Isolation and Molecular Identification of Culturable Rhizosphere Bacteria Associated with the Medicinal Plant Justicia Adhatoda

The rhizosphere of medicinal plants represents a chemically rich and ecologically selective microhabitat that supports distinctive bacterial communities, yet the culturable diversity associated with many traditionally important species remains poorly documented. Justicia adhatoda (Malabar nut), a shrub widely used in Ayurvedic and Unani medicine for respiratory ailments, is one such plant whose rhizosphere microbiome has received little dedicated attention. In this study, rhizosphere soil was collected from healthy J. adhatoda plants and processed by serial dilution and spread-plating on nutrient agar. Twelve morphologically distinct colony types were recovered, of which six representative isolates were purified for identification. Preliminary screening by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) yielded only one highconfidence and one moderate-confidence species-level match, with the remaining four isolates falling below the reliable identification threshold. Three representative isolates spanning the confidence range were therefore subjected to 16S rRNA gene sequencing (Sanger platform, 27F/1492R primers) and BLASTn comparison against the NCBI database. This confirmed the isolates as Paenarthrobacter nitroguajacolicus (98.88% identity), Kocuria rosea (99.77% identity), and Micrococcus aloeverae (100% identity). The results demonstrate that combining rapid mass-spectrometric screening with confirmatory molecular sequencing provides a reliable, tiered workflow for characterising culturable rhizosphere bacteria of medicinal plants, and establish a taxonomically verified isolate collection suitable as a starting point for genome-level bioprospecting, pursued in a companion study.