Production Of Plant Secondary Metabolites Research Articles

Review: Regulation of Phytohormones and Environmental Stresses to Increase the Production of Plant Secondary Metabolites

Secondary metabolites are synthesized by plants when subjected to abiotic and biotic stresses as a defense mechanism and play an ecologically vital role both as pollination attractants and as repellents. These compounds are not essential for the process of growth and development but provide a significant impact on human welfare in various aspects as pharmaceuticals and therapeutics, aroma and taste, biopesticides, and agrochemicals. This natural product has high economic and commercial value, so it is important to explore it along with the increasing needs of consumers and industry at a large scale. Various extensive studies were carried out to improve and increase the production and accumulation of secondary metabolites. One of the strategies adopted in this review is the role of exogenous phytohormones in triggering the production of important secondary metabolites based on their classification, namely terpenoids, phenols, and flavonoids, as well as alkaloids and nitrogen-containing compounds. Bioregulators work as elicitors to induce the production of secondary metabolites through up-regulation of specific gene expression. In some cases, the application of phytohormones combined with exposure to extreme abiotic stresses showed overexpression of secondary metabolite synthesis compared to treatment with only those subjected to phytohormone. The abiotic stress of irradiation, UV-B, temperature, drought, ultrasonic, salinity, and metal stress was discussed in this study. Oxidative conditions under environmental changes trigger the plant to produce reactive oxygen species (ROS), which induce various secondary metabolites as part of the regulation of antioxidant systems. Micropropagation of medicinal plants by the addition of exogenous growth regulators could enhance the production and accumulation of important secondary metabolites.

Practical applications of jasmonates in the biosynthesis and accumulation of secondary metabolites in plants

Secondary metabolites of plant origin are the potential bioactive compounds, which are extensively exploited in pharmacological industry because of their anticancer, antidiabetic, antidepressant, antiangiogenic, antiinflammatory, antihelminth, antimalarial, antibacterial, antiviral, antituberculosis and antioxidant attributes. Besides their utility in pharmaceuticals, they are also used as nutraceuticals and agrochemicals. They also act as pollination facilitators and herbivore repellents. Therefore, various strategies such as plant tissue culture, biotechnological approaches, efficient bioreactors, and use of different biotic and abiotic elicitors have been explored to enhance their production. Among the abiotic elicitors are the phytohormones including jasmonate analogues; jasmonic acid (JA), methyl jasmonates (MeJA) and jasmonoyl isoleucine (JA-Il). Jasmonates exert a profound influence on diverse physiological responses in plants and also confer resistance against various biotic and abiotic stresses. They are also known to affect the secondary metabolite production in a large number of plant species. These secondary metabolites include terpenes, phenolics, essential oils and alkaloids. All the related studies are presented here to assess the role of jasmonates in the accumulation of secondary metabolite production in plants, both under in vivo and in vitro conditions. Application of JA is an efficient strategy that elicits the secondary metabolite production in plants, which is the primary focus of this review. Additionally, a brief overview of the underlying mechanism depicting the biosynthesis of these important bioactive compounds is also presented.

Cascading Effects of Root Microbial Symbiosis on the Development and Metabolome of the Insect Herbivore Manduca sexta L.

Root mutualistic microbes can modulate the production of plant secondary metabolites affecting plant–herbivore interactions. Still, the main mechanisms underlying the impact of root mutualists on herbivore performance remain ambiguous. In particular, little is known about how changes in the plant metabolome induced by root mutualists affect the insect metabolome and post-larval development. By using bioassays with tomato plants (Solanum lycopersicum), we analyzed the impact of the arbuscular mycorrhizal fungus Rhizophagus irregularis and the growth-promoting fungus Trichoderma harzianum on the plant interaction with the specialist insect herbivore Manduca sexta. We found that root colonization by the mutualistic microbes impaired insect development, including metamorphosis. By using untargeted metabolomics, we found that root colonization by the mutualistic microbes altered the secondary metabolism of tomato shoots, leading to enhanced levels of steroidal glycoalkaloids. Untargeted metabolomics further revealed that root colonization by the mutualists affected the metabolome of the herbivore, leading to an enhanced accumulation of steroidal glycoalkaloids and altered patterns of fatty acid amides and carnitine-derived metabolites. Our results indicate that the changes in the shoot metabolome triggered by root mutualistic microbes can cascade up altering the metabolome of the insects feeding on the colonized plants, thus affecting the insect development.

Transcriptomics analysis of genes induced by melatonin related to glucosinolates synthesis in broccoli hairy roots

ABSTRACT Glucoraphanin (GRA) is found in the seeds and vegetative organs of broccoli (Brassica oleracea L. var. italica Planch) as the precursor of anti-carcinogen sulforaphane (SF). The yield of GRA obtained from these materials is weak and the cost is high. In recent years, the production of plant secondary metabolites by large-scale hairy roots culture in vitro has succeeded in some species. Melatonin (MT) is a natural hormone which existed in numerous organisms. Studies have demonstrated that MT can improve the synthesis of secondary metabolites in plants. At present, it has not been reported that MT regulates the biosynthesis of glucoraphanin in broccoli hairy roots. In this study, the broccoli hairy roots that grew for 20 d were respectively treated by 500 µM MT for 0, 6, 12, 20 and 32. To explore the reason of changes in secondary metabolites and reveal the biosynthetic pathway of glucoraphanin at transcriptional level. Compared with 0 h, the yield of GRA under other treatments was increased, and the overall trend was firstly increased and then decreased. The total yield of GRA reached the highest at 12 h, which was 1.22-fold of 0 h. Then, the genome of broccoli as the reference, a total of 13234 differentially expressed genes (DEGs) were identified in broccoli hairy roots under treatment with 500 µM MT for 0, 6, 12, 20 and 32 h, respectively. Among these DEGs, 6266 (47.35%) were upregulated and 6968 (52.65%) were downregulated. It was found that the pathway of ‘Glucosinolates biosynthesis (ko00966)’ was enriched in the 16th place by Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the upregulated DEGs. The expression of key genes in the GRA biosynthesis pathway was upregulated at all time points, and a deduced GRA biosynthesis pathway map was constructed for reference.

The Opportunity of Spent Bleaching Earth (Bentonite) and Silica Solubilizing Bacteria as Silica Source for Induction of Secondary Metabolites Production in Plants

Background: CPO refining which produces solid waste namely spent bleaching earth (SBE) in large quantities can pollute the environment. SBE from bentonite ores contains large amounts of silica, so it can be an alternative source of silica minerals. Silica plays an important role in increasing plant resistance and bioactive plant compound products. Methods: The application of Si in plants can increase secondary metabolites such as phenolic and anti-fungal compounds in response to disease pathogens. However, the low solubility of silica makes silica not sufficiently available for plants. Using microorganisms as silica solubilizing bacteria helps increasing solubility of silica in the soil. Bacteria dissolve silica by removing organic acids and producing indole acetic acid (AAI), which stimulates root hairs. Results: This review presents the results of a study on the utilization of silica-rich SBE waste as a source of available silica for plants with solubilizing method using bacteria to increase plant growth and resistance, as well as increase plant secondary metabolite compounds. Conclusions: The application of silica solubilizing bacteria has been known to play an important role in providing silica for plants, through enzymatic mechanisms, namely the production of organic acids and extracellular polysaccharides.

Exploiting endophytic microbes as micro-factories for plant secondary metabolite production.

Plant secondary metabolites have significant potential applications in a wide range of pharmaceutical, food, and cosmetic industries by providing new chemistries and compounds. However, direct isolation of such compounds from plants has resulted in over-harvesting and loss of biodiversity, currently threatening several medicinal plant species to extinction. With the breakthrough report of taxol production by an endophytic fungus of Taxus brevifolia, a new era in natural product research was established. Since then, the ability of endophytic microbes to produce metabolites similar to those produced by their host plants has been discovered. The plant "endosphere" represents a rich and unique biological niche inhabited by organisms capable of producing a range of desired compounds. In addition, plants growing in diverse habitats and adverse environmental conditions represent a valuable reservoir for obtaining rare microbes with potential applications. Despite being an attractive and sustainable approach for obtaining economically important metabolites, the industrial exploitation of microbial endophytes for the production and isolation of plant secondary metabolites remains in its infancy. The present review provides an updated overview of the prospects, challenges, and possible solutions for using microbial endophytes as micro-factories for obtaining commercially important plant metabolites.Key points• Some "plant" metabolites are rather synthesized by the associated endophytes.• Challenges: Attenuation, silencing of BGCs, unculturability, complex cross-talk.• Solutions: Simulation of in planta habitat, advanced characterization methods.

Vibrational Spectroscopy Combined with Chemometrics as Tool for Discriminating Organic vs. Conventional Culture Systems for Red Grape Extracts.

Food plants provide a regulated source of delivery of functional compounds, plant secondary metabolites production being also tissue specific. In grape berries, the phenolic compounds, flavonoids and non-flavonoids, are distributed in the different parts of the fruit. The aim of this study was to investigate the applicability of FTIR and Raman screening spectroscopic techniques combined with multivariate statistical tools to find patterns in red grape berry parts (skin, seeds and pulp) according to grape variety and vineyard type (organic and conventional). Spectral data were acquired and processed using the same pattern for each different berry part (skin, seeds and pulp). Multivariate analysis has allowed a separation between extracts obtained from organic and conventional vineyards for each grape variety for all grape berry parts. The innovative approach presented in this work is low-cost and feasible, being expected to have applications in studies referring to the authenticity and traceability of foods. The findings of this study are useful as well in solving a great challenge that producers are confronting, namely the consumers’ distrust of the organic origin of food products. Further analyses of the chemical composition of red grapes may enhance the capability of the method of using both vibrational spectroscopy and chemometrics for discriminating the hydroalcoholic extracts according to grape varieties.

Role of Biotic Elicitors Arbuscular Mycorrhizal Fungi (AMF) In Improving Growth, Development and Production of Plant Secondary Metabolites

Role of Biotic Elicitors Arbuscular Mycorrhizal Fungi (AMF) In Improving Growth, Development and Production of Plant Secondary Metabolites

Biotic elicitors: a boon for the in-vitro production of plant secondary metabolites

Biotic elicitors: a boon for the in-vitro production of plant secondary metabolites

​Insect Plant Interaction with Reference to Secondary Metabolites: A Review

Plant and herbivorous insect share a contradictory but everlasting relationship. To cope with the herbivores, plant invested its energy in the production of various defensive chemicals which are known as plant secondary metabolites (PSMs). During coevolution with plants, insects also adapted themselves to counter plant defensive mechanisms. The secreted saliva and ovipositional fluids of insects induce the production of secondary metabolites in plants. Plants also produce compounds constitutively in the tissues that are susceptible to insect attack. These compounds reduce insect growth and development of insects through interfering with the developmental phases, acting as feeding deterrent, reducing nutritive value, affecting protein digestion and the production of substances toxic to them. On the contrary, insect’s adaptations to PSMs include the detoxification of plant toxins, conversion of the toxic compounds into conducible form for its growth and development, development of feeding choice based on the concentration of PSMs, quicker absorption and elimination as feces and obtaining the help of symbiotic intestinal microorganism to inactivate toxic materials. Furthermore, insects also utilize PSMs as chemical cues mediated through their chemoreceptive systems. The selection of host plants by monophagous insect is sometimes concentrated on the production of particular PSMs. Secondary metabolites also act as attractant to insect pollinators by exhibiting distinctive odors. Insects also store toxic PSMs which are used by them against their predators and in giving protection to eggs. However, this intrinsic capability of plants to produce defensive chemicals in response to insect attack should be explored to a wide extent to optimize the performance of PSMs.

Transcriptomic analyses of cacao flavonoids produced in photobioreactors

BackgroundTheobroma cacao is a major source of flavonoids such as catechins and their monomers proanthocyanidins (PAs), widely studied for their potential benefits in cardiovascular diseases. Light has been shown to promote plant secondary metabolite production in vitro. In this study, cacao cells cultured in 7.5 L stirred tank photobioreactors (STPs) were exposed to a change of white to blue LED lights for 28 days (d).ResultsTranscriptomic analyses were performed in three time points comparing changing expression patterns, after cell exposure to white light (d0-VS-d14), after a shift from white to blue light (d14-VS-d15), and after an extended period of blue light for the following 15 days (d15-VS-d28). Under white light, there was enrichment in metabolic pathways associated with cell growth (carbon, glycolysis, and amino acid biosynthesis) accompanied by a significant increase in the PAs content. In the shift to blue light, further increase in PAs content was observed concomitantly with the significant expression of TWO-COMPONENT RESPONSE REGULATOR genes involved in the early stress responses via circadian clock and hormone pathways. Under blue light exposure, we observed a depletion of PAs content associated with ROS-mediated stress pathways.ConclusionsLight effects on large-scale cell cultures in photobioreactors are complex and pleiotropic; however, we have been able to identify key regulatory players upstream cacao flavonoid biosynthesis in STPs, including TWO-COMPONENT SYSTEM and ROS-signaling genes. The crosstalk between flavonoid biosynthesis and regulatory networks led to understand the dynamics of flavonoid production and degradation in response to light-driven ROS signals. This can be used to optimize the time, and the yield of in vitro targeted metabolites in large-scale culture systems.

Stimulation of plant secondary metabolites synthesis in soilless cultivated strawberries (Fragaria × ananassa Duchesne) using zinc-alginate microparticles

An innovative approach to stimulate the production of plant secondary metabolites (PSM) is the use of encapsulated bioactive agents. Zinc-alginate microparticles [microspheres (ALG/Zn) and microcapsules (CS/ALG/Zn)] were applied near the root zone of soilless cultivated strawberries (Fragaria × ananassa Duchesne) to ensure a constant supply of an essential micronutrient zinc ions throughout the whole period of maturation. Both, ALG/Zn and CS/ALG/Zn had a positive effect on PSM (total polyphenols, total flavonoids, total anthocyanins, and flavan-3-ols) synthesis and increased antioxidant activity in two strawberry varieties (Albion and San Andreas). Treatments with Zn-alginate microparticles resulted in no visible damages on the plant and fruits. A two-year study revealed an increase of ~34% in total polyphenolic compounds and ~23% in total flavonoids in the San Andreas variety. The highest increase in antioxidant activity [ABTS (~37%) and DPPH (~38%)] was observed in the San Andreas variety treated with CS/ALG/Zn. Respectively high increase in anthocyanins was observed in both varieties (~54% and ~61%, respectively) treated with CS/ALG/Zn. Treatments with zinc-alginate microparticles resulted in a successful stimulation of PSM synthesis and increased strawberries' nutritive value. Encapsulation of zinc ions proved to be simple, sustainable, and environmentally favorable to produce strawberries fortified with important bioactive compounds.

Enhanced inulin production by hairy root cultures of Cichoriumintybus in response to Pi and Fe starvation.

Cichorium intybus is rich in inulin and has several pharmacological applications. Hairy roots culture is a valuable biotechnological tool used to produce plant secondary metabolites. Agrobacterium rhizogenes-mediated genetic transformation of chicory to hairy roots was investigated using Agrobacterium Strains A4, A13, A7, and ATCC15834. Several hairy roots were tested, from which 17 lines were selected based on their fast-growing characteristics. Results of PCR analysis revealed foreign DNA integration into the selected transgenic hairy root lines. Finally, four Adventitious roots that contained the highest ratio of total sugar to total weight (µg/gr DW), were selected. This study investigated the effects of various levels of minerals and sucrose on the production of inulin in Cichorium hairy root culture. Different levels of sucrose, phosphate (Pi) and Iron (Fe) were evaluated, separately. It was found that an increase in sucrose levels from 3 to 5% could decrease the root growth; however, 60 g/l sucrose remarkably enhanced the inulin production rate in all the examined lines. The highest biomass was achieved by applying 3.75 mM Pi but it ended in the decreasing the inulin content per unit weight. In contrast, the highest inulin accumulation and the lowest amount of biomass were observed in 0.5 mM Pi. Fe starvation caused the biomass decrease and a significant increase in inulin accumulation. Results of this study suggest a successfully optimized culture medium to initiate the induction of Cichorium intybus hairy root cells to produce inulin as a valuable medicinal secondary metabolite.

Polyploidy‐promoted phenolic metabolism confers the increased competitive ability of Solidago canadensis

Allelopathy plays an important role in the successful invasion of alien invasive plants. Phenolic compounds are one of the primary classes of secondary metabolites with strong allelopathic activities. Polyploidy can cause significant effects on the production and yield of plant secondary metabolites. However, the effect of allelopathic potential of phenolic compounds on the competitiveness of different geographical cytotypes of Solidago canadensis remains unknown. We conducted a common garden field experiment in the invasive habitat of China to compare the competitive ability of native and introduced diploid, tetraploid and hexaploid S. canadensis against intentionally planted local weeds. Results showed that introduced tetraploids and hexaploids outcompeted local plants and formed a monodominant community due to superior competitive ability. Phenolic metabolism analysis showed these populations had a high accumulation of total phenolic compounds in aerial parts and rhizosphere soil throughout three developmental stages, which contributed to significant allelopathic effects on tested weeds. However, diploids and native polyploids showed less allelopathic activities due to the low level of total phenolic content. Together, these results suggest polyploidization is one of the reasons that driving the differentiation of phenolic metabolism in different geo‐cytotypes of S. canadensis, and the allelopathic abilities of introduced polyploids are further enhanced through a post‐introduction adaptive evolution. Polyploidy‐promoted allelopathic potential makes introduced polyploids more competitive against local plants than diploids and native polyploids, which contribute to its successful invasion.

Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites.

Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by Alkanna tinctoria might be influenced by its endomicrobiome. To study the interaction between this medicinal plant and its bacterial endophytes, we isolated bacteria from the roots of wild growing Alkanna tinctoria collected near to Athens and Thessaloniki in Greece. Representative strains selected by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The most abundant genera recovered were Pseudomonas, Xanthomonas, Variovorax, Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested in vitro for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed pectinase and cellulase activity in vitro. In addition, bacterial responses to alkannin and shikonin were investigated through resistance assays. Gram negative bacteria were found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives were sensitive. A selection of bacteria was then tested for the ability to induce A/S production in hairy roots culture of A. tinctoria. Four strains belonging to Chitinophaga sp., Allorhizobium sp., Duganella sp., and Micromonospora sp., resulted in significantly more A/S in the hairy roots than the uninoculated control. As these bacteria can produce cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with the plant-bacteria interaction during colonization.

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities.

PAL (phenylalanine ammonia lyase) is important for secondary metabolite production in plants and microorganisms. There is broad interest in engineering PAL for its biocatalytic applications in industry, agriculture, and medicine. The production of quantities of high-activity enzymes has been explored by gene cloning and heterogeneous expression of the corresponding protein. Here, we cloned the cDNA of Rhodotorula glutinis PAL (RgPAL) and introduced codon optimization to improve protein expression in Escherichia coli and enzyme activities in vitro. The RgPAL gene was cloned by reverse transcription and named pal-wt. It had a full-length of 2,121 bp and encoded a 706-amino-acid protein. The pal-wt was inefficiently expressed in E. coli, even when the expression host and physical conditions were optimized. Therefore, codon optimization was used to obtain the corresponding gene sequence, named pal-opt, in order to encode the same amino acid for the RgPAL protein. The recombinant protein encoded by pal-opt, named PAL-opt, was successfully expressed in E. coli and then purified to detect its enzymatic activity in vitro. Consequently, 55.33 ± 0.88 mg/L of PAL-opt protein with a specific activity of 1,219 ± 147 U/mg and Km value of 609 μM for substrate L-phenylalanine was easily obtained. The enzyme protein also displayed tyrosine ammonia lyase (TAL)–specific activity of 80 ± 2 U/mg and Km value of 13.3 μM for substrate L-tyrosine. The bifunctional enzyme RgPAL/TAL (PAL-opt) and its easy expression advantage will provide an important basis for further applications.

An Efficient Method for the Genetic Transformation of Acmella oleracea L. (Spilanthes acmella Linn.) with Agrobacterium tumefaciens.

Acmella oleracea L. is an important medicinal plant, commonly known as the toothache plant. It is a rich source of secondary metabolites used for the treatment of different human disorders. The demand for Acmella oleracea L. has increased due to its putative health benefits (in terms of both biomass quantity and bioactive compound purification). In vitro plant cultures have allowed the rapid increase of raw material availability through the use of suitable regeneration and multiplication systems. On the other hand, there is a general lack of methods for Acmella genetic transformation as a promising new technological approach for the improvement of secondary metabolites. In this work, an efficient transformation protocol has been established using the Agrobacterium tumefaciens LBA4404 strain bearing the binary vector pBI121 containing the NPTII gene for the resistance to kanamycin. Plant genetic transformation has been verified by direct polymerase chain reaction and GUS assay on regenerants. Transformation efficiency has been affected by the high level of the selection agent kanamycin. To our knowledge, this is the first report on the genetic transformation of A. oleracea, paving the way to further studies to improve in vitro plant growth and secondary metabolite production.

Closed polybag foliar methyl-jasmonate treatment: New technology for rapid enhancement of bioactive withanolide biosynthesis in field-grown plants of Withania somnifera

Withania somnifera (L.) Dunal, is an important medicinal plant with tremendous use in the herbal drug industry. Withanolides are the major bioactive metabolites present in the leaves of W. somnifera. However, due to the low abundance of withanolides, it is difficult to fulfil the market demand. The rationale of this study was to develop a simple technology for enhanced withanolide biosynthesis in field-grown plants of W. somnifera. Exogenous elicitor-treatment to the field-grown plants is an effective strategy to increase the production of many plant secondary metabolites. In this study, a high-performance liquid chromatography method was developed for the rapid separation of withanolides from the leaves along with phenolic acid and flavonoids. Field-grown plants of W. somnifera were directly used for methyl jasmonate elicitor treatment using a novel closed polybag approach. In this approach, branches were covered with a polybag and then MeJa was injected using a specially designed vial. Methyl-jasmonate (20μM) treatment for 4 h resulted in 2.2-fold, 1.9-fold, 2.0-fold, and 2.2-fold increase in withaferin A, withanolide A, withanolide D, and withanone production, respectively, over the control treatment. Methyl-jasmonate -treatment increased the transcript levels of four key withanolide biosynthetic genes, namely 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, and cycloartenol synthase. Methyl-jasmonate -treated leaf extracts and isolated pure withanolides also showed a significant cytotoxic effect against selected human breast cancer cell line. These results suggest that MeJa-treatment using a closed polybag system is an effective and easy-to-perform elicitation strategy for the enhanced production of bioactive withanolides using field-grown plants of W. somnifera. Farmers and industry can easily adapt this technology to meet the growing demand for bioactive withanolides.

NMR-based metabolomic analysis of the seasonal and regional variability of phytochemical compounds in Curtisia dentata stem bark

Traditional medicine markets are provided with medicinal plant material throughout the year, however, internal (e.g. plant age, genetic variability and differential expression of genes) and external factors (e.g. water and nutrient availability, rainfall, photoperiod and herbivory), affect secondary metabolite production in plants. In this study, seasonal variability in metabolite production in Curtisia dentata trees from two geographically separated regions in South Africa are compared. NMR analysis of C. dentata stem bark samples yielded spectral data which were processed in MestReNova to perform multivariate data analysis using Soft Independent Modeling of Class Analogy (SIMCA) software. This study shows that there are not only seasonal, regional and yearly differences in secondary metabolite production in C. dentata trees, but that production patterns of hydrophilic and lipophilic chemical compounds in individual trees also vary. Sucrose, isoeugenol and betulinic acid have been used in a targeted analysis to show the variation in individual compounds in the individual trees as a response to seasonal and geographical differences. Therefore, the season and year, as well as the region, harvesting site and specific trees from which plant material is collected affect the concentrations of chemical compounds extracted from C. dentata stem bark for the preparation of remedies.

Microbial inoculants modulate growth traits, nutrients acquisition and bioactive compounds accumulation of Cyclocarya paliurus (Batal.) Iljinskaja under degraded field condition

Cyclocarya paliurus (Batal.) Iljinsk is an important medicinal plant for treating chronic diseases, but it is difficult to obtain high yields when growing on low-fertility soil. Inoculation with soil beneficial microorganism has suggested an effective means of stimulating plant growth and secondary metabolite production, but effect on plant performance when competing degraded field condition remains unclear. We combined controlled laboratory experiments with field trials to investigate the effects of co-inoculation with phyto-stimulatory strains (Azospirillum brasilense and Pseudomonas fluorescens) and nutrient-enhancing strains (Bacillus megaterium and Azotobacter chroococcum). Bacteria were applied with organic fertilizer at different fertilizer levels, and we tracked effects on soil nutrient availability as well as C. paliurus morphological traits, photosynthesis, growth and bioactive compounds during cultivation on barren land. Amendment of beneficial microbes with organic fertilizer enhanced the soil nutrient availability with high fertilizer showing greatest stimulation under controlled conditions, with the medium fertilizer giving best results in improving plant performance in the field. All fertilization regimes expanded the 3D root architecture, and bacterial additions increased the proportion of lateral roots compared to a single organic fertilizer treatment, which led to higher nutrient uptake. Inoculations at medium fertilizing level modified the root system and increased the photosynthesis rate, nutrient acquisition and plant growth. The co-inoculation with B. megaterium and P. fluorescens at medium fertilizer level stimulated the accumulation of flavonoids and polysaccharides, while co-inoculation with A. chroococcum and A. brasilense at low fertilizing level facilitated the production of flavonoids and triterpenoids. The biosynthesis of secondary metabolites exhibited strong correlations with leaf C/N and C/P ratios. Thus, manipulation of bioactive compounds in C. paliurus leaves can be affected by internal nutrient balance, which is associated with reformed root system morphology that modulated by bacterial inoculation.