Introduction

The genus Streptomyces was proposed by Waksman and Henrici (1943) and is a unique source of novel antibiotics (Bérdy 2005; Goodfellow and Fiedler 2010). At present, the genus Streptomyces contains the large number of described species and nearly 600 were validly published (Euzéby 2012). Endophytes that live within the healthy plants are now considered as an important component of biodiversity. In recent years, endophytic actinomycetes have attracted significant interest for their capacity to produce a vast array of secondary metabolites exhibiting a wide variety of biological activities (Qin et al. 2011 ). In the course of investigating endophytic actinomycetes associated with medicinal plants in Sichuan Province, China, one streptomycete strain KLBMP 4601T was isolated. This strain showed characteristics different from other members of the Streptomyces genus by the polyphasic characterization. Here we report a polyphasic taxonomic study and showed that isolate KLBMP 4601T belonged to a new Streptomyces species, Streptomyces phytohabitans sp. nov.

Materials and methods

Isolation and maintenance of isolate

Strain KLBMP 4601T was isolated from the healthy roots of a medicinal plant Curcuma phaeocaulis collected from Sichuan Province, south-west China, in 2009. The root samples were firstly surface sterilized according to the procedure described previously (Qin et al. 2009). Subsequently, the surface sterilized samples were aseptically crumbled into smaller fragments using a commercial blender (Joyoung), spread onto tap water-yeast extract agar (TWYE, Crawford et al. 1993) and colonies were picked up after incubation for 4 weeks at 28 °C. The purified isolate was routinely cultured on yeast extract-malt extract agar (ISP 2) (Shirling and Gottlieb 1966) and maintained as a glycerol suspension (20 %, w/v) at −80 °C.

Phenotypic characterization

Cultural characteristics of strain KLBMP 4601T were determined using various agar media: ISP 2, oatmeal agar (ISP 3), inorganic salts-starch agar (ISP 4), glycerol-asparagine agar (ISP 5) (Shirling and Gottlieb 1966), potato-dextrose agar, Czapek’s agar and nutrient agar (Waksman 1967) for 14 days at 28 °C. The ISCC-NBS color charts were used to determine the designations of colony colors (Kelly 1964). Morphological characteristics were observed by light microscopy (SA3300-PL) and scanning electron microscopy (Hitachi; S-3400N) using cultures grown on ISP 2 medium at 28 °C for 14 days. The growth temperature (4, 10, 15, 20, 28, 37, 45 and 55 °C) and NaCl tolerance (0–15 %, at intervals of 1 %) was determined on ISP 2 agar at 28 °C for 14 days. The pH range (pH 4.0–12.0, at intervals of 1.0 pH units) for growth was determined using both solid and liquid ISP 2 medium at 28 °C for 14 days. Carbon-source utilization was tested by using ISP 9 medium (Shirling and Gottlieb 1966) supplemented with 1 % (final concentration) carbon sources. The utilization of amino acids as sole nitrogen sources was tested as described by Williams et al. (1983). Production of acid and other physiological and biochemical characteristics were tested by using the well established procedures (Gordon et al. 1974). The reference strains Streptomyces armeniacus JCM 3070T, Streptomyces pharmamarensis PM267T and Streptomyces artemisiae YIM 63135T were tested at the same condition in this study.

Chemotaxonomy

Biomass used for chemotaxonomic analyses was obtained from cultures grown in ISP 2 broth on a rotary shaker (about 150 rpm) for 8 days at 28 °C until good growth was obtained. Amino acids and sugars in whole-cell hydrolysates were analyzed according to the standard procedures (Lechevalier and Lechevalier 1980). Menaquinones were extracted and purified according to the method of Collins et al. (1977) and then analyzed by HPLC (Kroppenstedt 1985). Polar lipids were extracted and identified by two-dimensional TLC according to the method described by Minnikin et al. (1984). Fatty acids were analyzed using the standard MIDI (Microbial Identification, Sherlock version 6.0) procedure (Sasser 1990) and the gas chromatograph Agilent GC 6850. The resulting profiles were identified using the database library TSBA6 version 6.0.

Molecular analysis

Extraction of genomic DNA, PCR amplification and sequencing of 16S rRNA gene were carried out using the method of Li et al. (2007). The 1,422 bp sequence of strain KLBMP 4601T was aligned with those most closely related species by using CLUSTAL_X (Thompson et al. 1997). The 16S rRNA gene sequence similarity values were calculated by using the EzTaxon server (http://www.Eztaxon.org) (Chun et al. 2007). Phylogenetic trees were constructed using the neighbour-joining (Saitou and Nei 1987), maximum-parsimony (Fitch 1971) and maximum-likelihood (Felsenstein 1981) methods using MEGA version 5.0 (Tamura et al. 2011). The topologies of the phylogenetic trees were evaluated by the bootstrap resampling method of Felsenstein (1985) with 1,000 replicates. Determination of DNA G+C content was performed according to Mesbah et al. (1989). Levels of DNA–DNA relatedness were determined according to the fluorometric micro-well method (Ezaki et al. 1989; He et al. 2005).

Nucleotide sequence accession number

The 16S rRNA gene sequence of strain KLBMP 4601T determined in this study has been deposited in GenBank under the accession number JQ345722.

Results and discussion

Strain KLBMP 4601T showed abundant growth on ISP 2, ISP 3, Czapek’s agar and nutrient agar, moderate on ISP 4 and ISP 5 media and poor growth on PDA agar. White to gray-white aerial mycelium was present on these media. Substrate mycelium of strain KLBMP 4601T was yellowish-white to dark green on all media tested. Pink soluble pigment was formed on ISP 4 agar plate. The strain produced extensively branched substrate and aerial hyphae that carried straight to flexuous spore chains. The surface of the spores was smooth (Fig. 1). Differences in cultural characteristics from its closest related type strains are given in Table 1. Growth of strain KLBMP 4601T occurred in the pH range 6.0–8.0 and 0–7 % NaCl (w/v), with optimum growth at pH 7.0 and 3 % NaCl (w/v). The temperature range for growth was 4–45 °C, with the optimum temperature being 28 °C. Other physiological characteristics are given in the type strain description and Table 2.

Fig. 1
figure 1

Scanning electron micrograph of strain KLBMP 4601T, showing aerial mycelium and spore chains after growth on ISP 2 medium agar at 28 °C for 2 weeks. Bar 10.0 μm

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Table 1 Differential cultural characteristics of strain KLBMP 4601T and its closest phylogenetic neighbours
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Table 2 Features that distinguish strain KLBMP 4601T from the closely related Streptomyces species
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The cell wall of the novel isolate contained ll-diaminopimelic acid, which is characteristic for the genus Streptomyces. The whole-cell sugars were detected as mannose and glucose. The diagnostic phospholipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylinositolmannoside (PIM), two unidentified glycolipids and an unknown phospholipid (Supplementary Fig. S1). The phospholipid type, PII (Lechevalier et al. 1977), agrees with that reported for the genus Streptomyces. The major menaquinone found was MK-9(H4) (89.4 %), with minor amounts of MK-9(H8) (6.5 %), MK-9(H6) (1.9 %) and MK-10(H2) (2.2 %). The major fatty acids (>5.0 %) were C16:0 (15.8 %), iso-C16:0 (9.3 %), C18:1ω9c (8.9 %), C16:1 iso G (8.2 %), C17:1ω8c (8.0 %), C17:0 10-methyl (7.7 %), C16:1ω7c/C16:1ω6c (7.4 %), C17:1 iso w9c/C16:0 10-methyl (7.3 %) and anteiso-C17:1 A (5.4 %). A detailed fatty acid profile comparison with its nearest neighbour species is given in Table 3. The chemical properties of strain KLBMP 4601T are consistent with its classification as a member of the genus Streptomyces. The G+C content of the DNA was 69.0 mol%.

Table 3 Fatty acid profiles (%) of strain KLBMP 4601T and its nearest neighbour S. armeniacus JCM 3070T
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An almost-complete 16S rRNA gene sequence (1,422 bp) was obtained for the isolate. The sequence similarities between strain KLBMP 4601T and its closest relatives, S. armeniacus JCM 3070T, S. pharmamarensis PM267T and S. artemisiae YIM 63135T, were 97.9, 97.6 and 97.5 %, respectively. The 16S rRNA gene sequence similarities between strain KLBMP 4601T and the other type strains in this genus were <97.5 %. It was evident from the neighbor-joining dendrogram shown in Fig. 2 that the new isolate formed a distinct branch with S. armeniacus JCM 3070T, S. pharmamarensis PM267T and S. artemisiae YIM 63135T by a high bootstrap value of 99 %. This distinct branch was also recovered from maximum-parsimony and maximum-likelihood trees (Supplementary Fig. S2–S3). The mean DNA–DNA hybridization values found between the isolate and type strains of S. armeniacus JCM 3070T, S. pharmamarensis PM267T and S. artemisiae YIM 63135T were 36.3 ± 4.2 %, 27.3 ± 0.6 % and 30.9 ± 2.5 %, respectively, all of which are below the 70 % threshold value proposed by Wayne et al. (1987), indicating that strain KLBMP 4601T should be identified as a novel species.

Fig. 2
figure 2

Neighbour-joining tree based on almost complete 16S rRNA gene sequences (1,422 nt), showing the relationship between strain KLBMP 4601T and its phylogenetic neighbours. Only bootstrap values above 50 %, expressed as percentages of 1,000 replications, are shown at the branch points. Kitasatospora setae KM-6054T was used as the outgroup. Asterisks indicate that the corresponding nodes were also recovered in the maximum-parsimony and maximum-likelihood trees. Bar 0.005 substitutions per nucleotide position

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The characteristics shown in Table 1, 2 clearly indicate that strain KLBMP 4601T possesses obvious distinct phenotypic and chemotaxonomic profiles that distinguish it from its closest phylogenetic relatives. For example, the different growth characteristics on ISP 2 and ISP 3 media and only strain KLBMP 4601T produced soluble pink pigment on ISP 4 medium, differences in utilization of carbon sources and the menaquinones composition. For another, the major fatty acids of strain KLBMP 4601T are clearly different from the nearest neighbour S. armeniacus JCM 3070T (Table 3). Moreover, the differences in DNA G+C content, low level of DNA–DNA relatedness can be used to distinguish strain KLBMP 4601T from their closely related phylogenetic neighbours. Therefore, strain KLBMP 4601T represents a novel species of the genus Streptomyces, for which the name S. phytohabitans sp. nov. is proposed.

Description of S. phytohabitans sp. nov

Streptomyces phytohabitans (Phy.to.ha’bi.tans. Gr. n. phyton, plant; L. part. adj. habitans, inhabiting; N. L. part. adj. used as a masc. n. phytohabitans, plant inhabiting, isolated from a plant).

Aerobic, Gram-positive, catalase-positive actinomycete that forms white aerial mycelia and yellowish-white substrate mycelia on ISP 2 medium. The substrate mycelium does not fragment. Pink diffusible pigments are produced on ISP 4 agar. Produces straight to flexuous spore chains with smooth-surfaced spores (about 0.8–1.2 × 0.5–0.7 μm). Develops well on ISP 2, ISP 3, Czapek’s and nutrient agar. Moderate growth on ISP 4 and ISP 5. Growth occurs at 4–45 °C, at pH 6.0–8.0 and in the presence of 0–7 % (w/v) NaCl. Uses d-arabinose, cellobiose, cellulose, d-fructose, d-galactose, d-glucose, mannose, d-raffinose, d-ribose, l-rhamnose, sucrose, trehalose, xylitol and d-xylose as sole carbon and energy sources. Uses l-arginine, l-glutamic acid, l-glycine, l-histidine, l-lysine and l-proline as sole nitrogen sources. Acid is produced from arabinose, d-fructose, d-glucose, mannose and d-xylose. Positive for urease production, milk peptonization and coagulation, but negative for gelatin liquefaction and H2S production. Cell wall contains ll-diaminopimelic acid. The whole-cell hydrolysates contain mannose and glucose. The phospholipid composition includes DPG, PE, PG, PI, PIM, two unidentified glycolipids and an unknown phospholipid. Menaquinones found are MK-9(H4), MK-9(H6), MK-9(H8) and MK-10(H2). The major cellular fatty acids are C16:0, iso-C16:0, C18:1ω9c, C16:1 iso G, C17:1ω8c, C17:0 10-methyl, C16:1ω7c/C16:1ω6c, C17:1 iso w9c/C16:0 10-methyl and anteiso-C17:1 A. The G+C content of the DNA is 69.0 mol%.

The type strain, KLBMP 4601T (=KCTC 19892T = NBRC 108772T) was isolated from surface-sterilized roots of Curcuma phaeocaulis collected from the city of Panzhihua, Sichuan Province, south-west China.