Integrating growth, photosynthetic, and metabolomic analyses reveals how rhizobacteria affect Acanthopanax senticosus

Root-knot nematodes (Meloidogyne spp.) are plant-parasitic nematodes that cause serious damage worldwide. There are many species of traditional Chinese medicine (TCM) plants, but only a few have been reported to be infected by Meloidogyne species. From 2020 to 2022, a survey was conducted in the Qinling mountain area, which is the main production region of TCM plants in China. Obvious galling symptoms were observed on the root systems of 15 species of TCM plants. Females were collected from diverse diseased TCM plants and subsequently identified at morphological and molecular levels. Among the 20 diseased root samples collected, Meloidogyne hapla populations were identified in 12 samples (60%), and M. incognita populations were identified in eight samples (40%). Among the 15 species of diseased TCM plants, eight species, namely, Scutellaria baicalensis, Leonurus japonicus, Dioscorea zingiberensis, Cornus officinalis, Viola philippica, Achyranthes bidentata, Senecio scandens, and Plantago depressa, were reported to be infected by Meloidogyne species for the first time. The host status of five species of TCM plants for two M. hapla isolates and one M. incognita isolate from TCM plants in this study was then evaluated. Differences in TCM plants' response to nematode infection were apparent when susceptibility was evaluated by the egg counts per gram of fresh root and the reproduction factor of the nematodes. Among the five species of TCM plants tested, Salvia miltiorrhiza and Gynostemma pentaphyllum were the most susceptible, while S. baicalensis and V. philippica were not considered suitable hosts for M. hapla or M. incognita.

Traditional Chinese Medicine (TCM) plants have been known to confer beneficial therapeutic effects in the traditional systemic Chinese Medicinal, but a science-based verification is important to offer evidence-based utilization. This review will cover or discuss the pharmacological prospect of TCM plants, including Andrographis paniculata, Glycyrrhiza glabra, Scutellaria baicalensis, astragalus membranaceus, ginkgo biloba and panax ginseng using the preclinical study in animals. These investigations indicate a high level of antiviral, anti-inflammatory, immuno-modulator, anti-oxidant, heendoguinative proteomin and neuro-protection, which have been explained through recent pharmacognostic strategies such as phytochemical screening, chromatographic studies, in-vitro tests, as well as molecular analyses. Although translation into humans is impeded by species-specific effects, complex, formulations and reproducibility, the incorporation of standardized extracts, high- fidelity disease models, and increasing ethical options all improve the translational significance. The results indicate the possible potential in TCM plants as evidence-based human and veterinary medicines and point to possible areas of future intensive preclinical and clinical studies.

Chapter One - Methods for the Study of Endophytic Microorganisms from Traditional Chinese Medicine Plants

Salidroside is a phenylpropanoid glycoside isolated from Rhodiola rosea L., a traditional Chinese medicinal (TCM) plant, and has displayed a broad spectrum of pharmacological properties, such as resisting anoxia, antiradiation and antifatigue. In addition, it has low toxicity as traditional Chinese medicinal plant. However, the bioavailability of salidroside is low because of its instability for hydrolase. Lots of researchers modified the structure of salidroside to enhance its bioactivity. This paper summarizes the structure modification and activities study of salidroside in recent ten years, and preliminary discuss the structure-activity relationship of salidroside analogues, which hope to provide new ideas of modification in future.

Atractylodes lancea is an important medicinal plant in traditional Chinese medicine, its rhizome is rich of volatile secondary metabolites with medicinal values and is largely demanded in modern markets. Currently, supply of high-yield, high-quality A. lancea is mainly achieved via cultivation. Certain soil microbes can benefit plant growth, secondary metabolism and induce resistance to environmental stresses. Hence, studies on the effects of soil microbe communities and isolates microorganisms on A. lancea is extremely meaningful for future application of microbes on cultivation. Here we investigated the effects of the inoculation with an entire soil microbial community on the growth, resistance to drought, and accumulation of major medicinal compounds (hinesol, β-eudesmol, atractylon and atractylodin) of A. lancea. We analyzed the interaction between A. lancea and the soil microbes at the phylum and genus levels under drought stress of different severities (inflicted by 0%, 10% and 25% PEG6000 treatments). Our results showed that inoculation with soil microbes promoted the growth, root biomass yield, medicinal compound accumulation, and rendered drought-resistant traits of A. lancea, including relatively high root:shoot ratio and high root water content under drought. Moreover, our results suggested drought stress was more powerful than the selectivity of A. lancea in shaping the root-associated microbial communities; also, the fungal communities had a stronger role than the bacterial communities in protecting A. lancea from drought. Specific microbial clades that might have a role in protecting A. lancea from drought stress were identified: at the genus level, the rhizospheric bacteria Bacillus, Dylla and Actinomadura, and rhizospheric fungi Chaetomium, Acrophialophora, Trichoderma and Thielava, the root endophytic bacteria Burkholderia-Caballeronia-Paraburkholderia, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Dylla and Actinomadura, and the root endophytic fungus Fusarium were closely associated with A. lancea under drought stress. Additionally, we acquired several endophytic Paenibacillus, Paraburkholderia and Fusarium strains and verified they had differential promoting effects on the medicinal compound accumulation in A. lancea root. This study reports the interaction between A. lancea and soil microbe communities under drought stress, and provides insights for improving the outcomes in A. lancea farming via applying microbe inoculation.

Salvia miltiorrhiza (Labiatae) is an important medicinal plant in traditional Chinese medicine. Tanshinones are one of the main active components of S. miltiorrhiza. It has been found that the intraspecific variation of S. miltiorrhiza is relatively large and the content of tanshinones in its roots of different varieties is also relatively different. To investigate the molecular mechanisms that responsible for the differences among these varieties, the tanshinones content was determined and comparative transcriptomics analysis was carried out during the tanshinones accumulation stage. A total of 52,216 unigenes were obtained from the transcriptome by RNA sequencing among which 23,369 genes were differentially expressed among different varieties, and 2,016 genes including 18 diterpenoid biosynthesis-related genes were differentially expressed during the tanshinones accumulation stage. Functional categorization of the differentially expressed genes (DEGs) among these varieties revealed that the pathway related to photosynthesis, oxidative phosphorylation, secondary metabolite biosynthesis, diterpenoid biosynthesis, terpenoid backbone biosynthesis, sesquiterpenoid and triterpenoid biosynthesis are the most differentially regulated processes in these varieties. The six tanshinone components in these varieties showed different dynamic changes in tanshinone accumulation stage. In addition, combined with the analysis of the dynamic changes, 277 DEGs (including one dehydrogenase, three CYP450 and 24 transcription factors belonging to 12 transcription factor families) related to the accumulation of tanshinones components were obtained. Furthermore, the KEGG pathway enrichment analysis of these 277 DEGs suggested that there might be an interconnection between the primary metabolic processes, signaling processes and the accumulation of tanshinones components. This study expands the vision of intraspecific variation and gene regulation mechanism of secondary metabolite biosynthesis pathways in medicinal plants from the “omics” perspective.

Alisma plantago-aquatica is an important medicinal plant in traditional Chinese medicine. In this paper, we have reported and characterized the complete chloroplast genome of A. plantago-aquatica. Its sequence was 167,642 bp in length, which contained a large single-copy region (LSC) of 90,005 bp, a small single-copy region (SSC) of 12,209 bp, and a pair of inverted repeat (IR) regions of 32,714 bp. The overall nucleotide composition of chloroplast genome sequence is: A (53,496 bp, 31.9%), T (54,364 bp, 32.4%), C (30,346 bp, 18.1%), G (29,436 bp, 17.6%) and the total G + C content of 35.7%. The complete chloroplast genome sequence contains 136 genes, including 91 encoding genes, 37 transfer RNA genes and 8 ribosomal RNA genes. For the phylogenetic analysis result, A. plantago-aquatica is closely related to A. gramineum of the family Alismataceae in the phylogenetic relationship in this study.

Anethum foeniculum is an important medicinal plant in traditional Chinese medicine. In this paper, we characterized and reported the chloroplast genome of Anethum foeniculum. Its sequence length is 153,618 bp, which contains a large single-copy (LSC) region of 86,651 bp, a small single-copy (SSC) region of 17,471 bp, and a pair of inverted repeats (IR) regions of 24,748 bp. The overall nucleotide composition of the chloroplast genome sequence is: A (30.8%), T (31.5%), C (19.2%), G (18.5%) and the total G + C content of 37.7%. The complete chloroplast genome sequence contains 130 genes, including 85 encoding genes, 37 transfer RNA genes and 8 ribosomal RNA genes. The phylogenetic analysis result of this study reveals that Anethum foeniculum is closely related to Anethum foeniculum (NC_029469) in the phylogenetic relationship.

Pogostemon cablin (Blanco) Benth. is an important medicinal plant in Traditional Chinese Medicine (TCM). Because of differences in the chemical composition, this species has been classified into two major chemotypes, i. e. the patchouliol-type and the pogostone-type; however, no quick and effective method is presently available for the precise identification of these two chemotypes. DNA barcoding, using a standardized DNA fragment, is a promising molecular diagnostic method for species identification. We have established a reliable and quick method for the identification of the P. cablin chemotypes. Of five potential barcodes [rbcL, psbA-trnH, rpoB, ITS (internal transcibed spacer), and ndhJ], tested among 103 samples, ITS was the best candidate, as comparative studies between patchouliol-type and pogostone-type P. cablin revealed that ITS had more variable regions among these five barcodes. We suggest that ITS can serve as the most suitable barcode for differentiating between the chemotypes of P. cablin.

Alisma orientale (Sam.) Juzep (A. orientale) is an important medicinal plant in traditional Chinese medicine. In this study, de novo RNA-seq of A. orientale was performed based on the cDNA libraries from four different tissues, roots, leaves, scapes and inflorescences. A total of 41,685 unigenes were assembled, 25,024 unigene functional annotations were obtained by searching against the five public sequence databases, and 3,411 simple sequence repeats in A. orientale were reported for the first time. 15,402 differentially expressed genes were analysed. The morphological characteristics showed that compared to the other tissues, the leaves had more chlorophyll, the scapes had more vascular bundles, and the inflorescences contained more starch granules and protein. In addition, the metabolic profiles of eight kinds of alisols metabolite profiling, which were measured by ultra-Performance liquid chromatography-triple quadrupole-mass spectrometry showed that alisol B 23-acetate and alisol B were the major components of the four tissues at amounts of 0.068~0.350 mg/g and 0.046~0.587 mg/g, respectively. In addition, qRT-PCR validated that farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-CoA reductase should be considered the critical candidate genes involved in alisol biosynthesis. These transcriptome and metabolic profiles of A. orientale may help clarify the molecular mechanisms underlying the medicinal characteristics of A. orientale.

ABSTRACTDendrobium officinale is an important medicinal plant in traditional Chinese medicine. The consumption of D. officinale has increased rapidly in recent years due to the health awareness among Chinese people. The present study aimed to determine trace elements levels in 42 D. officinale samples and to utilize the elemental data for identifying the cultivation technique. Eighteen trace elements (B, V, Cr, As, Mn, Fe, Mo, Cu, Zn, Se, Sr, Sn, Ti, Al, Co, As Pb, Cd, and Bi) were determined by inductively coupled plasma–mass spectrometry. Of the 11 essential elements, the mean concentrations ranged from 30.6 µg kg−1 (Mo) in D. officinale with greenhouse cultivation to 161 mg kg−1 (Fe) in D. officinale with rock cultivation, and the contributions of Mn may exceed the dietary reference intake value by the uptake of 20 g of D. officinale. Of the seven toxic elements, the mean concentrations ranged from 3.29 µg kg−1 (Bi) in the D. officinale with greenhouse cultivation to 342 mg kg−1 (Al) in the D. officinale with rock cultivation, and the contributions of Al may exceed the provisional tolerable daily intake value by the uptake of 20 g of D. officinale. The concentrations of seven metals (Bi, Pb, Fe, Al, Cr, As, and Mo) were utilized to identify the cultivation technique of the analyzed D. officinale samples through linear discriminant analysis.

The production of bioactive compounds from traditional Chinese medicine and medicinal plants mainly depends on the extraction and separation from medicinal materials, which is time-consuming, laborious, and requires large amounts of medicinal resources. As the market demand for bioactive compounds increases, the shortage of medicinal resources tops the list. For a stable and sustainable supply of affordable bioactive compounds, it is necessary to optimize chemosynthetic and biosynthetic pathways. Although some progress has been made in chemocatalysis and biosynthesis, there are drawbacks and bottlenecks in current approaches. We hold the opinion that the combination of chemosynthesis and biosynthesis will be the key direction to efficiently produce bioactive compounds. Chemo-enzymatic synthesis, a strategy that combines biosynthesis and chemosynthesis, is an alternative approach for the heterologous production of bioactive compounds. This paper reviews the recent advances in the chemo-enzymatic synthesis of bioactive compounds derived from traditional Chinese medicine and medicinal plants, highlights the potential application, and presents our perspectives for future research.

Plant growth-promoting rhizobacteria (PGPR) are beneficial bacteria that colonize plant roots and enhance plant growth by a wide variety of mechanisms. The use of PGPR is steadily increasing in agriculture and offers an attractive way to replace chemical fertilizers, pesticides, and supplements. Here, we have isolated and characterized the PGPR from the rhizosphere soil of rice field for the enhancement of growth of rice. Rhizosphere soils were collected from different areas of Mymensingh in Bangladesh. Ten isolates of bacteria, designated as PGB1, PGB2, PGB3, PGB4, PGB5, PGT1, PGT2, PGT3, PGG1 and PGG2, were successfully isolated and characterized. Subsequently, to investigate the effects of PGPR isolates on the growth of rice, a pot culture experiment was conducted. Prior to seeds grown in plastic pots, seeds were treated with PGPR isolates and seedlings were harvested after 21 days of inoculation. Isolates PGB4, PGT1, PGT2, PGT3, PGG1 and PGG2 induced the production of indole acetic acid (IAA), whereas only PGT3 isolate was able to solubilize phosphorus. Most of isolates resulted in a significant increase in plant height, root length, and dry matter production of shoot and root of rice seedlings. Furthermore, PGPR isolates remarkably increased seed germination of rice. Among the ten isolates, PGB4 and PGG2 were found almost equally better in all aspects such as dry matter production, plant height and root length of rice, and IAA production. Isolate PGT3 was also found to be promising in IAA production having an additional property of phosphate solubilization. The present study, therefore, suggests that the use of PGPR isolates PGB4, PGG2 and PGT3 as inoculant biofertilizers might be beneficial for rice cultivation as they enhanced growth of rice, and induced IAA production and phosphorus solubilization.

Salt stress can adversely affect plant seed germination, growth and development, and eventually lead to slow growth and even death of plants. The purpose of this study was to investigate the effects of different concentrations of NaCl and Na2SO4 stress on the physicochemical properties, enzyme activities, rhizosphere microbial community and seven active components (L-phenylalanine, Protocatechuic acid, Eleutheroside B, Chlorogenic acid, Caffeic acid, Eleutheroside E, Isofraxidin) of Acanthopanax senticosus rhizosphere soil. Statistical analysis was used to explore the correlation between the rhizosphere ecological factors of Acanthopanax senticosus and its active components. Compared with Acanthopanax senticosus under NaCl stress, Na2SO4 generally had a greater effect on Acanthopanax senticosus, which reduced the richness of fungi in rhizosphere soil and adversely affected the content of multiple active components. Pearson analysis showed that pH, organic matter, ammonium nitrogen, available phosphorus, available potassium, catalase and urease were significantly correlated with active components such as Caffeic acid and Isofraxidin. There were 11 known bacterial genera, 12 unknown bacterial genera, 9 known fungal genera and 1 unknown fungal genus significantly associated with the active ingredient. Salt stress had great changes in the physicochemical properties, enzyme activities and microorganisms of the rhizosphere soil of Acanthopanax senticosus. In conclusion, different types and concentrations of salts had different effects on Acanthopanax senticosus, and the active components of Acanthopanax senticosus were regulated by rhizosphere soil ecological factors.

Modulating butyric acid-producing bacterial community abundance and structure in the intestine of immunocompromised mice with neutral polysaccharides extracted from Codonopsis pilosula