Field experiment was conducted in Wenxi County, Yuncheng City, Shanxi Province (111°13’27’’ E, 35°26’26’’ N), the primary production region for Polygala tenuifolia. A biennial P. tenuifolia field with consistent growth was selected. The cultivated P. tenuifolia was identified by Professor Jianping Han of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College following the standards outlined in the Pharmacopoeia of the People’s Republic of China (PPRC-2020). The specimens were preserved at the same institution. The region has a temperate continental monsoon climate, with an average annual temperature of 12.5 °C. The annual precipitation is 506 mm, and this region is susceptible to drought. The tested soil type is sandy loam soil. The plowing layer soil (0-20 cm) had a pH of 8.11, an organic matter content of 15.80 g/kg, a total nitrogen content of 1.08 g/kg, a total phosphorus content of 0.59 g/kg, a total potassium content of 25.70 g/kg, an alkali-hydrolyzable nitrogen content of 74.19 mg/kg, an available phosphorus content of 11.50 mg/kg, and an available potassium content of 215.00 mg/kg.

Seven treatments were established as shown in Table 1 : (1) CF: 100% chemical fertilizer; (2) PMI1: -20% chemical fertilizer + polyglutamic acid microbial inoculant; (3) PMI2: -40% chemical fertilizer + polyglutamic acid microbial inoculant; (4) SMI1: -20% chemical fertilizer + solid microbial inoculant; (5) SMI2: -40% chemical fertilizer + solid microbial inoculant; (6) MF1: -20% chemical fertilizer + microalgae fertilizer; (7) MF2: -40% chemical fertilizer + microalgae fertilizer. The chemical fertilizer (N: P: K = 16: 5: 24) was purchased from Liuguo Chemical Co. Ltd (Anhui, China) with an application rate of 750 kg/ha. The application rate of chemical fertilizer used in treatments with a 20% and 40% reduction in chemical fertilizer is 600 kg/ha and 450 kg/ha, respectively. The Gulefeng 8.8 billion^® polyglutamic acid microbial inoculant (PMI, viable count ≥ 8.8×10⁹/mL) was purchased from Xuankai Biotechnology (Xuankai Biotechnology Co. Ltd., Nanjing, China) and utilized in accordance with the manufacturer’s instructions, at a dosage of 75L diluted microbial inoculant per hectare (diluted at a ratio of 1:100). PMI primarily consists of polyglutamic acid (PGA), Bacillus amyloliquefaciens, B. subtilis and Brevibacillus laterosporu. The solid microbial inoculant (SMI, viable count ≥ 2.0×10⁸/g) was produced by Jintu Biotechnology (Jintu Biotechnology Co. Ltd., Hebei, China) and utilized at a dosage of 150 kg/ha. Its main components are Bacillus amyloliquefaciens and Brevibacillus laterosporu. The Titubang^® microalgae fertilizer (MF, viable count ≥ 1.0×10⁵/mL) was purchased from Bailing Biotechnology (Bailing Biotechnology Co. Ltd., Beijing, China) and utilized at a dosage of 50 L diluted microbial inoculant per hectare (diluted at a ratio of 1:100). Chlorella pyrenoidosa, nitrogen-fixing cyanobacteria and Tolypothrix tenuis are the main components. Each treatment consisted of three biological replicates, with each replicate covering an area of 12 m² (2 m×6 m). To minimize the marginal effect, protection lines (0.5 m) were placed between each replicate cell. The experiment was initiated in April 2023 with the first application of biennial P. tenuifolia, followed by subsequent applications of the same dosage of BOFs administered every two months, for a total of three times.

After six months, P. tenuifolia were sampled, and plant traits were collected from at least ten plants in each biological replicate. The underground biomass of each P. tenuifolia root was determined, while the root diameter was measured using vernier scale. All root samples were processed by removing the woody core (xylem), dried at 55 °C, powdered, and sieved through a 50-mesh sieve. The contents of polygalaxanthone III (POL) and 3,6’-disinapoylsucrose (DISS) in P. tenuifolia were determined by high-performance liquid chromatography (HPLC). 0.5 g of P. tenuifolia powder was mixed with 10 mL of 70% methanol, weighed, and subjected to ultrasonic extraction (400 W, 40 kHz) for 45 min. After cooling and re-weighing, any weight loss was compensated by adding additional 70% methanol. The mixture was centrifuged at a speed of 5000 r/min for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane. A Shimadzu LC-2030 HPLC system (Shimadzu, Kyoto, Japan) equipped with an Agilent ZORBAX Extend-C18 column (46×250 mm, 5 μm) was employed for isocratic elution. The mobile phase consisted of acetonitrile and 0.05% phosphoric acid solution at a ratio of 18:82 (v/v) with a flow rate of 1.0 mL/min. Detection was conducted at a wavelength of 320 nm, while the column temperature was 30 °C and the injection volume was 10 μL. A series of working standard solutions were set up with a mixed standard solution comprising POL and DISS, both of which were purchased from Yuanye Biotechnology Co., Ltd (Shanghai, China).

When harvesting P. tenuifolia plants, rhizosphere soil tightly adhering to the root surface were collected. One sample of P. tenuifolia rhizosphere soil was collected per replicate, with three replicates per treatment. Each sample represented a composite of soil collected from the rhizosphere of plants within its respective replicate plot. All rhizosphere soil samples were sieved and stored at -80 °C for microbial analysis. In each replicated plot, bulk soil samples weighing approximately 500 g were collected using a five-point sampling method at depths ranging from 0 to 20 cm around the plants. Three replicates were taken for each treatment. The collected soil samples were air-dried, sieved through a 100-mesh sieve, and subjected to analysis for pH and the contents of soil organic matter (SOM), alkali-hydrolyzable nitrogen (AN), available potassium (AK) and available phosphorus (AP). The soil pH was tested by the potentiometric method, while the organic matter content was determined using the K₂CrO₇/H₂SO₄ oxidation method (Marks et al., 2017; Shrestha et al., 2022). The alkaline hydrolysis diffusion method was employed to determine the soil alkali-hydrolyzable nitrogen content (Wang et al., 2022). The molybdenum antimony anti-colorimetric method was used to measure the soil available phosphorus content (Chu and Grogan, 2010). The flame spectrophotometer technique was utilized to assess the soil available potassium content (Li et al., 2022).

Total genome DNA from all rhizosphere soil samples were extracted using CTAB method (Kamdem et al., 2023; Zheng et al., 2024). DNA concentration and purity was monitored on 1% agarose gels. The 16S rRNA gene of bacteria was amplified using the primers 341F (5’-CCTAYGGGRBGCASCAG-3’)/806R (5’-GGACTACNNGGGTATCTAAT-3’), and the ITS region of the fungi was amplified with ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’)/ITS2R (5’-GCTGCGTTCTTCATCGATGC-3’) primers (Wei et al., 2021). The polymerase chain reaction (PCR) system was composed as follows: 15 μL of Phusion^® High – Fidelity PCR Master Mix (New England Biolabs), 0.2 μM of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, elongation at 72°C for 30 s; then ended with an extension at 72°C for 5 min. The integrity and concentration of PCR products were verified by 2% agarose gel electrophoresis. PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq^® DNA PCR-Free Sample Preparation Kit (Illumina, USA) and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq platform by Novogene Co., Ltd (Beijing, China) and 250 bp paired-end reads were generated.

Paired-end reads were first assigned to samples based on unique barcodes, then trimmed to remove barcode and primer sequences, and merged using FLASH. Quality filtering was done with Fastp (v. 0.23.1) to obtain high-quality clean tags. These tags were compared against the Silva database (16S) and Unite database (ITS) using the UCHIME Algorithm to detect and remove chimera sequences, resulting in effective tags. Denoising was then performed using the DADA2 module in QIIME2 (Version QIIME2-202202) to obtain initial amplicon sequence variants (ASVs). Species annotation was conducted using the Silva database (16S) and Unite database (ITS) within QIIME2. Multiple sequence alignment was done using QIIME2 to investigate phylogenetic relationships and differences in predominant species among sample groups. The absolute abundance of ASV was normalized based on the sample with the fewest sequences. Subsequent alpha and beta diversity analyses were performed using the normalized data.

GraphPad Prism (V10.0.3) was used to plot histograms representing the average values of the parameters, with bars indicating the standard error of the mean (SEM). The statistical differences were analyzed using one-way analysis of variance (ANOVA) with SPSS V26.0 statistical software (IBM, USA). Alpha diversity indices were measured through QIIME2 to analyze the diversity and richness. Principal coordinate analysis (PCoA) of rhizosphere microbial community composition were performed using Bray-Curtis distance. Biomarkers were identified by the Linear discriminant analysis effect size (LefSe) with a linear discriminant analysis (LDA) score > 2.0 and p < 0.05. Redundancy analysis (RDA) was used to explore the relationships between soil properties and microbial composition using R (V4.3.1). R (V4.3.1) was also used to estimate the correlation among plant growth indicators, quality indicators, soil properties and rhizosphere microbial communities by Pearson and Mantel correlation analysis.

In this study, combined application of reduced chemical fertilizer and BOF increased P. tenuifolia underground growth traits including underground biomass and root diameter ( Figure 1 ). MF showed the greatest efficacy in promoting both underground biomass and root diameter of P. tenuifolia ( Figure 1B ). The underground biomass of P. tenuifolia increased by 23.05% and 29.30% under MF1 and MF2 respectively, while the root diameter experienced respective increments of 6.82% and 4.78%. Reducing the application of chemical fertilizer and combining it with SMI and PMI did not result in a significant difference in the underground growth of P. tenuifolia when compared to applying 100% chemical fertilizer.

Microbial inoculants with Bacillus as the core microorganism exert a significant effect on increasing production (Chen et al., 2021; Shi et al., 2022). In this study, the underground biomass of P. tenuifolia in PMI and SMI groups were comparable to CF under reduced chemical fertilizer usage conditions, indicating that MIs partially substituting chemical fertilizer can ensure efficient production ( Figure 1B ). This effect may be attributed to the nitrogen fixation and phosphorus solubilization functions of the main functional bacteria including Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus mucilaginosus added in MIs, as well as their abilities to release growth-promoting hormones. Furthermore, poly-γ-glutamic (γ-PGA), a novel fertilizer synergist and one of the main components in PMI, not only exhibits remarkable water and fertilizer retention abilities but also enhances plant nutrient absorption by increasing root biomass and activity (Yin et al., 2018; Bai et al., 2022). Researchers had also discovered that the combination of microalgae with organic fertilizers or their partial substitution for chemical fertilizer can improve crop yield (Álvarez-González et al., 2022; Cao et al., 2023). This study confirmed that among different BOFs applied in this study, co-application of microalgae and chemical fertilizer has the best promotion effects on P. tenuifolia growth compared to 100% chemical fertilizer group ( Figure 1B ). Additionally, as shown in Figure 1A , MF significantly enhanced the growth of lateral and fibrous roots. This may be attributed to the pivotal role of indole-3-acetic acid (IAA) produced by various microalgae in primary and lateral root development (Zhang et al., 2024). Overall, partial substitution of chemical fertilizer with BOF provided a feasible strategy to reduce chemical fertilizer usage, thereby promoting underground growth of P. tenuifolia and increasing yield.

As shown in Figure 1C , MF significantly increased the content of DISS in the roots of P. tenuifolia, MF1 and MF2 increased the content of DISS by 10.45% and 19.72%, respectively, with the increase in MF2 being statistically significant (p < 0.05). While other BOFs treatments as partial substitutes for chemical fertilizer achieved comparable effectiveness to 100% chemical fertilizer in terms of DISS and POL contents in P. tenuifolia.

POL has anti-inflammatory properties, and DISS is known for its significant anxiolytic and antidepressant effects (Hu et al., 2011; Yang et al., 2022; Zhang et al., 2022). They are major bioactive components of P. tenuifolia, playing a crucial role in determining the clinical efficacy of P. tenuifolia roots, which serve as important quality control indicators (Xu et al., 2016). The content of oligosaccharide esters including DISS in P. tenuifolia is significantly influenced by soil type and origin, while there is minimal variation in xanthones (Pu et al., 2017; Ji et al., 2023). This suggested a close relationship between the accumulation of DISS during the growth process of P. tenuifolia and environmental factors such as climate, soil properties, and rhizosphere microbial communities. These findings supported our experimental results and confirmed that the application of MF may create more favorable conditions for DISS accumulation in P. tenuifolia. Our previous investigations have demonstrated that Bacillus sp. can facilitate the accumulation of total tanshinone in Salvia miltiorrhiza (Wei et al., 2023). Furthermore, many studies had shown that MF can increase the nutrient contents of crops, vegetables and fruits, including soluble sugars, vitamin C, protein, chlorophyll and carotenoids (Coppens et al., 2016, 2016; Zhang et al., 2024). MF also has the ability to increase the content of secondary metabolites in plants (Díaz et al., 2024). For instance, applying Coelastrella sp. can elevate most organic acids and phenolic compounds in strawberries by more than 10% (Žunić et al., 2024). The above results demonstrated that microalgae could improve plant quality while promoting plant growth, indicating its potential for further study and application in the cultivation of medicinal plants ( Figure 1C ). Meanwhile, based on the results of this study, further research is needed to investigate the mechanism by which microalgae promote DISS accumulation in P. tenuifolia.

As shown in Figure 2 , the partial substitution of chemical fertilizer with BOFs had a significant impact on soil physicochemical properties. PMI2 and MF1 exhibited a substantial increase in SOM by 11.32% and 15.68%, respectively (p < 0.05). CF group displayed the highest AN content, while soils treated with 80% chemical fertilizer application demonstrated higher AN level than those treated with 60% chemical fertilizer application, suggesting that inorganic nutrients provided by chemical fertilizer are primarily responsible for increasing inorganic nitrogen content in the soil. All the six treatments with BOFs showed a higher availability of P in soils as compared to CF. Among them, the AP content processed by PMI1 and PMI2 is 1.92 times and 2.27 times that of CF, respectively (p < 0.05).

In addition to the reduction in chemical fertilizer usage, the decrease of AN may also be attributed to the incorporation of BOFs, which enhanced nitrogen absorption by P. tenuifolia (Zhou et al., 2023). Beneficial microorganisms present in MIs can effectively colonize soils and secrete organic acids that dissolve and release nitrogen nutrients adsorbed onto soil particles, while also modifying indigenous bacterial communities involved in nitrogen cycling (van der Heijden et al., 2008; Lei et al., 2012; Huang et al., 2022). Application of microalgae can enhance soil enzyme activity and promote life activities of microorganisms related to nitrogen cycling, while nitrogen-fixing cyanobacteria can provide fixed-nitrogen through biological fixation or conversion between different forms of nitrogen fertilizers (Renuka et al., 2018; Zhou et al., 2023). Hence, the synergistic application of chemical fertilizer and MIs confers greater advantages on nitrogen uptake by plants compared to their individual applications. In addition to N nutrition, the inoculation of BOFs can augment the availability and translocation of various micro- and macronutrients, including Zn, Cu, Fe, C, P, K within the soils and plants (Coppens et al., 2016; Renuka et al., 2018). Various BOFs including Bacillus sp. and microalgae have been found to possess phosphate solubilization ability, which can lower soil pH and convert soil-bound phosphorus into soluble forms that are readily accessible for plant growth. This transformation is also facilitated by metabolites released during microbial metabolism and low molecular weight organic acids. The partial substitution of chemical fertilizer with BOFs primarily stimulate plant growth through microbial activities and interactions with soil or plants rather than directly supplying various nutrients to the soil. Consequently, changes in properties of the soil may be influenced by factors such as fertilization amount, fertilizer ratio, and duration of application. In conclusion, the partial substitution of chemical fertilizer with BOFs have shown potential to enhance nitrogen nutrient uptake by P. tenuifolia in soil and can significantly improve the availability of phosphorus, which is closely related to the beneficial effects of microorganisms.

Pearson correlation analysis were conducted to investigate the relationship among plant traits (growth parameters and bioactive components contents) and soil properties ( Supplementary Figure S2 ). A significant positive correlation was observed between the content of DISS and both underground biomass and root diameter of P. tenuifolia (p < 0.05). Studies on the active components and medicinal specifications of P. tenuifolia have shown that the content of DISS was positively correlated with root length but highly negatively correlated with plant height (Zhang et al., 2022). Saponins in P. tenuifolia are mainly distributed in the root parenchyma and stored within the secondary phloem (Teng et al., 2009). This may explain the significant correlation between the active components content in P. tenuifolia and its underground parts, especially the biomass of phloem. Based on our research findings, it can be inferred that the application of microalgae may enhance the medicinal quality by increasing the underground biomass of P. tenuifolia.

The effects of soil properties on the rhizosphere microbial community of P. tenuifolia upon fertilizer application were investigated by RDA analysis. It was found that soil properties had more significant effects on rhizosphere bacterial structure than fungi ( Figures 6A, B ). AP and AK significantly influenced soil bacterial community composition, while pH, AN and AK had a remarkable impact on soil fungal community composition ( Table 2 ). In conclusion, pH and available nutrients were all important factors influencing the structure of rhizosphere microbial communities. Further analysis revealed a close association between certain rhizosphere microorganisms and available soil nutrients ( Figures 6C, D ). The content of AP showed a significant positive correlation with Streptomyces (p < 0.05), which had the highest RAs in PMIs and were identified as biomarkers for PMI2. These microorganisms are capable of solubilizing nutrients sequestered in the crystalline lattice of soil mineral fraction through the secretion of low molecular weight organic acids such as gluconic acid, citric acid, succinic acid, and oxalic acid (Rajput et al., 2013; Timofeeva et al., 2022). As a result, the AP content of PMI1 and PMI2 were significantly increased (p < 0.05). Consequently, the application of PMIs promoted the growth of P. tenuifolia by recruiting Streptomyces to enhance phosphorus availability in soil. The content of pH and AN exhibited positive correlation with the RAs of Agromyces, which related to nitrogen-converting. Some Agromyces sp. have been identified to possess the nitrogen fixation gene (nifH) and have been experimentally verified to exhibit nitrogen-fixing ability (Zhou et al., 2014). AK was significantly positively correlated with Blastococcus and Solirubrobacter (p < 0.05). Solirubrobacter acts as a PGPR and also facilitates the mobilization of potentially toxic elements (Jiang et al., 2023; Cebekhulu et al., 2024). In short, using bio-organic fertilizer as a partial substitution for chemical fertilizer can recruit beneficial microorganisms and improve soil fertility, with complex interactions observed between rhizosphere microbial activity and soil properties.

According to the Pearson test between plant traits and microbial community, underground biomass and DISS content were positively correlated with the RA of Chloroflexi (p < 0.01) and negatively correlated with the RA of Proteobacteria (p < 0.05) ( Supplementary Figure S3 ). Rhizosphere microbial communities exhibited a response to reduced chemical fertilizer application and the utilization of BOFs by promoting the proliferation of oligotrophs (e.g., some Chloroflexi) while suppressing copiotrophs (e.g., some Proteobacteria) (Dai et al., 2018). Oligotrophs such as Chloroflexi exhibited a pronounced substrate affinity and preferentially decompose resistant carbon (Hug et al., 2013; Banerjee et al., 2016). Chloroflexi plays a central role in the symbiotic relationships between soil bacteria, fungi, and plants. Specifically, Chloroflexi can fix CO₂ and convert inorganic carbon into biodegradable organic matter, and it also acts as producers of nutrients such as phosphorus and nitrogen (Narsing Rao et al., 2022; Freches and Fradinho, 2024). In this study, Chloroflexi exhibited the highest RAs in MFs, showing a significant increase of 31.06% and 38.27% compared to CF in MF1 and MF2, respectively. Additionally, SOM content in MF1 was significantly higher than CF (p < 0.05). Therefore, the application of microalgae may positively stimulate specific species within Chloroflexi, thereby improving soil carbon cycling, enhancing soil fertility, promoting nutrient absorption by plants, and ultimately increasing biomass accumulation and bioactive component production in P. tenuifolia.