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

Abstract

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 high-confidence 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.

Introduction

This study investigates the culturable bacterial diversity in the rhizosphere of the medicinal plant Justicia adhatoda and evaluates the effectiveness of MALDI-TOF MS and 16S rRNA gene sequencing for bacterial identification. The rhizosphere is a biologically active soil region enriched by plant root exudates, making it a valuable source of microorganisms capable of producing antibiotics and other bioactive compounds. Because J. adhatoda produces medicinal phytochemicals such as vasicine and vasicinone, its rhizosphere is expected to harbor unique bacterial communities with potential pharmaceutical applications.

Previous research has shown that rhizosphere bacteria, particularly genera such as Streptomyces, Bacillus, and Pseudomonas, produce antimicrobial and other beneficial metabolites. However, the rhizosphere microbiome of J. adhatoda has received little scientific attention. While MALDI-TOF MS offers rapid and inexpensive bacterial identification, its accuracy is limited for environmental isolates because reference databases mainly contain clinical organisms. Therefore, 16S rRNA gene sequencing remains the most reliable method for species-level identification.

The study aimed to isolate bacteria from the rhizosphere of J. adhatoda, characterize them morphologically, evaluate MALDI-TOF MS as a screening tool, and confirm bacterial identities using 16S rRNA sequencing and BLAST analysis. Soil samples were collected from healthy plant roots, cultured on nutrient agar, and purified into distinct bacterial isolates. These isolates underwent morphological and biochemical characterization before being analyzed using MALDI-TOF MS. Selected isolates were further identified through PCR amplification and sequencing of the 16S rRNA gene.

Twelve morphologically distinct bacterial colonies were initially isolated, from which six representative isolates were selected for identification. MALDI-TOF MS produced reliable identification for only one isolate (Deinococcus wulumuqiensis) and a low-confidence identification for another (Micrococcus luteus), while the remaining isolates could not be confidently identified. Overall, only 17% of isolates were accurately identified using MALDI-TOF MS, demonstrating the limitations of current spectral databases for environmental bacteria.

In contrast, 16S rRNA gene sequencing successfully identified all selected isolates with 98.88–100% sequence identity. The identified species were Paenarthrobacter nitroguajacolicus, Kocuria rosea, and Micrococcus aloeverae. Sequencing also revealed that some MALDI-TOF identifications were incorrect or incomplete, confirming that molecular methods provide more reliable species-level identification.

The findings indicate that the rhizosphere of Justicia adhatoda contains a diverse community of potentially valuable bacteria and may represent an underexplored source of bioactive microorganisms. The study concludes that MALDI-TOF MS is useful for rapid preliminary screening, but 16S rRNA gene sequencing is essential for accurate identification, particularly in environmental microbiology and bioprospecting studies. This combined identification strategy provides a reliable foundation for future research on the antimicrobial and pharmaceutical potential of rhizosphere bacteria associated with medicinal plants.

Conclusion

This study establishes a taxonomically verified collection of culturable rhizosphere bacteria from Justicia adhatoda, obtained through a tiered identification workflow combining rapid MALDI-TOF MS screening with confirmatory 16S rRNA gene sequencing. Of six morphologically distinct isolates purified from rhizosphere soil, three were confirmed by sequencing as Paenarthrobacter nitroguajacolicus, Kocuria rosea, and Micrococcus aloeverae, each with high sequence identity (>98%) to reference database entries. The comparatively poor performance of MALDI-TOF MS for these environmental isolates (one high-confidence match out of six) reinforces the need for molecular confirmation when characterising rhizosphere and other non-clinical bacterial communities. As genera within Micrococcaceae are known elsewhere to harbour biosynthetic potential for antimicrobial and anti-inflammatory compounds, the isolates identified here represent a promising, taxonomically secure starting point for genome mining and natural product discovery, pursued in a companion genome-based study of the same three isolates.

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Copyright

Copyright © 2026 Dr. Mehul P. Dave, Dr. Naman N. Trivedi, Deep G. Patel. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.