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Streptomyces Halophytocola Sp. Nov., an Endophytic Actinomycete Isolated from the Surface-Sterilized Stems of a Coastal Halophyte Tamarix Chinensis Lour

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Streptomyces Halophytocola Sp. Nov., an Endophytic Actinomycete Isolated from the Surface-Sterilized Stems of a Coastal Halophyte Tamarix Chinensis Lour International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2770–2775 DOI 10.1099/ijs.0.047456-0 Streptomyces halophytocola sp. nov., an endophytic actinomycete isolated from the surface-sterilized stems of a coastal halophyte Tamarix chinensis Lour. Sheng Qin,1 Guang-Kai Bian,1 Tomohiko Tamura,2 Yue-Ji Zhang,1 Wen-Di Zhang,1 Cheng-Liang Cao1 and Ji-Hong Jiang1 Correspondence 1The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, School of Life Sheng Qin Science, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, PR China [email protected] 2NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, 2-5-8 Ji-Hong Jiang Kazusakamatari, Kisarazu, Chiba 292-0818, Japan [email protected] A novel actinomycete, designated KLBMP 1284T, was isolated from the surface-sterilized stems of a coastal halophyte Tamarix chinensis Lour. collected from the city of Nantong, Jiangsu Province, east China. The strain was found to have morphological and chemotaxonomic characteristics typical of members of the genus Streptomyces. Analysis of the 16S rRNA gene sequence of strain KLBMP 1284T revealed that the strain formed a distinct clade within the phylogenetic tree based on 16S rRNA gene sequences and the highest sequence similarity (99.43 %) was to Streptomyces sulphureus NRRL B-1627T. 16S rRNA gene sequence similarity to other species of the genus Streptomyces was lower than 97 %. Based on DNA–DNA hybridization values and comparison of morphological and phenotypic data, KLBMP 1284T could be distinguished from the closest phylogenetically related species, Streptomyces sulphureus NRRL B-1627T. Thus, based on these data, it is evident that strain KLBMP 1284T represents a novel species of the genus Streptomyces, for which the name Streptomyces halophytocola sp. nov. is proposed. The type strain is KLBMP 1284T (5KCTC 19890T5NBRC 108770T). The genus Streptomyces was proposed by Waksman & eastern China, an endophytic actinomycete strain, KLBMP Henrici (1943) to accommodate actinobacteria that 1284T, was isolated from the halophyte Tamarix chinensis develop branched substrate mycelium and aerial hyphae, Lour. The present polyphasic study was designed to establish present LL-diaminopimedic acid without characteristic the taxonomic status of this strain. sugars in the cell wall (wall chemotype I; Lechevalier & Healthy samples of the halophyte Tamarix chinensis Lechevalier, 1970). At the time of writing, the genus Strep- Lour., collected from Nantong coast (121u 09 38.960 E tomyces contains a large number of described species 32u 349 24.910 N), Jiangsu Province, east of China in and more than 600 have validly published names (http:// October 2010, were used for isolation. In accordance with www.bacterio.cict.fr/s/streptomycesa.html). Endophytic the surface-sterilized method described previously (Qin et Streptomyces have been isolated from many higher plants al., 2009), the actinobacteria strains were isolated from the in recent years and many of them have been demonstrated stems on modified tap water-yeast extract agar [3 % (w/v) to have capacity to produce a vast array of secondary NaCl added to this agar] (TWYE, Crawford et al., 1993) metabolites exhibiting a wide range of biological activity, after a 3-week incubation at 28 uC. Strain KLBMP 1284T such as antibiotic anti-tumour and anti-infection, plant was picked, purified and then maintained on yeast extract- growth promoters and enzymes (Strobel et al., 2004; malt extract agar [International Streptomyces Project Hasegawa et al., 2006; Qin et al., 2011). In an effort to medium 2 (ISP 2); Shirling & Gottlieb, 1966] slants at discover novel actinomycetes from coastal halophytes in 4 uC and as glycerol suspensions (20 %, v/v) at –80 uC. Abbreviation: ISP, International Streptomyces Project. Genomic DNA was extracted from the biomass prepara- The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene tions and PCR amplification and 16S rRNA gene sequen- sequence of strain KLBMP 1284T is JQ819259. cing was performed using procedures described by Li et al. A supplementary table and a supplementary figure are available with the (2007). The PCR product was purified and sequenced online version of this paper. directly by an automated DNA sequencing system (ABI 2770 047456 G 2013 IUMS Printed in Great Britain Streptomyces halophytocola sp. nov. 3730XL). The resultant sequences were first aligned via the algorithms, neighbour-joining (Saitou & Nei, 1987), max- BLAST search program of NCBI (http://blast.ncbi.nlm.nih. imum-likelihood (Felsenstein, 1981) and maximum- gov/). Phylogenetic neighbours were identified and pair- parsimony (Kluge & Farris, 1969). The stability of the wise 16S rRNA gene sequence similarities were calculated clades in the trees was appraised using a bootstrap value with using the EzTaxon-e database (http://eztaxon-e.ezbiocloud. 1000 repeats (Felsenstein, 1985). A distance matrix was net/; Kim et al., 2012). Multiple alignments with sequences generated using Kimura’s two-parameter model (Kimura, of the most closely related taxa and the construction of the 1980). All positions containing gaps and missing data were phylogenetic trees were carried out using MEGA software eliminated from the dataset (complete deletion option). The version 5.0 (Tamura et al., 2011), and three treeing G+C content of the genomic DNA was determined using Streptomyces lilacinus NBRC 3944T (AB184819) 0.005 Streptomyces coerulescens NBRC 12758T (AB184122) Streptomyces abikoensis NBRC 13860T (AB184537) Streptomyces varsoviensis NRRL B-3589T (DQ026653) 93* Streptomyces blastmyceticus NRRL B-5480T (AY999802) Streptomyces ardus NBRC 13430T (AB184864) 67* Streptomyces cinnamoneus NBRC 12852T (AB184850) Streptomyces hiroshimensis NBRC 3839T (AB184802) Streptomyces lacticiproducens GIMN4.001T (GQ184344) LMG 20074T (AJ781349) 60* Streptomyces morookaense 55 Streptomyces thioluteus LMG 20253T (AJ781360) Streptomyces kasugaensis M338-M1T (AB024441) Streptomyces youssoufiensis X4T (FN421338) 65* Streptomyces yatensis NBRC 101000T (AB249962) ‘Streptomyces bingchenggensis’ BCW-1 (CP002047) Streptomyces sclerotialus DSM 43032T (AJ621608) Streptomyces ascomycinicus DSM 40822T (EU170121) Streptomyces ramulosus NRRL B-2714- T (DQ026662) 79* Streptomyces platensis JCM 4662T (AB045882) 93* Streptomyces fulvissimus NBRC 3717T (AB184787) Streptomyces variegatus LMG 20315T (AJ781371) 75* 99* Streptomyces spectabilis NBRC 13424T (AB184393) DSM 44293T(U94490) 65* Streptomyces thermocarboxydus T 55* Streptomyces albogriseolus NRRL B-1305- (AJ494865) 100* Streptomyces viridodiastaticus NBRC 13106T (AB184317) 100* Streptomyces halophytocola KLBMP 1284T (JQ819259 ) 64* Streptomyces sulphureus NRRL B-1627-1627T (DQ442546) Streptomyces gibsonii NBRC 15415T (AB184663) Streptomyces almquistii NBRC 13015T (AB184258) 100* Streptomyces rangoonensis LMG 20295T (AJ781366) 98* Streptomyces megasporus NBRC 14749T (AB184617) Streptomyces macrosporus NBRC 14748T (AB184616) 99* Streptomyces radiopugnans R97T (DQ912930) GIMN4.003T (GU356598) 85* Streptomyces fenghuangensis 69 Streptomyces nanhaiensis SCSIO 01248T (GQ871748) Streptomyces thermolineatus DSM 41451T (Z68097) Kitasatospora setae KM-6054- T (AB022868) Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic positions of strain KLBMP 1284T and some related species of the genus Streptomyces. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branch points. Asterisks indicate branches of the trees that were also found using the maximum- parsimony and maximum-likelihood tree-making algorithms. Bar, 0.005 substitutions per nucleotide position. http://ijs.sgmjournals.org 2771 S. Qin and others the method of Mesbah et al. (1989). DNA–DNA hybridiza- DNA–DNA hybridization revealed 46±1.4 % relatedness tions were performed using the fluorometric micro-well between strain KLBMP 1284T and S. sulphureus NRRL B- method (Ezaki et al., 1989; He et al., 2005). 1627T, which is below the commonly accepted threshold value for the phylogenetic definition of different species as The morphology of the spore chain and the spore surface recognized by Wayne et al. (1987). ornamentation of strain KLBMP 1284T were observed by light microscopy (SA3300-PL) and scanning electron Morphological observation of a 21-day culture of strain microscopy (S-3400N; Hitachi) using cultures grown on KLBMP 1284T grown on ISP 2 medium revealed that strain ISP 2 agar for 21 days at 28 uC. Cultural characteristics KLBMP 1284T had the typical characteristics of members were observed on a number of standard media (Table S1, of the genus Streptomyces. The isolate formed a highly available in IJSEM Online) after 2 weeks at 28 uC. Colours branched substrate mycelium and aerial hyphae which were determined by the methods described by Kelly (1964). differentiated into spiral spore chains with smooth spores The growth temperature (4, 10, 15, 20, 28, 37, 45 and (Fig. 2). Growth of the organism was good on ISP 2, ISP 4, 55 uC) and NaCl tolerance [0–15 % (w/v), at intervals of nutrient agar (NA) and potatoe-dextrose agar (PDA) 1%] was determined on ISP 2 agar at 28 uC for 14 days. media, moderate on ISP media 3 and 5, but poor on The pH range for growth (pH 4.0–13.0, at intervals of 0.5 Czapek’s agar. The colour of the aerial mycelium was pH units) was determined using ISP 2 medium broth adjusted with 1 M HCl, 20 % (w/v) Na2CO3 and 1 M u NaOH solution after sterilization, and incubated at 28 C (a) for
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