BIODIVERSITAS ISSN: 1412-033X Volume 22, Number 10, October 2021 E-ISSN: 2085-4722 Pages: 4131-4138 DOI: 10.13057/biodiv/d221002 Agarwood formation in Aquilaria beccariana and Aquilaria microcarpa in response to inoculation of newly isolated fungi from Brunei Darussalam YUMNI HAZIQAH MOHAMMAD, POOJA SHIVANAND  , FAIZAH METALI, HUSSEIN TAHA, NUR BAZILAH AFIFAH MATUSSIN, ABDUL MUIZZ AL-AZIM ABDUL-HALIM, ABDUL ZUL’ADLY MOHAIMIN Environmental and Life Sciences Program, Faculty of Science, Universiti Brunei Darussalam. Jln. Tungku Link BE1410, Brunei Darussalam.  email: pooja.shivanand@ubd.edu.bn Manuscript received: 26 April 2021. Revision accepted: 4 September 2021. Abstract. Mohammad YH, Shivanand P, Metali F, Taha H, Matussin NBA, Abdul-Halim AMA, Mohaimin AZ. 2021. Agarwood formation in Aquilaria beccariana and Aquilaria microcarpa in response to inoculation of newly isolated fungi from Brunei Darussalam. Biodiversitas 22: 4131-4138. Aquilaria is an important agarwood-producing tree, but naturally occurring agarwood is rare. Artificial induction method is the most commonly used to produce agarwood in a short time. Out of the 21 species of Aquilaria, Brunei Darussalam houses two species that produce high quality agarwood, namely Aquilaria beccariana and A. microcarpa. The aims of this study are to artificially induce agarwood formation using eight different induction treatments: six fungal inoculant treatments with two control treatments, and to isolate and identify fungal strains from naturally infected agarwood tree using DNA barcoding. Agarwood formation was observed at four different time intervals i.e. 1, 3, 6 and 9 months of post-inoculation. Results exhibited that the area and the total length of discoloration zone were significantly greater at 6 and 9 months of incubation period. Induction treatment did not significantly affect the area and the total length of discoloration. FTIR analysis revealed that two aromatic compounds (C – H and C – C stretching) were detected at the wavelength ranges of 3000 – 2800 cm -1 , and 1500 – 1400 cm -1 respectively, and OH group was detected at 3600 – 3400 cm -1 . Nine fungal strains from Aspergillus, Fusarium, Mucor and Trichosporon were isolated and identified from a naturally infected agarwood, and the last two fungi (Mucor and Trichosporon) were never been previously reported. Keywords: Agarwood formation, Aquilaria, FTIR analysis, fungal diversity, fungal inoculants, incubation period INTRODUCTION Agarwood is a fragrant dark resinous wood, derived from the heartwood of wounded trees of several species of Aquilaria and Gyrinops species belonging to Thymeleaceae (Faizal et al. 2020). Synonymously, it is known by many names, such as chenxiang, eaglewood, gaharu or jinkoh, and is notable worldwide because of its high-priced resin (Cui et al. 2013). The genus Aquilaria is endemic to the Indomalesia region and it is currently protected under the Convention on International Trade in Endangered Species (CITES) and the International Union for Conservation of Nature (IUCN) (Lee and Mohamed 2016). From the natural phenomenon to the event of technology associated with agarwood formation, many countries around the world show endless efforts in unveiling the underlying mechanisms for developing the best quality of agarwood due to its immense value. Agarwood is famous for its essential oils, expensive handicrafts products, cosmetics products and most important for its medicinal values (Naziz et al. 2019). Without the wound, agarwood is of little value. A wound can be defined as injury to trees from natural causes and man-made. Wounds are caused by many natural activities, such as insect infestations, lightning, and disease infections caused by bacteria and fungi (Xu et al. 2013; Zhang et al. 2014). Injuries to trees may also be man-made such as making a deliberate scratch or holes to tree stems and any other types of intentional physical wounding to trees. With present technology, humans can induce infection in a relatively shorter time frame than natural infection using culturable microorganisms such as bacteria and fungi in laboratories. Such microorganisms play a crucial role in the development of agarwood. Several studies have reported that microbial inoculants induced agarwood formation in various Aquilaria species. Tian et al. (2013) previously inoculated uninfected A. sinensis with several inoculants of fungal species, namely Phomopsis, Botryosphaeria, Cylindrocladium and Colletotrichum. While Faizal et al. (2017) inoculated A. malaccensis with Fusarium solani. This study explores the potential of agarwood development using two Aquilaria species (A. beccariana and A. microcarpa) by employing artificial induction of microbial inoculants in Brunei Darussalam. According to the IUCN Red List, A. beccariana and A. microcarpa are categorized as vulnerable and endangered, respectively (IUCN Red List of Threatened Species 2020). Therefore, the aims of present study are to induce and assess artificial agarwood formation in two Aquilaria species using eight different induction treatments consisting of six fungal strains and two control treatments, and also to investigate the fungal diversity in a naturally infected A. beccariana and A. microcarpa.