Aquilaria Species Research Articles

Profiling of microRNAs under wound treatment in Aquilaria sinensis to identify possible microRNAs involved in agarwood formation.

Agarwood, a kind of highly valued non-timber product across Asia, is formed only when its resource trees -- the endangered genus Aquilaria are wounded or infected by some microbes. To promote the efficiency of agarwood production and protect the wild resource of Aquilaria species, we urgently need to reveal the regulation mechanism of agarwood formation. MicroRNAs (miRNAs) are a group of gene expression regulators with overwhelming effects on a large spectrum of biological processes. However, their roles in agarwood formation remain unknown. This work aimed at identifying possible miRNAs involved in the wound induced agarwood formation. In this study, the high-throughput sequencing was adopted to identify miRNAs and monitor their expression under wound treatment in the stems of A. sinensis. The miR171, miR390, miR394, miR2111, and miR3954 families remained at the reduced level two days after the treatment. 131 homologous miRNAs in the 0.5 h library showed over three-fold variation of read number compared with the control library, of which 12 exhibiting strong expression alterations were further confirmed by real-time quantitative PCR. Target prediction and annotation of the miRNAs demonstrated that the binding, metabolic process, catalytic activity, and cellular process are the most common functions of the predicted targets of these newly identified miRNAs in A.sinensis. The cleaveage sites of three newly predicted targets were verified by 5'RACE.

Development of an enzyme-linked immunosorbent assay for determination of iriflophenone 3-C-β-D-glucoside from Aquilaria spp. using a specific polyclonal antibody

Aquilaria species (agarwood) are cultivated in Asian countries for the source of agarwood oil and endangered due to the scent industry. The leaves of Aquilaria spp. are used ethnomedically to treat anti-inflammatory conditions, diabetes, and as laxative [1 – 3]. The active compounds in Aquilaria spp. leaves are benzophenone glycosides including iriflophenone 3-C-β-D-glucoside (IP3G), iriflophenone 2-O-α-L-rhamnopyranoside and iriflophenone 3,5-C-β-D-diglucopyranoside [4]. Among them, IP3G is major active compound found in Aquilaria spp. leaves. Thus, a reliable and sensitive analysis method is necessary for quality control of the plant raw material and product. An enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal antibody (PAb) against IP3G. The indirect competitive ELISA using anti-IP3G PAb was high specific for IP3G. There were low cross-reactivity with iriflophenone 3,5-C-β-D-diglucopyranoside (13%), genkwanin 5-O-β-primeveroside (3.55%) and no cross reactivity found in other compounds. The range of ELISA assay extends from 100 – 1,560 ng/ml with coefficient of variation (CV) 1.19 – 2.07% for intra-assay and 3.76 – 5.70% for inter-assay precision levels. The percentages of recovery in the range of 96.0 – 99.0% with CV of 4.50 – 5.32%, which indicated that the method is accurate in agarwood samples. The correlation between the result from the ELISA and HPLC had a coefficient of determination (r 2) of 0.9321. The developed immunoassays using the PAb against IP3G are simple, reliable, sensitive and cost-effective assay for quality control of agarwood raw material and product.

Whole-tree Agarwood-Inducing Technique: An Efficient Novel Technique for Producing High-Quality Agarwood in Cultivated Aquilaria sinensis Trees

Agarwood is the fragrant resin-infused wood derived from the wounded trees of Aquilaria species. It is a valuable non-timber forest product used in fragrances and as medicine. Reforestation for Aquilaria trees in combination with artificial agarwood-inducing methods serves as a way to supply agarwood and conserve of wild Aquilaria stock. However, the existing agarwood-inducing methods produce poor-quality agarwood at low yield. Our study evaluated a novel technique for producing agarwood in cultivated Aquilaria trees, called the whole-tree agarwood-inducing technique (Agar-Wit). Ten different agarwood inducers were used for comparison of Agar-Wit with three existing agarwood-inducing methods. For Aquilaria trees treated with these ten inducers, agarwood formed and spread throughout the entire tree from the transfusion point in the trunk to the roots and branches of the whole tree. Agarwood yield per tree reached 2,444.83 to 5,860.74 g, which is 4 to 28 times higher than that by the existing agarwood-inducing methods. Furthermore, this agarwood derived from Agar-Wit induction was found to have a higher quality compared with the existing methods, and similar to that of wild agarwood. This indicates Agar-Wit may have commercial potential. Induction of cultivated agarwood using this method could satisfy the significant demand for agarwood, while conserving and protecting the remaining wild Aquilaria trees.

Isolation and identification of bacterial endophytes from pharmaceutical agarwood-producing Aquilaria species

Background:Resins and gums are used in traditional medicine and do have potential applications in pharmacy and medicine. Agarwood is the fragrant resinous wood, which is an important commodity from Aquilaria species and has been used as a sedative, analgesic, and digestive in traditional medicine. Endophytic bacteria are potentially important in producing pharmaceutical compounds found in the plants. Hence, it was important to understand which types of endophytic bacteria are associated with pharmaceutical agarwood-producing Aquilaria species.Objective:This study was undertaken to isolate and identify endophytic bacteria associated with agarwood-producing seven (7) Aquilaria species from Malaysia.Materials and Methods:Botanical samples of seven Aquilaria species were collected, and endophytic bacteria were isolated from surface-sterilized-tissue samples. The 16S rRNA gene fragments were amplified using PCR method, and endophytic bacterial isolates (EBIs) were identified based on 16S rRNA gene sequence similarity based method.Results:Culturable, 77 EBIs were analyzed, and results of 16S rRNA gene sequences analysis suggest that 18 different types of endophytic bacteria are associated with (seven) Aquilaria species. From 77 EBIs, majority (36.4%) of the isolates were of Bacillus pumilus.Conclusion:These findings indicate that agarwood-producing Aquilaria species are harboring 18 different types of culturable endophytic bacteria.

Evaluating agarwood products for 2‐(2‐phenylethyl)chromones using direct analysis in real time time‐of‐flight mass spectrometry

Agarwood is the resinous material harvested from threatened Aquilaria species. We investigated how many protonated 2-(2-phenylethyl)chromone ions were sufficient to make an accurate identification of agarwood. Analysis of 125 reference samples was carried out by direct analysis in real time time-of-flight mass spectrometry (DART-TOFMS). The identification criteria developed were applied to commercial samples. We developed a technique that uses DART-TOFMS to detect 2-(2-phenylethyl)chromones. Additionally, we developed a set of criteria to infer the presence of Aquilaria in commercial samples of wood chips, sawdust, incense and liquids. Additionally, we examined other fragrant woods to determine if they contained a chemical profile that could be falsely identified as agarwood. Analysis of reference and commercial samples (n = 151) established that DART-TOFMS provides reproducible mass spectra that are useful for inferring the genus of suspected agarwood samples. We identified 17 ions which were useful for authenticating agarwood. Comparison of the number of chromone ions detected by direct analyses of dry wood chips versus eluent analysis of methanol-extracted wood showed that results were similar. Lastly, analysis of 25 scented woods of other species did not give false positive results. Reliable criteria for inferring agarwood include the presence of diagnostic ions, m/z 319.118 or 349.129, in addition to ten or more ions characteristic of 2-(2-phenylethyl)chromones. Wood anatomists challenged with difficult morphological identifications can use this tool to assist in their analyses. Published 2012. This article is a US Government work and is in the public domain in the USA.

Selection and validation of reference genes for studying stress-related agarwood formation of Aquilaria sinensis

Agarwood is a high-valued woody material for medicine, perfume, and incense production in Asia, Middle East, and Europe. The wild resources of agarwood-producing tree species, e.g., Aquilaria sinensis have been greatly threatened. The formation of agarwood is considered to be associated with the plant stress and defensive responses, thus it would be urgent and significant to investigate the molecular mechanism of these species responding to a variety of stresses. This is the first report regarding the reference gene selection of Aquilaria species for studying the molecular mechanism of stress-related agarwood production. Candidate reference genes were selected according to previous reports and the sequences were obtained from the 454 EST library of A. sinensis. To obtain the robust genes, we applied three independent programs depending on distinct assumptions and combined these results by a rank aggregation algorithm. The result supports tubulin, ribosomal protein, and glyceraldehyde-3-phosphate dehydrogenase to be the most stable reference genes for quantification of target gene expression in the overall samples examined. Validation of these genes through normalizing the expression of a terpene synthase demonstrated that these three genes are reliable. The selective usage of three algorithms based on their characteristics was underlined. However, more robust genes could be identified if the results of all algorithms were combined by a proper method such as the rank aggregation algorithm. Reference genes which are critical in gene expression studies are recommended for future molecular studies of stress response and agarwood production in the endangered Aquilaria and other tree species.

Performance of agarwood ( Aquilaria crassna) seedling transplants improved by shade and fertiliser

While Aquilaria species have had a long history of exploitation and trade, experience with their establishment and management in plantations is only recent. Significant transplant stress during establishment has been a feature of contemporary plantings in Australia. It is therefore important to determine the nature of this stress to mitigate its effect in future plantings. This study examined the influence of antitranspirant and fertiliser applied after planting in open conditions on the relative mean leaf chlorophyll content, survival and growth (height and diameter) of Aquilaria crassna seedlings when compared with a shade control. Additional field-applied fertiliser (without antitranspirant) had a significantly positive effect on mean chlorophyll index and height and diameter growth after transplanting, but a leaf application of antitranspirant had no such effect. Despite the benefit of additional fertiliser (without antitranspirant) this treatment had significantly greater mortality when compared with the shade treatment. The immediate increase in mean chlorophyll index in shade and its initial decline in full-sun exposed seedlings after planting indicates that the transplant stress is influenced, in part, by sun-exposure. Seedlings in the shade treatment were significantly taller than those exposed to full-sun after 60-weeks but no significant differences were found for mean basal diameter. With the exception of one treatment (fertiliser with antitranspirant) it is evident that taller seedlings with thicker stems have a greater survival after transplanting than shorter seedlings with thinner stems. This study reveals that the planting of A. crassna seedlings with mean height and stem diameter of 35 cm and 4.0 mm respectively and providing fertiliser and shade after planting can reduce transplant stress and improve survival and growth of this species. Areas requiring further research including other potential factors influencing transplant stress are explored in this report.

Genomic organization of δ-guaiene synthase genes in Aquilaria crassna and its possible use for the identification of Aquilaria species

The resinous portions of Aquilaria plants, called agarwood, have been used as medicines and incenses. Agarwood contains a great variety of sesquiterpenes, and a study using cultured cells of Aquilaria crassna showed that the production of sesquiterpenes (α-guaiene, α-humulene, and δ-guaiene) was induced by treatment with methyl jasmonate, which led to the cloning of δ-guaiene synthases. In the present study, analyses of genomic organization and Southern blotting of δ-guaiene synthase in A. crassna were performed in order to examine the genomic background of δ-guaiene synthases in Aquilaria plants. Genomic cloning and sequencing revealed five types of sequence in putative δ-guaiene synthases sharing more than 96% identity in exon regions, and that these enzymes belonged to the class III TPS subfamily with seven exons and six introns. Furthermore, Southern blotting revealed that at least five copies of δ-guaiene synthase existed in A. crassna. The hybridization of digested DNA of A. crassna and A. sinensis with probes made with a δ-guaiene synthase cDNA fragment resulted in different banding patterns for these two species. It may be possible to identify Aquilaria species by restriction fragment length polymorphism analyses with δ-guaiene synthase cDNA probes.

Genetic Variation and Molecular Authentication of Selected Aquilaria Species from Natural Populations in Malaysia Using RAPD and SCAR Markers

Aquilaria (Thymelaeaceae) is an endangered agarwood-producing tropical tree that is endemic to the Indomalesia region. Molecular information on genetic diversity of Aquilaria is limited. The aims of this research were to study genetic diversity among three Aquilaria species, growing in natural and distant populations in Malaysia using RAPD markers and to develop SCAR markers for easy identification of A. malaccensis, the major agarwood producer. By analyzing 23 RAPD primers, a total of 368 bands were scored. Multi-populations Descriptive statistics revealed that 333 (90.49%) polymorphic bands were found at species level, where A. malaccensis had 107 (29.08%) bands, A. hirta had 56 (15.22%) and Aquilaria sp.1 had 11 (2.99%), for the percentage of polymorphic loci in a single population/species. Nei's unbiased measurement indicated moderate similarities among populations/species. Out of the 23 RAPD primers, three were found specific to A. hirta and one was specific to each A. malaccensis and Aquilaria sp.1. RAPD-based SCAR markers generated a total of five species-specific amplicons: three for A. hirta, one each for A. malaccensis and Aquilaria sp.l. SCAR markers for A. malaccensis were used to distinguish five other different A malaccensis populations in Malaysia. SCAR markers for A. malaccensis tested in five other different A. malaccensis populations in Malaysia yielded positive and consistent results. The DNA fingerprints identified for each Aquilaria sp. will be useful for Aquilaria identification in natural population, young plantation and even at seedling and seed stages in the nursery, as it is rapid and cost-effective and does not rely on morphology.

The volatile and semi‐volatile constituents of agarwood, the infected heartwood of Aquilaria species: a review

AbstractA compilation of the volatile and semi‐volatile constituents of agarwood, the infected fragrant heartwood of some Aquilaria species, mainly A. malaccensis, A. sinensis and A. crassna, is presented. This high‐priced wood has a long tradition in religious ceremonies as incense and its essential oil is well appreciated in fine perfumery. More than 150 compounds have been identified so far, mostly sesquiterpenoids, chromones and volatile aromatic compounds. The scarce knowledge of their organoleptic properties is summarized. Studies on the chemistry of agarwood smoke are discussed. Emphasis is given to structural and analytical aspects. Copyright © 2011 John Wiley & Sons, Ltd.

Polymorphic microsatellites for forensic identification of agarwood ( Aquilaria crassna)

Tropical agarwood ( Aquilaria) is in danger of extinction in the wild due to illegal logging. Its resin (Gaharu) is used for the production of highly valued incense throughout Asia. We have isolated and characterized microsatellite loci of Aquilaria crassna to detect the geographic origin of agarwood for forensic applications using a modified enrichment procedure based on the capture of repetitive sequences from restricted genomic DNA. We assessed the polymorphisms of five microsatellites amplified from fresh leaves of 22 trees from seven plantations in Vietnam and Thailand and dried leaves of a herbarium specimen of one wild tree. Cross specificity of these markers was confirmed on two related Aquilaria species occurring in China and Vietnam and one microsatellite locus was successfully amplified from wood and incense samples. Four of the loci were polymorphic and the number of alleles ranged from 3 to 15. The loci characterized here can provide a starting point for forensic identification of traded material and certification of sustainably produced agarwood.

GENETIC VARIATIONS AMONG AQUILARIA SPECIES AND GYRINOPS VERSTEEGII USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS

Aquilaria sp Thymelaeaceae is the most valuable non wood production of forestry plant in Indonesia It produces a fragrant resin when subjected to fungal attack and has been traded internationally known as gaharu Knowledge of genetic diversity and relationship among species and genus is important for breeding purposes and species conservation In this study genetic variability of six Aquilaria species were analyzed using the AmplifiedFragment Length Polymorphism AFLP markers Ten AFLP primer combinations amplified 1353 DNA fragments ranging in size from100 to 350 bp of which 1285 95 of them were polymorphic Genetic similarities among Aquilaria sp consisted of A malaccensis A beccariana A microcarpa and A crassna ranged from 6390 to 7200 based on Dice coefficient The dendrogram derived by the unweighted pair group method with arithmetic mean of germplasm analysiswere clustered into two main groups Hence a genetic variation among species is quiet high Bootstrap values for the groups supported 70 of the cluster using a linear relationship equation of r 0724 P 00001 was observedbetween known pedigrees and AFLPderived genetic similarity for 136 pairwise comparisons of Aquilaria species For example A malacensis and A microcarpa have the highest genetic similarity 7200 compared with another Aquilaria species Primer pairs EACGMCTA produced a specific fragment for A beccariana 850 bp A crasna 550 bp 180 bp and 140 bp A malaccencis 1500 bp A microcarpa 250 bp and Gyrinops versteegii 150 bp Primer pairs EACGMCAA produced a specific DNA fragment only for A beccariana 1500 bp and 100 bp Primer pairs EACCMCAC also produced only specific fragment for A crassna 1500 bp Study showed the usefulness of AFLP analysis in Aquilaria sp and its potential application for breeding and species conservation Further molecular diversity estimated in the present study combined with the datasets on other morphologicalagronomic traits will be useful for selecting the appropriate accessions for plant improvement through conventional and molecular breeding approaches.Key words Gaharu Aquilaria sp Gyrinops sp genetic diversity molecular marker amplifiedfragment length polymorphism AFLP

Production of agarwood fragrant constituents in Aquilaria calli and cell suspension cultures

Calli and suspension cell culture were established from Aquilaria species whose resinous portion was called agarwood and used as medicine and incense. Four different strains of calli were analyzed for fragrant compounds such as sesquiterpenoids and chromone derivatives which were the major components of agarwood. Main sesquiterpenoids detected from calli were α-guaiene, α-humulene and δ-guaiene, and those of chromone derivatives were phenylethylchromones (AH3, AH4, AH5, AH6). Amount of these compounds differed among the four strains, indicating that Aquilaria plants may have variation in capacity for fragrant compound production. Incubation temperature analysis was also done from 20°C to 40°C and resulted that cell growth was the best at 25°C, whereas the amount of fragrant compounds was largest at 20°C. Salicylic acid (SA) and methyl jasmonate (MJ) were added to calli and suspension cell culture respectively in order to induce production of fragrant compounds. Both SA and MJ apparently induced production of three sesquiterpenoids, α-guaiene, α-humulene, and δ-guaiene at early stage of treatment of SA or MJ, but did not induce that of chromone derivatives directly. Further studies of time course of chromone production and cell viability suggested that cell death may take part in chromone production, and that phenylethylchromones would be produced via oxydoagarochromones (OACs). These results indicate that sesquiterpenoids are synthesized in living cells, but chromone derivatives may be produced from debris of dying cells.

STUDI POLA SPASIAL PERSEBARAN GAHARU ( Aquilaria spp.) DAN KETERKAITANNYA DENGAN KONDISI HABITAT DI TAMAN NASIONAL KUTAI KALIMANTAN TIMUR

Agarwood is an important forest product due to its high economic value. However, the natural population of agarwood species has been decreasing rapidly because of over exploitation, therefore the international trade of the most important agarwood species (i.e. Aquilaria malaccensis) is restricted to just a small quota. This research was conducted in an attempt to provide basic information for the conservation of Aquilaria spp. by identifying the species spatial distribution and habitat characteristics and developing a linkage model between their abundance and habitat characteristics. Data were collected through a field survey at Kutai National Park in August 2006 with Aquilaria spp. as the target species. Data on the species localities were used to determine the species densities and the value of Distance Index of Dispersion. The ordinance levels between the target species and the surrounding other species were identified using a correspondence analysis, while the linkage model between the species abundance and habitat characteristics was established using an ordinal multinomial logit model. The results showed that Aquilaria spp. had a low density (0.01/ha) and were spatially distributed in a clump pattern. The abundance of Aquilaria species had a close association with soil texture, humidity and acidity, land elevation, air temperature and humidity, and light intensity. The abundance decreased with increasing the proportion of sandy clay loam content, soil humidity and acidity, land elevation, and light intensity. In contrast, the abundance increased with increasing sandy loam content, air temperature and air humidity. The existence of A. malaccensis was likely to associate with the existence of Goniothalamus sp., Macaranga sp., Vordia splendidissima, Lygopodium sp., and Stachyphrynium borneensis.

Arbuscular Mycorrhizal Fungi Increased Early Growth of Gaharu Wood Of Aquilaria malaccencsis and A. crasna Under Greenhouse Conditions

Gaharu wood stand has an important source of profits to the forest community in South and Southeast Asia tropical forest countries, but Aquilaria species have reduced in number and turn out to be endangered due to overexploitation. Today, the planting stocks of Aquilaria species are not sufficient to sustain the yield of gaharu wood and promote forest conservation. The objective of this study was to determine the effect of five arbuscular mycorrhizal (AM) fungi: Entrophospora sp., Gigaspora decipiens , Glomus clarum, Glomus sp. ZEA, and Glomus sp. ACA , on the early growth of Aquilaria malaccensis and A. crasna under greenhouse conditions. The seedlings of Aquilaria spp. were inoculated with Entrophospora sp., Gi. decipiens , Glomus clarum, Glomus sp. ZEA, Glomus sp. ACA and uninoculated (control) under greenhouse conditions. Then, percentage AM colonization, plant growth, survival rate and nitrogen (N) and phosphorus (P) content and mycorrhizal dependence (MD) were measured. The percentage AM colonization of A. malaccensis and A. crasna ranged from 83 to 97% and from 63 to 78%, respectively. Colonization by five AM fungi increased plant height, diameter, and shoot and root dry weights. N and P content of the seedlings were also increased by AM colonization. Survival rates were higher in the AM-colonized seedlings at 180 days after transplantation than those in the control seedlings. The MD of Aquilaria species was higher than 55 %. The results suggested that AM fungi can be inoculated`to Aquilaria species under nursery conditions to obtain vigorous seedlings, and the field experiment is underway to clarify the role of AM fungi under field conditions.

Conservation and sustainable use of tropical trees in the genus Aquilaria I. Status and distribution in Indonesia

Tropical trees in the genus Aquilaria Lam. are the principal source of gaharu, one of the most valuable forest products traded internationally. Although these species are the focus of increasing conservation concern, information on their status and distribution is lacking. Information from herbarium accessions, a national forest inventory (NFI), field surveys and gaharu traders was used to assess the distribution of Aquilaria species in Indonesia, indicating population concentrations in Sumatra and eastern Kalimantan. Analysis of NFI data indicated that population densities are relatively low, with values generally <1.2 individuals ha −1; mean values recorded in field surveys were <0.6 individuals ha −1. Analysis of probability distributions emphasized the rarity of Aquilaria species, with a probability of encountering one or more individuals of Aquilaria in 1 ha being less than 0.2 in field assessments. Calculation of Morisita's index of dispersion indicated that the species are generally clumped, with Ip typically <0.6. Analysis of stand structure suggested continuous recruitment in some areas, but a general absence of larger individuals. Given current deforestation rates, these data suggest that all Aquilaria species in Indonesia classify as Vulnerable according to the IUCN Red List criteria.