Phenotypic analysis
Morphological observation of 3-week-old cultures of strain HDS12T grown on Gause's synthetic medium revealed that it had the typical characteristics of the members of the genus Nocardiopsis. Cells of strain HDS12T were Gram-stain-positive and aerobic. The isolate formed smoke gray substrate hyphae and well-developed light grayish olive aerial mycelium on Gause's synthetic medium which produced straight to flexuous spore chains spore chains arranged in rod spores with smooth-surfaced (Fig.1). Strain HDS12T was found to grow well and produce well-developed aerial mycelia on ISP 2, 3, 5, 6, 7 and Gause's synthetic medium, but no growth was observed on ISP 4 agar medium. Soluble pigments and melanin pigments were not produced on all tested media. The strain was found to grow at a temperature range of 20–37°C (optimum temperature 28°C), pH 6.0–8.0 (optimum pH of 7.0) and NaCl tolerance of 0–11% (optimum 0–1%). The physiological and biochemical properties of strain HDS12T were shown in Table 1.
Chemotaxonomic characterization
All the chemotaxonomic data were consistent with the assignment of strain HDS12T to the genus Nocardiopsis. Whole-cell hydrolysates of strain HDS12T contained meso-diaminopimelic acid as the diagnostic diamino acid and no diagnostic sugars. The menaquinones were MK-10(H2) (45.6%), MK-10(H4) (30.9%) and MK-10(H6) (22.1%). The major cellular fatty acid profile (>10.0%) was composed of iso-C16:0 (43.2%) and C18:1ω9c (19.0%). The polar lipids included diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), hydroxyl phosphatidylethanolamine (OH-PE), phosphatidyl choline (PC), phosphatidylinositol mannosides (PIM), phospholipids (PLS), phospholipids of unknown structure containing glucosamine (NPG) and unidentified phospholipids (L1 and L2) (Fig. S1).
Phylogeny and DNA-DNA correlation analysis
A blast search of the 16S rRNA gene sequence from the EzBioCloud database indicated that strain HDS12T was grouped into the genus Nocardiopsis and exhibited 99.79% similarity to N. dassonvillei subsp. dassonvillei CGMCC 4.1231T, 99.73% similarity to N. deserti H13T, 99.66% similarity to N. alborubida NBRC 13392T, 99.64% similarity to N. dassonvillei subsp. crassaminis D1T, 99.45% similarity to N. synnemataformans DSM 44143T, 99.04% similarity to N. lucentensis DSM 44048T, 98.90% similarity to N. aegyptia DSM 44442T, 98.76% similarity to N. flavescens CGMCC 4.5723T, 98.69% similarity to N. alba DSM, 98.63% similarity to N. halotolerans DSM 44410T and <98.60% similarities to various other strains. The ML phylogenetic analysis based on 16S rRNA gene sequences showed that strain HDS12T formed an independent subclade (Fig. 2). These results were also supported by NJ and MP phylogenetic trees (Figs S2 and S3), which suggested that strain HDS12T could belong to potential novel species. Considering that the phylogenomic analysis exhibited better resolution than the phylogenetic analysis based on 16S rRNA gene sequence (Rodriguez et al. 2018), so, in the present work, the phylogenomic analysis was carried out in order to clarify taxonomic status of strain HDS12T. It was shown in Fig. 3, strain HDS12T was clustered together with N. dassonvillei subsp. dassonvillei CGMCC 4.1231T and N. dassonvillei subsp. crassaminis D1T, suggesting that it was closely related to N. dassonvillei subsp. dassonvillei CGMCC 4.1231T and N. dassonvillei subsp. crassaminis D1T. However, the average nucleotide identity value (ANI) and the digital DNA-DNA hybridization (dDDH) value between HDS12T and N. dassonvillei subsp. dassonvillei CGMCC 4.1231T were 94.52% and 60.40%, 94.27% and 60.50%, respectively, less than 95-96% and 70% cut-off points recommended for delineating specie (Richter and Rosselló-Móra 2009; Wayne et al. 1987), which further supported that strain HDS12T represented a novel species of the genus Nocardiopsis. Furthermore, given that N. dassonvillei subsp. dassonvillei CGMCC 4.1231T and N. dassonvillei subsp. crassaminis D1T belong to the same species, the difference comparisons were only carried out between HDS12T and N. dassonvillei subsp. dassonvillei CGMCC 4.1231T. Results indicated that the cultural, physiological and biochemical characteristics were dissimilar enough to distinguish strain HDS12T from N. dassonvillei subsp. dassonvillei CGMCC 4.1231T (Table 1). Meanwhile, according to the description of Stackebrandt and Eber (Stackebrandt and Ebers 2006), if 16S rRNA gene sequence similarity between two strains ≥98.7 %, ANI and dDDH or DDH values need to be calculated to evaluate their DNA–DNA relatedness in delineating new species. In the present work, considering that strain HDS12T had 99.73%, 99.64%, 99.45%, 99.04%, 98.90%, 98.76%, 98.69% similarities to N. deserti H13T, N. alborubida NBRC 13392T, N. synnemataformans DSM 44143T, N. lucentensis DSM 44048T, N. aegyptia DSM 44442T, N. flavescens CGMCC 4.5723T, and N. alba DSM, respectively, the ANI and dDDH were calculated to clarify taxonomic relations between strain HDS12T and them. Results indicated that the ANI and dDDH values between them were 78.67%-94.27% and 23.50%-60.50% (Table 2), respectively, below the 95–96 and 70 % cutoff points recommended for delineating species (Richter and Rosselló-Móra 2009; Wayne et al. 1987). All these results confirmed that strains HDS12T should belong to a potential novel species.
Taxonomic conclusion
According on the above data, strain HDS12T can be clearly distinguished from phylogenetically related Nocardiopsis species and thus should be regarded as a new species of the genus Nocardiopsis, for which the name Nocardiopsis akebiae sp. nov. is proposed.
DESCRIPTION OF NOCARDIOPSIS AKEBIAE SP. NOV.
Nocardiopsis akebiae (a.ke'bi.ae. N.L. gen. n. akebiae referring to come from Akebia trifoliata).
Aerobic, non-motile and Gram-stain positive actinomycete that produces smoke gray substrate hyphae and well-developed light grayish olive aerial mycelium which produced straight to flexuous spore chains spore chains arranged in rod spores with smooth-surfaced. Good growth occurs on ISP 2, 3, 5, 6 and 7 and Gause's synthetic medium, while no growth occurs on ISP 4 agar medium. No soluble pigments and melanin pigments are produced on the above media. Growth occurs at a temperature range of 20–37°C (optimum temperature 28°C), pH 6.0–8.0 (optimum pH of 7.0) and in the maximum presence of 11% (w/v) (optimum 0–1%). Positive for starch hydrolysis, but negative for reduction of nitrate, production of urease, coagulation and peptonization of milk and degradation of Tweens (20, 40 and 80). d-Fructose, d-galactose, d-xylose, l-arabinose, l-rhamnose and myo-inositol are utilized as sole carbon sources, but not cellulose, d-mannose, lactose, raffinose and trehalose. Cell walls contain meso-diaminopimelic acid as the diagnostic diamino acid and no diagnostic sugars. The menaquinones are MK-10(H2), MK-10(H4) and MK-10(H6). The major cellular fatty acid profile (>10.0%) is iso-C16:0 and C18:1ω9c. The polar lipids contain diphosphatidylglycerol, phosphatidylethanolamine, hydroxyl phosphatidylethanolamine, phosphatidyl choline, phosphatidylinositol mannosides, phospholipids, phospholipids of unknown structure containing glucosamine (NPG) and unidentified phospholipids.
The type strain, HDS12T (=MCCC 1K06173T = JCM 34708T) was isolated from fruits of Akebia trifoliate, in Changde of Hunan Province, China. The genomic DNA G+C content of the type strain is 72.4mol%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and genome sequence of strain HDS12T are OM368592 and CP074132, respectively.