Shiou Yih Lee

DNA Barcoding of the Endangered Aquilaria (Thymelaeaceae) and Its Application in Species Authentication of Agarwood Products Traded in the Market.

The identification of Aquilaria species from their resinous non-wood product, the agarwood, is challenging as conventional techniques alone are unable to ascertain the species origin. Aquilaria is a highly protected species due to the excessive exploitation of its precious agarwood. Here, we applied the DNA barcoding technique to generate barcode sequences for Aquilaria species and later applied the barcodes to identify the source species of agarwood found in the market. We developed a reference DNA barcode library using eight candidate barcode loci (matK, rbcL, rpoB, rpoC1, psbA-trnH, trnL-trnF, ITS, and ITS2) amplified from 24 leaf accessions of seven Aquilaria species obtained from living trees. Our results indicated that all single barcodes can be easily amplified and sequenced with the selected primers. The combination of trnL-trnF+ITS and trnL-trnF+ITS2 yielded the greatest species resolution using the least number of loci combination, while matK+trnL-trnF+ITS showed potential in detecting the geographical origins of Aquilaria species. We propose trnL-trnF+ITS2 as the best candidate barcode for Aquilaria as ITS2 has a shorter sequence length compared to ITS, which eases PCR amplification especially when using degraded DNA samples such as those extracted from processed agarwood products. A blind test conducted on eight agarwood samples in different forms using the proposed barcode combination proved successful in their identification up to the species level. Such potential of DNA barcoding in identifying the source species of agarwood will contribute to the international timber trade control, by providing an effective method for species identification and product authentication.

The Origin and Domestication of Aquilaria, an Important Agarwood-Producing Genus

The Aquilaria (Thymelaeaceae) tree is a well-known important agarwood-producing genus, which is endemic to the Indomalesia region. The genus is currently protected under CITES regulation and the IUCN Red List due to its heavy declination in the natural population in various sourcing countries. Derived from its precious non-wood fragrant products, the genus was given different names throughout the history until it was finalized in 1783. To date, there are 21 recognized Aquilaria species recorded, of which 13 are reportedly fragrant resin producers, and the status of the remaining eight Aquilaria species is yet to be investigated. Aquilaria is heavily exploited in the wild due to the destructive agarwood harvesting technique that requires hacking of the wood parts to induce agarwood production. Various conservation efforts have been carried out to avoid further destruction toward its gene pool. This includes introducing the species for cultivation and planting the trees in large plantations or home gardens, which further provide a sustainable agarwood production in the industry and indirectly contribute to the local economy. At present, an accurate classification of Aquilaria species is yet to be achieved; misidentification happens frequently, either genuinely because of lack of information and training or intentionally for business gains. In conclusion, a proper taxonomy and classification system are essential for conserving Aquilaria species genetic diversity and for identifying species origin of agarwood products aimed at international trade control.

Rediscovery of Aquilaria rostrata (Thymelaeaceae), a species thought to be extinct, and notes on Aquilaria conservation in Peninsular Malaysia

After more than 100 years since the first discovery, Aquilaria rostrata, a critically endangered species listed in the IUCN Red List and presumably extinct, has been rediscovered in Terengganu State of Peninsular Malaysia. Here, we describe the history, taxonomy, ecology and conservation status of this endemic species, and compare our findings with the species description made from the first and only collection produced prior to this study. In addition, we present the similarities between A. rostrata and several Aquilaria species occurring in Peninsular Malaysia and neighbouring regions using molecular sequence data from the nuclear ribosomal DNA (Internal Transcribed Spacer) and chloroplast intergenic spacer region (trnL-trnF). Our morphological and sequence analyses support the separate status of A. rostrata, a long-lost endemic species of Malaysia.

Keeping Up Appearances: Agarwood Grades and Quality

Agarwood has many grades and goes by countless different names in both the sourcing and consuming countries. The different grades and classes of agarwood result from long-standing grading practices adopted by the people of each country. No standard method is available partly due to the intricacy during the hierarchical process of selling and buying. The foremost reason is the appearance of the traded agarwood itself, which can come in many forms from raw, such as chips, blocks, and flakes, to finished products such as oil, incenses, perfumes, accessories, and carvings. Agarwood in raw forms is of mixed quality; thus, the price and grade depend on this blended appearance. As the product is passed down from collectors to various levels of traders and finally to the buyers, the grade can be readjusted and the price inflated or understated depending on the interest. Therefore, buyers, traders, and collectors heavily rely upon time-honored trust when concluding a business deal. Authorities have not found the formula to standardize the grading system of agarwood trade, and this leads to the lack of coordination and regulation at international level. Nevertheless, several sourcing and consuming countries have made the effort to grade their agarwood according to their own local market, which can be used as a benchmark in formulating a more contemporary method that could be acceptable to all countries.

Phylogenetic Relatedness of Several Agarwood-Producing Taxa (Thymelaeaceae) from Indonesia

Indonesia is home to several tree taxa that are harvested for agarwood. This highly valuable oleoresin ironically was the cause for some species to become vulnerable due to gluttonous human activity. However, information on the genetic diversity of these endangered trees is limited. In this study, 28 specimens representing eight species from two genera, Aquilaria and Gyrinops, were collected from ex-situ and in-situ populations in Indonesia. Phylogenetic analysis conducted on DNA sequences of the nuclear ribosomal internal transcribed spacer (ITS) and the trnL-trnF intergenic spacer regions, revealed that Aquilaria and Gyrinops are paraphyletic when Aquilaria cumingiana is excluded. The phylogenetic analysis for ITS and trnL-trnF showed capability to categorise agarwood-producing species based on their regions: East Indonesia and West Indonesia, using Wallace’s Line as the divider. In addition, we discuss challenges in species identification and taxonomy of agarwood-producing genera, and their conservation efforts in Indonesia.

History and perspectives of induction technology for agarwood production from cultivated Aquilaria in Asia: a review

Agarwood, the resinous product of Aquilaria spp. (Thymelaeaceae), is highly valued for medicinal and fragrant purposes. Unsustainable forest harvesting contributes to the declining population in the wild, threatening their existence. As a protection effort, cultivation occurred in range countries, mainly in Asia, effectively establishing mass plantations. The success of domesticating Aquilaria relies heavily on technological advancement in agarwood induction, without which the entire thriving industry will collapse. In this paper, we describe efforts since 1929 and current progress and variation in induction as practiced in Asia. The methods include traditional practices and artificial induction via inoculum and chemical inducer, experimented and patented or otherwise. Artificial induction methods are being developed to intensify agarwood production in terms of yield and quality. While traditional methods are commonly applied by rural planters, artificial methods with the use of specific agents/chemicals are seen as more appealing for inducing agarwood, and have a higher demand from entrepreneurs and plantation owners. Several issues related to induction technology faced by stakeholders in agarwood cultivation are presented, such as safety levels, cost, yields, and quality. We conclude by highlighting remaining challenges in induction methods and their associated technologies.

Genome Size, Molecular Phylogeny, and Evolutionary History of the Tribe Aquilarieae (Thymelaeaceae), the Natural Source of Agarwood

The tribe Aquilarieae of the family Thymelaeaceae consists of two genera, Aquilaria and Gyrinops, with a total of 30 species, distributed from northeast India, through southeast Asia and the south of China, to Papua New Guinea. They are an important botanical resource for fragrant agarwood, a prized product derived from injured or infected stems of these species. The aim of this study was to estimate the genome size of selected Aquilaria species and comprehend the evolutionary history of Aquilarieae speciation through molecular phylogeny. Five non-coding chloroplast DNA regions and a nuclear region were sequenced from 12 Aquilaria and three Gyrinops species. Phylogenetic trees constructed using combined chloroplast DNA sequences revealed relationships of the studied 15 members in Aquilarieae, while nuclear ribosomal DNA internal transcribed spacer (ITS) sequences showed a paraphyletic relationship between Aquilaria species from Indochina and Malesian. We exposed, for the first time, the estimated divergence time for Aquilarieae speciation, which was speculated to happen during the Miocene Epoch. The ancestral split and biogeographic pattern of studied species were discussed. Results showed no large variation in the 2C-values for the five Aquilaria species (1.35–2.23 pg). Further investigation into the genome size may provide additional information regarding ancestral traits and its evolution history.

Diversity and Characterization of Endophytic Fungi Isolated From the Tropical Mangrove Species, Rhizophora mucronata, and Identification of Potential Antagonists Against the Soil-Borne Fungus, Fusarium solani

Rhizophora mucronata is an important ecosystem entity of the Malaysian mangrove forest. Since the species grows in a harsh environment, any organism that is isolated from this species would be of huge interest due to its potential in having novel bioactive compounds. In the present work, we isolated, identified and characterized, a total of 78 fungal isolates harboring inside the leaf tissues of R. mucronata. Molecular identification using the nuclear ribosomal DNA internal transcribe spacer (ITS) sequences returned with high similarity matches to known sequences in the GenBank. Maximum likelihood analysis revealed the phylogenetic relationship of all isolates from this study. Most of the dominating fungal endophytes were from the genera Pestalotiopsis, followed by Alternaria and Cladosporium. Six isolates representing the genera Alternaria, Fusarium, Nigrospora, Pestalotiopsis, Phoma, and Xylaria, were further screened for their antagonism activities. Dual culture test assay revealed their inhibition percentages against the phytopathogenic fungus Fusarium solani between 45–66%, and 0.8–23% when using non-volatile test assay. Of the six isolates, only Fusarium lateritium and Xylaria sp. showed antibacterial activities against the pathogenic bacteria, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, with the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) ranging from 0.5 to 2 mg/mL. The DPPH radical scavenging assay recorded a high level of antioxidant activity in Xylaria sp., 3-fold above that of F. lateritium. We demonstrate for the first time, two members belonging to the endophytic fungal community in the tropical mangrove species that have potential use as antagonists and antibacterial agents for future biotechnological applications.

Rapid species identification of highly degraded agarwood products from Aquilaria using real-time PCR

Aquilaria species are well known for their expensive agarwood, which is utilized as it is, or used as ingredients in many consumer products. Species validation in agarwood products is important because agarwood price is source-species-dependent. The best approach to establish species identity depends on DNA, as conventional methods (i.e. through morphology) are unable to tell apart products from different Aquilaria sources. However, genomic DNA from processed agarwood is often under poor condition. To overcome this challenge, we adopted real-time PCR technology coupled with species-specific primers derived from single nucleotide polymorphisms (SNPs) in the chloroplast DNA matK and trnL-trnF sequences. We targeted three commercial Aquilaria species: Aquilaria crassna, Aquilaria malaccensis, and Aquilaria sinensis. Dissociation curves and melting points from real-time analysis were found to be distinct across the species tested. In this study, we demonstrate that the real-time PCR-based technique using species-specific primers is capable of differentiating the three major commercial species, i.e. A. crassna, A. malaccensis, and A. sinensis, even when using highly degraded agarwood products as starting material.