Isolation, Characterization and Testing of Fungi Inoculant for Inducement of Resinous Agarwood in Aquilaria malaccensis

Magarate Rita, Elvis Sulang (2017) Isolation, Characterization and Testing of Fungi Inoculant for Inducement of Resinous Agarwood in Aquilaria malaccensis. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Agarwood or popular known as “Gaharu” is a non-timber forest product that produce a unique aroma. This unique scent of agarwood oil can be commercialized as perfumery, while the powder or extract of agarwood is used in incense and medicinal application. Agarwood is produced by diseased tree normally from the genus Aquilaria (Thymelaeaceae). However, overexploitation of gaharu from natural forest has led Aquilaria spp. to be listed in Appendix II of CITES since 2004. Therefore, in order to fulfill the market demands and to sustain gaharu production, proper agarwood inducement techniques is needed. Fungi is one of the microbe that can be isolated from Aquilaria trees that produce agarwood and this fungi can be used to induce resin formation in the Aquilaria trees. The objectives of this study were to isolate and identify fungi from wild gaharu and to elucidate the phylogenetics relationships among fungi isolated from agarwood resin and determines fungi inoculants for inoculation. A total of 17 fungi isolates were cultured from gaharu wood chip taken from Kota Samarahan and Ulu Baram. All fungi isolates were morphological characterized under compound microscope followed by molecular characterization by using ITS 1 and ITS 4 primer. Based on the results of molecular identification, 9 genera of fungi were identified which are Trichoderma, Curvularia, Aspergillus, Hypocrea, Grammothele, Fusarium, Lasiodiplodia, Whitfordia and Penicillium. Most of the isolates have ≥ 99% similarity to a known sequence in NCBI database. The neighbour-joining phylogenetic analysis shown that all fungi species descended from a single common ancestor eventough the fungi species isolated from two different sites. Second part of this study was to test the fungi inoculants in 2 gaharu farms, which were Matang Gaharu Farm and Kpg Raeh Gaharu Farm. The main objectives of this part were to describe the effects of inoculation with selected isolates on Aquilaria malaccensis tree and to determine the chemical constituents of agarwood extracted from inoculated Aquilaria malaccensis. The selected fungi inoculants were G4-B2 (Trichoderma spirale), G3-B2 (Curvularia affinis), G4-B3 (Aspergillus flavus), GB 4 (Penicillium citrinum), GKS 2 (Fusarium solani) and Mixture (combination of 5 fungi isolates). Field testing was conducted by injecting 1 mL of the fungi inoculants and negative control (sterile distilled water) into each holes of the drilled Aquilaria tree in the farms. After 2 and 6 months inoculation, the infected zone were measured longitudinally and the colour changes were observed. The whitish fresh stem of Aquilaria tree in the earlier stage became either dark brown or light brown in second and sixth month of the inoculation. While, the mean length of infection among fungi inoculants were significantly difference (P<0.05) only in second month of the inoculation. The samples of infected zone of Aquilaria stem were taken for chemical compound analysis by GC-MS. Prior GC-MS analysis, the samples were extracted by using methanol as a solvent in Soxhlet extractor within 3 hours. A total of 44 compounds were identified in 6 inoculants and negative control. Different chemical components were found in inoculated Aquilaria trees. Two identified sesquiterpenes namely alpha-caryophyllene (alpha-humulene) and ar-curcumene were found in mixture and G4-B2 inoculants with 0.68% and 0.32%, respectively. Besides, 1, 2-benzenedicarboxylic acid, bis (2- methylpropyl) ester, hexadecanoic acid, benzylacetone, caryophyllene oxide and anisylacetone were also the major components found in the inoculated trees. This results revealed that fungi inoculants used in this study have a potential in inducing the resin in Aquilaria spp.

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