Identification of antifungal compound from endophytic bacteria of Taxus sumatrana

Abstract

In recent decades, there has been an increase in fungal infection caused by resistance of some species of fungus to fungicides used in medicinal treatment. The use of antifungal in the treatment of fungal infections should considering the toxicity, efficacy, cost and frequent use had led to emergence of resistant strains. One approach to developing antifungal drugs is use of plants as sources of medicine (Abad et al. 2007). Plants are host for the presence of endophytic microbes. Some of these endophytes produce bioactive compound that are involved in the host-microbes relationship. Taxus is one of the hosts plant for endophytic microbes. An endophytic fungus Paecilomyces sp. was isolated from Taxus mairei, produce metabolites brefeldin A, which has several activities such as antifungal, antiviral and anticancer drugs (Wang et al. 2002). Tayung et al (2007) also reported that a bacterium Pseudomonas fluorescens was isolated from Taxus batacca rhizosphere, produce antimicrobial compounds. Many endophytic microbes associated with Taxus have been demonstrated to produce biactive compound that was an opportunity to explore endophytic microbes of Taxus from Indonesia. Taxus sumatrana was known as the Sumatran Yew that was a needle tree, grown naturally in Indonesia. T. sumatrana was recognized for producing a number of active compounds such as taxol, cephalomannine, 7-epi-10-deacetyltaxol, 7-epi-10- deacetylcephalomannine, baccatin III, 19-hydroxybaccatin III and 10-deacetyl-13- oxobaccatin III taxol, cepalomanin, 7-epi-10-deasetil. Shen et al. (2003) reported, two new taxane diterpenoid, tasumatrol A and B, have been isolated from extract of the leaves and twigs of Taiwanese T. sumatrana. However, the potential of endophytic microbes especially endophytic bacteria from T. sumatrana for producing antifungal compounds is still limited. The aim of this studies were to identify antifungal compound against Candida albicans which produced by endophytes bacteria of T. sumatrana and characterize the selected isolates. The results of this study were expected to receive pharmacological compound which have high value and increase scientific information of antifungal compound from endophytic bacteria of T. sumatrana. Endophytic bacteria were isolated from the stems and leaves of T. sumatrana (Tomita 2003). Selection of isolates was done by paper disk diffusion and point inoculation method. Sixteen isolates were found at isolation of endophytic bacteria. Among 16 isolates, isolate Tx 4 showed antagonistic activity against C. albicans. The culture broth of Tx 4 isolate was centifuged and supernatant was extracted using n-hexane solvent three times. Extracts which have antifungal activity was analyzed by Thin Layer Chromatography using n-hexane: ethyl acetate solvent. Extracts was fractionated by column chromatography using nhexane: ethyl acetate solvent as a gradient. Fractions were obtained, tested antagonistic to C. albicans. Fractions which have antifungal activity were analyzed by preparative TLC. Fractions which showing antifungal activity, were analyzed by Infra Red Spectrophotometry (IR) and Gas Chromatography - Mass Spectrometry (GC-MS). The IR results were wavenumber 1740.64 cm-1 (carbonyl group), 2919.06 cm-1 (aromatic group), 1521.73 cm-1 (alkene group) and 1081,03 cm-1 (carboxyl group). Identification by GC-MS estimated that bioactive compounds contain of 1,2-benzenedicarboxylic acid, diisooctyl ester. Identification of Tx 4 isolate was done base on morphology and molecular. The results of morphology identification showed that Tx 4 isolate is rod-shaped and positive Gram. The results of 16S rRNA gene sequences were compared to available databases using the BLAST-N program. Based on BLAST analysis, Tx 4 isolate had 100% similarity with Bacillus amyloliquefaciens NBRC 101586.