Multi-locus sequence analysis (MLSA) has been proved to be a useful method for Streptomyces identification and MLSA distance of 0.007 is considered as the boundary value. However, we found that MLSA distance of 0.007 might be insufficient to act as a threshold according to the correlations among average nucleotide identity based on MuMmer ultra-rapid aligning tool (ANIm), digital DNA–DNA hybridization (dDDH) and MLSA from the 80 pairs of Streptomyces species; in addition, a 70% dDDH value did not correspond to a 95∼96% ANIm value but approximately to 96.7% in the genus Streptomyces. Based on our analysis, it was proposed that when the MLSA distance value between a novel Streptomyces and a reference strain was < 0.008, the novel strain could be considered as a heterotypic synonym of the reference strain; when the MLSA distance value was ≥ 0.014, the novel strain could be regarded as a new Streptomyces species; when the MLSA distance value was between 0.008 and 0.014 (not included), the dDDH or ANIm value between a new strain and a reference strain must be calculated in order to determine the taxonomic status of a novel strain. In this context, a 70% dDDH or 96.7% ANIm value could act as the threshold value in delineating Streptomyces species, but if the dDDH or ANIm value was less than but close to 70 or 96.7% cut-off point, the taxonomic status of a novel strain could only be determined by a combination of phenotypic characteristics, chemotaxonomic characteristics and phylogenomic analysis.

Two strains of Actinobacteria, designated CRXT-Y-14^T and CRXT-G-22^T, were isolated from the healthy leaves and seeds, respectively, of a medicinal plant Xanthium sibiricum. Their taxonomic positions were determined using a polyphasic approach. Strain CRXT-Y-14^T produced flexuous chains of smooth-surfaced spores. Strain CRXT-G-22^T produced straight chains of smooth-surfaced spores. Their morphological features were consistent with the diagnostic characteristics of members of the genus Streptomyces . The results of 16S rRNA gene sequence analyses indicated two strains represented members of the genus Streptomyces . CRXT-Y-14^T shared 99.3, 98.9, 98.8 % sequence similarities to Streptomyces atriruber NRRL B-24165^T, Streptomyces avermitilis MA-4680^T and Streptomyces davaonensis JCM 4913^T, respectively. Whilst CRXT-G-22^T exhibited highest similarity to Streptomyces acidiscabies ATCC 49003^T (98.9 %). The results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the closest phylogenetic neighbours of strains CRXT-Y-14^T and CRXT-G-22^T were S. atriruber NRRL B-24165^T and S. acidiscabies ATCC 49003^T, respectively. The phylogenomic analyses further confirmed the relative relationship between strain CRXT-G-22^T and S. acidiscabies ATCC 49003^T, but indicated that CRXT-Y-14^T could represent a novel species of the genus Streptomyce. However, the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between CRXT-Y-14^T and strain CRXT-G-22^T, between CRXT-Y-14^T and S. atriruber NRRL B-24165^T, and between CRXT-G-22^T and S. acidiscabies ATCC 49003^T were 85.4 and 23.2 %, 85.8 and 23.9 % and 89.1 and 34.1 %, respectively, far below the 95~96 and 70 % cut-off points recommended for delineating species. Furthermore, these two novel isolates were distinctly differentiated from their relatives in the genus Streptomyces with respect to phenotypic and chemotaxonomic characteristics. On the basis of these data, CRXT-Y-14^T and CRXT-G-22^T clearly represent two novel species within the genus Streptomyces , for which the names Streptomyces xanthii sp. nov. (type strain CRXT-Y-14^T = MCCC 1K04966^T= JCM 34527^T) and Streptomyces roseirectus sp. nov. (type CRXT-G-22^T = MCCC 1K04979^T= JCM 34565^T) are proposed.

At present, it is widely believed that a 95–96% average nucleotide identity (ANI) value is equivalent to a 70% digital DNA–DNA hybridization (dDDH) value in the prokaryotic taxonomy. However, in the present study, comparative genome analysis of 29 pairs of Amycolatopsis type strains revealed that a 70% dDDH value did not correspond to a 95–96% ANI based on the MuMmer ultra-rapid aligning tool (ANIm) but approximately corresponded to a 96.6% ANIm value in the genus Amycolatopsis. Based on this corresponding relationship, phenotypic and chemotaxonomical characteristics, as well as phylogenetic analysis, an actinobacterial strain HUAS 11-8^T isolated from the rhizosphere soil of Cynara scolymus, was subjected to a polyphasic taxonomic characterization. Based on EzBioCloud alignment, it was found that strain HUAS11-8^T had the 16S rRNA gene similarities of 99.78% with A. rhizosphaerae JCM 32589^T, 97.8% with A. dongchuanensis YIM 75904^T, and < 97.8% sequence similarities to other Amycolatopsis species. Phylogenetic analysis of 16S rRNA gene sequences and whole-genome sequences revealed that strain HUAS 11-8^T was closely related to A. rhizosphaerae JCM 32589^T. ANIm and dDDH values between strains HUAS 11-8^T and A. rhizosphaerae JCM 32589^T were 96.3 and 68.5%, respectively, lower than the 96.6 and 70% thresholds recommended for the delineation of a novel Amycolatopsis species. Consequently, strain HUAS 11-8^T should represent a novel Amycolatopsis species, for which the name Amycolatopsis cynarae sp. nov. (type strain HUAS 11-8^T = MCCC 1K08337^T = JCM 35980^T) is proposed. Furthermore, based on comparative genomic analysis and rule 42 of the Prokaryotic Code, we propose that Amycolatopsis niigatensis is a later heterotypic synonym of Amycolatopsis echigonensis.

A novel actinomycete, designated strain QMT-28T, was isolated from rhizosphere soil of Fagopyrum dibotrys collected from Shuangfeng, Hunan Province, PR China. Strain QMT-28T grew well on International Streptomyces Project series media and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into loose spiral spore chains consisting of cylindrical spores with smooth surfaces. The diagnostic diamino acid was ll-diaminopimelic acid and the whole-cell sugars were galactose and glucose. The predominant fatty acids were C18 : 1 cis9, summed feature 6 (C18 : 2 cis 9,12/C18 : 0 a) and C16 : 0. The polar lipids included diphosphatidylglycerol, hydroxy phospatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides, phospholipids of unknown structure containing glucosamine and several unidentified phospholipids. The major menaquinones were MK-9, MK-9(H2), MK-9(H4), MK-9(H6) and MK-9(H8). The genome size of strain QMT-28T was about 8.7 Mbp with a G+C content of 71.2 mol%. Phylogenetic analysis showed that the novel strain was closely related to Streptomyces olivochromogenes DSM 40451T (99.5 % similarity), Streptomyces mirabilis NBRC 13450T (98.9 %), Streptomyces kanamyceticus NBRC 13414T (98.9 %), Streptomyces kaempferi I37T (98.9 %) and Streptomyces arcticus ZLN234T (98.8 %). However, the average nucleotide identity values, the digital DNA–DNA hybridization values and the multilocus sequence analysis evolutionary distances between this strain and closely related strains showed that it belonged to a distinct species. In addition, these results were also supported by differences in the phenotypic characteristics between QMT-28T and five closely related type strains. Consequently, strain QMT-28T should represent a novel species of the genus Streptomyces , with the suggested name Streptomyces fagopyri sp. nov. The type strain is QMT-28T (=CICC 24808T=JCM 33796T).