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.

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.

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.

[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.

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.

[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.

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.