Diversity and Bioactivities of Endophytic Fungi from Medicinal Plant Andrographis paniculata (Hempedu Bumi)
thesisposted on 15.03.2017 by Tan Wei Shang
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In recent years, immense interest is placed on the bioactivities of endophytic fungi due to the possibility of these microorganisms synthesizing pharmacologically active compounds with vast biotechnological applications. This project aimed to study the biodiversity and explore the bioactivities of endophytic fungi from a local medicinal plant Andrographis paniculata (Hempedu Bumi) which is rich with various ethnobotanical properties. It was hypothesized that endophytic fungi from this medicinal plant possess potentials as bioactive compound producers. The biodiversity of endophytic fungi from this plant was successfully established, with 50 of the isolates clustered into 15 different genera, mainly Colletotrichum, Guignardia, Fusarium, Phomopsis and Diarporthe. Root tissues have the highest number of isolates (21 isolates), followed by stem (11 isolates), leaf (15 isolates), and finally flower tissues (3 isolates). Bioprospecting studies revealed different bioactivities of the endophytes. Four of the 50 isolates showed antibacterial activity, with one isolate HBR18 (Aspergillus sclerotiorum) showing broad-spectrum activity against 10 bacterial pathogens tested. For antioxidant assays, 18 endophytic isolates demonstrated 2.2’-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity greater than 50%. This study is the first to report Guignardia sp. as potential free radical scavenger (92.27% scavenging rate). Three Colletotrichum sp. isolates (HBR4, HBR14, HBL8) demonstrated the highest ferric ion reducing potential (FRP) with FRP value greater than 1.900. Total phenolic content (TPC) of all 50 isolates were between 0.12 – 10.85 mg/mL culture filtrate with no specific association found between TPC, FRP and DPPH values. Enzymatic screening showed all 50 isolates were capable of producing at least four of the eight enzymes screened, with one isolate HBR1 (Corynespora cassiicola) producing seven enzymes. For antifungal potential, three and seven endophytic isolates inhibited the growth of Ganoderma boninense and Fusarium oxysporum f. sp. cubense race 4 (FOCR4) up to 50%. G. boninense and FOCR4 are the causal agents for basal stem rot disease and Fusarium wilt in oil palm and banana plantations, respectively. Isolate HBR9 (Aspergillus nomius) exhibited promising inhibitory activities towards both fungal pathogens and hence was selected for further studies. The growth conditions of isolate HBR9 was optimized to be 1% sucrose, 1% sodium nitrate in pH 5 Czapek Dox medium cultured at static condition at room temperature (25± 2 oC). Investigation of the antifungal mechanisms of isolate HBR9 revealed two groups of antifungal compounds (heat-sensitive and heat-stable) contributing to its inhibitory activities. Heat-sensitive metabolites were determined to be potent fungal cell wall-degrading enzymes, namely β-glucanase and chitinase. These antifungal enzymes were characterized based on the optimum incubation time, pH stability, thermos-stability, effect of substrate concentration and kinetic studies. Heat-stable antifungal metabolites, on the other hand, constituted of various phytochemicals tested in this study (saponins, phenolic compounds, cardiac glycosides, steroids, flavonoids, terpenoids, and anthraquinone). Isolate HBR9 was also found to produce hydrolytic enzymes such as amylase, cellulase, xylanase, and lipase which advantage this isolate to procure nutriment from host plants while eliciting protective mechanisms against pathogens especially G. boninense and FOCR4. However, genome analysis of isolate HBR9 revealed complete aflatoxin-producing gene clusters. Production of aflatoxin, a potent liver carcinogen, was tested positive in isolate HBR9. Future work should concentrate on silencing aflatoxin gene expression before further phytochemical purification and application of the metabolites of HBR9 in the field to avoid aflatoxin contamination in oil palm and banana crops.