International Journal of Systematic and Evolutionary Microbiology (2013), 63, 224–229
DOI 10.1099/ijs.0.035725-0
Streptomyces endophyticus sp. nov., an endophytic actinomycete isolated from Artemisia annua L. Jie Li,1,23 Guo-Zhen Zhao,13 Wen-Yong Zhu,1 Hai-Yu Huang,1 Li-Hua Xu,1 Si Zhang2 and Wen-Jun Li1,3 Correspondence Wen-Jun Li
[email protected] or
[email protected]
1
Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming, 650091, PR China
2
Key Laboratory of Marine Bio-resources Sustainable Utilization CAS, RNAM Center for Marine Microbiology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, PR China
3
Key Laboratory of Biogeography and Bioresource in Arid Land, CAS, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, U¨ru˝mqi 830011, China
Three filamentous actinomycetes, strains YIM 65594T, YIM 65638 and YIM 65642, were isolated from the surface-sterilized roots of Artemisia annua L. collected from Yunnan province, southwest China. These strains were found to have morphological and chemotaxonomic characteristics typical of the genus Streptomyces. The organisms formed an extensively branched substrate mycelium, with abundant aerial hyphae that differentiated into spores. The cell wall of the isolates contained LL-diaminopimelic acid and the menaquinones were MK-9(H8) and MK-9(H6). The major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Phylogenetic analysis of the 16S rRNA gene sequences of these strains revealed that the strains clustered together and were most closely related to Streptomyces kunmingensis NBRC 14463T, with 98.5–98.6 % 16S rRNA gene sequence similarity. The results of DNA–DNA hybridization and physiological tests allowed the genotypic and phenotypic differentiation of strains YIM 65594T, YIM 65638 and YIM 65642 from related species. However, the high level of DNA–DNA relatedness between them showed that these three strains belong to the same species. Strain YIM 65594T (5DSM 41984T5CCTCC AA 209036T) was selected as the type strain to represent this novel species, for which the name Streptomyces endophyticus sp. nov. is proposed.
The genus Streptomyces was introduced by Waksman & Henrici (1943) to encompass aerobic, spore-forming actinomycetes. Members of the genus Streptomyces are able to form an extensively branched substrate mycelium and are also able to produce aerial hyphae that typically differentiate into chains of spores. The genus has LLdiaminopimelic acid in the cell-wall peptidoglycan, but has no characteristic sugars (Lechevalier & Lechevalier, 1970) and possesses DNA rich in G+C (Manfio et al., 1995; Williams et al., 1983). Streptomyces species are abundant in soil and are well known for their ability to produce biologically active secondary metabolites, including antibiotics, enzymes, enzyme inhibitors, antitumour agents and antifungal compounds (Be´rdy, 2005). 3These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains YIM 65594T, YIM 65638 and YIM 65642 are GU367154, GU367160 and GU367159, respectively. Five supplementary tables and six supplementary figures are available with the online version of this paper.
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Endophytic bacteria can be defined as bacteria that colonize the internal tissue of plants, but show no external sign of infection or negative effect on their host (Schulz & Boyle, 2006). There are more than 300 000 plant species on the earth and each individual plant is host to one or more endophytes (Strobel et al., 2004). The opportunity to find novel and beneficial endophytic micro-organisms among the diversity of plants in different ecosystems is considerable (Ryan et al., 2008). During our investigations of the diversity and taxonomy of actinomycetes associated with medicinal plants from Yunnan Province, south-west China, strains YIM 65594T, YIM 65638 and YIM 65642 were isolated from the surface-sterilized roots of Artemisia annua L. The objective of the present study was to determine the taxonomic position of strains YIM 65594T, YIM 65638 and YIM 65642. The roots of Artemisia annua L., a traditional Chinese medicinal plant, were collected in Yunnan Province, southwest China. Roots were washed in running water to remove soil particles and sterilized by an established procedure (Li 035725 G 2013 IUMS Printed in Great Britain
Streptomyces endophyticus sp. nov.
et al., 2008), then sliced into pieces, followed by plating on HV agar plates (Hayakawa & Nonomura, 1987), containing nalidixic acid (25 mg l21), nystatin (50 mg l21) and cycloheximide (50 mg l21) to inhibit growth of bacteria and fungi. The plates were incubated at 28 uC for 4– 6 weeks until outgrowth of endophytic actinomycetes was discerned. Colonies originating from the segments were selected and pure cultures were obtained by repeated streaking on TWYE (0.25 g yeast extract, 0.5 g K2HPO4 and 18 g agar per litre tap water, pH 7.2) plates. The purified strain was routinely cultured on yeast extract-malt extract agar medium (ISP 2; Shirling & Gottlieb, 1966) at 28 uC and stored as a glycerol suspension (20 %, v/v) at 280 uC. Morphological characteristics were assessed using light (BH-2; Olympus) and scanning electron microscopes (Philips XL30; ESEM-TMP) following incubation on ISP 2 agar medium for 14 days at 28 uC. Cultural characteristics of the strains were recorded on ISP (International Streptomyces Project) media (Shirling & Gottlieb, 1966), Czapek’s agar, potato-glucose agar and nutrient agar prepared as described by Dong & Cai (2001). Colours of the aerial mycelium, substrate mycelium and diffusible pigments were determined by using colour chips from the ISCC–NBS colour charts (standard samples, no. 2106) (Kelly, 1964). Growth at different temperatures (4, 10, 20, 28, 37, 40, 45, 50 and 55 uC) was tested on trypticase soy agar (TSA) plates incubated for 21 days. The pH range for growth (pH 4, 5, 6, 7, 8, 9 and 10, using the buffer system described by Xu et al., 2005) were tested at 28 uC for 14– 21 days in trypticase soy broth (TSB) medium. The NaCl tolerance for growth (0, 1, 3, 5, 7, 10, 11, 13, 15 and 20 %, w/v) was tested at 28 uC for 14–21 days on TSA plates. Catalase activity, oxidase and gelatinase activities, starch hydrolysis, nitrate reduction and urease activity were assessed as described by Smibert & Krieg (1994). Other physiological and biochemical tests were analysed as described by Gordon et al. (1974). Biomass for chemical and molecular studies was obtained by cultivation in shake flasks (about 200 r.p.m.) using TSB at 28 uC for 1 week. The isomers of diaminopimelic acid and sugars of the whole-cell hydrolysate were determined according to the procedures described by Hasegawa et al. (1983), Lechevalier & Lechevalier (1970) and Tang et al. (2009). Phospholipids were extracted, examined by twodimensional TLC and identified using previously described procedures (Minnikin et al., 1979; Collins & Jones, 1980). Menaquinones were isolated according to Collins et al. (1977) and separated by HPLC (Tamaoka et al., 1983). Cellular fatty acids were extracted, methylated and analysed by using the Sherlock Microbial Identification System (MIDI) according to the manufacturer’s instructions. The fatty acid methyl esters were analysed by using the Microbial Identification software package (Sherlock Version 6.1; MIDI database: TSBA6). The G+C content of the genomic DNA was determined by using the HPLC method according to Mesbah et al. (1989). http://ijs.sgmjournals.org
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were performed as described by Li et al. (2007). The 16S rRNA gene sequences of the strains were compared against a database of cultured species via BLAST analysis (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) and the EzTaxon-e server Database (http:// eztaxon-e.ezbiocloud.net/; Kim et al., 2012) of type strains in order to retrieve most similar sequences of recognized bacteria. Multiple alignments with sequences of the most closely related actinobacteria and calculations of levels of sequence similarity were carried out using CLUSTAL X (Thompson et al., 1997). Phylogenetic trees were constructed using the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and minimum-evolution (Rzhetsky & Nei, 1992) tree-making algorithms from MEGA version 4.0 (Tamura et al., 2007). The PHYLIP version 3.6 (Felsenstein, 2002) and PHYML (Guindon & Gascuel, 2003) software packages were used to construct the maximum-likelihood (Felsenstein, 1981) trees. The topologies of the phylogenetic trees were evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. The morphological and chemical properties of strains YIM 65594T, YIM 65638 and YIM 65642 were consistent with assignment to the genus Streptomyces (Williams et al., 1989; Manfio et al., 1995). The isolates grew at 10–37 uC, pH 6.0–9.0 and with 0–10 % (w/v) NaCl. Optimal growth was observed between 20 and 28 uC and at pH 7.0. Strains YIM 65594T, YIM 65638 and YIM 65642 grew well on ISP 2, ISP 3, ISP 4, ISP 5, Czapek’s agar, nutrient agar and potato-glucose agar media at 28 uC. The strains formed white or yellow–white or orange–yellow or olive brown aerial mycelia and yellow–white or orange–yellow or yellowish brown or olive brown substrate mycelia on the media tested. Soluble pigments were not produced on the media tested (Tables S1–S3 available in IJSEM Online). The organisms formed an extensively branched substrate mycelium, with abundant aerial hyphae that differentiated into smooth cylindrical spores (0.40–0.4460.70–0.85 mm) in straight spore chains (Fig. S1). The morphological, physiological and biochemical characteristics of strains YIM 65594T, YIM 65638 and YIM 65642 are detailed in Table 1 and Table S4. Whole-cell hydrolysates of the isolates contained LLdiaminopimelic acid as the diamino acid in the peptidoglycan, and the whole-cell sugars detected were rhamnose, galactose, glucose and mannose (cell wall type I of Lechevalier & Lechevalier, 1980). The predominant menaquinones were MK-9(H8) and MK-9(H6). The major phospholipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine, phosphatidylethanolamine, hydroxylated phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylinositol mannosides and some unknown phospholipids (Fig. S2). The fatty acid profile of the strains contained anteiso-C15 : 0, anteiso-C17 : 0 and 225
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Table 1. Differential characteristics between strain YIM 65594T and S. kunmingensis NBRC 14463T Strains: 1, YIM 65594T; 2, S. kunmingensis NBRC 14463T. Both strains were Gram-positive, non-motile and catalase- and urease- positive, and grew under aerobic conditions and at 5 % (w/v) NaCl, pH 6.0–9.0 and at temperatures between 10 and 37 uC. Both strains were able to utilize Larabinose, D-fructose, D-galactose, glucose, glycerol, lactose, maltose, L-rhamnose, ribose and xylose, but not dulcitol, sodium acetate or D-sorbitol as sole carbon sources. Acid is produced from D-fructose, L-rhamnose and ribose. +, Positive or present; W, weakly positive; 2, negative or absent. For testing physiological properties, strain YIM 65594T and S. kunmingensis NBRC 14463T were grown under the same conditions. Characteristic Spore chains Utilization as sole carbon source Cellobiose myo-Inositol D-Mannitol D-Mannose Raffinose Sucrose Oxidase Gelatin liquefaction Nitrate reduction Growth at/on: 10 % NaCl 40 uC pH 5.0 pH 9.0 PhospholipidsD
1
2
Straight
Spirals*
+
2 + +
W
2 + 2 2 2 + 2 W
2 2 + DPG, PG, PME, PE, PE-OH, PC, PI, PIM, PLs
W
+ + + 2 + 2 + + W
DPG, PI, PIM, PE, PE-OH*
*Data from Ruan et al. (1985). DDPG, Diphosphatidylglycerol; PME, phosphatidylmethylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PE-OH, hydroxylated phosphatidylethanolamine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PIM, phosphatidylinositol mannosides; PLs, unknown phospholipids.
Almost-complete 16S rRNA gene sequences of strains YIM 65594T (1522 bp), YIM 65638 (1363 bp) and YIM 65642 (1422 bp) were determined. Preliminary comparison of the 16S rRNA gene sequences of the isolates against the NCBI nucleotide collection database using the default BLAST algorithm indicated that the strains are members of the genus Streptomyces. Strains YIM 65594T, YIM 65638 and YIM 65642 had 16S rRNA gene sequence similarities of 98.55, 98.53 and 98.59 % with Streptomyces kunmingensis NBRC 14463T, respectively. Levels of 16S rRNA gene sequence similarity between strains YIM 65594T, YIM 65638 and YIM 65642 and the type strains of all other recognized Streptomyces species with validly published names were ¡97.81 %. Sequence similarities between the
three strains were higher than 99.0 %, indicating that they may belong to the same species. Therefore, DNA–DNA hybridization studies of the novel strains and the type strains of closely related species were performed by using the microplate hybridization method (Ezaki et al., 1989; Christensen et al., 2000; He et al., 2005), and the hybridizations were performed with six replications. Levels of DNA–DNA between strain YIM 65594T and strains YIM 65638 and YIM 65642 were 79.0 % (SD, 1.7 %) and 86.0 % (SD, 0.8 %), respectively, which were higher than the recommended threshold value for the delineation of genomic species and also indicated that these three isolates should be classified as members of the same species. However, the level of DNA–DNA relatedness between strain YIM 65594T and S. kunmingensis NBRC 14463T was 31.4 % (SD, 2.2 %), indicating that the group of strains under study constitutes a novel species (Stackebrandt & Goebel, 1994). DNA–DNA hybridization studies were not carried out between strain YIM 65594T (YIM 65638 and YIM 65642) and other Streptomyces species because of their relatively high evolutionary distances (.2 %) and the distinct phylogenetic position of these strains; this is because representatives of a number of Streptomyces species with much shorter 16S rRNA gene sequence evolutionary distances have DNA–DNA
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iso-C16 : 0; the detailed fatty acid profile is given in Table S5. The qualitative and quantitative combination of fatty acids for the novel strains is diagnostic for species of the genus Streptomyces, corresponding to fatty acid pattern 2c of Kroppenstedt (1985). The G+C contents of the DNA of these strains were 72.0 mol%. All chemotaxonomic properties of strains YIM 65594T, YIM 65638 and YIM 65642 were consistent with their classification in the genus Streptomyces (Ka¨mpfer, 2006; Kroppenstedt & Evtushenko, 2006).
Streptomyces endophyticus sp. nov.
90 *
Streptomyces koyangensis VK-A60T (AY079156) 99 *
95 *
0.005
Streptomyces tacrolimicus ATCC 55098T (FN429653)
88 *
Streptomyces fulvissimus NBRC 3717T (AB184787) Streptomyces flavofungini NBRC 13371T (AB184359)
T 99 * Streptomyces violarus NBRC 13104 (AB184316) T Streptomyces arenae ISP 5293 (AJ399485)
74 *
Streptomyces kunmingensis NBRC 14463T (AB184597) 99 *
Streptomyces endophyticus YIM 65642 (GU367159) Streptomyces endophyticus YIM 65638 (GU367160) Streptomyces endophyticus YIM 65594T (GU367154) Streptomyces niveiscabiei S78T (AF361786)
84 *
*
Streptomyces psammoticus NBRC 13971T (AB184554) Streptomyces lanatus NBRC 12787T (AB184845) Streptomyces cinnabarinus NBRC 13028T (AB184266) Streptomyces avermitilis MA-4680T (BA000030)
63*
Streptomyces atriruber NRRL B-24165T (EU812169) 99* Streptomyces curacoi NRRL B-2901T (EF626595) Streptomyces coeruleorubidus NBRC 12761T (AB184841) Streptomyces regensis NRRL B-11479T (DQ026649) Streptomyces corchorusii NBRC 13032T (AB184267) Streptomyces capoamus JCM 4734T (AB045877) Streptomyces bungoensis NBRC 15711T (AB184696) 89*
Streptomyces novaecaesareae NBRC 13368T (AB184357)
96*
50
Streptomyces galbus DSM 40089T (X79852)
Streptomyces phaeoluteigriseus NRRL ISP-5182T (AJ391815) 57
*
Streptomyces chartreusis NBRC 12753T (AB184839) Streptomyces resistomycificus NBRC 12814T (AB184166)
T 96* Streptomyces bobili JCM 4624 (AB045876) Streptomyces galilaeus JCM 4757T (AB045878)
Streptomyces cyaneus NRRL B-2296T (AF346475) 94*
Streptomyces phaeofaciens NBRC 13372T (AB184360) Streptomyces rishiriensis NBRC 13407T (AB184383) Kitasatospora setae KM-6054T (U93332)
Fig. 1. Phylogenetic position of strains YIM 65594T, YIM 65638, YIM 65642 and members of related species within the genus Streptomyces. The phylogenetic tree was based on a CLUSTAL X alignment of almost-complete 16S rRNA gene sequences (1361 unambiguous nucleotide positions) and was reconstructed with the neighbour-joining method. Asterisks indicate clades that were also conserved when maximum-parsimony, minimum-evolution and maximum-likelihood methods were used to construct phylogenetic trees. GenBank accession numbers are given in parentheses. The sequence of Kitasatospora setae KM6054T (U93332) was used as outgroup. Bootstrap values (expressed as percentages of 1000 replications) of ¢50 % are shown at branch points. Bar, 0.005 substitutions per nucleotide position.
hybridization values well below the 70 % cut-off point recommended for the delineation of genomic species (Zhao et al., 2010; Hamedi et al., 2010; Reddy et al., 2010). In addition, strain YIM 65594T formed a robust clade with strains YIM 65638 and YIM 65642, and this clade clustered tightly with the nearest clade comprising S. kunmingensis NBRC 14463T, with the grouping being supported by high bootstrap values (Fig. 1). The same phylogenetic relationship among these strains was also found in the phylogenetic trees obtained by the maximum-parsimony and maximum-likelihood http://ijs.sgmjournals.org
algorithms (Figs S3–S6). The phylogenetic distinctiveness and DNA–DNA hybridization data are sufficient to categorize strain YIM 65594T (and thus YIM 65638 and YIM 65642) as distinct from recognized species of the genus Streptomyces. In view of the combination of morphological, physiological, chemotaxonomic and phylogenetic data discussed here, it is evident that strains YIM 65594T, YIM 65638 and YIM 65642 belong to the genus Streptomyces. Differences in some phenotypic characteristics and their phylogenetic 227
J. Li and others
distinctiveness distinguish the three strains from previously described Streptomyces species. For example, the novel isolates differ from S. kunmingensis NBRC 14463T in that they produce long and straight spore chains. There are differences in utilization of cellobiose, D-mannitol, raffinose and sucrose. They can also be differentiated based on ability to degrade compounds such as gelatin and nitrate, and based on temperature (40 uC), NaCl concentration (10 %) and different pH ranges for growth (Table 1). On the basis of the data described above, strains YIM 65594T, YIM 65638 and YIM 65642 are considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces endophyticus sp. nov. is proposed. Description of Streptomyces endophyticus sp. nov. Streptomyces endophyticus (en.do.phy9ti.cus. Gr. pref. endo within; Gr. n. phyton plant; L. masc. suff. -icus adjectival suffix used with the sense of belonging to; N.L. masc. adj. endophyticus within plant, endophytic, pertaining to the isolation from plant tissues). Forms extensively branched substrate mycelia and aerial mycelia which carry smooth cylindrical spores (0.40– 0.4460.70–0.85 mm) in straight spore chains. Good growth occurs on ISP 2, ISP 3, ISP 4, ISP 5, Czapek’s agar, potato-glucose agar and nutrient agar media. Diffusible pigments are not observed on the media tested. Temperature range for growth is 10–37 uC, with optimal growth at 20–28 uC. The pH range for growth is 6.0–9.0 (optimum, pH 7.0). The NaCl concentration range for growth is 0–10 %. Positive for catalase, urease, milk coagulation, milk peptonization and gelatin liquefaction, but negative for nitrate reduction, oxidase, cellulose hydrolysis and H2S production. Hydrolyses Tweens 20, 40 and 80. Utilizes L-arabinose, cellobiose, D-fructose, Dgalactose, glycerol, lactose, maltose, D-mannose, L-rhamnose and ribose as sole carbon sources, but not dulcitol, D-sorbitol or sucrose. Acid is produced from D-fructose. L-Alanine, L-arginine, L-asparagine, glycine, L-hydroxyproline, hypoxanthine, L-lysine, L-phenylalanine, L-serine, Ltyrosine, L-valine and xanthine are used as sole nitrogen sources. Whole-cell hydrolysates are rich in LL-diaminopimelic acid, rhamnose, galactose, glucose and mannose. The phospholipids are diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine, phosphatidylethanolamine, hydroxylated phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylinositol mannosides and some unknown phospholipids. The menaquinones are MK-9(H8) and MK-9(H6). The major fatty acids are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. T
T
Acknowledgements This research was supported by the National Natural Science Foundation of China (no. U0932601).
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