*3.1. Polyphasic Taxonomic Characterization of NEAU-H2<sup>T</sup>*

Morphological observation of the two-week culture of strain NEAU-H2T grown on ISP 3 medium revealed that it had characteristics typical of the genus *Streptomyces* [47]. Aerial and substrate mycelium were well developed without fragmentation. Spiral spore chains with spiny surfaced spores (0.8–1.0 <sup>×</sup> 1.0–1.3 <sup>μ</sup>m) were borne on the aerial mycelium (Figure 1). Strain NEAU-H2<sup>T</sup> exhibited good growth on ISP 1–4, ISP 7, and Nutrition agar media; moderate growth on Bennett's and Czapek's agar media; and poor growth on ISP 5 and ISP 6 media. The colony colors varied from white to moderate yellow. A dark grayish olive pigment was produced on ISP 6 medium. The detailed cultural characteristics of strain NEAU-H2<sup>T</sup> are shown in Table S1. Strain NEAU-H2<sup>T</sup> was found to grow at a temperature range of 4 to 40 ◦C (optimum temperature 28 ◦C), pH 5 to 10 (optimum pH 7), and NaCl tolerance of 0% to 9% (optimum NaCl of 0% to 1%). The physiological and biochemical properties of strain NEAU-H2<sup>T</sup> are given in Table 1 and the species description.


**Table 1.** Differential characteristics of strain NEAU-H2T and its most closely related *Streptomyces* species. Strains: 1, NEAU-H2T; 2, *S. rhizosphaerihabitans* 109807T;3,*S.populi*A249T;4,*S.siamensis*NBRC108799T.Alldataarefromthisstudyexceptwheremarked.+,positive;negative.‡DatafromLeeetal.

NBRC

**Figure 1.** Scanning electron micrograph of strain NEAU-H2T grown on International *Streptomyces* Project (ISP) medium 3 (ISP 3 ) for 2 weeks at 28 ◦C; Bar 1 μm.

Chemotaxonomic analyses revealed that strain NEAU-H2<sup>T</sup> also exhibited the typical characteristics of the genus *Streptomyces* [47]. It contained LL-diaminopimelic acid as cell wall diamino acid, indicating that the strain is of cell wall chemotype I [51]. The whole-cell sugar was found to contain glucose. The phospholipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylinositolmannosides (PIM), and an unidentified phospholipid (PL), corresponding to phospholipid type II [52] (Figure S1). The major cellular fatty acids (>10%) were iso-C16:0 (21.6%), anteiso-C15:0 (19.4%), iso-C15:0 (16.9%), and anteiso-C17:0 (13.0 %), which is fatty acid type IIc [53]; minor amounts of C16:1ω7c (8.5 %), C16:0 (7.5 %), iso-C14:0 (7.2%), C17:0 cyclo (2.1%), C17:1ω8c (2.1%), C18:0 (1.2%), and C15:0 (0.5%) were also present. The menaquinones detected were MK-9(H8) (57.5%), MK-9(H6) (32.3%), and MK-9(H4) (10.2%), which have been reported for most species of the genus *Streptomyces* [47].

EzBioCloud analysis suggests that strain NEAU-H2<sup>T</sup> belongs to the genus *Streptomyces*. The novel strain shared the highest 16S rRNA gene sequence similarities with *S. rhizosphaerihabitans* NBRC 109807<sup>T</sup> (98.8%), *S. populi* A249T (98.6%), and *S. siamensis* NBRC 108799<sup>T</sup> (98.6%). In the neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, strain NEAU-H2T formed a separate clade with *S. populi* A249<sup>T</sup> (Figure 2), a relationship also recovered by the maximum likelihood algorithm (Figure S2). To further clarify the affiliation of strain NEAU-H2T to closely related strains, phylogenetic trees were constructed from the concatenated sequence alignment of the five housekeeping genes based on the neighbor-joining and maximum likelihood algorithms (Figure 3 and Figure S3), which showed the same topology as the 16S rRNA gene tree. Furthermore, the concatenated sequences of *atp*D-*gyr*B-*rec*A-*rpo*B-*trp*B were used to calculate pairwise distances well above 0.007 (Table S2) for the related species, which was considered to be the threshold for species determination [54]. Based on the 16S rRNA gene sequence similarities and phylogenetic trees, *S. rhizosphaerihabitans* NBRC 109807T, *S. populi* A249T, and *S. siamensis* NBRC 108799<sup>T</sup> were selected as the closely related strains for subsequent comparative analysis.

**Figure 2.** Neighbor-joining tree based on 16S rRNA gene sequences (1418 bp) showing the relationship of strain NEAU-H2<sup>T</sup> (in bold) with related taxa, which are the top 50 type strains of *Streptomyces* species of gene sequence similarities based on analysis using EzTaxon-e. Filled circles indicate branches that were also recovered using the maximum likelihood methods. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. *Allostreptomyces psammosilenae* YIM DR4008<sup>T</sup> (KX689228) was used as an outgroup. Bar, 0.01 nucleotide substitutions per site.

**Figure 3.** Neighbor-joining tree based on multilocus sequence analysis (MLSA)analysis of the concatenated partial sequences (2060 bp) from five housekeeping genes (*atpD*, *gyrB*, *recA*, *rpoB*, and *trpB*) of strain NEAU-H2<sup>T</sup> (in bold) with related taxa. Filled circles indicate branches that were also recovered using the maximum likelihood methods. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. *Kitasatospora setae* KM-6054<sup>T</sup> was used as an outgroup. Bar, 0.02 nucleotide substitutions per site.

The assembled genome sequence of strain NEAU-H2<sup>T</sup> was found to be 9,921,301 bp long and composed of 135 contigs with an N50 of 167,996 bp, a DNA G+C content of 71.5 mol%, and a coverage of 152.0×. It was deposited into GenBank under the accession number WBKG00000000. Detailed genomic information is presented in the Table S3. DNA–DNA hybridization was employed to further clarify the relatedness between strain NEAU-H2T and *S. rhizosphaerihabitans* NBRC 109807T and *S. siamensis* NBRC 108799T. The DNA–DNA relatedness values were 33.3 <sup>±</sup> 2.5% and 44.5 <sup>±</sup> 3.5%, respectively. Digital DNA–DNA hybridization was employed to clarify the relatedness between strain NEAU-H2T and *S. populi* A249T. The level of DNA–DNA relatedness between them was 56.5 to 62.1%. According to the description proposed by Wayne et al. [55], the relatedness values are below the threshold value of 70% for assigning bacterial strains to the same genomic species.

Besides the genotypic evidence above, some obvious differences can also be found between strain NEAU-H2<sup>T</sup> with its closely related strains regarding several phenotypic and chemotaxonomic characteristics. Strain NEAU-H2T could be easily distinguished from its most closely related species by cultural characteristics, such as colony colors and diffusible pigment production (Table S1 and Figure S4). Morphological characteristics, including spore chain and surface ornamentation, could also distinguish the isolate from its closely related strains (Table 1). In addition, the isolate was able to grow at 4 ◦C, in contrast to its closely related strains, which could not. The novel strain could not utilize l-serine, l-tyrosine, l-arabinose, and meso-inositol while the closely related species could. Strain NEAU-H2<sup>T</sup> was found to contain both PI and PIM in its phospholipid profile, which could distinguish it from *S. siamensis* NBRC 108799T and *S. populi* A249T. The presence of MK-9(H4) could differentiate the isolate from *S. rhizosphaerihabitans* NBRC 109807T. Most notably, the whole-cell sugar of strain NEAU-H2<sup>T</sup> was evidently different from that of *S. rhizosphaerihabitans* NBRC 109807T and *S. populi* A249T, with the only presence of glucose. The detailed characteristics of strain NEAU-H2T in comparison with its closely related strains are listed in Table 1.

Therefore, it is evident from the genotypic, phenotypic, and chemotaxonomic data that strain NEAU-H2T represents a novel species of the genus *Streptomyces*, for which the name *Streptomyces triticiradicis* sp. nov. is proposed.
