Antimicrobial activity of endophytic fungi isolated from curcuma mangga, with emphasize on Ceratobasidium ramicola IBRLCM127.

Medicinal plant that serves as a prime source of therapeutic agent to treat various diseases is becoming scarce, thus endophyte-host plant interaction is receiving considerable exploration as endophytes possess capability to produce diverse compounds including those originated from their host plant. This research was aimed to study the antimicrobial activity of fungal endophytes isolated from various parts of medicinal plant Curcuma mangga. A total of 125 fungal endophytes were obtained, including 90 from leaves, 23 from stems and 12 from rhizomes. Overall, 118 out of 125 isolates demonstrated significant antimicrobial activity against at least on one test microorganisms. Isolate IBRLCM127 showed the most significant antimicrobial activity and was identified as Ceratobasidium ramicola IBRLCM127 according to macro-, micro-morphology and molecular identification. Result from enhancement of physical parameters for cultural condition revealed that the optimum antimicrobial activity of C. ramicola IBRLCM127 was obtained with the usage of 2 fungal agar plugs of six day-old of preculture age, inoculated into culture medium consisting of host plant extract and incubated at 200 C in the dark using agitation speed of 120 rpm for 12 days. The ethyl acetate extract of fermentative broth of C. ramicola IBRLCM127 demonstrated a significant antimicrobial activity against three representative groups of test microorganisms, namely Gram positive bacteria (MRSA), Gram negative bacteria (Yersenia enterocolitica) and yeast (Candida albicans) with minimal inhibitory concentration (MIC) values ranged from 0.25 to 2.5 mg/mL. The time kill study results also indicated that the extract exerted a “static” effect at ½ MIC and MIC values, whereas a “cidal” effect was exerted at 2 MIC value. The effect of extract at concentration of 2MIC value towards microbial cells of the three selected test microorganisms showed that the cells were crumpled, dented, distorted, collapsed, formed debris and even death under observation of Scanning Electron Microscope. The prolonged exposure to the extract resulted to the wavy cell structures, partially disintegration of cell membrane and leakage of cytoplasmic content that led to the cell lysis under Transmission Electron Microscope observation. Thin layer chromatography of dichloromethane partitionated extract with significant antimicrobial activity using the combination of solvent system hexane: diethyl ether: ethyl acetate (2:4:4) gave out eight fractions. Bioautography assay disclosed that the light yellow of fourth sport with Rf value of 0.49 contributed to the antimicrobial activity on all selected test microorganisms. Qualitative chemical analysis of the extract probably consists of phenol and unsaturated compounds. All fractions were eluted using column chromatography and the activity of the targeted fraction was reduced compared to its partition extract with MIC value ranging from 0.625 to 6.000 mg/mL. The analysis of gas chromatography-mass spectrometry divulged that the major compound was suggested to be 5H-Pyrrolo(3,2-d)pyrimidin-4-amine. The toxicity assay using brine shrimp lethality test indicated that the ethyl acetate extract was not toxic at acute toxicity (1627.18 μg/mL) but toxic at chronic toxicity (111.16 μg/mL). The antioxidant activity of extract also showed that it possessed moderate antioxidant activity. In short, C. mangga serves as a great warehouse of endophytic fungi with remarkable antimicrobial activity that can be further developed as natural antimicrobial agent.