Species Of Aquilaria Research Articles

Statistical analysis of agarwood oil chemical compound exists in four species of Aquilaria

Statistical analysis of agarwood oil chemical compound exists in four species of Aquilaria

Population genomic analyses of protected incense trees Aquilaria sinensis reveal the existence of genetically distinct subpopulations

The incense tree Aquilaria sinensis (Thymelaeaceae) can produce agarwood with commercial values and is now under threat from illegal exploitation in Hong Kong, impairing the local population and biodiversity. Together with other species of Aquilaria, it is listed in the CITES Appendix II, which strictly regulates its international trade. To understand the population structure of A. sinensis and to make relevant conservation measures, we have sequenced 346 individuals collected in Hong Kong and southern mainland China. Population genomic analyses including principal component analysis, neighbor-joining tree construction, ADMIXTURE, and hierarchical pairwise-FST analyses suggested that genetically distinct populations are contained in certain areas. Genomic scan analyses further detected single-nucleotide polymorphism (SNP) outliers related to plant defense, including the CYP71BE gene cluster. In addition to the population analyses, we have developed a modified hexadecyltrimethyl-ammonium bromide (CTAB) DNA extraction protocol for obtaining DNA from agarwood samples in this study, and resequencing of DNA extracted from two agarwood samples using this method allows us to successfully map to the sample corresponding localities in the phylogenetic tree. To sum up, this study suggested that there is a genetically distinct subpopulation of incense tree in Hong Kong that would require special conservation measures and established a foundation for future conservation measures.

DNA barcoding identification of IUCN Red listed threatened species in the genus Aquilaria (Thymelaeaceae) using machine learning approaches

Aquilaria Lam. is a remarkable genus that produces agarwood, which is widely used as an herbal medicine, perfume, and incense sticks, as well as in interior decoration. However, illegal harvesting pose threats to endangered Aquilaria species, and the adulteration in agarwood products causes potential medical safety. In this study, 306 authentic sequences of five candidate barcodes (ITS, matK, psbA-trnH, rbcL, and trnL-trnF) downloaded from GenBank and 330 sequences taken from our samples were collected to establish a DNA barcode reference dataset for 11 species of Aquilaria listed in the IUCN Red List of Threatened Species. Machine learning approaches, including BLOG, Naïve Bayes, SMO, Jrip, and J48, were compared with distance-based (TaxonDNA) and tree-based (neighbor-joining tree [NJ tree]) methods based on the accuracy of discrimination across the five single barcodes and their combinations. The results indicated that BLOG and SMO could successfully identify the six species of Aquilaria with ITS + matK, ITS + rbcL, and ITS + trnL-trnF. However, when TaxonDNA and the NJ tree were used, only ITS + matK could identify these six species. Additionally, greater species resolution was found in machine learning approaches (16.67%−66.67%) compared with classical methods (0–50%) in managing low variation barcodes, such as psbA-trnH, and chloroplast-locus combinations. Compared with traditional analytical methods, machine learning approaches are reliable and efficient tools for DNA barcoding analysis of Aquilaria, which can be utilized to combat the adulteration of agarwood products and facilitate the conservation of Aquilaria species.

A review on biological induction of agarwood in Aquilaria, with special reference to India

There are thirteen species of Aquilaria producing agarwood, a product of plant and microbe interaction. The process of natural formation of agarwood is slow and only 10 percent of the trees in wild get infected forming agarwood. Since commercial cultivation of agarwood has begun, there is a need for a supporting programme of artificial induction to make the programme viable. The paper reviews the methods of biological induction in Aquilaria sp., the role of endophytes especially species of Fusarium and the availability of inocula for inducing agarwood formation.

Leaf morphoanatomical character variation of Gyrinops and Aquilaria (Thymelaeaceae) in Indonesia region at east Wallace line

There are seven species of Gyrinops and two species of Aquilaria (Thymelaeaceae), which are distributed in Eastern Indonesia at the east of the Wallace line. The anatomical character of the paradermal tissue, midrib and petiole were the important characteristics possessed by plants that can be used as data for identification. The purpose of this article was to study leaf morphoanatomical character variation of Gyrinops and Aquilaria in Indonesia Region at East Wallace Line. The Gyrinops specimen used in the research was the collection of the Agarwood Study Center, Universitas Mataram, Mataram, Indonesia. The paradermal slide was made with the whole-mount slide method and the slide cross-section of midrib and petiole was made by using a hand-free section. Their preparation process used a permanent slide method and mounted with glycerine jelly. Based on the results of the research conducted, it can be seen that the anatomical characters of paradermal tissues, midrib and petiole of Gyrinops and Aquilaria have general characteristics such as hypostomatic leaves, stomata type anomocytic, non-glandular and unicellular trichome. While other characters of structure paradermal, midrib and petiole could be used as a taxonomy diagnostic marker to identify at the level genus and species on Gyrinops and Aqilaria.

Rapid Discrimination of Aroma Components in Natural and Artificial Agarwood Incense by Static Headspace Sampling – Gas Chromatography – Mass Spectrometry (SHS-GC-MS) with Principal Component Analysis (PCA) and Partial Least Squares – Discrimination Analysis (PLS-DA)

Agarwood is a resinous, fragrant wood produced by tropical tree species of the genus Aquilaria, and is highly valued for its wide range of uses in medicine, incense, and perfume. In this study, the quality of agarwood and patterns of chemical constituents induced in natural and artificial agarwood by physical injury, chemical inducer, and chemical plus fungal infection were compared using static headspace sampling gas chromatography – mass spectrometry (SHS-GC-MS). The optimal agarwood heating temperature was determined to be 180 °C. The fingerprints of 40 batches of agarwood were established. Multivariate statistical analysis was utilized to identify 15 common and ten differentiating components, respectively. Agarwood induced by the physical method was more similar to natural agarwood. Exploration of the pyrolysis products of chromone derivatives and sesquiterpenes was undertaken to discover the causes of differences in chemical compositions between artificial and natural agarwood. Our findings revealed that benzylacetone, baimuxinal, and guaiol were significant for agarwood incense and these compounds may be used as markers for classification.

A Review of Aquilaria malaccensis Propagation and Production of the Secondary Metabolite from Callus

Aquilaria is an evergreen non-timber agarwood obtained from the 15 species of Aquilaria belonging to the family Thymelecea. There are two species endemic to Northeast India, A. malaccensis and A. khasiana. A. malaccensis generate a high-grade degree of resin as compared to the other Aquilaria species, and it contributes to the economy of the Northeast states of India and the country as a whole. Due to its profoundly valuable sources, it is overexploited, which impacted its availability in genetic environments. The cultivation of the tree is challenging due to some environmental factors like the sensitivity of the seeds to desiccation, high light intensity, low shelf life, slow growth rate, and the effect of insects and microorganisms. Therefore, conservation and proliferation are urgently required for environmental sustainability and prevention from the stage of extinction. The objective of this paper is to compile the major research works on the conservation, production of the secondary metabolite from callus of A. malaccensis and updated information on its developments and approaches that are rapidly taking place in recent years so that further novel research can be envisaged.

Agarwood formation in Aquilaria beccariana and Aquilaria microcarpa in response to inoculation of newly isolated fungi from Brunei Darussalam

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.

Mining and Analysis of Chloroplast Simple Sequence Repeats (SSRs) from Eight Species of Aquilaria

Mining and Analysis of Chloroplast Simple Sequence Repeats (SSRs) from Eight Species of Aquilaria

Inhibition of pancreatic lipase by gallic acid and quercetin equivalent in ultrasonicated Malaysian grown Aquilaria spp. leaves of different particle size

Pancreatic lipase inhibitory compounds (gallic acid and quercetin) and their inhibition properties were determined in the crude extract of matured leaves from widely grown Aquilaria spp. in Malaysia, which are Aquilaria malaccensis and Aquilaria subintegra. The leaves were dried at 60 °C for 24 hours and undergone a pre-treatment method by using ultrasonication, within 30 minutes of reaction. Dried matured leaves were ground and sieved into particle sizes of 250, 300, 400, 500, and 1000 µm. Each particle size was soaked in distilled water with a ratio of 1% (w/v) for 24 hours, prior to ultrasonication process. Particle size of 250 µm resulted in the highest concentration of gallic acid and quercetin equivalent for both species of Aquilaria, wherein 89.99 mg/ml of gallic acid equivalent and 0.0295 mg/ml of quercetin equivalent were obtained in A. malaccensis crude leaves extract and in A. subintegra crude extract, 101.27 mg/ml and 0.0373 mg/ml were obtained, respectively. Thus, this sample was used in the inhibition study of pancreatic lipase. The concentrations of substrate and enzyme activity prepared were varied in the inhibition kinetics study. Based on the inhibition of pancreatic lipase, the crude leaves extract of both Aquilaria species exhibited the mixed-inhibition which is also known to be the non-competitive inhibition because the reaction mechanism showing the binding of inhibitor to the free pancreatic lipase and pancreatic lipase-substrate complex. This research is one of the fundamental works for future studies on the natural source to control obesity, which is a new direction of the key research in lipase inhibition.

Metabolite Profiling of Aquilaria malaccensis Leaf Extract Using Liquid Chromatography-Q-TOF-Mass Spectrometry and Investigation of Its Potential Antilipoxygenase Activity In-Vitro

The Aquilaria malaccensis species of the genus Aquilaria is an abundant source of agarwood resin and many bioactive phytochemicals. Recent data regarding the chemical constituents and biological activities of Aquilaria leaves led us to attempt to qualitatively profile the metabolites of Aquilaria malaccensis leaves from a healthy, noninoculated tree through phytochemical screening, GC-MS, and LC/Q-TOF-MS. The present work is also the first to report the antilipoxygenase activity of A. malaccensis leaves from healthy noninoculated tree and investigate its toxicity on oral mucosal cells. A total of 53 compounds were tentatively identified in the extract, some of which have been described in literature as exhibiting anti-inflammatory activity. A number of compounds were identified for the first time in the extract of A. malaccensis leaf, including quercetin, quercetin-O-hexoside, kaempferol-O-dirhamnoside, isorhamnetin-O-hexoside, syringetin-O-hexoside, myricetin, tetrahydroxyflavanone, hesperetin, sissotrin, and lupeol. The antilipoxygenase assay was used to determine the lipoxygenase (LOX) inhibitory potential of the extract, while a WST-1 assay was conducted to investigate the effect of the extract on oral epithelial cells (OEC). The extract implied moderate anti-LOX activity with IC50 value of 71.6 µg/mL. Meanwhile, the cell viability of OEC ranged between 92.55% (10 µg/mL)–76.06% ± (100 µg/mL) upon treatment, indicating some potential toxicity risks. The results attained encourage future studies of the isolation of bioactive compounds from Aquilaria malaccensis leaves, as well as further investigation on the anti-inflammatory mechanisms and toxicity associated with their use.

Study of a chemotaxonomic marker able to identify the genus Aquilaria (Thymelaeaceae)

Le genre Aquilaria Lam. (Thymelaeaceae) comprend 21 espèces d’arbres (The Plant List, 2013) et se trouve principalement en Asie du Sud-Est. Lorsque l’arbre est infecté (champignons, bactéries), son bois devient brunâtre ou noirâtre (appelé bois d’agar) en raison de la sécrétion d’une oléorésine en réaction à l’infection. La résine est très parfumée et a été recherchée et utilisée pendant des siècles par les bouddhistes, les hindous et les musulmans pour faire de l’encens pour les cérémonies religieuses. Cette oléorésine se trouve principalement dans les espèces du genre Aquilaria, mais aussi dans quelques espèces des genres Gyrinops Gaertner et Gonystylus Teijsmann & Binnendijk. Il est difficile de faire la distinction entre ces espèces, et ce manque de connaissances taxonomiques a conduit à une surutilisation des arbres, mettant en danger ces espèces endémiques inscrites à l’Annexe II de la CITES. Nous avons utilisé la chimiotaxonomie comme outil de discrimination pour analyser les molécules polyphénoliques, métabolites secondaires, qui sont connus pour agir comme marqueurs taxonomiques dans d’autres plantes. En utilisant une technique d’analyse simple, efficace et peu coûteuse (chromatographie bidimensionnelle en couche mince), nous avons trouvé la même molécule polyphénolique dans les six espèces du genre Aquilaria étudiées, qui a été identifiée comme mangiférine par analyse colorimétrique et chromatographique en comparaison avec la mangiférine de contrôle. Au cours de ces analyses, nous avons trouvé une relation entre les genres Aquilaria et Gyrinops (botaniquement démontrée) par l’existence occasionnelle de mangiférine chez trois espèces du genre Gyrinops. Nous avons également constaté qu’un laps de temps de cent-quarante ans entre deux échantillons d’herbier de la même espèce n’avait pratiquement aucun effet sur la concentration de mangiférine dans la plante. Ces travaux ont montré les mérites de la chimiotaxonomie dans la recherche de marqueurs taxonomiques et l’originalité de l’analyse colorimétrique d’un métabolite de plante chromatographié en couche mince.

The complete chloroplast genome of agarwood producing species, Aquilaria sinensis (Lour.) Gilg: a species on IUCN red list

The entire chloroplast genome of Aquilaria sinensis (Lour.) Gilg was identified as a circular molecule of 174,885 bp length with a typical tetrad structure, including a pair of inverted repeats (42,103 bp each), a large single copy (87,331 bp) and a small single copy (3,348 bp) regions. The A. sinensis cp genome encoded 8 rRNAs, 39 tRNAs, and 90 proteins. A phylogenetic tree was reconstructed using the 43 protein-coding genes of eight Thymelaeaceae. Two other Malvales, Abelmoschus esculentus and Durio zibethinus, were selected as the outgroup. Our phylogenetic analysis suggests that the five examined species of Aquilaria appeared a monophyletic group with robust support.

Antioxidant and Antifungal Activity of Endophytic Fungi Associated with Agarwood Trees

Several species of Aquilaria and Gyrinops are native to Indonesia and well known as agarwood-producing trees with a high economic value. Their bioactive compounds have a wide spectrum of uses, such as in medicine and cosmetics. These genera have undergone extensive search for novel bioactive compounds. The purpose of this study was to isolate, identify, and characterize the endophytic fungi community associated with Aquilaria malaccensis, A. microcarpa, Gyrinops versteegii, and A. crassna trees and investigate their bioactive properties as antioxidant agents and antagonists. A total of 50 fungi were successfully isolated from different tissues of the four species of agarwood-producing trees. Two isolates exhibited strong antioxidant activity, namely, Apodus oryzae (R2MC3A, IC50 60.92 mg/mL) and Diaporthe sp. (P1DS1[C], IC50 76.65 mg/mL). Two isolates, Pestalotiopsis theae (P3BS3[B]) and Curvularia sp. (P2CD3A), showed >75% antifungal activity against pathogenic Fusarium solani. The results revealed that endophytic fungi associated with the studied agarwood-producing trees had potential antioxidant and antifungal activities for further applications in biotechnology.

The complete chloroplast genome of a critically endangered agarwood tree, Aquilaria crassna (Thymelaeaceae)

Aquilaria crassna Pierre ex Lecomte is a principal agarwood tree resource. It has been listed as a critically endangered species. The complete chloroplast genome of A. crassna was assembled based on next-generation sequencing. The plastome was a quadripartite circular shape with 174,830 bp in length, including a large single-copy (LSC) region of 87,450 bp and small single-copy (SSC) region of 3346 bp, separated by two inverted repeat (IR) regions of 42,017 bp each. The chloroplast genome contained 125 genes, including 79 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The overall GC content of the whole genome was 36.7%. The phylogenetic tree showed that three species of Aquilaria formed one monophyletic clade with 100% bootstrap value.

Phylogenetic analysis of Aquilaria Lam. (Thymelaeaceae) based on DNA barcoding

Abstract Aquilaria Lam. is an important group of trees that produce agarwood, which is widely used for manufacturing medicine, perfumes and incense. The members of the genus Aquilaria are close to being extinct due to illegal harvesting and are now protected in many countries. In this study, five DNA barcodes [internal transcribed spacer (ITS), maturase K (matK), psbA-trnH, ribulose bisphosphate carboxylase (rbcL) and trnL-trnF] and their combinations were evaluated for the discrimination of the major (16 out of 21) species of Aquilaria based on three criteria: sequence variation, genetic distances and the discrimination ability. In addition, we attempted to determine the phylogenetic relationship between Aquilaria and Gyrinops species using three phylogenetic analysis methods. We observed that the combination barcode ITS+trnL-trnF had a good discrimination ability based on the best match and best close match methods, provided more genetic information, and clearly indicated the comprehensive phylogenetic relationship between most Aquilaria and Gyrinops species. Considering the overall performance of these barcodes, the ITS+trnL-trnF is a suitable barcode for the identification of Aquilaria species.

Isolation of Associated Fungal Species in Aquilaria and Gyrinops Species of Family Thymelaeaceae

Certain tree species of Aquilaria and Gyrinops of family Thymelaeaceae are capable of producing a valuable resin called agarwood as a result of a self-defense mechanism to protect the internal tissue damage caused by pathogenic microorganisms entering via wounds occurred due to natural or artificial causes. The aromatic properties of agarwood oil are extraordinary high and therefore it is extensively used for incense, perfume and for traditional medicine in different parts of the world. Among the microorganisms which are capable of causing agarwood formation inducement, fungal species are believed to be the most influential agents. The ability of agarwood production in Gyrinops walla, believed to be endemic to Sri Lanka was recently identified. Due to the legal constraints of exporting agarwood and other products extracted from G. walla, Aquilaria crassna was introduced to the country as a high income plantation crop by private sector companies. However, studies were not conducted to identify the associated fungi in these species when grown under the climatic and geological conditions of Sri Lanka, which could be potential inocula for producing agarwood in artificial manner. Therefore the present study attempted to identify the associated fungal species in various parts of G. walla and A. crassna grown in the wet zone in Sri Lanka. For this reason, leaves, un-infected normal stem tissues and agarwood formed stem tissues were extracted from G. walla and A. crassna trees growing in Neboda and Ingiriya region respectively by non-destructive sampling. Those tissues were surface sterilised and separately grown in Potato Dextrose Agar at room temperature for 7 days in the laboratory. The morphological and genetic characteristics were then used for the identification of different fungal species. Trichoderma harzianum, Botryosphaeria laricina, Aspergillus terreus, A. sojae, A. niger, Fusarium sp, one unidentified Botryosphaeria sp and Sarcinomyces sp were identified in the agarwood resinous tissues extracted from G. walla. B. laricina, A. terreus, one unidentified Botryosphaeria sp and F. equiseti were identified from non-resinous stem tissue samples while T. harzianum, Colletotrichum gloeosporioide sand A. terreus were identified from the leaves of G. walla. B. laricina and one unidentified Aspergillus sp were identified from resinous tissue samples of A. crassna while B. laricina and Diaparthe amygdale were identified from non-resinous tissues and leaf samples respectively. The tests are currently underway to identify the most influential fungal species among the above to form agarwood in artificial manner. Keywords: Gyrinops walla, Aquilaria, Associated fungi, Agarwood, Fungal species Acknowledgement: Sadaharitha Plantations Limited and the Centre for Forestry and Environment, University of Sri Jayewardenepura.

Impacts of Selected Fungal Toxins on the Inducement of Agarwood in Gyrinops walla

Agarwood is a resinous wood substance that is produced as a non-specific host response to wounding, insects or microbial invasion by certain trees of family Thymelaeaceae. Most species of Aquilaria and a few species of Gyrinops, Aetoxylon and Gonystylus are capable of producing agarwood. Gyrinops walla is the only agarwood producing tree growing in Sri Lanka which is believed to be endemic. Previous studies have identified that the two fungal species Aspergillus niger and Fusarium soloni are capable of inducing agarwood formation in G. walla. Since those fungi form toxins, it can be assumed that the agarwood production is induced by fungal toxins. Although the ability of fungal species in agarwood production inducement was studied previously, the impact of toxins on agarwood production was never tested. Therefore the present study is aimed to investigate the ability of these mycotoxins to induce the formation of agarwood in G. walla. Secondary metabolites were extracted from one selected isolate of A. niger and F. soloni. The presence of toxins in those metabolites was confirmed through a structured leaf bioassay. Volumes of 5 ml and 10 ml of extracted toxins were inoculated into live healthy G. walla trees and the formation of agarwood resin within the stem was observed using tissue samples collected at 10 cm above and below the inoculation points, 3 months after inoculation. Agarwood resins were extracted from those tissues and resin constituents were analysed by GC-FID method. According to the results, tissue samples obtained from trees inoculated with toxins of F. solani showed higher average resin content (1.69%±0.33) than the trees inoculated with toxins of A. niger (0.81%±0.26) though there was no significant difference (F=2.34, p=0.099). The highest resin percentage was observed with 5 ml inoculation of F. solani toxins (2.00%±0.82). Several resin constituents considered as important markers in identifying agarwood aroma were present in the resins extracted from inoculated trees. Among them, 6-acetyl-7-hydroxy-2,2-dimethyl benzopyran showed the highest abundance. However, the presence of common resin constituents was higher in the inoculation of toxins of A. niger when compared to toxins of F. solani. This study identified that fungal toxins can be used as an effective inoculant to trigger the formation of agarwood in G. walla. However, further research is recommended to verify above observations. Keywords: Agarwood, Gyrinops walla, Fungal toxins, Aspergillus niger, Fusarium solani

A Review on the Malaysian Aquilaria species in Karas Plantation and Agarwood Production

Malaysia is one of the countries which is rich in its flora and fauna bio-diversity. Malaysian forest products have been exploited since ancient times for its high economic values. One of these high economic value products is agarwood. Thymelaeacea is one medium-size family tree where it is estimated to consist of 50 genera and 900 species of which many can be found in Africa, Australia and Asia. Most species of Aquilaria in Thymelaeaceae family produced agarwood or ‘gaharu’. Aquilaria malaccensis is a species of the highest recorded number of planting by karas tree farmers and known as the primary producer of agarwood in Malaysia. Agarwood is non timber forest product it is one of the precious wood on earth and prized for its rich and wonderful fragrance. Agarwood is formed due to the phathological process as respond to fungal infection or chemically stimulated. This paper seek to understand in general the type of karas trees species most favoured by karas farmers and to know what other similar species that are capable of producing resin for gaharu products. This research is to review the natural process of producing gaharu and its importance to the diverse related industries. This research also found the high commercial value and its potential for further development.

Agarwood Formation Induced by Fermentation Liquid of Lasiodiplodia theobromae, the Dominating Fungus in Wounded Wood of Aquilaria sinensis

Agarwood is broadly used in incense and medicine. Traditionally, agarwood formation is induced by wounding the trunks and branches of some species of Aquilaria spp., including A. sinensis. As recently evidenced, some fungi or their fermentation liquid may have the potential of inducing agarwood formation. The present study aimed to analyze the fungi isolated from an agarwood-producing A. sinensis tree and subsequently identify the fungi capable of promoting agarwood formation. We identified a total of 110 fungi isolates based on their morphological characteristics and rDNA ITS sequences. These isolates came from four different layers (namely the decomposing layer, agarwood layer, transition layer, and normal layer) near the agarwood formation site of the trunk. According to the experimental results, most of them belonged to Dothideomycetes (81.82%), while the others to Sordariomycetes (13.64%) or Eurotiomycetes (4.55%). Of note, 88 isolates were shown belonging to the species of Lasiodiplodia theobromae that are most frequently isolated from different layers. In addition, when the fermentation liquid of two isolates of L. theobromae (AF4 and AF12) and one isolate of Fusarium solani (AF21) was inoculated into the A. sinensis wood using the Agar-Wit technique, promoted agarwood formation was observed; however, the effect of AF21 did not keep stable in the later test, while AF4 and AF12 still functioned 1 year later. This study may lay a foundation for exploring the underlying mechanism of agarwood formation as well as fungi application in agarwood production.