Isolation and Molecular Identification of Culturable Rhizosphere Bacteria Associated with Azadirachta indica (Neem)

Abstract

The rhizosphere of medicinal plants represents a unique ecological niche that harbours diverse microbial communities with important ecological and biotechnological significance. Among medicinal plants, Azadirachta indica (Neem) is widely recognised for its antimicrobial, anti-inflammatory, and therapeutic properties; however, comparatively little information is available regarding its culturable rhizosphere bacterial diversity. The present study aimed to isolate and molecularly identify culturable bacteria associated with the rhizosphere of healthy A. indica plants using a combination of conventional microbiological and molecular techniques. Rhizosphere soil samples were collected aseptically and processed by serial dilution followed by spread-plating on nutrient agar. Twelve morphologically distinct bacterial colonies were isolated, purified, and subjected to preliminary identification using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). High-confidence identification was obtained for two isolates, while three isolates produced low-confidence identification scores, indicating the limited representation of environmental bacteria in commercial spectral databases. Three representative isolates were subsequently selected for confirmatory identification through 16S rRNA gene sequencing and BLAST analysis against the NCBI database. Molecular analysis identified the isolates as Kocuria flava, Kocuria salina, and Staphylococcus capitis. The comparison between MALDI-TOF MS and 16S rRNA sequencing demonstrated that molecular techniques provide reliable species-level confirmation for environmental bacterial isolates, particularly when proteomic databases are incomplete. The study establishes a taxonomically verified collection of culturable rhizosphere bacteria associated with A. indica, providing a valuable microbial resource for future investigations involving plant–microbe interactions and microbial biotechnology.