Metabolic Analysis of Medicinal Dendrobium officinale and Dendrobium huoshanense during Different Growth Years

Metabolomics technology has enabled an important method for the identification and quality control of Traditional Chinese Medical materials. In this study, we isolated metabolites from cultivated Dendrobium officinale and Dendrobium huoshanense stems of different growth years in the methanol/water phase and identified them using gas chromatography coupled with mass spectrometry (GC-MS). First, a metabolomics technology platform for Dendrobium was constructed. The metabolites in the Dendrobium methanol/water phase were mainly sugars and glycosides, amino acids, organic acids, alcohols. D. officinale and D. huoshanense and their growth years were distinguished by cluster analysis in combination with multivariate statistical analysis, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Eleven metabolites that contributed significantly to this differentiation were subjected to t-tests (P<0.05) to identify biomarkers that discriminate between D. officinale and D. huoshanense, including sucrose, glucose, galactose, succinate, fructose, hexadecanoate, oleanitrile, myo-inositol, and glycerol. Metabolic profiling of the chemical compositions of Dendrobium species revealed that the polysaccharide content of D. huoshanense was higher than that of D. officinale, indicating that the D. huoshanense was of higher quality. Based on the accumulation of Dendrobium metabolites, the optimal harvest time for Dendrobium was in the third year. This initial metabolic profiling platform for Dendrobium provides an important foundation for the further study of secondary metabolites (pharmaceutical active ingredients) and metabolic pathways.     

Specificity and preference of mycorrhizal associations in two species of the genus Dendrobium (Orchidaceae)

Dendrobium is a large genus of tropical epiphytic orchids. Some members of this genus are in danger of extinction across China. To investigate orchid mycorrhizal associations of the genus Dendrobium, plants from two Dendrobium species (Dendrobium officinale and Dendrobium fimbriatum) were collected from two habitats in Guangxi Province, China, and clone libraries were constructed to identify the mycorrhizal fungi of individual plants. A low and high degree of specificity was observed in D. officinale and D. fimbriatum, respectively. Phylogenetic analysis revealed that the majority of Dendrobium mycorrhizal fungi are members of the Tulasnellaceae, but, in some plants, members of the Ceratobasidiaceae and Pluteaceae were also found. In D. officinale, individual plants associated with more than three fungi simultaneously, and, in some cases, associations with five fungi at the same time. One fungus was shared by individual plants of D. officinale collected from the two habitats. In D. fimbriatum, only one fungal partner was found in each population, and this fungus differed between populations. The two species of Dendrobium sampled from the same habitat did not share any fungal taxa. These results provide valuable information for conservation of these orchid species.     

Enhancement of polysaccharides accumulation in <Emphasis Type="Italic">Dendrobium officinale</Emphasis> by exogenously applied methyl jasmonate

The accumulation of polysaccharides, activities of sucrose metabolism enzymes, and the expression of sucrose biosynthetic genes in Dendrobium officinale were significantly affected by exogenous methyl jasmonate (MeJA). Application of MeJA increased the content of polysaccharides and the highest polysaccharide production occurred in the samples treated with 200 μM MeJa. The MeJA application influenced polysaccharide biosynthesis rather than degradation because the activities of sucrose metabolism enzymes and the expressions of sucrose biosynthetic genes were upregulated by MeJA. Interestingly, low MeJA concentrations promoted accumulation of Dendrobium polysaccharides, while high MeJA amounts played an inhibitory role. The content of major constituent of polysaccharides, glucose and mannose, also increased after MeJa treatment.     

Extractive compounds of betulaceae family birch buds (Betula pendula Roth.): IV. Composition of sesquiterpene diols, triols, and flavonoids

This report is devoted to study of the hydrocarbon composition of the extract of buds of birch (family Betulaceae). We identified 3,5-dihydroxy-7,4??-dimethoxyflavone, 5,7-dihydroxy-3,4??-dimethoxyflavone, 7-methoxy-4??,5-dihydroxyflavanone, 4??-methoxy-5,7-hydroxyflavanone, 5??,6??-dihydroxycaryophyllen-4(14),8(15)-diene ((1R,5R,6R,9S)-5,6-dihydroxy-11,11-dimethyl-4,8-methylenebicyclo[7.2.0]-undecane), 5??,6??-dihydroxycaryophyllen-3,8(15)-diene ((1R,5R,6R,9S)-5,6-dihydroxy-11,11-dimethyl-8-methylenebicyclo[7.2.0]undec-3-ene), 6??,9??-dihydroxy-??-humulene((1E,4E,6R,9S)-6,9-dihydroxy-4,11,11-trimethyl-8-methylene-1,4-cycloundecanediene), 5??,6??,8??-trihydroxycariolan, and (1R,5R,6R,8R,9S)-trihydroxycariolan. The gas chromatographic retention indices of all identified compounds were determined.     

不同栽培方式的铁皮石斛根、茎中内生细菌多样性及其抑菌活性研究

【目的】分析树干仿生栽培和大棚种植的铁皮石斛根、茎中内生细菌的组成与分布,并挖掘潜在的微生物资源及功能。【方法】利用Illumina MiSeq高通量测序技术,对不同栽培方式铁皮石斛根、茎中免培养内生细菌丰富度及多样性进行比较分析;通过传统的内生菌分离法以及16S rRNA基因测序技术对可培养内生细菌进行分离鉴定;采用滤纸片法筛选拮抗石斛病原菌的内生菌株。【结果】高通量测序分析中内生细菌以Pseudomonas(30.52%)为主,其次是Mycobacterium(10.22%)以及Brachybacterium(8.32%),树干仿生栽培石斛内生细菌组成丰富度及多样性高于大棚种植,石斛根中内生细菌组成丰富度及多样性高于石斛茎。可培养内生细菌中Bacillus(18.37%)所占比例最高,其次是Curtobacterium (8.16%),同时筛选得到8株具有抑制石斛病原菌活性的菌株。【结论】铁皮石斛中蕴含着丰富的内生细菌资源,菌群组成和分布受其不同栽培方式和组织部位的影响。此外,筛选得到8株具有抑制病原菌活性的菌株,表明铁皮石斛内生细菌代谢产物具有抗菌活性。本试验对铁皮石斛内生细菌多...     

干旱-复水对两种石斛属植物叶水势的影响

石斛属植物多附着在其他植物体或岩石上,水分获取困难,其特殊的水分利用策略是其生存和发展的重要保证.为弄清石斛属植物对干旱胁迫的适应能力和机制,该文选用3年生金钗石斛和铁皮石斛,通过盆栽控水进行干旱胁迫和复水处理,探讨在不同干旱历时和干旱后复水条件下两种石斛的叶水势变化情况.结果表明:随着干旱时间的延长,两种石斛叶水势均...     

Endophytic fungi,host genotype,and their interaction influence the growth and production of key chemical components of Dendrobium catenatum

To examine how host plant genotype, endophytic fungal species, and their interaction may affect growth and key chemical content and composition in an important orchid species, we assessed four Dendrobium catenatum cultivars co-cultured with three fungi previously isolated from D. catenatum. Fungal endophytes (Tulasnella sp., Leptosphaeria microscopica, and Guignardia sp.) specifically affected the growth and chemical composition of the four cultivars. Fungal infection significantly increased certain growth traits, especially mid-stem thickness, stem biomass, stem polysaccharide and ethanol-soluble extractive content, and leaf flavonoid and phenol content. Presence or abundance of some key chemical components was also altered by fungal treatment. These increases and alterations were highly dependent on the host genotype. The findings of this study contribute to our understanding of Dendrobium and endophytic fungi interactions, and provide vital information for improving the development and use of endophytic fungi in D. catenatum breeding.     

Comparison of metabolomics of Dendrobium officinale in different habitats by UPLC-Q-TOF-MS

Dendrobium officinale has been considered over past centuries to be extremely valuable for use as an herbal medicine in South Asian countries. In this work, the chemical profiles of D. officinale from different habitats were systematically characterized using ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and multivariate analysis. The principal component analysis (PCA), partial least squares discriminate analysis (PLS-DA) and orthogonal partial least squares analysis (OPLS-DA) of UPLC-Q-TOF-MS data displayed an obvious separation. Several flavonoids and terpenoids derivatives contribute to the quantitative chemotypic variation within and between the samples as observed. These findings lead to a better understanding of the phytochemical variation of D. officinale which can aid in quality control of raw material.     

Widely Targeted Metabolomics Analysis of Dendrobium officinale at Different Altitudes

In order to investigate the differences in the chemical composition of Dendrobium officinale at different altitudes, we collected Dendrobium officinale from different altitudes in Guizhou Province, China, and firstly determined its polysaccharide content by using a sulfuric acid-phenol color development method with reference to the Chinese Pharmacopoeia, and further determined its metabolites by using widely targeted metabolomics, and explored the differences in the chemical composition of Dendrobium officinale at different altitudes using multivariate statistical analysis. We found that the polysaccharide content was higher in plants growing at 1122 m, a total of 902 secondary metabolites were detected by wildly targeted metabolomics, and amino acids and their derivatives were more highly expressed at 1122 m, while other metabolites were more highly expressed at 835 m. Furthermore, we found that the phenolic acid compound nerugein was only present in plants at 835 m, and two lipid compounds, namely Lyso PE 20 : 4 and its isomer, were only present at 1122 m. Taken together, these results may provide a basis for the selection and clinical application of D. officinale cultivated at different altitudes.     

In situ seed baiting techniques in <Emphasis Type="Italic">Dendrobium officinale</Emphasis> Kimuraet Migo and <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl. : the endangered Chinese endemic <Emphasis Type="Italic">Dendrobium</Emphasis> (Orchidaceae)

The new in situ seed baiting method using seed packets to assess germination of orchid species within soil provides a means of locating, collecting, and identifying specific fungi that are involved in the lifecycle of orchids in the wild and investigating their relationship with the orchids under natural field conditions. Two isolates (SHH44, SHH53) originating from the seedlings found in the seeds packets in situ were demonstrated to support seed germination and seedling development (in vitro) of two endangered Chinese endemic herbs, Dendrobium officinale and Dendrobium nobile (Orchidaceae). Advanced protocorm development (Stage 3 and greater) in this study of these species of Dendrobium only occurred under the 12/12 h L/D photo-period indicated that illumination may play an important role in seedling recruitment of terrestrial orchid species in their natural environment. The information provided in this study may prove invaluable in the conservation of D. officinale and D. nobile and congener species in China, especially given their rare status.     

Diversity and Taxonomy of Endophytic Xylariaceous Fungi from Medicinal Plants of Dendrobium (Orchidaceae)

Dendrobium spp. are traditional Chinese medicinal plants, and the main effective ingredients (polysaccharides and alkaloids) have pharmacologic effects on gastritis infection, cancer, and anti-aging. Previously, we confirmed endophytic xylariaceous fungi as the dominant fungi in several Dendrobium species of tropical regions from China. In the present study, the diversity, taxonomy, and distribution of culturable endophytic xylariaceous fungi associated with seven medicinal species of Dendrobium (Orchidaceae) were investigated. Among the 961 endophytes newly isolated, 217 xylariaceous fungi (morphotaxa) were identified using morphological and molecular methods. The phylogenetic tree constructed using nuclear ribosomal internal transcribed spacer (ITS), large subunit of ribosomal DNA (LSU), and beta-tubulin sequences divided these anamorphic xylariaceous isolates into at least 18 operational taxonomic units (OTUs). The diversity of the endophytic xylariaceous fungi in these seven Dendrobium species was estimated using Shannon and evenness indices, with the results indicating that the dominant Xylariaceae taxa in each Dendrobium species were greatly different, though common xylariaceous fungi were found in several Dendrobium species. These findings implied that different host plants in the same habitats exhibit a preference and selectivity for their fungal partners. Using culture-dependent approaches, these xylariaceous isolates may be important sources for the future screening of new natural products and drug discovery.     

四川石斛属一新种

描述了四川夹江（兰科）石斛属植物一新种,即夹江石斛。     

Genetic diversity assessment and ex situ conservation strategy of the endangered Dendrobium officinale (Orchidaceae) using new trinucleotide microsatellite markers

Dendrobium officinale (Orchidaceae) is an endangered plant species with important medicinal value. To evaluate the effectiveness of ex situ collection of D. officinale genetic diversity, we developed 15 polymorphic trinucleotide microsatellite loci of D. officinale to examine the genetic diversity and structure of three D. officinale germplasm collections comprising 120 individuals from its germplasm collection base and their respective wild populations consisting of 62 individuals from three provinces in China. The three germplasm collections showed reductions in gene diversity and average number of alleles per locus, but an increase in average number of rare alleles (frequency?≤?0.05) per locus in comparison to their wild populations. However, the differences in gene diversity between the germplasm collections and wild populations were not statistically significant. The analysis using STRUCTURE revealed evident differences in genetic composition between each germplasm collection and its wild population, probably because the D. officinale individuals with distinct genotypes in each wild population were unevenly selected for establishing its germplasm collection. For conservation management plans, we propose that D. officinale individuals with rare alleles need to be conserved with top priority, and those individuals with the most common alleles also should be concerned. The 15 new microsatellite loci may be used as a powerful tool for further evaluation and conservation of the genetic diversity of D. officinale germplasm resources.     

Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh

Background and Aims

Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera.

Methods

In order to isolate highly repetitive sequences, a c₀t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH).

Key Results

Characterization of the c₀t-1 library resulted in the detection of repetitive sequences including the (GA)_n dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S–5·8S–25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species.

Conclusions

The repeated sequences isolated from D. moschatum c₀t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species.     

Molecular Cloning and Characterization of a Mannose-binding Lectin Gene from Dendrobium officinale

Using RNA extracted from Dendrobium officinale young leaves and primers designed according to the conservative regions of Orchidaceae lectins, the full-length cDNA of Dendrobium officinale agglutinin (DOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of doa was 768 bp and contained a 498 bp open reading frame (ORF) encoding a lectin precursor of 165 amino acids. Through comparative analysis of doa gene and its deduced amino acid sequence with those of other Orchidaceae species, it was found that doa encoded a precursor lectin with signal peptide. DOA was a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that doa mRNA expression was detected in all tested tissues including root, stem and leaf, however, the expression was higher in stem, lower in leaf. As the doa mRNA was detected in all the tested plant tissues, the doa was considered to be a constitutively expressed gene.     

A high-throughput flow cytometry system for early screening of in vitro made polyploids in <Emphasis Type="Italic">Dendrobium</Emphasis> hybrids

Orchids of the genus Dendrobium hold a high economical value in the international markets both as pot plants or cut flowers, and for the production of some metabolites with antioxidant and anti-tumoral activities. Manipulating ploidy levels of Dendrobium species is one of the possible methods to develop new varieties with increased ornamental value and a higher production of secondary metabolites. In this work, we present a new and fast flow cytometry approach to obtain and select Dendrobium phalaenopsis?×?Dendrobium loddigesii polyploids, through an early in vitro screening on protocorm like bodies (PLBs) after antimitotic treatment. Our approach allows the identification of the best time of treatment on control PLBs and the assessment of best conditions for polyploid recovery just one month after treatments, by using Cycle Value. We were able to discard about the two-third of the unchanged material by drastically reducing the explants to work with and the corresponding costs. Different conditions, regarding concentrations and exposition time, were tested using colchicine or amiprophos-methyl (APM). A high polyploids recovery, up to 80%, were obtained with both antimitotic agents, and those materials were further characterized by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS), to identify independent polyploids explants with increased levels in high-value molecules as shihunidine and hircinol, together with stochastic events and genotype-specific metabolite fluctuations.