Isolation of secondary metabolites from endophytic fungi of syzygium cordatum (myrtaceae) for the control of bean bacterial pathogens

Abstract

Common bean (Phaseolus vulgaris L.) is the most consumed leguminous crop among all communities in Kenya. Its productivity is declining due to bacterial infections; common bacterial blight and halo blight. Synthetic agrochemicals are currently used in curbing these bacterial infections in beans but studies have reported lots of problems associated to their usage. Medicinal plants host important microorganism (endophytes) that mutualistically correlate with the host plant and are considered a prolific source of important secondary metabolites that can be used as leads in production of drugs. S. cordatum is among these medicinal plants that host important endophytic fungi. This study was carried out to evaluate bioactive endophytic fungi isolated from S. cordatum plant and their extractives against bean bacterial pathogens; Pseudonomas syringae pv phaseolicola and Xanthonomas axonopodis pv phaseoli. Endophytic fungi were isolated from sterilized leaves and stem bark plated on Potato Dextrose agar (PDA) modified with 20 mg/l of streptomycin sulphate, incubated and sub-cultured to their pure fungal cultures. Seven (7) pure cultures were isolated from the plant materials, however, three (3) of them were identified. All isolated fungal endophytes were active against the test organism, with SC-S (9), SC-S (11) and SC-L (7) all identified as Diaporthe species showing the highest antibacterial activities against the selected test organism. The three (3) most active fungal endophytes were subjected to fermentation on rice media and secondary metabolites extraction done using methanol. Methanol crude extracts were then suspended inwater then thereafter partition between hexane and ethyl acetate yielding hexane and ethyl acetate crude extracts. Purification of SC-S (11) ethyl acetate crude extract on column chromatographic techniques thereafter in preparative High Performance Liquid Chromatography (HPLC) yielded three new compounds; Two geometric isomers, named (2Z,4Z, 8Z)- 5-MethylDec-2, 4, 8-triene-1, 6, 7-triol (20), (2E,4E, 8E)- 5-MethylDec-2, 4, 8-triene-1, 6, 7-triol (21) and one phenolic derivate; 2-methyl-5-(1-(3-methylaziridin-2-yl)ethyl)phenol (22). Similarly, SC-S (9) yielded one pure compound named; 3-mthoxy-5-mehtylnaphalene-1, 7-diol (23). The pure compounds were identified using 1D and 2D NMR analysis and molecular mass confirmation done using High Resolution Mass Spectrometer (HRMS). Agar disc diffusion assay was used to evaluate antibacterial activities of extracts. Agar disc diffusion assay was used to evaluate antibacterial activities of extracts. It showed that all the fungal extracts had activities against X. axonopodis pv phaseoli with F2 of Diaporthe sp3, showing the least MIC value of 2.5 mg/ml corresponding to zones of inhibition of 8.00 ± 0.58 mm. A weak antibacterial activity of fungal extracts was recorded against P. syringae pv phaseolicola with F4 of Diaporthe sp3 showing the lowest MIC value of 1.25 mg/ml corresponding to zones of inhibition of 7.33 ± 0.33 mm. The results from the study showed a diverse source of antibacterial secondary metabolites from endophytic fungi housed by most medicinal plants and the potentiality of these compounds to be used as leads in control of bean bacterial pathogens especially X. axonopodis pv phaseolicola. Therefore toxicological and cytotoxicity tests should be done to evaluate their safety in crop protection application.