- Research
- Open access
- Published:
Biochar-induced microbial and metabolic reprogramming enhances bioactive compound accumulation in Panax quinquefolius L.
BMC Plant Biology volume 25, Article number: 669 (2025)
Abstract
Panax quinquefolius L., with a history of over 300 years in traditional Chinese medicine, is notably rich in ginsenosides—its primary bioactive components. Although our previous study found that biochar application could enhance the content of ginsenoside Re, Rg and other contents in P. quinquefolius, its effect on the overall secondary metabolism of P. quinquefolius and its mechanism are still unclear. In this paper, the correlation between plant microbiome and secondary metabolites was studied from the perspective of plant rhizosphere microorganisms and endophytes, and the mechanism of biochar-induced metabolic reprogramming of P. quinquefolius was revealed. The results showed that biochar treatment significantly increased the accumulation of various substances in P. quinquefolius, including nucleosides, glycerophosphocholines, fatty acyls, steroidal glycosides, triterpenoids, and other bioactive compounds. Additionally, biochar treatment significantly enriched beneficial rhizosphere microorganisms such as Bacillus, Flavobacterium, and Devosia, while reducing the relative abundance of harmful fungi like Fusarium. Furthermore, it promoted endophytic Flavobacterium, Acaulospora, and Glomus, and suppressed pathogenic genera such as Plectosphaerella, Cladosporium, and Phaeosphaeria. These shifts in rhizosphere microbial community and endophytes structure and function were closely linked to the accumulation of secondary metabolites (e.g. ginsenosides Rg3, F2) in P. quinquefolius. Overall, our findings suggest that biochar may influence key endophytes and rhizosphere microorganisms to regulate the accumulation of secondary metabolites in P. quinquefolius. Therefore, this study provides valuable insights into the potential application of biochar in Chinese medicine agriculture.
Introduction
Panax quinquefolium L., also known as American ginseng, has been a staple in traditional Chinese medicine (TCM) for over three centuries [1]. TCM theory posits that P. quinquefolius replenishes qi, nourishes yin, clears heat, and promotes body fluid production [2,3,4]. Modern clinical studies have validated these uses, confirming its anti-cancer, anti-diabetes, immunomodulatory, and neuroprotective effects [5,6,7]. P. quinquefolius contains a wealth of active ingredients, including saponins, organic acids, sugars, and volatile oils [8,9,10]. Among these, ginsenoside is the primary active component and the key indicator of its medicinal quality [11, 12]. In recent years, P. quinquefolius has gained widespread use in health foods, cosmetics, and other industries across Europe, America, and Asia [13]. In 2023, China officially recognized P. quinquefolius as both a medicine and a food. With the increasing market demand, ensuring the herb’s quality becomes crucial. Currently, most P. quinquefolius on the market is cultivated, with China being a major producer, accounting for 30% of global production - approximately 2100 tonnes [14]. Therefore, it is essential to implement effective measures to enhance the content of active metabolites in P. quinquefolius.
Biochar is a carbon-rich, porous, and alkaline solid produced from the pyrolysis of biomass waste [15]. It possesses a large specific surface area, abundant pores, and various physical properties, which enhance its adsorption capacity and improve soil microbial community structure [16,17,18]. Studies have shown that biochar can boost crop yield and the accumulation of secondary metabolites in medicinal plant, such as maize, tobacco, Aloe vera (L.) Burm. f., Fritillaria thunbergii Miq.and so on [19,20,21,22]. Our team discovered that biochar increases the accumulation of ginsenosides Re, Rg in P. quinquefolius [23], although its effect on the overall secondary metabolism of P. quinquefolius and its mechanism are still unclear, and further research is needed.
Recently, the role of rhizosphere microorganisms and endophytes in remodeling plant metabolism has gained significant attention. These microorganisms are crucial for the accumulation of host metabolites induced by various environmental factors [24, 25]. Our group previously found that rhizosphere arbuscular mycorrhizal fungi (AMF) promote the accumulation of ginsenosides in P. quinquefolius and elucidated the signal transduction mechanism [26,27,28]. Biochar, when introduced into the soil, can influence rhizosphere microorganisms [17, 29], potentially affecting the accumulation of secondary metabolites. Further study is needed to determine if biochar can regulate the accumulation of these metabolites by modifying the rhizosphere microecological environment.
Additionally, endophytes are microorganisms that live harmlessly within the healthy tissues or organs of plants [30]. They originate from the rhizosphere or are influenced by rhizosphere microorganisms [31]. Endophytes significantly impact the quality of medicinal materials [32], by altering the metabolite composition of their host plants [33, 34]. Our team discovered correlations between endophytes in P. quinquefolius and their secondary metabolites, indicating that endophytes influence accumulation of secondary metabolites [35]. Further study is needed to determine if biochar’s effect on P. quinquefolius’s secondary metabolite accumulation is related to endophytes. It can be seen that endophytes and rhizosphere microorganisms play an important role in the accumulation of secondary metabolites in plants. Therefore, it is worth studying whether biochar regulates secondary metabolism by modulating endophyte communities and rhizosphere microorganisms. To investigate this, ultra high-performance liquid chromatography (UPLC-MS/MS) was employed to detect metabolites in P. quinquefolius roots, and 16 S and ITS microbial sequencing was used to analyze P. quinquefolius rhizosphere microorganisms and endophyte.
By integrating microbiome and metabolomics analyses, we elucidated the correlation between rhizosphere microorganisms, endophytes, and secondary metabolites. Our objectives were twofold: (1) To determine the effects of biochar application on metabolic reprogramming in P. quinquefolius. (2) To reveal how biochar influences the rhizosphere microbial community and endophyte structure composition in P. quinquefolius, as well as their relevance to secondary metabolism. This study aims to uncover the mechanism behind biochar’s regulation of P. quinquefolius secondary metabolism from the perspective of rhizosphere microorganisms and endophytes. Furthermore, it also provides a reference for biochar application in plant production.
Materials and methods
Experimental design
The experiment was carried out at a test station in Wendeng District, Shandong Province, China (121°49 ‘E, 37°05’ N), a major production area for P. quinquefolius. Biochar provided by Jiangsu Huafeng Agricultural Bioengineering Co., Ltd., was applied at a rate of 1.8%. Prior to the experiment, P. quinquefolius seeds were surface-disinfected following the method detailed in Yang et al. [23]. A pot experiment was conducted outdoors from March 2022 to October 2022. Before initiating the pot experiment, surface soil (0–20 cm) was collected from the experimental site, air-dried, ground, and sieved through a 2 mm screen. Biochar was then mixed with the soil at concentrations of 0% (CK) and 1.8% (biochar), with the selected concentration based on previous laboratory optimizations. Both the soil and biochar were autoclaved at 121 °C and 0.10 MPa for 2 h before the experiment to eliminate microorganisms. Six P. quinquefolius seeds were evenly placed on the surface of 2.0 kg of the soil-biochar mixture in each pot. An additional 100 g of the mixture was spread over the seeds to ensure uniform depth. Thirty pots were prepared for each treatment. In October 2022, all potted plants were watered to 70% of the field water volume and then buried in the same soil at the test site. Each treatment was replicated thrice.
Sample collection
Plant and soil samples were collected in October 2022. Each plant sample was divided into two sub-samples. One was extracted with liquid nitrogen and stored in a refrigerator at -80 ℃ for endophytic bacteria and plant metabolism analysis. The other sub-sample was stored in dry ice and immediately shipped back to the laboratory for saponin analysis. Soil samples were collected from a depth of 0–11 cm, approximately 2 cm from the roots of each plant, using a sterile spatula. Excess soil was vigorously shaken off the roots, leaving about 1 mm of soil. The root surface was then carefully wiped with a sterile brush, and the collected soil was stored in a disinfected bag. Soil samples were treated with liquid nitrogen and stored at -80 ℃ for microbiological analysis.
Microbial community analysis
For soil samples, DNA was extracted using the Magnetic Soil and Stool DNA Kit. For plant root samples, DNA was extracted using the CTAB extraction method. DNA purity and concentration were measured by agarose gel electrophoresis. Suitable DNA samples were placed into a centrifuge tube and diluted to 1 ng/µL with sterile water. The following primers were used for PCR amplification:
-
Bacterial 16 S v4 region: 515 F (GTGCCAGCMGCCGCGGTAA) and 806 R (GGACTACHVGGGTWTCTAAT).
-
ITS1-5 F region: ITS5-1737 F (CTTGGTCATTTAGAGGAAGTAA) and ITS2-2043R(GCTGCGTTCTTCATCGATGC).
-
Plant bacterial 16S gene: 799F (5’-AACMGGATTAGATACCCKG-3’) and 1193R (5’-ACGTCATCCCCACCTTCC-3’).
-
Plant fungal ITS genes: ITS1-1 F-F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS1-1 F-R (5’-GCTGCGTTCTTCATCGATGC-3’).
The library was constructed using the TruSeq® DNA PCR-Free Sample Preparation Kit, and the constructed library was quantified by Qubit and Q-PCR. The qualified libraries were sequenced on the Illumina NovaSeq6000 platform at Novogene Biotechnology Co., Ltd., Beijing, China. Final data presentation: T1 is CK and T2 is biochar treatment. The same below.
Metabonomics analysis
100 mg plant root samples (six replicates per group) were weighed, frozen in liquid nitrogen, and the homogenate was resuspended with prechilled 80% methanol by well vortex. The samples were incubated on ice for 5 min and then were centrifuged at 15,000 g, 4 °C for 20 min. Some of supernatant was diluted to final concentration containing 53% methanol by LC-MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 15,000 g, 4 °C for 20 min. Finally, the supernatant was injected into the LC-MS/MS system analysis. UHPLC-MS/MS analyses were conducted using a Vanquish UHPLC system coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher, Germany) at Novogene Co., Ltd. (Beijing, China). The compounds were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg.jp/kegg/compound/). The differentially abundant metabolites (DAMs) were identified with a variable importance in project (VIP) value ≥ 1 and an absolute log2 (fold change [FC]) ≥ 1. VIP values were obtained from the orthogonal partial least squares discriminant analysis (OPLS-DA) results using the R package MetaboAnalystR (https://www.metaboanalyst.ca/).
Determination of ginsenoside content
The ginsenoside content was determined as described previously [36]. Each root sample (0.5 g) was crushed and extracted using 70% methanol. The suspension was then subjected to ultrasonic treatment at 50 °C for 30 min, followed by filtration. The combined filtrate was dried at 45 °C using a rotary evaporator, and the residue was redissolved in 5 mL of methanol. After further filtration through a 0.22 μm membrane filter, ginsenosides were analyzed using an Agilent 1200 HPLC system (Agilent Technologies Co., Ltd.). Separation was achieved on a YMC-PACK ODA-A column (250 mm × 4.6 mm, 5 μm, YMC Co., LTD., Kyoto, Japan) with a mobile phase comprising acetonitrile (A) and 0.1% phosphoric acid solution (B). The gradient elution procedure was as follows: 0–25 min, A: 19–20%; 25–60 min, A: 20–40%; 60–90 min, A: 40–55%; 90–100 min, A: 55–60%. The mobile phase flow rate was set to 1 mL·min− 1. The detection wavelength was 203 nm, and the sample size was 20 µL. Ginsenoside standards (Rg1, Re, Rf, Rb1, Rb2, Rd, Rh1, Rh2) were obtained from Chengdu Manset Biotechnology Co., Ltd.
Statistical analysis
Statistical analysis was performed using SPSS 26.0. Tukey’s test was used to assess differences in means, with data expressed as mean ± standard deviation. Differences between the two groups were analyzed using Student’s t-test with a significance level of < 0.05.
Result
Effects of Biochar on the metabolome of P. quinquefolius roots
To assess the impact of biochar on the metabolome of P. quinquefolius roots, a partial least squares discriminant analysis (PLS-DA) was performed on the differential metabolites (DAMs). The PLS-DA scores indicated significant metabolic differences between treatments (Fig. 1A). Compared to the CK, biochar treatment up-regulated 98 metabolites and down-regulated 101 metabolites (Fig. 1B).
Effects of biochar on rhizome metabolomics of P. quinquefolius roots. A: PLS-DA score chart. Each point in the diagram represents a sample, with samples from the same group represented by the same color. B: Volcano map. A dot in the figure represents a metabolite, with red and green colors indicating up-regulation and down-regulation, respectively. C: Heatmap analysis between different treatments (T2 vs. T1). D: Classification of 98 upregulated differential metabolites between different treatments (T2 vs. T1). F: KEGG enrichment analysis (T2 vs. T1). T1 is CK, T2 is biochar treatment
Biochar application resulted in differences in the relative abundance of 199 DAMs (Tab S1). To analyze the response to biochar, heat maps of two sets of metabolite classes were generated (Fig. 1C). These included mainly 37 lipids and lipid-like molecules, 15 nucleosides, nucleotides, and analogues, and 8 organoheterocyclic compounds (Fig. 1D). First, lipids, which are key components of cell membranes and act as energy reserves [37], showed significant up-regulation. This includes glycerophospholipids, fatty acyls, steroids, steroid derivatives, and prenol lipids. Membrane lipid remodeling is an important adaptation strategy for plants under abiotic stress [38]. Notably, many of the up-regulated lipids have biological and medicinal activity, such as oleic acid, prohydrojasmon, jervine, brassinolide, timosaponin A1, vitamin A, (20R) ginsenoside Rg3, ginsenoside F2, poricoic acid B, notoginsenoside Fe, ginsenoside Rb1, protopanaxadiol, and ziyuglycoside II. Secondary nucleotides, crucial for energy metabolism and plant physiological processes [39], were also up-regulated. Examples include cytidine, isoguanosine, guanosine, adenosine, and uridine. Among these, purine nucleotides are precursors of cytokinin biosynthesis, which regulate plant growth and development and enhance abiotic stress tolerance. Finally, organoheterocyclic compounds were up regulated after biochar application. These include adenine, cytosine, guanine, 5-hydroxymeloxicam, 4-methylaminoantipyrine, (-)-chimonanthine, and indole-3-carboxylic acid. Additionally, biochar application increased other bioactive metabolites like targinine, schisandrin B, and galanthamine.
Using the KEGG database and previous studies, a metabolic pathway was constructed for the top 20 differential metabolites, highlighting the relationship between these compounds in the metabolic spectrum of differently treated P. quinquefolius. Figure 1E displays the significance of each pathway, determined by p-value and abundance factors; larger, darker bubbles indicate more significant pathways. Key pathways included purine and pyrimidine metabolism, zeatin biosynthesis, valine, leucine, and isoleucine degradation, propanoate metabolism, terpenoid backbone biosynthesis, brassinosteroid biosynthesis, and plant hormone signal transduction. These pathways help explain the differences between biochar and the root metabolites of P. quinquefolius. Notably, among the detected saponins, the relative content of ginsenosides increased with biochar treatment.
Ginsenoside is the primary active component of P. quinquefolius and a key indicator of its medicinal quality. As shown in Table 1, biochar treatment significantly increased the contents of ginsenosides Re, Rb1, Rb2, and Rh2 by 22.20%, 16.97%, 44.00%, and 24.78% respectively, compared to the CK (P < 0.05). While the contents of ginsenosides Rg1, Rh1, and Ro also increased, these changes were not statistically significant (P > 0.05). Additionally, the content of ginsenoside Rd decreased significantly by 12.8% (P < 0.05).
Effects of Biochar on rhizosphere soil microorganisms of P. quinquefolius
The effects of biochar application on the rhizosphere soil microorganisms of P. quinquefolius are shown in Fig. 2. Figure 2A displays the top 10 amplicons of 16 S rRNA detected in all samples, mainly Proteobacteria, Acidobacteriota, and Bacteroidota. Compared to the CK, biochar treatment significantly increased the relative abundance of Bacteroidota by 60.55% (P < 0.05). The relative abundance of Acidobacteriota, Verrucomicrobiota, and Actinobacteria also increased. Figure 2B showed the top 10 amplicons of ITS detected in all samples, with Basidiomycota, Ascomycota, and Mortierellomycota being the main phyla.
Heat maps were used to describe the trend, distribution and abundance of the first 35 bacteria genera (Fig. 2C). Compared to the CK, biochar treatment increased the relative abundance of Steroidobacter, Sphingomonas, Dongia, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Bacillus, Hirschia, Flavobacterium, Sphingobium, Terrimonas, Devosia, and Subgroup_10. Conversely, it decreased the relative abundance of Massilia, MND1, Candidatus_Koribacter, Burkholderia-Caballeronia-Paraburkholderia, Mucilaginibacter, Candidatus_Nitrosotalea, and Duganella. The heat map described the trends, distribution, and abundance of the top 35 fungi genera (Fig. 2D). Compared to the CK, biochar treatment significantly enhanced the levels of Tausonia, Solicoccozyma, Naganishia, Thanatephorus, and Pseudogymnoascus but significantly reduced the levels of Mortierella, Fusarium, Pseudaleuria, and Trichoderma (P < 0.05).
Effects of Biochar on endophytic diversity of P. quinquefolius roots
The effects of biochar on the endophytic diversity of P. quinquefolius are shown in Fig. 3. After clustering sample sequences with 97% similarity, the OTUs sparse curve becomes sufficiently saturated to reveal the diversity of microbial communities (Figs. 3A, B). A Venn diagram was constructed at the OTU level to analyze the composition of species in the samples. As shown in Figs. 2D and 3C, the number of bacterial and fungal OTUs in plant tissues was relatively large after biochar treatment, with a relatively small number of shared OTUs. This indicates that the composition of endophytic bacteria and fungi differed greatly between treatments.
Effects of biochar on endophytic bacterial diversity in P. quinquefolius roots. Rarefaction curves of endophytic bacteria (A) and fungi (B); Venn diagram of OTU distribution of endophytic bacteria (C) and fungi (D) in the different tissues of P. quinquefolius; Multi-sample NMDS analysis of endophytic bacteria (E) and fungi (F) in P. quinquefolius samples. T1 is control, T2 is biochar treatment. Each treatment has three replicates
The analysis of the sample species’ abundance and diversity was conducted using the α diversity index (Table 2). The Chao1 richness index estimated species richness, and the Shannon (H’) index quantitatively described biodiversity based on species richness. Table 2 showed that, after applying biochar, the α diversity index (Chao1 and Shannon) of endophytic bacteria in P. quinquefolius increased. However, the Shannon index for fungi decreased significantly (P < 0.05). Beta diversity was evaluated at the OTU level to compare the endophytic community structures under different treatments. NMDS analysis was used to illustrate the between- and within-group differences of the samples (Figs. 3E, F). The results indicated that the P. quinquefolius samples could be well separated, and the endophytic bacterial communities were significantly different (P < 0.05).
Effect of Biochar on endophytes composition of P. quinquefolius roots
The effect of biochar on the endophyte composition of P. quinquefolius roots is shown in Fig. 4. The top 10 amplicons of 16 S rRNA detected in the two treatments are predominantly Proteobacteria, followed by Cyanobacteria, accounting for over 84% (Fig. 4A). The endophytic fungal communities of two treated P. quinquefolius samples were analyzed at the phylum, order, family, and genus levels. As illustrated in Fig. 4B, the primary phyla were Glomeromycota, Ascomycota, and Basidiomycota. Notably, the abundance of Glomeromycota significantly increased with biochar addition.
Heat maps describe the trend, distribution, and abundance of the top 35 bacteria genera (Fig. 4C). After biochar addition, the abundance of endophytic bacteria in P. quinquefolius roots, including Neochlamydia, Subgroup_10, Bosea, Shinella, Reyranella, Polaromonas, and Flavobacterium, increased significantly (P < 0.05). The heat map in Fig. 4D displays the trends, distribution, and abundance of the top 35 fungi genera. Compared to the CK, biochar treatment significantly increased the relative abundance of beneficial fungi such as Rhizophagus and Glomus (P < 0.05). Conversely, it significantly reduced the relative abundance of certain pathogenic fungi, including Plectosphaerella, Cladosporium, and Phaeosphaeria (P < 0.05).
Correlation analysis between soil microorganism diversity and metabolites of P. quinquefolius
To study the impacts of soil microorganisms on the quality of P. quinquefolius, we performed a correlation analysis between soil microorganisms and plant metabolites. Figure 5 showed that fatty acyls, steroids and their derivatives, and prenol lipids positively correlated with Mycobacterium, Shinella, Reyranella, Polaromonas, and Corymbiglomus, while Chloroplast and Sphingomonas significantly and negatively correlated with these metabolites. Additionally, steroids and their derivatives positively correlated with Sphingobium, Novosphingobium, Neochlamydia, Bosea, Rozellomycota_gen_Incertae_sedis, Acremonium, and Alloleptosphaeria. Schisandrin B and galanthamine showed significant positive correlations with Subgroup_10, Polaromonas, Methylotenera, Thanatephorus, and Zopfiella (P < 0.05).
Correlation analysis between endophytic diversity and metabolites of P. quinquefolius roots
To understand the response of P. quinquefolius root metabolites to endophytic bacteria, we identified key differential secondary metabolites and conducted a correlation analysis with endophytic bacteria and fungi. Figure 6 reveals that prenol lipids positively correlated with Chloroplast, Prevotella, Acidibacter, Sphingomonas, Armatimonadales, Burkholderia-Caballeronia-Paraburkholderia, and Pyrenochaetopsis. Fatty acids and conjugates positively correlated with Chloroplast, Sphingomonas, Armatimonadales, and Burkholderia-Caballeronia-Paraburkholderia, but negatively correlated with Pyrenochaetopsis. Steroids and their derivatives positively correlated with Chloroplast, Phenylobacterium, Prevotella, Armatimonadales, Burkholderia-Caballeronia-Paraburkholderia, Tausonia, and Acremonium. Notably, these bacteria and fungi (Chloroplast, Prevotella, Sphingomonas, Armatimonadales, Burkholderia-Caballeronia-Paraburkholderia, Psathyrella, Glomus) significantly related to most metabolites in the plants, suggesting their role in the synthesis and accumulation of metabolites in P. quinquefolius.
Discussion
The application of biochar was found to promote the reprogramming of secondary metabolites in P. quinquefolius. Notably, ginsenosides, which are key components for evaluating the quality of P. quinquefolius due to their immune-boosting, anti-tumor, and antioxidant properties [13], were generally up-regulated. Specific ginsenosides that showed increased levels include (20R) ginsenoside Rg3, ginsenoside F2, notoginseng saponin Fe, and ginsenoside Rb1. Besides ginsenosides, the relative contents of vitamin A, brassinolide, and galantamine also increased. Brassinolide plays a crucial role in enhancing plant stress resistance and quality by regulating the contents of unsaturated fatty acids, proline, phenolic compounds, and flavonoids [40, 41]. Galantamine is a primary ingredient in medications used to treat Alzheimer’s disease, helping to reduce some of its symptoms [42]. In conclusion, biochar treatment can reprogram secondary metabolites of P. quinquefolius and improve the quality of P. quinquefolius.
The microbiome is a critical component for maintaining plant biomass production, soil fertility, and overall plant health [43]. Following biochar treatment, the soil bacterial community changed significantly. The relative abundance of Sphingomonas, Bacillus, and Flavobacterium increased, while Bacteroides and Solicoccozyma increased significantly.They can enhance soil health by improving the soil environment. Specifically, Sphingomonas and Solicoccozyma can degrade chemical pesticides like glyphosate [44, 45], and Bacteroides thrives in conditions of lower salinity and soil moisture [46], indicating an improved cultivation environment for P. quinquefolius. Bacillus and Flavobacterium promote plant growth, inhibit plant diseases, and enhance plant resistance to abiotic stress [47,48,49,50]. In addition, the addition of biochar also decreased significantly the relative abundance of Fusarium. The decline of Fusarium, a major cause of soil-borne diseases in P. quinquefolius, can reduce the incidence of plant diseases [20]. Correlation studies reveal that Mycobacterium, Reyranella, Flavobacterium, and Corymbiglomus in soil are positively correlated with active secondary metabolites. Manero et al. found that rhizosphere probiotics can enhance the secondary metabolism of the host plant Hypericum perforatum, increasing its medicinal components [51]. After biochar treatment, rhizosphere microbial community changes were significantly correlated with the accumulation of secondary metabolites in P. quinquefolius. This suggests that shifts in the rhizosphere microecology may be a key mechanism by which biochar stimulates the secondary metabolism of P. quinquefolius.
Endophytes are initially adapted by soil bacteria to the plant rhizosphere [52] and then colonize the plant root surface and some rhizodermal cells [53]. Changes in soil microorganisms may induce structural changes in endophytes. Biochar treatment increased the abundance of certain endophytic bacteria (Pseudomonas and Flavobacterium) and fungi (Rhizophagus, Cladophialophora, Acaulospora, and Glomus) in P. quinquefolius. Pseudomonas and Flavobacterium effectively colonize the plant environment, promoting growth and antagonizing plant pathogens [54, 55]. Cladophialophora enhances seedling and plant growth [56]. Arbuscular mycorrhizal fungi (AMF), including Rhizophagus, Acaulospora, and Glomus, promote plant growth and development, and affect the biosynthesis of plant secondary metabolites like phenols, flavonoids, and terpenes [28, 57]. Conversely, the abundance of Plectosphaerella and Phaeosphaeria decreased significantly. The reduction of these pathogens suggests improved plant health after biochar treatment. Correlation studies reveal that Chloroplast, Sphingomonas, Cladophialophora, and Glomus are positively correlated with ginsenosides, while Tausonia and Acremonium show significant correlation with brassinolide. During the co-evolution of endophytes and plants, endophytes can produce unique secondary metabolites and induce their synthesis in host plants, as demonstrated in Salvia miltiorrhiza Bunge, Bletilla striata, and Codonopsis pilosula [58,59,60]. After biochar treatment, changes in endophytes were significantly related to the accumulation of secondary metabolites in P. quinquefolius. This suggests that alterations in endophytes may be a crucial mechanism by which biochar influences the secondary metabolism of P. quinquefolius.
Conclusion
In this study, it was found that biochar application up-regulated the accumulation of key metabolites such as terpenoids and nucleosides, glycerophosphocholines, fatty acyls, steroidal glycosides. In addition, the structure and function of rhizosphere microorganisms were changed after biochar treatment, especially Flavobacterium showed a positive correlation with secondary metabolites such as ginsenoside Rb1. Moreover, the diversity structure and function of endophytes were also changed with biochar treatment, especially Sphingomonas was positively correlated with secondary metabolism such as ginsenosides Rb1, Rg3, F2. In summary, biochar facilitates a complex microbe-plant metabolic reprogramming that improves the overall quality of medicinal materials. This finding provides valuable insights into the potential use of biochar in traditional Chinese medicine agriculture.
Data availability
The sequencing data generated in the study are deposited to the NCBI SRA database under Bioproject No. PRJNA1254450, PRJNA1254922, PRJNA1255382 and PRJNA1255842.
References
Zhang Z, Lei H, Qian J, Wu C, Zhang Y. Introduction history of Panax quinquefolium L. Ginseng Res. 2020;32(02):59–62. https://doi.org/10.19403/j.cnki.1671-1521.2020.02.017
China Pharmacopoeia Committee. Pharmacopoeia of peoples Republic of China. Beijing: China Medical Science and Technology; 2020. pp. 136–7.
Huang X, Wang R, Wang Y, Chen C, Liu S. Investigation on property differences of ginseng and American ginseng by Spatial metabolomics of neurochemicals with desorption electrospray ionization mass spectrometry imaging. J Ethnopharmacol. 2023;303:116006. https://doi.org/10.1016/j.jep.2022.116006
Yang L, Hou A, Zhang J, Wang S, Man W, Yu H. Panacis quinquefolii Radix: a review of the botany, phytochemistry, quality control, pharmacology, toxicology and industrial applications research progress. Front Pharmacol. 2020;11:602092. https://doi.org/10.3389/fphar.2020.602092
Xie L, Yan H, Han L, Cui L, Hussain H, Feng Q, et al. Structural characterization and anti-inflammatory activity of neutral polysaccharides from American ginseng. Int J Biol Macromol. 2023;248:125586. https://doi.org/10.1016/j.ijbiomac.2023.125586
Jen C, Hsu B, Chen B. A study on anti-fatigue effects in rats by nanoemulsion and liposome prepared from American ginseng root residue extract. Food Bioscience. 2022;50:102130. https://doi.org/10.1016/j.fbio.2022.102130
Jovanovski E, Smircic L, Komishon A, Au-Yeung F, Zurbau A, Jenkins A, et al. Vascular effects of combined enriched Korean red ginseng (Panax Ginseng) and American ginseng (Panax Quinquefolius) administration in individuals with hypertension and type 2 diabetes: a randomized controlled trial. Complement Ther Med. 2020;49:102338. https://doi.org/10.1016/j.ctim.2020.102338
Szczuka D, Nowak A, Zakłos-Szyda M, Kochan E, Szymańska G, Motyl I, et al. American ginseng (Panax quinquefolium L.) as a source of bioactive phytochemicals with pro-health properties. Nutrients. 2019;11(5):1041. https://doi.org/10.3390/nu11051041
Chen L, Zhang Y, Yang X, Xu J, Wang Z, Sun Y, et al. Application of UPLC-Triple TOF-MS/MS metabolomics strategy to reveal the dynamic changes of triterpenoid saponins during the decocting process of Asian ginseng and American ginseng. Food Chem. 2023;424:136425. https://doi.org/10.1016/j.foodchem.2023.136425
Mancuso C, Santangelo R. Panax ginseng and Panax quinquefolius: from Pharmacology to toxicology. Industrial Biol Res Association. 2017;107(A):362–72. https://doi.org/10.1016/j.fct.2017.07.019
Zhong Y, Yuan J, Liu F, Zhao B, Chen K, Su J. Research progress on chemical constituents, Pharmacological effects and quality control of American ginseng. Chin Med Mod Distance Educ China. 2020;18(07):130–3.
Ran Z, Duan W, Chen X, Yu H, Zhang Y, Guo L, et al. Research status and analysis on key cultivation techniques of Panacis quinquefolii radix: A Chinese medicine of ‘Lu Shi Wei’ in Shandong Province. China J Traditional Chin Med. 2023;38(08):3719–24.
Li Y. Study on the analysis of flavor components and activity evaluation of P. quinquefolius beverage. Chengdu Univ. 2023. https://doi.org/10.27917/d.cnki.gcxdy.2023.000132
Zhang S, Li D, Yu Z. Global ginseng trade pattern and suggestions for is industrial development. Mod Chin Med. 2022;24(08):1568–73. https://doi.org/10.13313/j.issn.1673-4890.20211209006
Ghodake G, Shinde S, Kadam A, Saratale R, Saratale G, Kumar M, et al. Review on biomass feedstocks, pyrolysis mechanism and physicochemical properties of Biochar: State-of-the-art framework to speed up vision of circular bioeconomy. J Clean Prod. 2021;297:126645. https://doi.org/10.1016/j.jclepro.2021.126645
Fu Q, Zhao H, Li H, Li T, Hou R, Liu D, et al. Effects of Biochar application during different periods on soil structures and water retention in seasonally frozen soil areas. Sci Total Environ. 2019;694:133732. https://doi.org/10.1016/j.scitotenv.2019.133732
Yuan M, Zhu X, Sun H, Song J, Li C, Shen Y, et al. The addition of Biochar and nitrogen alters the microbial community and their cooccurrence network by affecting soil properties. Chemosphere. 2023;312(1):137101. https://doi.org/10.1016/j.chemosphere.2022.137101
Sheng Z, Qian Y, Meng J, Tao J, Zhao D. Rice hull Biochar improved the growth of tree peony (Paeonia suffruticosa Andr.) by altering plant physiology and rhizosphere microbial communities. Scientia Hortic. 2023;322:112204. https://doi.org/10.1016/j.scienta.2023.112204
Helaoui S, Boughattas I, Mkhinini M, Ghazouani H, Jabnouni H, Kribi-Boukhris S, et al. Biochar application mitigates salt stress on maize plant: study of the agronomic parameters, photosynthetic activities and biochemical attributes. Plant Stress. 2023;9:100182. https://doi.org/10.1016/j.stress.2023.100182
Yan S, Wang P, Cai X, Wang C, Zwieten L, Wang H, et al. Environ Technol Innov. 2025;37:103964. https://doi.org/10.1016/j.eti.2024.103964. Biochar-based fertilizer enhanced tobacco yield and quality by improving soil quality and soil microbial community.
Lazzara S, Carrubba A, Fascella G, Marceddu R, Napoli E, Sarno M. Biochar enhances root development and Aloin content of mature leaves in containerized Aloe arborescens mill. South Afr J Bot. 2023;163:703–14. https://doi.org/10.1016/j.sajb.2023.11.019
Zhang Z, Wang B, Yang S, Liu Y. Effect of Biochar on yield and quality of Fritillaria thunbergii Miq and soil properties. J Zhejiang Agricultural Sci. 2022;63(03):492–4. https://doi.org/10.16178/j.issn.0528-9017.20212836
Yang X, Ran Z, Li R, Fang L, Zhou J, Guo L. Effects of Biochar on the growth, ginsenoside content, and soil microbial community composition of Panax quinquefolium L. J Soil Sci Plant Nutr. 2022;22:2670–86. https://doi.org/10.1007/s42729-022-00835-7
Etalo D, Jeon J, Raaijmakers J. Modulation of plant chemistry by beneficial root microbiota. Nat Prod Rep. 2018;35(5):398–409. https://doi.org/10.1039/c7np00057j
Korenblum E, Aharoni A. Phytobiome metabolism: beneficial soil microbes steer crop plants’ secondary metabolism. Pest Manag Sci. 2019;75:2378–84. https://doi.org/10.1002/ps.5440
Ran Z, Yang X, Zhang Y, Zhou J, Guo L. Effects of arbuscular mycorrhizal fungi on photosynthesis and biosynthesis of ginsenoside in Panax quinquefolius L. Theoretical Experimental Plant Physiol. 2021;33:235–48. https://doi.org/10.1007/s40626-021-00208-y
Ran Z, Yang X, Zhang Y, Zhou J, Guo L. Transcriptional responses for biosynthesis of ginsenoside in arbuscular mycorrhizal fungi-treated Panax quinquefolius L. seedlings using RNA-seq. Plant Growth Regul. 2021;95:83–96. https://doi.org/10.1007/s10725-021-00727-3
Ran Z, Ding W, Cao S, Fang L, Zhou J, Zhang Y. Arbuscular mycorrhizal fungi: effects on secondary metabolite accumulation of traditional Chinese medicines. Plant Biol. 2022;24(6):932–8. https://doi.org/10.1111/plb.13449
Zhang M, Riaz M, Liu B, Xia H, El-Desouki Z, Jiang C. Two-year study of Biochar: achieving excellent capability of potassium supply via alter clay mineral composition and potassium-dissolving bacteria activity. Sci Total Environ. 2020;717:137286. https://doi.org/10.1016/j.scitotenv.2020.137286
Compant S, Cambon M, Vacher C, Mitter B, Samad A, Sessitsch A. The plant endosphere world-bacterial life within plants. Environ Microbiol. 2021;23(4):1812–29. https://doi.org/10.1111/1462-2920.15240
Taulé C, Vaz-Jauri P, Battistoni F. Insights into the early stages of plant-endophytic bacteria interaction. World J Microbiol Biotechnol. 2021;37(1):13–21. https://doi.org/10.1007/s11274-020-02966-4
Jia M, Chen L, Xin H, Zheng C, Rahman K, Han T, et al. A friendly relationship between endophytic fungi and medicinal plants: a systematic review. Front Microbiol. 2016;7:906. https://doi.org/10.3389/fmicb.2016.00906
Ju M, Zhang Q, Wang R, Yan S, Li Z, Li P, et al. Correlation in endophytic fungi community diversity and bioactive compounds of Sophora alopecuroides. Front Microbiol. 2022;13:955647. https://doi.org/10.3389/fmicb.2022.955647
Li Y. Astragalus mongholicus associated Microbiome and its effects on the secondary metabolism. Northwest A&F Univ. 2023. https://doi.org/10.27409/d.cnki.gxbnu.2022.001458
Li R, Duan W, Ran Z, Chen X, Yu H, Fang L, et al. Diversity and correlation analysis of endophytes and metabolites of Panax quinquefolius L. in various tissues. BMC Plant Biol. 2023;23:275. https://doi.org/10.1186/s12870-023-04282-z
Yu L, Zhou C, Fan J, Shanklin J, Xu C. Mechanisms and functions of membrane lipid remodeling in plants. Plant Journal: Cell Mol Biology. 2021;107(1):37–53. https://doi.org/10.1111/tpj.15273
Cockcroft S. Mammalian lipids: structure, synthesis and function. Essays Biochem. 2021;65(5):813–45. https://doi.org/10.1042/EBC20200067
Willers C, Rensburg P, Claassens S. Can a metabolomics-based approach be used as alternative to analyse fatty acid Methyl esters from soil microbial communities? Soil Biol Biochem. 2016;103:417–28. https://doi.org/10.1016/j.soilbio.2016.09.021
Stasolla C, Katahira R, Thorpe T, Ashihara H. Purine and pyrimidine nucleotide metabolism in higher plants. J Plant Physiol. 2003;160(11):1271–95. https://doi.org/10.1078/0176-1617-01169
Zhang L, Cao X, Wang Z, Zhang Z, Li J, Wang Q, et al. Brassinolide alleviated chilling injury of banana fruit by regulating unsaturated fatty acids and phenolic compounds. Scientia Hortic. 2022;297:110922. https://doi.org/10.1016/j.scienta.2022.110922
Wang S, Zhao H, Zhao L, Gu C, Na Y, Xie B. Application of Brassinolide alleviates cold stress at the booting stage of rice. J Integr Agric. 2020;19(4):975–87. https://doi.org/10.1016/S2095-3119(19)62639-0
Prvulovic D, Hampel H, Pantel J. Galantamine for Alzheimer’s disease. Expert Opin Grug Metabolism Toxicol. 2010;6(3):345–54. https://doi.org/10.1517/17425251003592137
Bertola M, Ferrarini A, Visioli G. Improvement of soil microbial diversity through sustainable agricultural practices and its evaluation by -omics approaches: a perspective for the environment. Food Qual Hum Saf Microorganisms. 2021;28(7):1400. https://doi.org/10.3390/microorganisms9071400
Zhou M, Liu Z, Wang J, Zhao Y, Hu B. Sphingomonas relies on chemotaxis to degrade polycyclic aromatic hydrocarbons and maintain dominance in coking sites. Microorganisms. 2022;10(6):1109. https://doi.org/10.3390/microorganisms10061109
Du T, He H, Zhang Q, Lu L, Mao W, Zhai M. Positive effects of organic fertilizers and biofertilizers on soil microbial community composition and walnut yield. Appl Soil Ecol. 2022;175:104457. https://doi.org/10.1016/j.apsoil.2022.104457
Kruczyńska A, Kuźniar A, Podlewski J, Słomczewski A, Grządziel J, Marzec-Grządziel A. Bacteroidota structure in the face of varying agricultural practices as an important indicator of soil quality-a culture independent approach. Agric Ecosyst Environ. 2023;342:108252. https://doi.org/10.1016/j.agee.2022.108252
Tsotetsi T, Nephali L, Malebe M, Tugizimana F. Bacillus for plant growth promotion and stress resilience: what have we learned? Plants. 2022;11(19):2482. https://doi.org/10.3390/plants11192482
Kumar M, Charishma K, Sahu K, Sheoran N, Patel A, Kundu A, et al. Rice leaf associated Chryseo bacterium species: an untapped antagonistic flavobacterium displays volatile mediated suppression of rice blast disease. Biol Control. 2021;161:10470. https://doi.org/10.1016/j.biocontrol.2021.104703
Kraut-Cohen J, Shapiro O, Dror B, Cytryn E. Pectin induced colony expansion of soil-derived flavobacterium strains. Front Microbiol. 2021;12:651891. https://doi.org/10.3389/fmicb.2021.651891
Wei Z, Gu Y, Friman V, Kowalchuk G, Xu Y, Shen Q, et al. Initial soil Microbiome composition and functioning predetermine future plant health. Sci Adv. 2019;5(9):0759. https://doi.org/10.1126/sciadv.aaw0759
Mañero F, Algar E, Martín G, Saco Sierra M, Solano B. Elicitation of secondary metabolism in Hypericum perforatum by rhizosphere bacteria and derived elicitors in seedlings and shoot cultures. Pharm Biol. 2012;50(10):1201–9. https://doi.org/10.3109/13880209.2012.664150
Gamalero E, Lingua G, Berta G, Lemanceau P. Methods for studying root colonization by introduced beneficial bacteria. Sustainable Agric. 2009;23(5–6):407–18. https://doi.org/10.1007/978-90-481-2666-8-37
Benizri E, Baudoin E, Guckert A. Root colonization by inoculated plant growth-promoting rhizobacteria. Biocontrol Sci Technol. 2001;11:557–74. https://doi.org/10.1080/09583150120076120
Pandey S, Gupta S. Evaluation of Pseudomonas Sp. for its multifarious plant growth promoting potential and its ability to alleviate biotic and abiotic stress in tomato (Solanum lycopersicum) plants. Sci Rep. 2020;10:20951. https://doi.org/10.1038/s41598-020-77850-0
Menon R, Kumari S, Viver T, Rameshkumar N. Flavobacterium Pokkalii Sp. nov., a novel plant growth promoting native rhizobacteria isolated from Pokkali rice grown in coastal saline affected agricultural regions of Southern India. Kerala Microbiol Res. 2020;240:126533. https://doi.org/10.1016/j.micres.2020.126533
Harsonowati W, Masrukhin N. Prospecting the unpredicted potential traits of Cladophialophora chaetospira SK51 to alter photoperiodic flowering in strawberry, a perennial SD plant. Sci Hort. 2022;295:110835. https://doi.org/10.1016/j.scienta.2021.110835
Liu X, Xie M, Hashem A, Abd-Allah E, Wu Q. Arbuscular mycorrhizal fungi and rhizobia synergistically promote root colonization, plant growth, and nitrogen acquisition. Plant Growth Regul. 2023;100:691–701. https://doi.org/10.1007/s10725-023-00966-6
Rilling J, Acuña J, Nannipieri P, Cassan F, Maruyama F, Jorquera M. Current opinion and perspectives on the methods for tracking and monitoring plant growth–promoting bacteria. Soil Biol Biochem. 2019;130:205–19. https://doi.org/10.1016/j.soilbio.2018.12.012
Chen J, Li L, Tian P, Xiang W, Lu X, Huang R, et al. Fungal endophytes from medicinal plant Bletilla striata (Thunb.) Reichb. F. promote the host plant growth and phenolic accumulation. South Afr J Bot. 2021;143:25–32. https://doi.org/10.1016/j.sajb.2021.07.041
Fan L, Wang J, Leng F, Li S, Ma X, Wang X, et al. Effects of time-space conversion on microflora structure, secondary metabolites composition and antioxidant capacity of Codonopsis pilosula root. Plant Physiol Biochem. 2023;198:107659. https://doi.org/10.1016/j.plaphy.2023.107659
Acknowledgements
We thank Prof. Lei Fang (Jinan University) for helping with the data analysis and Prof.Jie Zhou (Jinan University) and Prof. Lanping Guo (Chinese Academy of Medical Sciences, China) for revising the manuscript.
Funding
This work was financially supported by the National Key Research and Development Project (2023YFC3503802), the Key Technology Research and Development Program of Shandong (2022TZXD0036), the construction project for sustainable utilization of valuable traditional Chinese medicine resources (2060302-2302-06), the Natural Science Foundation of Shandong Province (ZR2022MH101).
Author information
Authors and Affiliations
Contributions
XL.C. performed the experiments, drafted the manuscript’s main sections and contributed to data analysis and interpretation of results. XY.M., Y.D., T.C., Y.W., J.B., and L.F. conducted data analysis, provided technical support for experimental methods, and reviewed the data for accuracy. LP.G. and J.Z. conceived the research topic, conducted the overall planning and supervision of the study.
Corresponding authors
Ethics declarations
Ethics approval and consent to participate
Plant materials (P. quinquefolius) used in the experiments were from Weihai Wendeng District Dao-di ginseng industry Development Co. LTD (Weihai, China). All plant materials (not endangered materials or species) were provided free of charge, and comply with local institutional guidelines and legislation.
Consent for publication
The authors have approved of publication, and there is no confict of interest. All the authors equally approve of publication.
Competing interests
The authors declare no competing interests.
Consent for publication
Not applicable.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Chen, X., Mao, X., Ding, Y. et al. Biochar-induced microbial and metabolic reprogramming enhances bioactive compound accumulation in Panax quinquefolius L.. BMC Plant Biol 25, 669 (2025). https://doi.org/10.1186/s12870-025-06656-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/s12870-025-06656-x