Isolation and Phylogenetic Diversity of Streptomyces from Underexplored Soils of Nepal

Abstract

The genus Streptomyces is a prolific source of bioactive secondary metabolites and remains central to antibiotic discovery; however, many ecologically diverse regions remain underexplored for Streptomyces diversity. In this study, soil samples collected from four ecologically distinct regions of Nepal were investigated for the isolation and molecular characterization of Streptomyces species. Selective isolation was performed using ISP4 medium supplemented with antibacterial and antifungal agents, followed by morphological and microscopic characterization. Genomic DNA was extracted from pure cultures, and the 16S rRNA gene was amplified, sequenced, and subjected to comparative sequence and phylogenetic analyses. Four isolates (PY1–PY4) exhibiting characteristic Streptomyces morphology were successfully recovered. High-molecular-weight genomic DNA was obtained from all isolates, enabling reliable amplification of approximately 1.5 kb 16S rRNA gene fragments. Sequence similarity searches and phylogenetic reconstruction confirmed that all isolates belonged to the genus Streptomyces, showing close relationships to previously described species while also exhibiting strain-level genetic variation, particularly in isolate PY3. The phylogenetic distribution of isolates from both lowland and high-altitude environments suggests that ecological heterogeneity may contribute to Streptomyces diversity in Nepalese soils. Overall, this study provides baseline molecular data supporting Nepalese soils as a valuable and underexplored reservoir of Streptomyces diversity and establishes a foundation for future genomic and functional investigations into their biosynthetic and antimicrobial potential.