Introduction

The genus Actinoplanes was proposed by Couch (1950) for actinomycetes that produced motile spores with spherical, cylindrical, digitate, lobate, bottle or flask-shaped or very irregular sporangia with tufts of polar flagella. Subsequently, a detailed phenotypic analysis of the genus has been given by Goodfellow et al. (1990), who determined the chemotaxonomic and phenotypic characteristics of species of Actinoplanes and reported that chemical and numerical taxonomic data supported the integrity of the genus. Furthermore, a comprehensive phylogenetic analysis of the genus has been given by Tamura and Hatano (2001). To date, 35 species of Actinoplanes with validly published names have been described. During a study on the ecological diversity of actinomycetes from soybean root, an aerobic actinomycete strain, NEAU-M9T, was isolated. In this study, the taxonomic status of this strain is reported based on phylogenetic, chemotaxonomic and phenotypic evidence. It is proposed that strain NEAU-M9T should be classified as representing a novel species of the genus Actinoplanes.

Materials and methods

Isolation and maintenance of the organism

Strain NEAU-M9T was isolated from soybean root collected from Hulin, Heilongjiang province, north China (46°16′N, 133°36′E). In the open, the plant was tagged and stored in a clean plastic bag until used (~24 h). The root sample was air dried for 24 h at room temperature and then washed with an ultrasonic step (160W, 15 min) (KH-160TDV, Hechuang, China) to remove the surface soils and adherent epiphytes completely. After drying, the sample was cut into pieces of 5–10 mm in length and then subjected to a seven-step surface sterilization procedure: a 60-s wash in sterile tap water containing cycloheximide (100 mg l−1) and nalidixic acid (20 mg l−1), followed by a wash in sterile water, a 5-min wash in 5 % NaOCl, a 10-min wash in 2.5 % Na2S2O3, a 5-min wash in 75 % ethanol, a wash in sterile water, and a final rinse in 10 % NaHCO3 for 10 min. The rinsed root sample was transferred temporarily onto sterile filter paper to eliminate excess moisture and then placed on a plate of humic acid-vitamin agar (HV) (Hayakawa and Nonomura 1987) supplemented with cycloheximide (50 mg l−1) and nalidixic acid (20 mg l−1). After 21 days of aerobic incubation at 28 °C, colonies were transferred and purified on oatmeal agar (ISP medium 3) and incubated at 28 °C for 2–3 weeks.

Morphological, cultural and physiological characteristics

Morphology of the sporangia and surface ornamentation of the sporangiospores were observed by scanning electron microscopy (Hitachi S-3400N) using cultures grown on ISP3 agar for 21 days. Spore motility was assessed by light microscopic (Nikon ECLIPSE E200) observation of cells suspended in phosphate buffer (pH 7.0, 1 mM). Cultural characteristics were determined after 3 weeks at 28 °C by methods used in the international Streptomyces project (ISP) (Shirling and Gottlieb 1966). Color determination was done with color chips from the ISCC-NBS Color Charts standard samples no 2106 (Kelly 1964). Growth over a range of temperatures, pH values and NaCl concentrations were tested using modified YEME medium (yeast extract 0.3 %, sucrose 10.3 %, tryptone 0.5 %, malt extract 0.3 %, glucose 1.0 % w/v, pH 7.2). Decomposition of cellulose, hydrolysis of starch, reduction of nitrate, liquefaction of gelatin and production of H2S were examined as described previously (Gordon et al. 1974). Carbon source utilization was tested using carbon source utilization (ISP9) medium (Shirling and Gottlieb 1966) supplemented with a final concentration of 1 % of the tested carbon sources. Nitrogen source utilization was examined using the basal medium recommended by Williams et al. (1983) supplemented with a final concentration of 0.1 % of the nitrogen sources tested.

Chemotaxonomic characterization

Biomass for chemical studies was prepared by growing the strain in GY medium (glucose 1.0 %, yeast extract 1.0 %, K2HPQ4·3H2O 0.05 %, MgSO4·7H2O 0.05 % w/v, pH 7.2–7.4) in Erlenmeyer flasks for 7 days at 28 °C. Cells were harvested by centrifugation, washed with distilled water and freeze-dried. The isomers of diaminopimelic acid (DAP) in the whole cell hydrolysates were analysed by HPLC method using Agilent TC-C18 Column (250 × 4.6 mm i.d. 5 μm) with a mobile phase consisting of acetonitrile: 0.05 mol l−1 phosphate buffer pH 7.2 = 15:85 at a flow rate of 0.5 ml min−1. The peak detection used an Agilent G1321A fluorescence detector with a 365 nm excitation and 455 nm longpass emission filters (McKerrow et al. 2000). The N-acyl group of muramic acid in peptidoglycan was determined by the method of Uchida et al. (1999). The whole-organism sugars were analyzed according to the procedures developed by Lechevalier and Lechevalier (1980). Polar lipids were examined by two-dimensional TLC and identified using the method of Minnikin et al. (1984). Menaquinones were extracted from freeze-dried biomass and purified according to Collins (1985). Extracts were analyzed by HPLC–UV method using Agilent Extend-C18 Column (150 × 4.6 mm, i.d. 5 μm), typically at 270 nm. The mobile phase was acetonitrile-propyl alcohol (60:40, v/v) and the flow rate was set to 1.0 ml min−1 and the run time was 60 min (Wu et al. 1989). Cellular fatty acids were analysed by GC–MS using the method of Xiang et al. (2011).

DNA base composition and DNA–DNA hybridization

The G+C content of the genomic DNA was determined by the thermal denaturation method as described by Mandel and Marmur (1968), and Escherichia coli JM109 was used as the reference strain. DNA–DNA relatedness test was performed in triplicate using the optical renaturation method as described by De Ley et al. (1970).

DNA preparation, amplification and determination of 16S rRNA sequence

Genomic DNA was extracted as described previously by Lee et al. (2003) and PCR amplification of 16S rRNA gene was carried out according to the procedures described by Loqman et al. (2009). The 16S rRNA gene sequence of strain NEAU-M9T was aligned with multiple sequences obtained from the GenBank/EMBL/DDBJ databases using Clustal X 1.83 software. The alignment was manually verified and adjusted prior to the construction of phylogenetic trees. Phylogenetic trees were generated with the neighbour-joining (Saitou and Nei 1987) and maximum-likelihood (Felsenstein 1981) algorithms using molecular evolutionary genetics analysis (MEGA) software version 5.05 (Tamura et al. 2011). The stability of the clades in the trees was appraised using a bootstrap value with 1,000 repeats (Felsenstein 1985). A distance matrix was generated using Kimura’s two-parameter model (Kimura 1980). All positions containing gaps and missing data were eliminated from the dataset (complete deletion option).

Results and discussion

Morphological observation of a 21-day-old culture of strain NEAU-M9T grown on ISP3 agar revealed it had the typical characteristics of genus Actinoplanes. Strain NEAU-M9T was observed to produce branched, non-fragmenting substrate hyphae which bore characteristic sporangia. The mature spore vesicles (4.56 × 10.72 μm) were found to be bell-shaped, and the surface of which were adorned by short hairs (Fig. 1). Sporangiospores were found to be motile. Strain NEAU-M9T was determined to grow well on ISP3 and ISP4 agar; moderate growth was observed on ISP2, ISP6 and ISP7 agar and poor growth on ISP5 agar, Aerial mycelia were not detected on any of the tested ISP media. The substrate mycelium of strain NEAU-M9T was observed to be pale yellow to deep reddish orange on all media tested. No diffusible pigment was detected on any of the tested media (Supplementary Table S1).

Fig. 1
figure 1

Scanning electron micrograph of strain NEAU-M9T grown on ISP3 medium for 21 days at 28 °C. Bar 5 μm

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Growth of strain NEAU-M9T was found to occur in the pH range 7–10 and 0–1 % NaCl (w/v), with optimum growth at pH 8.0 and 0 % NaCl (w/v). The temperature range for growth was determined to be 18–32 °C, with the optimum temperature being 28 °C. Strain NEAU-M9T contained meso-diaminopimelic acid as diamino acid. Whole-cell hydrolysates were found to contain galactose, mannose and ribose (Supplementary Fig. S1). The acyl type of the cell wall polysaccharides was determined to be glycolyl. The phospholipid profile was found to consist of phosphatidylethanolamine and phosphatidylinositol (phospholipid type PII; Lechevalier et al. 1977) (Supplementary Fig. S2). The menaquinones detected were MK-9(H4) (59.8 %), MK-9(H6) (40.3 %). The cellular fatty acid profile was determined to be composed of C14:0 (4.7 %), C15:0 (1.1 %), C16:0 (50.0 %), C16:1ω7c (1.5 %), C17:0 cyclo (1.2 %), C18:1ω9c (5.0 %) and C18:0 (35.2 %) (fatty acids representing <1.0 % of the total are not reported) (Supplementary Table S2).

The almost-complete 16S rRNA gene sequence (1,511 nt) of strain NEAU-M9T was determined and deposited as JQ073723 in the GenBank/EMBL/DDBJ databases. Blast sequence analysis of the 16S rRNA gene sequence showed that the strain was affiliated to the genus Actinoplanes. The closest phylogenetic relatives were Actinoplanes campanulatus DSM 43148T (98.85 %), Actinoplanes capillaceus DSM 44859T (98.70 %), Actinoplanes lobatus DSM 43150T (98.30 %), Actinoplanes auranticolor DSM 43031T (98.23 %) and Actinoplanes sichuanensis 03-723T (98.06 %); lower sequence similarities (<97.56 %) were found with the type strains of all other members of the genus Actinoplanes with validly published names. The phylogenetic tree (Fig. 2) based on 16S rRNA gene sequences showed that strain NEAU-M9T formed a distinct phyletic line with A. campanulatus DSM 43148T and A. capillaceus DSM 44859T, an association that was supported by maximum-likelihood algorithm employed (Supplementary Fig. S3) and by a 86 % bootstrap value in the neighbour-joining analysis. To establish the precise taxonomic position of strain NEAU-M9T, DNA–DNA hybridizations performed between the novel isolate and A. campanulatus DSM 43148T, A. capillaceus DSM 44859T and A. lobatus DSM 43150T; the levels of DNA–DNA relatedness between them were 52.2 ± 0.7, 50.8 ± 0.7 and 15.2 ± 1.4 %, respectively. These values were below the threshold value of 70 % recommended by Wayne et al. (1987) for assignment of strains to the same species. Strain NEAU-M9T was noted to share many morphological and chemotaxonomic characteristics with other species of the genus Actinoplanes, including the development of motile sporangiospores; the scanty presence of aerial hyphae; MK-9(H4) as the predominant menaquinone, the presence of meso-diaminopimelic acid in the whole-cell peptidoglycan; and phosphatidylethanolamine as the diagnostic phospholipid (Couch 1950; Goodfellow and Cross 1984; Kothe 1987; Vobis 1989; Kämpfer et al. 2007). However, the whole-cell sugar patterns (absence of arabinose and xylose) and major cellular fatty acid compositions (C16:0 and C18:0) of strain NEAU-M9T could differentiate it from its most related type strains of the genus Actinoplanes, which contain arabinose and xylose as characteristic sugars and iso-C15:0, iso-C16:0 and C17:1ω9c as predominant fatty acids (Supplementary Table S2). In addition, the isolate could also be clearly distinguished from the most closely related type strains of the genus Actinoplanes based on physiological and biochemical characteristics as summarized in Table 1. Data from the present polyphasic taxonomic study, together with the DNA–DNA hybridization data, indicated that strain NEAU-M9T represents a novel species of the genus Actinoplanes, for which we propose the name Actinoplanes hulinensis sp. nov.

Fig. 2
figure 2

Neighbour-joining tree showing the phylogenetic position of strain NEAU-M9T and related taxa based on 16S rRNA gene sequences. Asterisks indicate branches that were also found using maximum-likelihood methods. Numbers at branch points indicate bootstrap percentages (based on 1,000 replicates); only values >50 % are indicated. Bar 0.005 substitutions per nucleotide position

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Table 1 Differential physiological characteristics of strain NEAU-M9T and the type strains of related Actinoplanes species
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Description of Actinoplanes hulinensis sp. nov.

Actinoplanes hulinensis (hu.lin.en′sis. N.L. masc. adj. hulinensis, pertaining to Hulin, China, from where the sample was collected).

Aerobic actinomycete which produces branched, non-fragmenting substrate hyphae which carry bell-shaped spore vesicles (4.56 × 10.72 μm). Sporangiospores are motile and the surface is covered by short hairs. Substrate mycelium is pale yellow to deep reddish orange on ISP2, ISP3, ISP4, ISP5, ISP6 and ISP7 media. Aerial mycelium is absent. Soluble pigment and melanin are not formed. Negative for decomposition of cellulose, reduction of nitrate and production of H2S and positive for hydrolysis of starch and liquefaction of gelatin. Arabinose, fructose, galactose, glucose, inositol, lactose, maltose, mannose, mannitol, raffinose, rhamnose, sorbitol and sucrose are utilized as sole carbon sources but ribose and xylose are not utilized. Alanine, arginine, asparagine, creatine, glycine, glutamine, threonine and tyrosine are utilized as sole nitrogen sources but arginine are not. Tolerates up to 1 % NaCl and grows at temperatures between 18 and 32 °C, with an optimum temperature of 28 °C. Growth occurs at initial pH values between 7 and 10, the optimum being pH 8.0. Whole-cell hydrolysates contains meso-DAP. Whole-cell hydrolysates comprise galactose, mannose and ribose. The acyl type of the cell wall polysaccharides is glycolyl. The major menaquinone are MK-9(H4) and MK-9(H6). The phospholipid profile comprises phosphatidylethanolamine and phosphatidylinositol (phospholipid type PII). Major fatty acids are C16:0 and C18:0. The G+C content of the DNA is 67.90 mol%.

The type strain is NEAU-M9T (= CGMCC 4.7036T = DSM 45728T), isolated from soybean root collected from Hulin, Heilongjiang province, China. The GenBank/EMBL/DDBJ database accession number of the 16S rRNA sequence of strain NEAU-M9T is JQ073723.