Antibacterial activity of an endophytic fungus Lasiodiplodia pseudotheobromae IBRL OS-64 residing in leaves of a medicinal herb, Ocimum sanctum Linn.

ABSTRACT

The study was carried out to investigate the antibacterial activity of an endophytic fungal isolate, Lasiodiplodiapseudotheobromae IBRL OS-64 residing in leaves of a medicinal herb, Ocimum sanctum Linn. Qualitative screeningof the antimicrobial activity was done using an agar plug assay, and the results showed that the fungal isolate wasable to inhibit all the 13 test bacteria. Three Gram-positive bacteria (methicillin-resistant Staphylococcus aureus[MRSA] ATCC 33591, Staphylococcus aureus, and Streptococcus mutans) were the most susceptible species withthe inhibition zones of ≥21 mm. The other three (Bacillus cereus ATCC 10876, Bacillus subtilis IBRL A3, andStreptococcus agalactiae) showed the inhibition zones of 11–≤20 mm of diameter. As for Gram-negative bacteria,Yersinia enterocolitica was the most susceptible to the fungal isolate with the size of inhibition zone of ≥21 mm,followed by Klebsiella pneumoniae ATCC 13883, Salmonella typhimurium, and Shigella boydii ATCC 9207 withthe inhibition zones of 11–≤20 mm, whereas Escherichia coli IBRL 0157, Proteus mirabilis, and Pseudomonasaeruginosa ATCC 27883 were the least susceptible with the inhibition zones of ≤10 mm. Quantitative screeningusing disc diffusion assay showed that the fungal ethyl acetate extract prepared from the fermentative broth(extracellular) exhibited better antibacterial activity compared to the methanolic extract prepared from the fungalbiomass (intracellular). The results showed that the ethyl acetate extract exhibited antibacterial activity against all the13 test bacteria with the inhibition zone sizes of 20.0 ± 0.3–31.3 ± 1.2 mm in diameter for Gram-positive bacteria and10.31 ± 0.6–20.1 ± 0.6 mm in diameter for Gram-negative bacteria. On the other hand, the methanolic extract onlyinhibited three Gram-positive bacteria (MRSA ATCC 33591, S. aureus, and S. mutans) with the inhibition zones of9.0 ± 0.6–11.0 ± 0.3 mm in diameter, whereas only one Gram-negative (S. typhimurium) with the inhibition zone sizeof 13.3 ± 1.5 mm diameter. The minimal inhibitory concentration (MIC) and minimum bactericidal concentration(MBC) values of the ethyl acetate extract on Gram-positive bacteria were in the range of 62.50–125.00 and 62.50–500.00 µg/mL, respectively, whereas for the Gram-negative bacteria, the MIC and MBC values were 125.00–250.00and 250.0–1000.00 µg/mL, respectively. On the other hand, the MIC and MBC values for methanolic extract againstGram-positive bacteria were 250.00–500.00 µg/mL and against Gram-negative bacteria were 1000.00 µg/mL,respectively. Both of the extracts exhibited bactericidal effects on test bacteria with the MBC/MIC ratio ≤4. Further,detail of the effects of the ethyl acetate extract on the bacterial cells was observed from the scanning electronmicroscopy photomicrographs which revealed the severity of the morphological deterioration experienced by theextract-treated cells were beyond repair, and the most possible mode of actions were by interrupting the cell wallbiosynthesis and cell membrane permeability