms (Holguin et al. 2001). During our study on iso- soluble pigments, were investigated after 7–15 days lation and diversity of bacteria with nematicidal incubation at 28 °C on oatmeal agar (DSMZ medium activity from mangrove sediments, several novel 609), glycerol-asparagine agar (Shirling and Gottlieb strains were obtained. In this study, the taxonomic 1966), Gause inorganic agar, inorganic salt starch (ISP positions of novel Pseudonocardia strains isolated 4) agar (Shirling and Gottlieb 1966), potato-dextrose from mangrove sediments in Dongzhaigang Man- agar (DSMZ medium 129) and on yeast extract-malt grove Forest were identified using a polyphasic extract agar (ISP 2), respectively. The morphological approach. characteristics of mycelia and spores were observed by light microscopy (DM6000B; Leica) and scanning electron microscopy (Phenom TM; Quantum Design). Materials and methods Gram staining was performed by using the Gram stain kit (Beijing Land Bridge). To test for aerotactic Isolation and maintenance of bacterial strains ability, cells were inoculated by mixing them with semisolid medium in tubes at 28 °C for 14 days. The Strains HA11164T and HA12591 were isolated from temperature (4, 10, 15, 20, 25, 30, 35, 40, 42 °C) and sediments in Bruguiera sexangula and Bruguiera pH (4–12, with 1 unit increments) range for growth, gymnorrhiza forest zones in Dongzhaigang Mangrove and the tolerance of NaCl [at 0–12 % with 1 % Forest (19°570N, 110°350E), respectively. 10 g air- increments (w/v)], were examined on ISP 2 medium. dried sediment samples were heated at 55 °C for Antibiotic susceptibility tests were performed on 6 min, then suspended in 100 mL sterile aged seawa- ISP 2 medium by Sensi-Discs (6 mm; BBL) with the ter and stirred for 30 min. Bacterial strains were following antibiotics (lg per disc): ampicillin (10), isolated by the standard dilution-plating technique on chloromycetin (30), erythromycin (15), gentamycin modified Gause inorganic agar with 20 g soluble (10), kanamycin (30), nalidixic acid (30), neomycin starch, 1 g KNO3, 0.5 g K2HPO4, 0.5 g MgSO4Á7H2O, (30), novobiocin (30), rifampicin (5), tetracycline 0.01 g FeSO4Á7H2O, 15 g agar, 1 L aged seawater (30). The physiological and biochemical characteris- (pH 7.2) (Gause et al. 1983). Plates were incubated at tics, such as utilisation of carbon source, gelatin 28 °C for 7 days. Single colonies were picked from liquefaction, H2S and melanin production, catalase the plates and purified on Gause inorganic agar. and oxidase activities, nitrate reduction, decomposi- The bacterial cultures were kept temporarily on tion of starch, cellulose and milk were tested as Gause inorganic slants and in glycerol suspensions previously described by Smibert and Krieg (1994). (20 %, v/v) at –80 °C for permanent preservation. For detection of nematicidal activities, each isolated Strain HA11164T has been deposited in the German bacterial strain was cultured in a liquid fermentation Collection of Microorganisms and Cell Cultures medium (yeast extract-malt extract broth) with shaking (DSMZ), Germany, as strain DSM 45940T and in the at 180 rpm at 28 °C. The fermentation broth was China General Microbiological Culture Collection centrifuged and the supernatant was tested for 123 Antonie van Leeuwenhoek (2015) 108:571–577 573 nematicidal activity using 24-well cell culture plates from a B. sexangula forest zone and strain HA12591 (Zeng et al. 2013). The target nematodes were from a B. gymnorrhiza forest zone). Both strains were Meloidogyne Arenaria and Meloidogyne incognita. found to have nematicidal activity. The strains were observed to be Gram-positive, aerobic, and to grow Chemotaxonomic characterization well on oatmeal agar, Gause inorganic agar, potato- dextrose agar and yeast extract-malt extract agar. On For chemotaxonomic and molecular systematic stud- Gause inorganic agar, the aerial mycelia were ies, the strains were grown in yeast extract-malt observed to be white and the substrate mycelia to be extract broth at 28 °C with shaking cultivation for pale yellow. The aerial mycelium was observed to 5 days. Biomass was harvested by centrifugation, fragment into rod-shaped spores with smooth surfaces washed with sterile distilled water three times, re- (Supplementary Fig. S1). No soluble pigment was centrifuged and freeze-dried. Polar lipids were anal- observed to be produced. The temperature and pH ysed according to the previous methods (Minnikin range for growth were determined to be 15–30 °C and et al. 1979; Collins and Jones 1980). Menaquinones 5–10, with the optimal growth at 25 °C and pH 7.0, were isolated according to Minnikin et al. (1984) and respectively. Growth of the strains was observed at analysed by HPLC (Collins 1994).The fatty acid profile NaCl concentrations between 0 and 8 %, with an was determined by using GC/MS (model GC-2010; optimum of 3 %. Nitrate reduction was found to be Shimadzu) (Kuykendall et al. 1988). negative. The strains are not able to degrade cellulose, gelatin and milk but starch was found to be hydrol- Phylogenetic analyses
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