*3.1. Isolation, Morphological, and Molecular Characterization of Actinobacterial Isolates*

In this study, from forest soil of Bisle Ghat and Virajpet of Western Ghats region of Karnataka, we targeted the isolation of different genera of actinobacteria in search of new natural products (Table 1). A total of 32 actinobacterial isolates were recovered on Starch casein agar and Actinomycetes isolation agar. Of them, seven isolates grown on starch casein agar medium showing marked antimicrobial activity against test organisms in primary screening were characterized on the basis of cultural, morphological, and molecular characteristics. The colonies of isolates revealed diverse morphological appearances with varied spore color, aerial and substrate mycelium, and colony morphology (Table 2 and Figure 1). Scanning electron microscope examination showed chains of smooth and spiny spores in oval, round, and spiral ornamentation (Figure 2). The molecular identification of isolates by amplification of 16S-rRNA gene was done by using universal primers 27F' and 1492R' (Figure S1). In 16S-rRNA sequencing, alignment of the nucleotide sequences of strain S1A, SS4, SS5, SS6, and SCA35 exhibited a similarity of 98.32%, 99.71%, 99.70%, 99.72%, and 99.35% with closely related *Streptomyces* species, respectively. The strain SCA11 was considered to represent a species of the genus *Nocardiopsis*, since it was closely related to *Nocardiopsis* species with 98.99% sequence similarity. The nucleotide sequence of strain SCA13 showed 98.28% similarity with the closely related *Nocardioides* sp. (Table 3). The phylogenetic relatedness of strains with their closely related species obtained by neighbor-joining method is shown in Figures 3–5.


**Table 1.** Actinobacterial isolates and sampling areas in Western Ghats regions of Karnataka, India.


**Table 2.** Morphological characteristics of isolated actinobacterial strains.

**Figure 1.** Morphological characterization of actinobacterial isolates on starch casein agar plates.

**Table 3.** Molecular identification (based on 16S rRNA amplification) of actinobacterial strains isolated from Western Ghats.


**Figure 2.** Scanning electron micrograph of strains (**A**) S1A, (**B**) SCA11, (**C**) SS4, (**D**) SS5, (**E**) SS6, (**F**) SCA35, (**G**) SCA13; scale bar represents 5 μm.

**Figure 3.** Phylogenetic analysis of isolate *Nocardiopsis* sp. strain SCA11. Neighbor-joining phylogenetic tree showing evolutionary relationship of selected isolate based on 16S r-RNA sequence alignments. Bootstrap values at the nodes indicate collated values based on 1000 resampled datasets. Bar indicates 0.0005 substitutions per nucleotide position.
