Differential Antimicrobial Activity of Endophytes in the External vs. Internal Bark of Neem (Azadirachta indica): Isolation, Characterization, and Bioactive Metabolite Analysis

Abstract

Introduction: Endophytic fungi are beneficial microbes living harmlessly within plants and producing useful bioactive compounds. Azadirachta indica (Neem) has strong antimicrobial properties, but the endophytes inside its inner and outer bark are not well explored. This study compares their antimicrobial activity to identify their potential benefits.

Methods: Healthy neem bark was collected, surface sterilized, and separated into outer and inner sections. Endophytic fungi were isolated using the tissue segment method and cultured on Potato Dextrose Agar. After microscopic morphological identification, fermentation was carried out in nutrient broth, and secondary metabolites were extracted using ethyl acetate. Antimicrobial activity of these extracts was evaluated against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa using the agar disc diffusion method, with Fosfomycin as the positive control.

Results: The fungal endophytes, C. cladosporioides (outer and inner bark) and P. marneffei (inner bark) were identified. The inner bark isolate P. marneffei showed the highest antimicrobial activity, particularly against P. aeruginosa and K. pneumoniae. In comparison, the C. cladosporioides isolate from the outer bark exhibited moderate inhibition, especially against S. aureus and P. aeruginosa.

Conclusion: Internal bark endophytes of neem showed stronger antimicrobial activity than outer bark isolates, suggesting that deeper tissues harbour more potent and stable endophytic associations. This indicates neem endophytes may serve as promising sustainable sources of novel antimicrobial compounds, aligning with Ayurvedic bioprospecting concepts. However, since this study involved isolates from only one tree, further research on larger samples from varied ecological regions is needed to confirm broader applicability.