Objective: To isolate and evaluate the antimicrobial activity of the active principle(s) from the ethyl acetate (EtOAc) extract of endophytic fungus Colletotrichum gloeosporioides (C. gloeosporioides) isolated from Sonneratia apetala. Methods: Water agar technique was used to isolate the fungus, and both microscopic and molecular techniques were used for identification of the strain. Potato dextrose broth was used to grow the fungus in large-scale. Reversed-phase preparative HPLC analysis was performed to isolate the major active compound, kojic acid. The EtOAc extract and kojic acid were screened for their antimicrobial activity against two Gram-positive and two Gram-negative bacteria as well as a fungal strain using the resazurin 96-well microtitre plate antimicrobial assay. Results: The fungus C. gloeosporioides was isolated from the leaves of Sonneratia apetala. Initial identification of the fugal isolate was carried out using spore characteristics observed under the microscope. Subsequently, the ITS1-5.8S-ITS2 sequencing was employed for species-level identification of the fungus C. gloeosporioides. Five litres of liquid culture of the fungus produced approximately 610 mg of a mixture of secondary metabolites. Kojic acid (1) was isolated as the main secondary metabolite present in the fungal extract, and the structure was confirmed by 1D, 2D NMR and mass spectrometry. The EtOAc extract and compound 1 exhibited considerable antimicrobial activity against all tested microorganisms. Whilst the minimum inhibitory concentration (MIC) values from the EtOAc extract ranged between 2.4× 10^-4 mg/mL and 2.5 mg/mL, those of kojic acid (1) were between 0.125 mg/mL and 1 mg/mL. The EtOAc extract and kojic acid (1) were most active against Pseudomonas aeruginosa (MIC = 2.4×10^-4 mg/mL) and Micrococcus luteus (MIC = 0.125 mg/mL), respectively. Conclusions: The results revealed that the endophytic fungus C. gloeosporioides could be a good source of commercially important kojic acid, which exhibited antimicrobial properties.

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