*2.3. Screening of Antagonistic Actinobacteria Strains*

The isolates were screened using the agar well diffusion method, and *R. solanacearum* was used as the indicator bacterium [27]. To further investigate the antibacterial components produced by the isolated cultures, these strains were cultured in ISP 2 medium [26] and the inhibitory activities of the supernatant and cell precipitate were tested. Initially, the isolated cultures were grown in ISP 2 medium and incubated at 28 ◦C on a rotary shaker. After 7 days of incubation, the supernatants were obtained by centrifugation at 8000 rpm and 4 ◦C for 10 min and subsequently filtrated with a 0.2 μm membrane filter. The cell precipitates were extracted with an equal volume of methanol for approximately 24 h [28]. A cell suspension (1 mL at 1 <sup>×</sup> <sup>10</sup><sup>8</sup> cfu mL<sup>−</sup>1) of *R. solanacearum* was aseptically plated onto Bactoagar-glucose (BG) media supplemented with 0.5% glucose [22]. Supernatant and methanol extracts were collected from each isolate and tested initially for antimicrobial activity against *R. solanacearum*; each well contained 200 μL of supernatant or methanol extract. The plates were incubated at 37 ◦C for 12 h to test antibacterial activity. The diameters of inhibition zones were measured by using vernier calipers [29]. The experiments were conducted twice. The isolates that showed activities against tested organisms were collected and maintained. Among the collected isolates, the potential isolate designated as NEAU-HV9 was selected for further studies.
