Isolation, Identification and Molecular Analysis of Fungi in the Study of Aquilaria SP

Sudaya, Ravi Varma (2023) Isolation, Identification and Molecular Analysis of Fungi in the Study of Aquilaria SP. Final Year Project (Bachelor), Tunku Abdul Rahman University of Management and Technology.

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Abstract

Aquilaria sp. which is known as agarwood, produces expensive resinous product after infected by specific endophytic fungi. The objectives of this study are to successfully identify the specific endophytic fungi that infect Aquilaria sp. to improve the yield and output of agarwood production and to determine the compatibility among different strains of fungus before inoculation. Four different types of fungal strains which were Fusarium sp., Aspergillus sp., Alternaria sp. and Penicillium sp. were isolated using potato dextrose agar (PDA) medium. These 4 fungi were identified and confirmed their genus strain via macroscopically and microscopically examination using lactophenol cotton blue (LCB) staining. Dual culture plate method carried out to determine the compatibility among the fungal strains and it is found that Fusarium sp. is compatible with Alternaria sp., Penicillium sp. is compatible with Aspergillus sp. and Alternaria sp. Furthermore, the species of the fungal strains determined using molecular analysis which involve DNA extraction. amplification, purification and sequencing. CTAB extraction method was used to extract the DNA and the purity of the DNA can be determined by using Nanodrop spectrophotometer. Then, polymerase chain reaction (PCR) promoted rapid amplification of DNA segment where the conditions programmed as follows: 5 minutes at 94°C for a first-order denaturation, 50 seconds at 94°C for denaturation with 40 cycles, 50 seconds at 54°C for annealing, 50 seconds at 72°C for extension and 10 minutes at 72°C for final extension. Gel electrophoresis technique was carried out to separate DNA mixtures by their molecular size. There were bands observed for Fusarium sp. and Alternaria sp. at 550 bp but no bands observed for Aspergillus sp. and Penicillium sp. The amplified DNA was purified using MEGAquick-spinTM Total DNA purification kit. The purified DNA of the fungus was sent for Sanger sequencing and the nucleotide sequences that obtained can be used to determine specific fungal species using Genbank BLAST database. Fusarium sp. found to be Fusarium solani and other three strains, Alternaria sp., Aspergillus sp. and Penicillium sp. were inconclusive since no similarity found in the Genbank BLAST database for the sequences obtained. Keywords: Agarwood, Inoculation, Fusarium sp., Aspergillus sp., Alternaria sp. and Penicillium sp.