Introduction

The genus Streptomyces was first proposed by Waksman and Henrici (1943). Since then, more than 700 species with validly published names have been reported (http://www.bacterio.net/index.html, 2016). The genus Streptomyces remains a unique source for novel antibiotics, novel bioactive products and pharmacologically active compounds (Watve et al. 2001; Abdalla et al. 2010; Goodfellow and Fiedler 2010; Rateb et al. 2011a, b; Kim et al. 2012a; Hayakawa et al. 2015; Také et al. 2015). Till date, more researchers focused on isolating novel Streptomyces from special habitats including hot springs, marine sediments, medicinal plants or their rhizospheric soil and speleothem, with the view for isolating new therapeutic compounds or secondary metabolites from novel strains (Liu et al. 2013; Carmona-Novillo et al. 2014; Han et al. 2015; Khieu et al. 2015; Marta et al. 2015). As part of a study focusing on isolation of actinomycetes from traditional and precious Chinese herbal and medicinal plants, roots and rhizospheric soil samples of Psammosilene tunicoides in Yunnan, China, were collected. More than 300 actinomycete strains were isolated during the process, of which many were found to belong to the genus Streptomyces. The isolate YIM T102T was one such Streptomyces strain isolated from rhizospheric soil. The strain YIM T102T was characterized by phenotypic, chemotaxonomic and phylogenetic analysis. Based on the findings of the polyphasic study, isolate YIM T102T is characterized as a novel species of the genus Streptomyces.

Materials and methods

Isolation and culture conditions

For the isolation of actinobacteria from the rhizospheric soil of P. tunicoides, 2 g of soil sample collected from Lijiang was pretreated at 50 °C for 2 h and taken into a conical flask containing 18 mL sterile water and several glass beads. The mixture was kept incubated under shaken conditions (28 °C, 200 rpm, 2 h). The sample suspensions were then diluted 100-fold, and 200 μL of the diluted suspension spread on oatmeal agar (International Streptomyces Project medium 3 or ISP 3; Shirling and Gottlieb 1966) plate. The isolation media were supplemented with nalidixic acid (25 mg L−1) and nystatin (50 mg L−1) to inhibit growth of fastidious bacteria and fungi. The plates were incubated at 28 °C for 15 days. Purified strain YIM T102T was routinely cultured on yeast extract–malt extract agar (ISP 2) medium (Shirling and Gottlieb 1966) at 28 °C and stored as a glycerol suspension (20 %, w/v) at −80 °C.

The reference type strain Streptomyces eurocidicus NBRC 13491T was obtained from NITE Biological Resource Center (NBRC), Japan. The strain was maintained routinely on ISP 2 medium (28 °C, 7 days). Biomass of strain YIM T102T and the reference type strain for chemical and molecular tests was harvested from cultures grown on ISP 2 and/or tryptic soy broth (TSB, Difco) (28 °C, 6 days).

Phenotypic characteristics

Morphological and cultural characteristics were tested on ISP 2, ISP 3, inorganic salts-starch agar (ISP 4), glycerol–asparagine agar (ISP 5) (Shirling and Gottlieb 1966), potato dextrose agar (PDA), Czapek’s agar and nutrient agar (Waksman 1967). The colors of the colony were determined by using the ISCC-NBS color charts (Kelly 1964). The morphological characteristics of strain YIM T102T were observed by a light microscope (BH-2; Olympus, Tokyo, Japan) and scanning electron microscopy (ESEM-TMP) from the cultures grown on ISP 2 medium at 28 °C for 7 days. The spore chain morphology, spore size and surface ornamentation of isolate were observed. Growth at various NaCl concentrations (0–12 % w/v, at intervals of 1 % units) and different temperatures (10–60 °C, at intervals of 5 °C units) was examined by growing the strain on ISP 2 plates. The pH range for growth [4–12, at intervals of 1 pH unit prepared by using the buffer system as described by Xu et al. (2005)] was tested at 28 °C for 30 days by culturing the strains with ISP 2 broth. Activities of oxidase, catalase and urease, gelatin liquefaction, milk peptonization and coagulation, nitrate reduction, H2S production, degradation of tweens 20, 40, 60 and 80, starch and cellulose were investigated according to the conventional procedures described by Williams et al. (1989) and Gordon et al. (1974). Carbon source utilization tests were performed according to the methods described by Shirling and Gottlieb (1966) and Athalye et al. (1985) using modified basal medium recommended by Pridham and Gottlieb (1948). Nitrogen sources utilization was observed according to Nie et al. (2012). Other physiological and biochemical characteristics were assessed by using the media and methods described by Gordon et al. (1974). Antibiotic susceptibility tests were performed by using antibiotic disks (μg per disk, unless indicated otherwise): amikacin (30), cefuroxime sodium (30), chloramphenicol (30), ciprofloxacin (5), erythromycin (15), ethylhydrocupreine (5), tetracycline (30), gentamicin (10), norfloxacin (10), novobiocin (30), oxacillin (1), penicillin (10 IU), piperacillin (100), polymyxin B (300 IU) sulfamethoxazole (300) and vancomycin (30). Disks were placed on ISP 2 agar plates spread with strain YIM T102, and the plates were incubated at 28 °C for 3 days.

Chemotaxonomy

Chemotaxonomic characteristics were determined following the standard procedures. The isomer of diaminopimelic acid of cell wall and sugars of whole-cell hydrolysates were analyzed as described by Hasegawa et al. (1983), Staneck and Roberts (1974) and Tang et al. (2009). Polar lipids were extracted, separated by two-dimensional thin-layer chromatography (TLC) and identified using the described procedures (Minnikin et al. 1979; Collins and Jones 1980). Menaquinones were extracted from lyophilized cells as described by Collins et al. (1977) and Minnikin et al. (1984) and analyzed by HPLC (Kroppenstedt 1982; Hu et al. 2001). For analysis of cellular fatty acids, strains YIM T102T and S. eurocidicus NBRC 13491T were cultured under shaking condition using TSB medium (180 rpm, 7 days, 28 °C). The cellular fatty acids were extracted, methylated and analyzed by using the protocol of the Sherlock Microbial Identification System (MIDI) (Sherlock Version 6. 1; MIDI database: TSBA6) (Sasser 1990). The G+C content of the genomic DNA was determined by HPLC (Mesbah et al. 1989) using Escherichia coli JM-109 as the reference strain.

Molecular analysis

Genomic DNA extraction and PCR amplification of the 16S rRNA gene sequences were performed as described by Li et al. (2007). The amplicon was purified using a Sangon PCR purification kit (China). Purified PCR amplicon was sequenced in Sangon Biotech, Shanghai, using the Sanger sequencing method. The full-length 16S rRNA gene sequence of strain YIM T102T was compared with cultured species from NCBI database via BLAST search (Altschul et al. 1990) and EzTaxon-e server database (Kim et al. 2012b). Multiple sequence alignments were performed using the CLUSTAL_X software package (Thompson et al. 1997). The Kimura two-parameter model (Kimura 1980, 1983) was used to calculate evolutionary distance. Phylogenetic trees were constructed by the neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) tree-making algorithms using the software packages MEGA version 5.0 (Tamura et al. 2011). Bootstrap analysis with 1000 resamplings was used to evaluate the topology of each tree (Felsenstein 1985). DNA–DNA relatedness was studied by applying the fluorometric micro-well method (Ezaki et al. 1989; Christensen et al. 2000; He et al. 2005) at the optimal hybridization temperature (50 °C). The experiments were set with eight replications between strain YIM T102T and the reference type strain S. eurocidicus NBRC 13491T.

Antimicrobial assay

Strain YIM T102T was evaluated for antimicrobial activities against seven test fungi and bacteria: Alternaria alternata, Alternaria brassicae, Colletotrichum nicotianae Averna, Escherichia coli, Monilia albican, Pseudomonas aeruginosa and Staphylococcus aureus Rosenbach, by dual-culture antagonistic bioassay method. The test organisms were obtained from Yunnan Institute of Microbiology, Yunnan University, China. Sterile disks (8 mm diameter) were impregnated with cultures of strain YIM T102T grown on ISP 2 agar (5 days, 28 °C). The culture disks were then placed at the center of PDA agar plates previously spread with test organism, viz. Alternaria alternata, Alternaria brassicae, Colletotrichum nicotianae Averna, Staphylococcus aureus Rosenbach, and LB agar plates with Escherichia coli, Monilia albican and Pseudomonas aeruginosa. All plates were incubated at 28 °C for 5 days. All the assays were performed in triplicate.

Results and discussion

YIM T102T grew well on ISP 2, ISP 3 and PDA, moderately on ISP 4, ISP 5 and Czapek’s agar, and weakly on nutrient agar. Aerial mycelium was produced on all tested media. Colors of the substrate mycelium varied between white, yellow-white, yellow-green cream-white or yellow, while the aerial mycelium had cream-white, white, light gray, medium gray or deep gray colors on the tested media. No diffusible pigment was produced on all media (Supplementary Table S1). A week-old culture of strain YIM T102T had morphological properties typical of the genus Streptomyces such as abundant aerial hyphae and vegetative mycelium, long spore chains with rhabditiform-shaped spores having smooth surfaces (Supplementary Fig. S1).

Growth was observed at 0–9 % (NaCl, w/v) (optimum 1–3 %), 10–40 °C (optimum 25–30 °C) and pH 6–9 (optimum pH 7). The strain was found to be positive for oxidase and catalase tests, but negative for urease activity. It gave positive results for coagulation and peptonization of milk, hydrolysis of starch, cellulose and gelatin, while negative results for nitrate reduction and H2S production tests. The strain could degrade Tweens 20, 40, 60 and 80. The strain was susceptible to the following antibiotics: amikacin, cefuroxime sodium, chloramphenicol, ciprofloxacin, tetracycline, gentamicin, novobiocin, penicillin, piperacillin, polymyxin B, sulfamethoxazole and vancomycin, while resistant to erythromycin, ethylhydrocupreine, norfloxacin and oxacillin. Characteristics that differentiate strain YIM T102T phenotypically from its closest related strain are listed in Table 1. The detailed physiological characteristics of strain YIM T102T are given in species description.

Table 1 Differential characteristics between strains YIM T102T and S. eurocidicus NBRC 13491T
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The diagnostic cell wall diamino acid of the strain YIM T102T was ll-diaminopimelic acid (ll-DAP), while glycine was also found in the peptidoglycan. The whole-cell sugars consisted of glucose, mannose, ribose and galactose. The polar lipids of strain YIM T102T comprised of diphosphatidylglycerol, phosphatidyl methyl ethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and three unidentified phospholipids (Supplementary Fig. S2). The respiratory menaquinones of strain YIM T102T were found to be MK-9 (H6) and MK-9 (H8). The fatty acids profile (>5 %) was summed feature 4 comprising iso-C17:1 I and/or anteiso-C17:1 B (38.0 %), anteiso-C15:0 (13.1 %), iso-C16:0 (10.1 %), summed feature 3 comprising C16:1 ω7c and/or C16:1 ω6c (9.8 %), C16:0 (9.0 %) and iso-C15:0 (5.2 %). Detailed fatty acid profiles of strain YIM T102T and the reference type strain are shown in Table 1. The DNA G+C content of strain YIM T102T was determined to be 70.7 mol%, which is in accordance with the level for the genus Streptomyces (67–78 mol%).

To determine the phylogenetic position, full-length 16S rRNA gene sequence (1518 nt; accession number KU936048) of strain YIM T102T was determined. Comparison of the sequence with the corresponding 16S rRNA gene sequences retrieved from GenBank/EMBL/DDBJ clearly demonstrated that strain YIM T102T was a member of the genus Streptomyces with highest sequence similarity with S. eurocidicus NBRC 13491T. In the neighbor-joining phylogenetic tree (Fig. 1), strain YIM T102T formed a clade with five Streptomyces strains, but their 16S rRNA gene sequence similarities were less than 98.5 % except for S. eurocidicus NBRC 13491T (98.9 % similarity). This phylogenetic relationship was also supported in the trees generated with maximum-parsimony phylogenetic tree and maximum-likelihood phylogenetic tree. Based on the phylogenetic analyses, sequence similarities profile and recommendation of Stackebrandt and Ebers (2006), the strain S. eurocidicus NBRC 13491T was considered for DNA–DNA hybridization study with strain YIM T102T. DNA–DNA relatedness value between strain YIM T102T and the type strains S. eurocidicus NBRC 13491T was determined to be 37.8 ± 1.8 %, which was notably lower than the threshold value (70 %) for the recognition of genomic species (Stackebrandt and Goebel 1994).

Fig. 1
figure 1

Neighbor-joining phylogenetic tree showing the phylogenetic relationship of strain YIM T102T and other closely related Streptomyces species based on 16S rRNA gene sequences. Asterisks indicate branches that were also recovered using the maximum-parsimony and maximum-likelihood methods. Bootstrap values (expressed as percentages of 1000 replications) of above 50 % are shown at branch points. Bar 0.005 substitutions per nucleotide position

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Strain YIM T102T exhibited antagonistic activity against the fungi Alternaria alternata, Alternaria brassicae and Colletotrichum nicotianae Averna, but not against Escherichia coli, Monilia albican, Pseudomonas aeruginosa or Staphylococcus aureus Rosenbach.

The phylogenetic analysis, morphological and chemotaxonomic characteristics support the characterization of strain YIM T102T as a member of the genus Streptomyces. However, the differences in biochemical characteristics, DNA–DNA relatedness values and fatty acid compositions distinguish strain YIM T102T from its closest related strain S. eurocidicus NBRC 13491T. Therefore, based on these results, strain YIM T102T is considered to represent a novel species of genus Streptomyces, for which the name Streptomyces zhihengii sp. nov is proposed.

Description of Streptomyces zhihengii sp. nov

Streptomyces zhihengii (zhi.hen’gi.i. N.L. gen. masc. n. zhiheng of Zhi-Heng, to Honor Zhi-heng Liu, a respected Chinese microbiologist, for his enormous contributions to the development of Streptomyces taxonomy in China).

Cells are Gram-staining positive and aerobic. Forms extensively branched substrate and aerial mycelia. Substrate mycelia range its colors from white, yellow-white, yellow-green, cream-white to yellow, while aerial mycelia are cream-white, white, light gray, medium gray or deep gray colors on tested media. No diffusible pigments are produced on the media tested. Growth occurs at 10–40 °C, pH 6.0–9.0 and in the presence of up to 9 % (w/v) NaCl. Utilizes cellobiose, d-galactose, d-glucose, maltose, d-mannose, d-xylitol as the sole carbon and energy sources, but not dulcitol, d-fructose, d-sucrose, sorbinose, l-arabinose, d-xylose and lactose. Utilizes l-alanine, l-arginine, l-asparagine, l-histidine, l-cystine, l-glutamic acid, hypoxanthine, l-lysine, l-phenylalanine, l-serine, l-threonine, l-tryptophan, l-tyrosine and l-valine as sole nitrogen sources. Positive for catalase and oxidase tests, milk coagulation and peptonization, hydrolysis of starch, cellulose and gelatin, but negative for of urease activity, nitrate reduction and H2S production tests. Degrades Tweens 20, 40, 60 and 80. The diagnostic cell wall diamino acid is ll-diaminopimelic acid (ll-DAP). The whole-cell hydrolysates contain glucose, mannose, ribose and galactose. The polar lipids consist of diphosphatidylglycerol, phosphatidyl methyl ethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and three unidentified phospholipids. MK-9 (H6) and MK-9 (H8) are the menaquinones detected. The fatty acids profile (>5 %) is composed of iso-C15:0, anteiso-C15:0, iso-C16:0, C16:0, summed feature 3 and summed feature 4. The DNA G+C content is 70.7 mol%.

The type strain YIM T102T (=KCTC 39115T = DSM 42176T = CGMCC 4.7248T) was isolated from rhizospheric soil of P. tunicoides W. C. Wu et C. Y. Wu in Lijiang, Yunnan Province, southwest China.

The 16S rRNA gene sequence of strain YIM T102T has been deposited in GenBank under the accession number KU936048.