*2.3. Phenotypic Characterization*

The macroscopic features of the 11 representative actinobacterial strains selected for this experiment are show in Table 3. In general, the colonies were mostly white in color, circular in shape, convex in elevation, with an entire edge, hard consistency and variable pigment production (Figure 2). Microscopic observation of Gram-stained bacterial cells showed stable branched mycelium bearing aerial hyphae, which differentiate into short or long spore chains. Microscopic characterization using the microculture technique revealed details of aerial and vegetative mycelium, mycelial fragmentation and clustering of spores. A spiral arrangement of spores was observed on most of the microculture slides of each sample. In addition, all the strains were characterized as Gram-positive suggesting that they belong to the genus *Streptomyces*.


**Table 3.** Macroscopic characteristics of colonies of 11 actinobacterial strains (*Streptomyces* spp.) grown on Casein Starch Agar at 28 ◦C in darkness for 10 days \*.

<sup>a</sup> (+):actinobacteria G+. <sup>b</sup> (+): Presence of aerial mycelium. \* The phenotypic characteristics of the colonies of the actinobacterial strains were selected according with [18,19].

**Figure 2.** Two-weeks-old colonies of *Streptomyces* strains CBQ-B-8 (**A**–**E**), and CBQ-EA-2 (**F**–**J**) growing on ACA medium (**A**–**C**,**F**–**H**) and on PDA medium (**D**,**E**,**I**,**J**) at 28 ◦C in the dark.

#### *2.4. Biochemical Characterization and Assimilation of Carbon Sources*

None of the eleven strains under study were positive for indole production and the Voges Proskauer test. Strains CBQ-J-4, -OSS-3, -EA-2 and -EBa-5, were positive for casein hydrolysis; and the latter two strains were also able to be positive for the methyl red test, in addition to strains CBQ-B-8, -CB-14, -EBa-21 and -Plat-2. Only the strains CBQ-EA-12 and -ESFe-4 did not hydrolyse gelatine. The strains CBQ-OSS-3 and -Plat-2 did not hydrolyse starch (Table 4).



\* (+): Positive reaction; (-): Negative reaction; (D): Dubious.

On the other hand, all the evaluated strains were positive for catalase citrate utilization, nitrate reduction and urea hydrolysis. Variability between strains was also observed for the assimilation and utilization of carbohydrates (Table 4).

#### *2.5. Molecular Characterization*

BLASTn searches on GenBank showed that the 16S rDNA sequences of the strains CBQ-EA-2 and CBQ-B-8 had 99.71 and 99.93% identity with strains of *Streptomyces* sp. HBUM206419 (MT540570) and MP47-91 (EU263063), respectively. The sequences logged in GenBank and Blast results of the two representative actinobacterial strains selected for their highest effectiveness in vitro in this study are shown in Table 5.

**Table 5.** Identification by sequencing the 16S rDNA gene of the two actinobacterial strains selected for molecular characterization with their corresponding GenBank accession numbers and data of Blast results obtained from GenBank.


<sup>a</sup> Corresponding GenBank accession numbers of our isolates. <sup>b</sup> GenBank accession numbers blasted with the isolates obtained in this study. <sup>c</sup> Number of spaces introduced into the alignment to compensate for insertions and deletions in our sequence relative to blasted sequences. <sup>d</sup> Number of nucleotides of our sequences/Number of nucleotides of blasted sequences.
