*3.1. Isolation and Identification of an Antimicrobial Compound Producing Strain*

More than 20 isolates from the soil samples were isolated, purified, and screened for bioactivity against *R. solanacearum*. Among them, only four isolates showed bioactivity against *R. solanacearum*. Since the methanol extract of the the cell pellet and supernatant of one isolate, designated as NEAU-HV9, revealed a higher activity (30.5 mm and 32.8 mm) against the tested bacterial strain (Table 1, Figure S2), this strain was selected for further studies.

**Table 1.** Bioactivities of the supernatant and cell pellet of NEAU-HV9 against *R. solanacearum*.


Data shown are the mean of two replications.

Strain NEAU-HV9 was aerobic, Gram-stain positive and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into spiral spore chains with oval spores (Figure 1). The spore surface was wrinkled. It had good growth on ISP 1, ISP 2, ISP 3, ISP 4, ISP 5, ISP 6, ISP 7, Bennett's agar and Nutrient agar, and poor growth on Czapek's agar (Figure S1). The data on the growth characteristics of NEAU-HV9 in different media are given in Table S1.

**Figure 1.** Scanning electron micrograph of strain NEAU-HV9 grown on International *Streptomyces* Project (ISP) 3 agar for 2 weeks at 28 ◦C.

Further characterization of NEAU-HV9 was performed by evaluating various biochemical tests (Table S2). Growth at 15 ◦C to 37 ◦C (optimum: 28 ◦C) and in the range of pH 5 to 9 (optimum: pH 7.0). Tolerate up to 7% (*w*/*v*) NaCl in the culture medium. Positive for hydrolysis of starch, production of H2S, hydrolysis of aesculin and decomposition of adenine, hypoxanthine, tyrosine and xanthine, negative for reduction of nitrate, coagulation and peptonization of milk, liquefaction of gelatin and decomposition of casein. D-Glucose, D-maltose, D-mannitol, D-galactose, inositol, D-mannose, L-rhamnose and D-sucrose are utilized as sole carbon sources, but not L-arabinose, dulcitol, D-fructose, lactose, D-ribose, D-sorbitol or D-xylose. L-Alanine, D-arginine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glutamine, glycine, L-proline, L-serine, L-threonine and L-tyrosine are utilized as sole nitrogen sources, but not creatine. The above growth data of isolate NEAU-HV9 denote that the isolate has the typical characteristics of the genus *Streptomyces*.

Recently, it has been suggested that the 16S rRNA gene can be used as a reliable molecular clock due to 16S rRNA sequences from distantly related bacterial lineages having similar functionalities [49]. Basically, the 16S rRNA gene sequence, comprising of about 1500 bp with hyper variable and conserved regions, is universal in all bacteria. According to Woese's report [50], comparing a stable part of the genetic code could determine phylogenetic relationships of bacteria. The hyper variable regions of the 16S rRNA gene sequences provide species-specific signature sequences, so it is widely used in

bacterial identification all over the world. Therefore, the almost-complete 16S rRNA gene sequence (1510 bp) of strain NEAU-HV9 was obtained and has been deposited as MN578143 in the GenBank, EMBL (European Molecular Biology Laboratory) and DDBJ (DNA Data Bank of Japan) databases. BLAST sequence analysis of the 16S rRNA gene sequence indicated that strain NEAU-HV9 was related to members of the genus *Streptomyces*. The EzBioCloud analysis showed that strain NEAU-HV9 was most closely related to *Streptomyces panaciradicis* 1MR-8T and *Streptomyces sasae* JR-39T with a gene sequence similarity of 98.90% and 98.89%, respectively. In conclusion, based on the 16S rRNA gene sequence and the genetic identity of isolate NEAU-HV9, the isolated strain was further identified by neighbor-joining tree (Figure 2), and was also found to belong to the genus *Streptomyces*.

**Figure 2.** Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships among strain NEAU-HV9 (bold) and members of the genus *Streptomyces*. Bootstrap percentages (≥50%) based on 1000 resamplings are listed at the nodes. *Actinospica robiniae* DSM 44927<sup>T</sup> was used as the out-group. Scale bar represents 0.01 nucleotide substitutions per site.

### *3.2. Structural Characterization of Compound*

The active component was isolated from fermentation medium (7.5 L) and one bioactive compound was obtained as red, amorphous powder. The compound had UV visible spectra at 215 nm, 440 nm in methanol. The compound showed absorptions at 220 nm and 254 nm with a retention time of 12.367 min (Figure S3), similar to that of actinomycin class of compounds [51,52]. The structure of the compound was further elucidated by 1H NMR, 13C NMR, and MS analysis as well as comparison with previously reported data. The ESI-MS of the isolated compound revealed molecular ion peaks at m/z 1277.6 [M+Na]<sup>+</sup> (Figure S4), which was identical to that of actinomycin D [53]; 1H and 13C spectra of the isolated compound in CD4O also showed great similarities to that of actinomycin D [52,53] (Figures S5 and S6). In addition, the retention time of commercial actinomycin D (Biotopped, purity: ≥98%) was 12.328 min (Figure S7), and the retention time of compound 2 was 12.367 min (Figure S3). Compound 2 and commercial actinomycin D have similar activity against *R. solanacearum* (Figure S8). The above results showed that the structure of the main active compound was confirmed to be actinomycin D (Figure 3).

**Figure 3.** Chemical structure of actinomycin D.

#### *3.3. Bioactivity of Isolated Compound*

#### 3.3.1. Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) of the antibacterial compound was determined as described by Rathod et al. [45]. The minimum inhibitory concentration of actinomycin D was determined as 0.6 mg L−<sup>1</sup> (0.48 μmol L<sup>−</sup>1) against *R. solanacearum* (Table 2).

**Table 2.** Minimum inhibitory concentration (MIC) values of actinomycin D against *R. solanacearum*.

