Evaluation of three different artificial agarwood-inducing methods from Aquilaria sinensis using antimicrobial activity.

The aim of this paper was to evaluate the effect of three different approaches for artificially inducing the formation of agarwood over time in young Aquilaria sinensis trees using antimicrobial activity. The antimicrobial activity was determined by a two-fold serial dilution method and minimum inhibitory concentration (MIC) against a panel of microorganisms (two bacterial strains, Staphylococcus aureus and anti-methicillin-resistant Staphylococcus aureus, and seven fungal strains: Penicillium melinii, Penicillium adametzi, Penicillium urticae, Penicillium notatum, Paecilomyces varioti, Mucor saturninus Hagem and Aspergillus niger). The results showed that artificial agarwood obtained by comprehensive stimulated method (formic acid plus fungal inoculation) and extended longer inducing time have better antimicrobial activity, which is similar to the result of chemical analysis. Therefore, it is a beneficial exploration to the first use of antimicrobial activity to evaluate artificial agarwood obtained by different producing methods and different culture time.

iTRAQ-Based Quantitative Proteomic Analysis of Chemically Induced Aquilaria sinensis Provides Insights into Agarwood Formation Mechanism.

Agarwood is a precious traditional Chinese medicine with a variety of pharmacological effects. Although efforts have been made in elucidating the mechanism of agarwood formation, little progress is obtained till now. Therefore, the molecular mechanism of agarwood formation needs to be further explored using different biological approaches. In this study, the quantitative proteomic analysis using iTRAQ technology combined with transcriptomic and metabolomic analyses on chemically induced Aquilaria sinensis is performed to elucidate the agarwood formation mechanism by formic acid stimulus. Data are available via ProteomeXchange with identifier PXD007586; 1884 proteins are detected, 504 differential proteins that show at least twofold differences in their expression levels are selected based on GO annotations, KEGG, STRING analysis, and quantitative RT-PCR analysis. The results indicate that sesquiterpene synthase, germin-like protein, pathogenesis-related protein, 6-phosphogluconate dehydrogenase, lipoyl synthase, and superoxide dismutase play important roles in the agarwood formation, suggesting that the proteins related to the plant defensive response, the removal of peroxide, the disease-resistance, the biosythesis of glycan, fatty acids, and sesquiterpene are crucial for agarwood formation.

Relationship between leaf functional traits and productivity in Aquilaria crassna (Thymelaeaceae) plantations: a tool to aid in the early selection of high-yielding trees.

Physiological traits are frequently used as indicators of tree productivity. Aquilaria species growing in a research planting were studied to investigate relationships between leaf-productivity traits and tree growth. Twenty-eight trees were selected to measure isotopic composition of carbon (δ13C) and nitrogen (δ15N) and monitor six leaf attributes. Trees were sampled randomly within each of four diametric classes (at 150 mm above ground level) ensuring the variability in growth of the whole population was represented. A model averaging technique based on the Akaike's information criterion was computed to identify whether leaf traits could assist in diameter prediction. Regression analysis was performed to test for relationships between carbon isotope values and diameter and leaf traits. Approximately one new leaf per week was produced by a shoot. The rate of leaf expansion was estimated as 1.45 mm day-1. The range of δ13C values in leaves of Aquilaria species was from -25.5‰ to -31‰, with an average of -28.4 ‰ (±1.5‰ SD). A moderate negative correlation (R2 = 0.357) between diameter and δ13C in leaf dry matter indicated that individuals with high intercellular CO2 concentrations (low δ13C) and associated low water-use efficiency sustained rapid growth. Analysis of the 95% confidence of best-ranked regression models indicated that the predictors that could best explain growth in Aquilaria species were δ13C, δ15N, petiole length, number of new leaves produced per week and specific leaf area. The model constructed with these variables explained 55% (R2 = 0.55) of the variability in stem diameter. This demonstrates that leaf traits can assist in the early selection of high-productivity trees in Aquilaria species.

Chemometric analysis reveals links in the formation of fragrant bio-molecules during agarwood (Aquilaria malaccensis) and fungal interactions.

Fragrant agarwood, arguably the costliest wood in the world, is formed by plant-fungal interactions in Aquilaria spp. However, very little is known about this fragrant outcome of interaction. Therefore, mimicking the ancient traditions of agarwood production in Assam (Northeast India), a chemometric assessment of the agarwood-fungus interaction was made by chemical profiling (GC-MS) coupled with statistical analysis (principal component, correlation network analysis) across three platforms, viz. callus, juvenile plants and resinous wood-chips with an associated Fusarium. In the study of callus-fungus interaction, increased accumulation of key aroma compounds such as pentatriacontane {fold change (log2FC) = 3.47)}, 17-pentatriacontene (log2FC = 2.95), tetradecane, 2-methyl- (log2FC = 1.10) over callus and activation of pathways related to defense and secondary metabolism indicated links to aroma production. Study on fungal interactions in juvenile plants and resinous wood-chips indicated formation of terpenoid precursors (e.g. farnesol, geranylgeraniol acetate) and agarwood sesquiterpenes (e.g. agarospirol, γ-eudesmol). Correlation network analysis revealed the possible regulation of sesquiterpene biosynthesis involving squalene. Also a direct role of fungus in aroma (e.g. dodecane, 4-methyl-, tetracosane) was highlighted. Appearance of fragrant molecules unknown to agarwood during interaction featured as a new possibility for future research.

[Extraction of spectral difference characteristics of Stellera chamaejasme in Qilian County of Qinghai Province, Northwest China].

Stellera chamaejasme is one of the main poisonous weeds distributed in alpine meadow of Qinghai Province. Rapid spreading of S. chamaejasme has done serious harm to local animal husbandry and caused continuous grassland ecosystem degradation. This paper focused on the spectral differences between S. chamaejasme and herbage, taking the typical degraded alpine meadow dominated by S. chamaejasme in Qilian County of Haibei Region as the test site and using the spectral measurements acquired in the full-blossom period of S. chamaejasme from 2012 to 2014. The results showed that the spectral behavior of flowers of S. chamaejasme differed significantly from green background that included leaves of S. chamaejasme and herbage within 350-900 nm of VIS-NIR wavebands. The biggest reflectance difference between flowers of S. chamaejasme and green background was located in the red valley, followed by the blue valley. The reflectance of S. chamaejasme community increased with the rising of coverage, the biggest reflectance difference between S. chamaejasme and herbage communities lied in the near-infrared peak, and the best separability between S. chamaejasme communities with different coverage was also at the point. The difference of first derivative spectra between flowers of S. chamaejasme and green background located in amplitude of yellow edge was remarkable, followed by amplitude of blue edge, the same as differences between S. chamaejasme and herbage communities. Linear regression analysis between coverage of S. chamaejasme and spectral feature parameters showed best result for red valley (R2 = 0.94). Finally, the red valley, the blue valley and the near-infrared peak were proposed for discriminating S. chamaejasme from herbage in the full-blossom period of S. chamaejasme, and the combination of corresponding red, blue and near-infrared bands could be used to build sensitive indices for S. chamaejasme recognition.

The biosynthetic activities of primary and secondary metabolites in suspension cultures of Aquilaria microcarpa.

Two types of suspension-cultured Aquilaria microcarpa cells, friable and aggregated, were selectively generated. The biosynthetic activities of primary and secondary metabolites in target cells were detected using laser scanning microscopy (LSM) imaging with diphenylboric acid 2-amino ethyl ester (DPBA) and 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) staining. Scanned friable cells produced weakly fluorescent images revealing low productivity of metabolites. On the other hand, scanning of aggregated cells produced clear fluorescent images depicting the accumulations of flavonoids and lipids. Furthermore, abundant deposition of an unknown resinous compound in extracellular portion of aggregated cells could be visualized. The resinous compound was white to whitish-gray in color and highly sedimented in the medium. Based on these observations, we focused our investigation of metabolite productivity on aggregated suspension cells. Some prominent extracellular compounds were detected in the used liquid medium, as well as in the resinous residue within the medium. The characteristics of these metabolites were investigated in detail via gas chromatography-mass spectrometry (GC-MS) analysis.

Isolation and characterization of bacteria from the rhizosphere and bulk soil of Stellera chamaejasme L.

This study is the first to describe the composition and characteristics of culturable bacterial isolates from the rhizosphere and bulk soil of the medicinal plant Stellera chamaejasme L. at different growth stages. Using a cultivation-dependent approach, a total of 148 isolates showing different phenotypic properties were obtained from the rhizosphere and bulk soil. Firmicutes and Actinobacteria were the major bacterial groups in both the rhizosphere and bulk soil at all 4 growth stages of S. chamaejasme. The diversity of the bacterial community in the rhizosphere was higher than that in bulk soil in flowering and fruiting stages. The abundance of bacterial communities in the rhizosphere changed with the growth stages and had a major shift at the fruiting stage. Dynamic changes of bacterial abundance and many bacterial groups in the rhizosphere were similar to those in bulk soil. Furthermore, most bacterial isolates exhibited single or multiple biochemical activities associated with S. chamaejasme growth, which revealed that bacteria with multiple physiological functions were abundant and widespread in the rhizosphere and bulk soil. These results are essential (i) for understanding the ecological roles of bacteria in the rhizosphere and bulk soil and (ii) as a foundation for further evaluating their efficacy as effective S. chamaejasme growth-promoting rhizobacteria.

[Establishment of a cell suspension culture system of endangered Aquilaria sinensis (Lour.) Gilg].

Aquilaria sinensis callus induced by stem tips were used to establish the suspension cell system. The results showed that the most suitable medium for callus induction and subculture is MS + 2.0 mg x L(-1) NAA + 1.0 mg x L(-1) 6-BA. After 12 times of subculture, the energetic and loose callus, which were appropriate for cell suspension culture, were cultured and shook in liquid medium MS + 2.0 mg x L(-1) NAA + 1.0 mg x L(-1) 6-BA + 500.0 mg x L(-1) casein hydrolysate (CH) to establish the suspension cell system. The growth curve of suspension cells showed a "S" type. At the beginning of the culture, cell density increased slowly; during 4 to 6 days, suspension cells reached logarithmic growth period; during 7 to 12 days, suspension cells were in the platform period; but after 12 days, cell density and activity went down obviously. Agarwood sesquiterpenes were not detected in the suspension cells during the growth period, however, they could be detected in MeJA treated suspension cells. In this study, a stable and active growing suspension cell system was established, which was a proper system to study the mechanism of agarwood sesquiterpene formation, and additionally provided a potential way to generate agarwood sesquiterpenes through application of cell culture.

[Historical records and modern studies on agarwood production method and overall agarwood production method].

Agarwood is a precious traditional Chinese medicine with the efficacy of promoting qi circulation and relieving pain, warming middle-jiao, controlling nausea and vomiting, governing inspiration and relieving asthma, therefore it is widely applied in the clinic. Meanwhile, agarwood is also a precious spice. Aquilaria sinensis is the only source of agarwood production in China. Under natural conditions, a healthy A. sinensis tree produces no agarwood. Only if being wounded or infected with fungus can it synthetize and accumulate agarwood. It takes a decade or even several decades to produce agarwood, thus natural agarwood can not meet market demands. The essay summarizes historical records of agarwood production method and modern agarwood production method, in order to provide basis and reference for large-scale production of agarwood.

Identification of genes related to agarwood formation: transcriptome analysis of healthy and wounded tissues of Aquilaria sinensis.

BACKGROUND: Agarwood is an expensive resinous heartwood derived from Aquilaria plants that is widely used in traditional medicines, incense and perfume. Only wounded trees can produce agarwood, and the huge demand for the agarwood products has led all Aquilaria spp. being endangered and listed in the Appendix II of the CITES (http://www.cites.org). The major components of agarwood are sesquiterpenes and phenylethyl chromones. Owing to a lack of genomic information, the molecular basis of wound-induced sesquiterpenes biosynthesis and agarwood formation remains unknown. RESULTS: To identify the primary genes that maybe related to agarwood formation, we sequenced 2 cDNA libraries generated from healthy and wounded A. sinensis (Lour.) Gilg. A total of 89,137 unigenes with an average length of 678.65 bp were obtained, and they were annotated in detail at bioinformatics levels. Of those associated with agarwood formation, 30 putatively encoded enzymes in the sesquiterpene biosynthesis pathway, and a handful of transcription factors and protein kinases were related to wound signal transduction. Three full-length cDNAs of sesquiterpene synthases (ASS1-3) were cloned and expressed in Escherichia coli, and enzyme assays revealed that they are active enzymes, with the major products being δ-guaiene. A methyl jasmonate (MJ) induction experiment revealed that the expression of ASS was significantly induced by MJ, and the production of sesquiterpenes was elevated accordingly. The expression of some transcription factors and protein kinases, especially MYB4, WRKY4, MPKK2 and MAPK2, was also induced by MJ and coordinated with ASS expression, suggesting they maybe positive regulators of ASS. CONCLUSIONS: This study provides extensive transcriptome information for Aquilaria spp. and valuable clues for elucidating the mechanism of wound-induced agarwood sesquiterpenes biosynthesis and their regulation.

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.

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

UNLABELLED: 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. KEY MESSAGE: 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.

[RP-HPLC fingerprint of milk Stellera chamaejasme].

OBJECTIVE: To establish a RP-HPLC fingerprint of milk Stellera chamaejasme. METHODS: The gradient elution mode was applied in chromatographic separation, and data were analysed by "Computer Aided Similarity Evaluation" software to compare the quality of milk Stellera chamaejasme samples from different habitats. RESULTS: Samples from different habitats were of high similarity, and analysied milk Stellera chamaejasme chromone and E15 in the atlas. CONCLUSION: The method is repeatable, and can be used in quality assessment of milk Stellera chamaejasme.

[Study on the physiological and biochemical characteristics of different types of leaves of Aquilaria sinensis].

OBJECTIVE: To study the physiological and biochemical characteristics of different types of leaves of Aquilaria sinensis. METHODS: Six types of leaves' physiological and biochemical indexes were studied and analysed, these indicators included: malondialdehyde (MDA), soluble sugar content, soluble protein content, peroxidase activity (POD), catalase activity (CAT) and superoxide dismutase activity (SOD). RESULTS: POD, CAT, SOD had no significant difference; POD, CAT, and SOD had a coordinating role on cleaning-up oxidation products MDA; The soluble protein contents of different types of leaves was negative corelatad with the centent of MDA; The contents of soluble sugar among them were signifiant different. CONCLUSION: Growth activity sequence of different types of Aquilaria sinensis is as follows: lobular type > large-leaved type, transitional leaf type > elliptical leaf type.

Arbuscular mycorrhizal fungi increased early growth of two nontimber forest product species Dyera polyphylla and Aquilaria filaria under greenhouse conditions.

Nontimber forest products (NTFPs) represent an important source of income to millions of people in tropical forest regions, but some NTFP species have decreased in number and become endangered due to overexploitation. There is increasing concern that the planting stocks of Dyera polyphylla and Aquilaria filaria are not sufficient to sustain the yield of NTFPs and promote forest conservation. The objective of this study was to determine the effect of two arbuscular mycorrhizal (AM) fungi, Glomus clarum and Gigaspora decipiens, on the early growth of two NTFP species, D. polyphylla and A. filaria, under greenhouse conditions. The seedlings of both species were inoculated with G. clarum or G. decipiens, or uninoculated (control) under greenhouse conditions. Percentage of AM colonization, plant growth, survival rate, and nitrogen (N) and phosphorus (P) concentrations were measured after 180 days of growth. The percentage of AM colonization of D. polyphylla and A. filaria ranged from 87 to 93% and from 22 to 39%, respectively. Colonization by G. clarum and G. decipiens increased plant height, diameter, and shoot and root dry weights. Shoot N and P concentrations of the seedlings were increased by AM colonization by as much as 70-153% and 135-360%, respectively. Survival rates were higher in the AM-colonized seedlings at 180 days after transplantation than in the control seedlings. The results suggest that AM fungi can accelerate the establishment of the planting stocks of D. polyphylla and A. filaria, thereby promoting their conservation ecologically and sustaining the production of these NTFPs economically.

Rapid in vitro propagation of medicinally important Aquilaria agallocha.

Aquilaria agallocha can produce fragrant agarwood used for incense, traditional medicine and other products. An efficient plant regeneration system was established via organogenesis from shoots developed from seedlings of Aquilaria agallocha. Shoots generated many buds on MS medium supplemented with 1.3 micromol/L BA (6-benzylaminopurine) in the first 7 weeks, and the buds elongated on MS medium with 1.3 micromol/L BA+0.5 micromol/L NAA (naphthaleneacetic acid) in another 7 weeks, 2.3 shoots 2 cm in length per explant were obtained within 14 weeks. Plantlets were rooted on 1/2 MS medium after being immersed in 5 micromol/L NAA for 48 h, 96.7% of the roots grew up two weeks later. All plantlets that survived acclimatization grew well in the pots.

[Judging method of individual age and age structure of Stellera chamaejasme population in degraded steppe].

Based on the minute observation of branches morphology of root-crown of Stellera chamaejasme in Cleistogenes squarosa community and its growth characteristics, this paper studied the age structure of S. chamaejasme population, and an individual age judging method "the times of quasi-dichotomous branching plus two" was put forward for the first time. Remnant stubbles, branch trace, and annular trace on the root crown were regarded as important morphological features, and used to confirm the times of quasi-dichotomous branching. The results showed that the oldest individuals at three grazing succession stages (i.e., heavy grazing, over grazing and extreme grazing) were 15, 16 and 19 years old, respectively. Among all age classes, the numbers of eight years old individuals were the largest, and the age ratio was 18.71%, 24.20% and 19.06%, respectively, at the different succession stages. There were no one- and two-year old individuals at heavy grazing stage, and no one-year old individuals at the other two grazing stages. The age structures of the populations were "early declining types", and the survival curves were similar to protuberant type or Deevey I type. The numbers of old age individuals (thirteen years old and more) at the three succession stages accounted for 4.83%, 2.84% and 14.02%, respectively. The age structure of the population tended to aging with the increase of grazing intensity.

[Seed germination characteristics and regeneration mechanism of Stellera chamaejasme population].

Stellera chamaejasme is a perennial herbaceous plant of Thymelaceae, as one of major poisonous plant species to animal, distributing in Northeast, Northwest, and Southeast of China. In some area on the degraded steppes, the plant grew with abnormal abundance and badly endangered the health of animals. The germination capacity of Stellera chamaejasme seed that collected from soil seed bank and immediately gathered from the plant was studied under different temperature, light condition and five pretreatments (rupture seed pericarp, remove seed pericarp, soaked in 98% H2SO4 for 5 min, soaked in 0.2% KNO3 for 24 h, and kept at 10 degrees C for a week). The seed germination percentage (GP) of the plant was lower in the mass and it was 13% under constant temperature 25 degrees C with dark condition. The suitable temperatures for germination of the seeds were constant 30 degrees C or alternative 10-30 degrees C. To rupture or to remove seed pericarp could significantly increase GP and it was 49% and 47%, respectively. GP under pretreatment of seed soaked in 98% H2SO4 for 5 min was 32%. The pretreatments of seed soaked in 0.2% KNO3 for 24 h and seed kept at 10 degrees C for a week had no significantly effects on increasing GP. The seed germination was not sensitive to light condition. The hardness of the seeds was mainly responsible for the seed dormancy and low germination rate. The GP of S. chamaejasme seeds in the soil seed bank was higher than that gathered in the immediate year. Based on the comprehensive analysis with the lower GP and the results of field observation, it was concluded that S. chamaejasme seeds did not germinate every year in nature, and chance of population regeneration was random or periodic.