Application of formononetin and novosphingobium in promoting soybean tolerance to acid aluminum

Abstract

The application discloses application of ononin and novosphingobium lindaniclasticum in promoting acid aluminum tolerance of soybean, and utilizes plant-derived metabolite ononin to enhance novosphingobium lindaniclasticum (Novosphingobium lindaniclasticum RS-01) to promote acid aluminum tolerance of soybean. 3+ Chelating ability, and improves the tolerance of soybean under acid aluminum stress.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology and plant stress resistance technology, specifically involving the application of sphingolipids and neosphingosine monocytogenes in promoting the aluminum tolerance of soybeans. Background Technology

[0002] Aluminum toxicity in acidic soils leads to reduced soybean yields by inhibiting root tip elongation, damaging root structure, and inducing oxidative damage. Traditional methods of lime remediation and aluminum-tolerant variety breeding are costly and time-consuming. Using beneficial rhizosphere microorganisms to chelate Al... 3+ Preventing aluminum from migrating to the root tips is a green alternative that has gained attention in recent years.

[0003] Isoflavones in legumes are mostly stored intracellularly in glycosidic form. After hydrolysis by β-glucosidase, they release aglycones. This transformation is considered an important mechanism for regulating rhizosphere signal intensity in plants. Existing research has mainly focused on the induction of the nod gene in rhizobia by genistein and daidzein. However, the effects of 7-O-glucosidic isoflavones on the extracellular polysaccharide metabolism and metal chelating ability of non-symbiotic rhizosphere bacteria have not been systematically studied, and there are no reports of applications to improve the aluminum tolerance of soybeans based on this. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide the application of ononin and Novosphingobium lindaniclasticum in promoting aluminum tolerance in soybeans. This method uses ononin to enhance the ability of Novosphingobium lindaniclasticum RS-01 to promote aluminum tolerance in soybeans, providing a new microbial regulation pathway for soybean production in acidic soils.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A microbial inoculant for improving the aluminum tolerance of soybeans comprises *Novosphingobium lindaniclasticum* RS-01 and its metabolite styrosin; the strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No. 68050, on April 3, 2026; the inoculant contains a viable count of not less than 1 × 10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL, with a content of 0.5~10 mg / g or 0.5~10 mg / mL of gentianin.

[0006] Furthermore, it also includes a carrier and excipients; by weight, the carrier is one or more of peat moss (50-70 parts), vermiculite (20-30 parts), and diatomaceous earth (5-15 parts), and the excipients are one or more of sodium alginate (1-3 parts), carboxymethyl cellulose (0.5-2 parts), skim milk (1-2 parts), and trehalose (0.3-1 parts).

[0007] A method for improving the aluminum tolerance of soybeans involves applying *Novosphingobium lindaniclasticum* RS-01 and its metabolite styrosinase to soybeans grown in acidic soil. The strain is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No. 68050) on April 3, 2026. The concentration of styrosinase is 5–80 mg / L, and the application rate of the strain is 1 × 10⁻⁶ mg / L. 7 ~1×10 9 CFU / mL bacterial culture or 1×10 7 ~1×10 9 CFU / g growth medium.

[0008] Furthermore, the application concentration of the gentianin is 10~40 mg / L.

[0009] Furthermore, the gentianin upregulates the expression level of the gene0959 gene in the strain, thereby enhancing the secretion of extracellular polysaccharides, organic acids, and Al in the strain. 3+ Chelating ability; the CDS sequence of the gene0959 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the CDS sequence is shown in SEQ ID NO:2.

[0010] Furthermore, the common application method is one or more of seed dressing, seed soaking, root irrigation, and soil application; the root irrigation or soil application is performed during the soybean seedling stage, which is 10 to 30 days after soybean emergence.

[0011] Furthermore, the acidic soil has a pH of 4.0–5.5 and an exchangeable aluminum content of 0.5–5.0 cmol(+) / kg, or an Al content in the soil solution of [missing information]. 3+ The concentration is 20~200 µmol / L.

[0012] The above-mentioned microbial agents are used to improve the aluminum tolerance of soybeans in acidic soil cultivation.

[0013] Application of any of the above methods to improve the aluminum tolerance of soybeans in acidic soil soybean cultivation.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides the application of ononin and *Novosphingobium lindaniclasticum* in promoting aluminum tolerance in soybeans. Ononin was enriched at 11.597 times in the roots of the aluminum-tolerant soybean variety HC2, demonstrating its high specificity as a biomarker. After treatment with ononin, the extracellular polysaccharides (EPS) and organic acids secreted by *Novosphingobium lindaniclasticum* RS-01 significantly increased. When the concentration of ononin was 20 mg / L, the Al of *Novosphingobium lindaniclasticum* RS-01 was significantly increased. 3+ The chelation rate increased from 19% to approximately 52%, and the extracellular polysaccharide (EPS) increased from 46 mg / L to 111 mg / L. The enhanced effect was lost upon knockout of the key gene gene0959 in *Novosphingobium lindaniclasticum* RS-01, but was restored upon gene replacement, proving it to be an essential gene. Combined application of *Novosphingobium lindaniclasticum* RS-01 and its metabolite ononin significantly improved soybean root elongation and reduced aluminum accumulation at the root tip in acidic soils. Attached Figure Description

[0015] Figure 1 Phenotypic diagrams of the aluminum-tolerant soybean variety HC2 and the aluminum-sensitive soybean variety LD2 in neutral / acidic soils; Figure 2 Volcano diagrams of differential metabolites in the roots of aluminum-tolerant soybean variety HC2 and aluminum-sensitive soybean variety LD2, with key differential metabolites marked in orange circles. Figure 3 A statistical chart showing the enhancing effects of key differential metabolites on the function of Novosphingobium lindaniclasticum RS-01; Figure 4 The concentration-response curve of ononin on Novosphingobium lindaniclasticum RS-01; Figure 5 Transcriptomic volcano plot of Novosphingobium lindaniclasticum RS-01 treated with ononin, where key functional genes that were significantly upregulated are marked in orange circles; Figure 6A bar chart showing the key upregulated genes induced by ononin (locus tag and functional annotation) and their upregulation folds; Figure 7 A bar chart showing the effects of knockout strains (Δgene0959) and complement strains of the key functional gene gene0959 on the function of Novosphingobium lindaniclasticum RS-01; Figure 8 A bar graph showing the promoting effect of the combined application of ononin and Novosphingobium lindaniclasticum RS-01 on the aluminum tolerance of soybeans under aluminum stress. Detailed Implementation

[0016] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are all within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc., used in the embodiments of the present invention are all commercially available; unless specifically specified, all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0017] In this invention, n=6 refers to 6 biological replicates per treatment. Data are expressed as mean ± standard deviation. Different lowercase letters in the bar chart indicate significant differences between treatments (P<0.05).

[0018] The YMA solid medium used in this invention has the following formulation: 10.0 g mannitol, 0.4 g yeast extract, 0.5 g K₂HPO₄, 0.2 g MgSO₄·7H₂O, 0.1 g NaCl, and 15.0 g agar. Distilled water is added to bring the volume to 1 L, and the pH is adjusted to 6.8 with 1 mol / L NaOH. The medium is then sterilized at 121 °C for 20 min. The YMB liquid medium has the same composition and proportions except for the absence of agar. Unless otherwise specified, the culture conditions for *Novosphingobium lindaniclasticum* RS-01 in all embodiments of this invention are as follows: inoculation with YMB liquid medium at a 2% (V / V) inoculum, and cultured at 28 °C with shaking at 150 rpm for 48 h.

[0019] Example 1: Identification of Aluminum Resistance Phenotypes in Soybean Varieties HC2 and LD2 Soybean varieties HC2 and LD2 were planted in neutral soil (pH≈6.8) and acidic soil (pH≈4.3, active Al) respectively. 3+ (enrichment), n=6.

[0020] To comprehensively assess the effects of acidic soil on soybean varieties HC2 and LD2, this example measured eight growth and physiological indicators, such as... Figure 1 As shown, Figure 1 The parameters are, in order: shoot dry weight, plant height, root length, relative root elongation, root-tip Al content, root MDA, leaf SPAD chlorophyll content, and root callose deposition. Leaf SPAD chlorophyll refers to the relative chlorophyll content of leaves measured using a SPAD meter.

[0021] The results are as follows Figure 1 As shown, there were no significant differences in various indicators between soybean varieties HC2 and LD2 under neutral soil. Under acidic soil, the aboveground dry weight, plant height, root length, and relative root elongation rate of soybean variety LD2 decreased significantly. Among them, the aboveground dry weight represents the biomass accumulated in the stems and leaves of soybean. The significant decrease under aluminum stress indicates that the damaged root system cannot transport water and nutrients to the aboveground parts, resulting in inhibited photosynthesis and reduced material synthesis. Plant height represents the elongation growth capacity of aboveground cells. The significant decrease in plant height indicates that aluminum toxicity inhibits cell division and elongation, and also interferes with the transport and signal transduction of hormones in the plant, resulting in the inability of internodes to elongate normally. Root length represents the potential for longitudinal root growth. The significant decrease in root length indicates that aluminum ions damage the extensibility of cell walls and inhibit microtubule assembly, directly leading to thickening and shortening of root tips. The relative root elongation rate reflects the intensity of aluminum stress. The value of the relative root elongation rate of soybean variety LD2 is less than 50%, indicating that aluminum is extremely toxic to soybean variety LD2. Meanwhile, in acidic soil, the root tip Al content, root MDA, and root callose of soybean variety LD2 were significantly increased, with the root tip Al content being particularly high. This is direct evidence of aluminum stress, as the plant itself does not require aluminum, and the Al accumulated in the root tip... 3+ The higher the concentration of aluminum in the root tip cells, the more aluminum is absorbed or adsorbed onto the root tip cell walls. Root MDA (malondialdehyde) content indicates higher cell membrane damage and greater plant stress. Root callose is negatively correlated with root length; a significant increase in callose indicates that callose blocks plasmodesmata, hindering nutrient transport and cell wall relaxation, thus preventing normal root elongation. Furthermore, in acidic soil, the relative chlorophyll content and SPAD value of soybean variety LD2 decreased, indicating reduced photosynthetic capacity. These experimental results suggest that soybean variety LD2 is an aluminum-sensitive variety.

[0022] Compared with soybean variety LD2, soybean variety HC2 was less affected by aboveground dry weight, plant height, root length, relative root elongation, root tip Al content, root MDA, relative chlorophyll content in leaves, and root callose in acidic soil, thus proving that soybean variety HC2 is an acid-tolerant variety.

[0023] Example 2: Root Differential Metabolite Analysis of Soybean Varieties HC2 and LD2 Root samples were collected from soybean varieties HC2 and LD2. Metabolites from the roots of both varieties were extracted with methanol. Untargeted metabolomics analysis of the root metabolites was performed using liquid chromatography-mass spectrometry (LC-MS), with electrospray ionization (ESI) used for mass spectrometry detection in both positive and negative ion modes. The raw LC-MS data were peak-aligned and normalized before being imported into SIMCA software for OPLS-DA (orthogonal partial least squares discriminant analysis). Differential metabolites between the two varieties were screened based on |log2(FC)|>1 (i.e., fold change > 2 or fold change < 0.5) and FDR (false detection rate) < 0.05.

[0024] Based on the screening criteria of |log2(FC)| > 1 and FDR < 0.05, a total of 30 significantly differentially regulated metabolites were identified, of which 25 were significantly upregulated and 5 were significantly downregulated. The volcano plot of the differentially regulated metabolites is shown below. Figure 2 As shown, the horizontal axis represents the difference factor log2(FC), and the vertical axis represents the significance level -log 10 (p-value), red dots represent upregulated differentially regulated metabolites, blue dots represent downregulated differentially regulated metabolites, gray dots represent metabolites with no significant difference, and orange circles mark key differentially regulated metabolites such as ononin. Ononin is a naturally occurring isoflavone glycoside found in various plants, including legumes such as astragalus, chicken blood vine, and kudzu root, as well as common foods such as soybeans, lupins, and broccoli. Ononin (molecular formula C...) 22 H 22 O9 (m / z 429.1191, neg mode) was significantly enriched in the aluminum-tolerant soybean variety HC2 (log2(FC) = 3.536, fold change 11.597, FDR < 0.001), which is a key differential metabolite of this invention.

[0025] Preparation of Ononin: This example uses commercially available ononin standard (purity ≥98%); alternatively, purification can be performed using the following method. Ononin is an isoflavone 7-O-β-D-glucoside. The glycosidic bond is easily hydrolyzed by strong acids; therefore, acidic conditions should be avoided throughout the preparation process. The dried root of Astragalus membranaceus was pulverized and extracted twice with 70% ethanol at a material-to-liquid ratio of 1:10, for 1.5 h each time. The extracts were combined, concentrated under reduced pressure until no alcohol odor remained, and diluted with water. The sample was loaded onto an AB-8 macroporous adsorption resin column. Sugars and water-soluble impurities were first washed away with distilled water, followed by stepwise elution with 10%, 30%, and 70% ethanol. Ononin was mainly concentrated in the 30% ethanol fraction; this step increased its content by approximately 10 times, with a recovery rate exceeding 80%. The 30% ethanol fraction was collected and concentrated under reduced pressure, then purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: C18 reversed-phase column, acetonitrile-0.1% formic acid-water gradient elution, and detection wavelength of 254 nm. The target component was then analyzed by LC-MS. 1 H-NMR, 13 The structure and glycosidic bond configuration were confirmed by C-NMR, and the purity was determined to be ≥98% before being used in subsequent experiments.

[0026] Example 3: Isolation, identification, and preservation of *Sphingosine Monoclonalella* strains First, rhizosphere soil was collected from the aluminum-tolerant soybean variety HC2. After shaking on a shaker, a rhizosphere soil suspension was obtained. Next, a dilution was prepared, and the rhizosphere soil suspension was diluted using a serial dilution method. The dilution was spread onto YMA (yeast extract-mannitol-agar) solid medium plates. After colonies grew, single colonies were picked based on morphological differences and further purified on YMA plates to obtain a pure culture. 16S rDNA sequencing and whole-genome sequencing identified it as *Novosphingobium lindaniclasticum* RS-01. The genome of this bacterium consists of two chromosomes (Chromosome1, Chromosome2) and two plasmids (PlasmidA, PlasmidB), containing approximately 4389 coding genes. The strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No. 68050, deposited on April 3, 2026, and has been confirmed to be viable.

[0027] Example 4: Enhancement effect of gentianin on the function of Neosphingomonas strains The metabolite ononin (20 mg / L) was added to a liquid culture system. After incubation at 28℃ and 150 rpm, various functional indicators were measured. A control group without ononin was used. (n=6) Results are as follows: Figure 3 As shown, Figure 3 The functional indicators, in descending order, are growth (OD600), extracellular polysaccharide (EPS), organic acid secretion, and Al. 3+ Chelation rate, biofilm, and siderophore were measured. Ononin was the treatment group treated with the metabolite ononin added to *Novosphingobium lindaniclasticum* RS-01, while CK was the control group without ononin. Figure 3 The results showed that the biomass OD600 value of the CK control group was 0.7, while that of the Ononin-treated group was 1.2, indicating a significant increase in biomass in the Ononin-treated group (P<0.001). The extracellular polysaccharide (EPS) content of the CK control group was 46.3 mg / L, while that of the Ononin-treated group was 110.9 mg / L, indicating a significant increase in EPS in the Ononin-treated group (P<0.001). The organic acid secretion of the CK control group was 37.1 mg / L, while that of the Ononin-treated group was 86.0 mg / L, indicating a significant increase in organic acid secretion in the Ononin-treated group (P<0.001). The Al content of the CK control group... 3+ The chelation rate was 19.0% in the Ononin-treated group. 3+ The chelation rate was 52.4%, indicating that the Al in the Ononin-treated group... 3+ The chelation rate was significantly increased (P<0.001); the OD570 value of the biofilm in the CK control group was 0.3, while that in the Ononin treatment group was 1.0, indicating that the amount of biofilm formed in the Ononin treatment group was significantly increased compared with that in the CK control group (P<0.001); the relative activity (SU) of the siderophores in the CK control group was 20.6%, while that in the Ononin treatment group was 53.8%, indicating that the relative activity (SU) of the siderophores in the Ononin treatment group was significantly increased (P<0.001).

[0028] Gene0959 encodes a 1061-amino acid autotransporter (Type VA secretory system). Its sequence exhibits the typical three-segment characteristic of this family: an N-terminal signal peptide (MPRYLLASTALLAMATAAHA, followed by a signal peptide cleavage site), a middle passenger domain rich in β-helical repeats, and a C-terminal β-barrel translocation domain ending in tryptophan residues. These proteins are located on the outer membrane, with the passenger domain exposed on the cell surface, mediating adhesion between cells and between cells and the root surface. While upregulating O-antigen ligase genes such as gene3140, styracin significantly upregulated gene0959, increasing cell surface adhesion and biofilm formation. This resulted in a continuous coating layer on the root surface, directly blocking aluminum migration to the root surface and creating a high-concentration extracellular polysaccharide and organic acid microenvironment on the root surface, chelating Al in situ. 3+ This reduces aluminum accumulation at the root tip.

[0029] The concentration gradient of ononin was set at 0 / 2.5 / 5 / 10 / 20 / 40 / 80 mg / L, with Al 3+ The chelation rate was the primary reading, n=6. Results are as follows: Figure 4 As shown, the horizontal axis represents the concentration of ononin, and the vertical axis represents Al. 3+ The chelation rate showed a typical curve of first increasing and then decreasing. The effective concentration of ononin was 5-80 mg / L, with the preferred concentration being 10-40 mg / L (i.e., the concentration range that achieves a maximum synergistic effect of more than 50%). The optimal concentration was about 20 mg / L, with a maximum Al3+ chelation rate of about 52.4%. Slight inhibition occurred at higher concentrations, and the upper limit of the concentration was defined accordingly.

[0030] Example 5: Effects of gentianin on the transcriptome of *Sphingosine monocytogenes* strains In this embodiment, *Novosphingobium lindaniclasticum* RS-01 was treated with the metabolite ononin as the Ononin treatment group, and the control group (CK) was not treated with ononin. RNA was extracted from both the Ononin treatment group and the CK control group, and strand-specific RNA-seq analysis was performed, with three biological replicates for each group. Differentially expressed genes were screened using a threshold of |log2FC|>1 and FDR<0.05. The results showed that ononin specifically upregulated a group of functional genes, such as... Figure 5 and Figure 6 As shown, the representative key upregulated genes are as follows: gene0959 (autotransporter domain-containi...), log2FC≈4.17 gene3092 (hypothetical protein), log2FC≈4.04 gene3294 (glycosyltransferase family 39 p...), log2FC≈3.85 gene3491 (outer membrane beta-barrel protein), log2FC≈3.84 gene3140 (O-antigen ligase family protein), log2FC≈3.82 gene0812 (O-antigen ligase family protein), log2FC≈3.82 The representative key gene of this invention is gene0959, located in Chromosome1 (positions 947439–944254, - chain) of *Novosphingobium lindaniclasticum* RS-01; functional annotation: autotransporter domain-containing protein; COG: Uncharacterized conserved protein, contains a C-terminal beta-barrel porin domain; KO: -; Pfam domain: -. Figure 5 and Figure 6 As shown, after treatment with ononin, the gene0959 was the functional gene with the largest upregulation in the transcriptome of *Novosphingobium lindaniclasticum* RS-01. Figure 7 As shown, the gene0959 gene was verified to be essential for this enhancement effect through gene knockout and complementation. Its encoded product is a self-transporter protein of the type VA secretory system, containing an N-terminal signal peptide, a middle β-helical passenger domain and a C-terminal β-barrel translocation domain, which mediates cell surface adhesion, biofilm formation and root colonization.

[0031] The complete coding sequence (CDS, from start codon to stop codon) of gene0959 is shown in SEQ ID NO:1, totaling 3186 bp.

[0032] The amino acid sequence of the protein encoded by the gene0959 gene is shown in SEQ ID NO:2, totaling 1061 amino acids.

[0033] In this embodiment, a knockout strain (Δgene0959) and a complement strain (Δgene0959-Comp) of the gene0959 gene were constructed according to the method in Example 8. The functions of the wild-type, knockout, and complement strains were compared under conditions of addition and non-addition of ononin, n=6. The methods for determining each functional index are described in Example 7. Extracellular polysaccharide EPS and Al... 3+ Taking chelation rate as an example, the results are as follows: Figure 7 As shown: the EPS of wild-type WT was 46.3 mg / L, and Al... 3+ The chelation rate was 19.0%; after treatment with thymol, the EPS of WT+Ononin increased to 110.9 mg / L, and Al... 3+ The chelation rate increased to 52.4%, both significantly higher; most of the above-mentioned increases in the knockout strain Δgene0959+Ononin were lost (EPS 57.0 mg / L, Al 3+ The chelation rate was 26.2%, equivalent to 16.6% and 21.6% of the induced increment in the wild type, respectively; the recombinant strain Δgene0959-Comp+Ononin partially recovered (EPS 88.6 mg / L, Al 3+ The chelation rate was 42.4%, equivalent to 65.5% and 70.1% of the induced increment, respectively. Based on this, gene0959 was confirmed to be essential for the enhancing effect of gentianin.

[0034] Furthermore, knocking out the gene0959 gene resulted in a significant loss of the synergistic effect of ononin, but it did not completely return to zero, indicating that ononin, while upregulating the gene0959 gene, also synergistically upregulated other genes such as ononin. Figure 6 The diagram shows multiple functional genes; these minor genes have weak individual effects, but under the induction of ononin, they work synergistically and cumulatively with the gene0959 gene to enhance the aluminum tolerance of soybean in acidic environments by Novosphingobium lindaniclasticum RS-01, with the gene0959 gene making the most significant contribution.

[0035] Example 6: Combined application of gentianin and Neosphingomonas strain to promote soybean's tolerance to aluminum acid. Using the aluminum-sensitive cultivar LD2 as the test material, five treatments were set up in a pot / hydroponic system, with each treatment containing six biological replicates: CK (normal, aluminum-free), Al (aluminum stress), Al + Novosphingobium lindaniclasticum RS-01, Al + Ononin, and Al + Novosphingobium lindaniclasticum RS-01 + Ononin. Root length, root tip Al content, aboveground dry weight, and root MDA were measured under aluminum stress conditions.

[0036] The results are as follows Figure 8 As shown, the root length of CK (no Al) was 12.7 cm and the root tip Al content was 85 µg / g DW. Compared with CK (no Al), the root length of Al decreased to 5.5 cm and the root tip Al content increased to 702 µg / g DW, indicating that aluminum stress inhibits soybean root length while increasing root tip Al content. The root length of Al + Novosphingobium lindaniclasticum RS-01 was 7.6 cm and the root tip Al content was 476 µg / g DW, indicating that soybean inoculation with Novosphingobium lindaniclasticum RS-01 alone provided some relief. The root length of Al + Ononin was 6.2 cm and the root tip Al content was 602 µg / g DW, indicating that the effect of applying Ononin alone was limited. Meanwhile, the effect of Novosphingobium lindaniclasticum... The combined application of *Novosphingobium lindaniclasticum* RS-01 and ononin showed the best alleviating effect, with root length increasing to 10.6 cm, root tip Al content decreasing to 297 µg / g DW, aboveground dry weight increasing to 4.2 g, and root MDA decreasing to 12.0 nmol / g. All indicators showed significant differences compared to inoculation alone or application of metabolites alone (P < 0.05 for different letters), indicating a synergistic effect between the two in improving soybean's tolerance to aluminum. This demonstrates that the combined application of *Novosphingobium lindaniclasticum* RS-01 and ononin to soybeans can significantly improve root elongation and reduce aluminum accumulation in root tips in acidic soils.

[0037] Example 7: Methods for determining various functional indicators This embodiment provides specific methods for determining the functional indicators involved in each embodiment of the present invention. Unless otherwise specified, all indicators are determined in a pure bacterial culture system, with 6 biological replicates for each treatment, and the results are expressed as mean ± standard deviation.

[0038] (a) Determination of growth. A bacterial culture solution after 48 h of culture was zeroed with uninoculated YMB liquid medium, and the absorbance was measured at 600 nm using a UV-Vis spectrophotometer, recorded as OD. 600 .

[0039] (II) Determination of extracellular polysaccharides (EPS): The phenol-sulfuric acid method was used. The culture medium was centrifuged at 10000×g for 15 min at 4 ℃, and the supernatant was collected. Three volumes of pre-cooled anhydrous ethanol were added, and the mixture was allowed to stand overnight at 4 ℃ to precipitate. The precipitate was collected by centrifugation at 10000×g for 20 min, and then reconstituted with deionized water and brought to a final volume. 1.0 mL of the reconstituted solution was taken, and 1.0 mL of 5% (m / V) phenol solution and 5.0 mL of concentrated sulfuric acid were added sequentially. After standing for 10 min, the mixture was vortexed and incubated in a water bath at 40 ℃ for 20 min. After cooling to room temperature, the absorbance was measured at 490 nm. A standard curve was plotted using 0–100 mg / L standard solutions prepared with anhydrous glucose. The EPS content was converted according to the standard curve and expressed in glucose equivalents, in mg / L.

[0040] (III) Determination of organic acid secretion: High performance liquid chromatography (HPLC) was used. The culture medium was centrifuged at 4 ℃ and 10000×g for 15 min. The supernatant was filtered through a 0.22 μm aqueous filter before injection. Chromatographic conditions: dedicated organic acid column (e.g., Aminex HPX-87H, 300 mm × 7.8 mm); mobile phase: 5 mmol / L sulfuric acid aqueous solution; flow rate: 0.6 mL / min; column temperature: 50 ℃; UV detection wavelength: 210 nm; injection volume: 20 μL. Standard curves were plotted using oxalic acid, citric acid, malic acid, and succinic acid standards. The sum of the contents of each component was recorded as the total organic acid secretion, expressed in mg / L.

[0041] (iv) Al 3+ Determination of chelation rate. The culture medium was centrifuged at 4 ℃ and 10000×g for 15 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain a sterile extracellular secretion solution. 5.0 mL of this solution was mixed with an equal volume of 20 mmol / L acetate-sodium acetate buffer (pH 4.3) containing AlCl3 to adjust the Al concentration in the system. 3+ The initial concentration was 100 μmol / L; the reaction was carried out at 25 ℃ and 150 rpm for 2 h with shaking. The reaction solution was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, centrifuged at 5000×g for 20 min, and the unchelated free Al was measured in the filtrate. 3+ Concentration – can be determined colorimetrically at 545 nm using chromium azure S (ECR) spectrophotometry, or by inductively coupled plasma optical emission spectrometry (ICP-OES). An equal volume of uninoculated sterile YMB liquid culture medium treated according to the same procedure was used as a control; the Al concentration in the filtrate was... 3+The concentration is denoted as C0, and the sample filtrate is denoted as C1. Al 3+ Chelation rate (%) = (C0-C1) / C0×100%.

[0042] (v) The biofilm formation amount was determined using the crystal violet staining method. The bacterial culture was diluted with YMB liquid medium to OD0.05. 600 =0.05, add 200 μL to a 96-well polystyrene microplate, and incubate at 28 ℃ for 48 h; discard the floating bacterial culture in the wells, wash 3 times with phosphate buffer, fix with methanol for 15 min and air dry, add 0.1% (m / V) crystal violet solution to stain for 20 min, wash with running water until the effluent is colorless; add 200 μL of 33% (V / V) glacial acetic acid to each well, dissolve at room temperature for 15 min, and measure the absorbance at 570 nm, recorded as OD. 570 .

[0043] (vi) The relative activity of siderophores was determined using the chromazurite S (CAS) assay. 0.5 mL of culture supernatant was mixed with 0.5 mL of CAS detection solution, and the mixture was reacted at room temperature in the dark for 1 h. The absorbance was measured at 630 nm and recorded as A_s. An equal volume of uninoculated sterile YMB liquid culture medium was used in the same manner, and the resulting absorbance was recorded as A_r. The relative activity of siderophores, SU (%), was calculated as (A_r - A_s) / A_r × 100%.

[0044] Example 8: Methods for constructing gene knockout and replacement strains This embodiment provides specific construction methods for the gene knockout and replacement strains involved in the various embodiments of the present invention, using a homologous recombination double exchange strategy based on suicide plasmids.

[0045] (I) Construction of the knockout vector. Using the genomic DNA of *Novosphingobium lindaniclasticum* RS-01 as a template, homologous arm fragments approximately 800 bp upstream and 800 bp downstream of the coding region of the target gene were amplified, respectively. After the two fragments were fused by overlap extension PCR, they were digested with EcoRI and HindIII and ligated into the same digested suicide plasmid pK18mobsacB (carrying the kanamycin resistance gene and the sacB sucrose lethal reverse selection marker). The ligation was then transformed into *Escherichia coli* DH5α, and the knockout vector was obtained by colony PCR and Sanger sequencing.

[0046] (II) Conjugation Transfer and Double Crossover Screening. The knockout vector was transformed into the donor bacterium *Escherichia coli* S17-1 λpir and mixed with logarithmically growing *Novosphingobium lindaniclasticum* RS-01 at a 1:1 bacterial ratio. The mixture was spotted onto antibiotic-free YMA plates and conjugated at 28 °C for 12 h. After elution, the bacterial colony was spread onto YMA plates containing 50 mg / L kanamycin and 20 mg / L nalidixic acid to screen for single crossovers that underwent the first homologous recombination. Single crossovers were picked, inoculated into antibiotic-free YMB liquid medium, and passaged for 12 h. They were then spread onto YMA plates containing 10% (m / V) sucrose and antibiotic-free. The sucrose lethality effect of sacB was used to reverse-select for double crossovers that underwent the second homologous recombination. Using kanamycin sensitivity and sucrose resistance as initial screening criteria, PCR amplification and sequencing were performed using primers located on the outer side of the homologous arm to confirm that the coding region of the target gene had been completely deleted, resulting in a knockout strain (denoted as Δ target gene).

[0047] (III) Construction of the complement strain. Using wild-type genomic DNA as a template, the complete CDS of the target gene and its promoter region of approximately 300 bp upstream were amplified and cloned into the multiple cloning site of the broad-host plasmid pBBR1MCS-2. The recombinant plasmid was transferred into the knockout strain via E. coli S17-1 λpir conjugation. Transformants were screened on YMA plates containing 50 mg / L kanamycin, and the transcriptional level of the target gene was verified to have been restored to the wild-type level by real-time quantitative PCR (with the 16S rRNA gene as the internal reference gene), thus obtaining the complement strain (denoted as Δtarget gene-Comp).

[0048] (iv) Control and exclusion. The growth curves of wild type, knockout strain and replenishment strain in YMB liquid medium showed no significant difference, indicating that the knockout and replenishment operations themselves did not cause growth defects, and the differences in functional indicators among strains could not be attributed to differences in biomass.

[0049] Example 9: Transcriptome Sequencing and Differential Expression Analysis Methods Total RNA was extracted from bacterial cells in both the treatment and control groups using the TRIzol method, followed by DNase I digestion to remove genomic DNA contamination. Ribosomal RNA was removed using the Ribo-Zero kit, and strand-specific libraries were constructed. PE150 paired-end sequencing was performed on the Illumina platform, with three biological replicates for each group. After quality filtering, the raw data were aligned to the complete genome sequence of *Novosphingobium lindaniclasticum* RS-01. Feature counts were used to count the number of reads for each gene, and differential expression analysis was performed using DESeq2. Differentially expressed genes were screened using a threshold of |log2FC|>1 and FDR<0.05. The expression levels of key upregulated genes were further verified using real-time quantitative PCR.

[0050] Example 10 Preparation of Microbial Inoculants (a) Preparation of the carrier. By weight, take 60 parts of peat moss, 25 parts of vermiculite and 10 parts of diatomaceous earth, crush them through a 40-mesh sieve and mix them evenly. Sterilize at 121 ℃ for 30 min and cool to room temperature for later use.

[0051] (II) Preparation of bacterial suspension. *Novosphingobium lindaniclasticum* RS-01 was inoculated into YMB liquid medium and cultured at 28 ℃ and 150 rpm for 48 h. The cells were collected by centrifugation at 4 ℃ and 8000×g for 10 min, washed twice with sterile physiological saline, and resuspended. The viable count was adjusted to 1×10⁻⁶. 10 CFU / mL.

[0052] (III) Preparation of the metabolite stock solution. Take the gentianin standard, dissolve it with a small amount of anhydrous ethanol, dilute it with sterile water to prepare a 1 g / L stock solution, filter it through a 0.22 μm filter membrane for sterilization, and store it at 4 ℃ in the dark.

[0053] (iv) Compounding and adsorption. Take the bacterial suspension from step (ii) and the mother liquor from step (iii), and add 2 parts sodium alginate, 1 part carboxymethyl cellulose, 1.5 parts skim milk and 0.5 parts trehalose (all by weight), and stir until completely dissolved; spray the resulting mixture and the carrier from step (i) evenly at a ratio of 1:4 (V / m) for adsorption, stirring while spraying.

[0054] (v) Drying and packaging. The adsorbed material is dried in a ventilated environment at a temperature below 30 °C until the moisture content is no more than 15%, passed through a 20-mesh sieve, and sealed in an aluminum foil composite bag to obtain a solid microbial agent.

[0055] (vi) Quality indicators. The number of viable bacteria in the finished microbial agent shall not be less than 1×10⁻⁶. 9The cFU / g concentration of gentianin is 0.5-10 mg / g; after storage at 4 ℃ in the dark for 6 months, the viable bacterial count decreases by no more than one order of magnitude. When using, mix 15-30 kg per hectare with the soil before sowing, or dilute with sterile water for seed dressing, seed soaking, or root irrigation during the seedling stage. The concentration of gentianin in the diluted system should be 5-80 mg / L, and the viable bacterial count should be 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

[0056] It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. A microbial agent for improving the aluminum resistance of soybeans, characterized in that, The strain includes *Novosphingobium lindaniclasticum* RS-01 and its metabolite styrosin; the strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No. 68050, and the deposit date is April 3, 2026; the viable count in the inoculum is not less than 1 × 10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL, with a content of 0.5~10 mg / g or 0.5~10 mg / mL of gentianin.

2. The microbial agent according to claim 1, characterized in that, It also includes a carrier and excipients; by weight, the carrier is one or more of peat moss (50-70 parts), vermiculite (20-30 parts), and diatomaceous earth (5-15 parts), and the excipients are one or more of sodium alginate (1-3 parts), carboxymethyl cellulose (0.5-2 parts), skim milk (1-2 parts), and trehalose (0.3-1 parts).

3. A method for improving the aluminum resistance of soybeans, characterized in that, Novosphingobium lindaniclasticum RS-01 was co-applied with its metabolite styrosin to soybeans grown in acidic soil. The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No. 68050) on April 3, 2026. The concentration of styrosin was 5–80 mg / L, and the application rate of the strain was 1 × 10⁻⁶ mg / L. 7 ~1×10 9 CFU / mL bacterial culture or 1×10 7 ~1×10 9 CFU / g growth medium.

4. The method according to claim 3, characterized in that, The application concentration of the gentianin is 10~40 mg / L.

5. The method according to claim 3, characterized in that, The gentianin upregulates the expression level of the gene0959 gene in the strain, thereby enhancing the secretion of extracellular polysaccharides, organic acids, and Al in the strain. 3+ Chelating ability; the CDS sequence of the gene0959 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the CDS sequence is shown in SEQ ID NO:

2.

6. The method according to claim 3, characterized in that, The combined application method is one or more of seed dressing, seed soaking, root irrigation, and soil application; the root irrigation or soil application is performed during the soybean seedling stage, which is 10 to 30 days after soybean emergence.

7. The method according to claim 3, characterized in that, The acidic soil has a pH of 4.0–5.5 and an exchangeable aluminum content of 0.5–5.0 cmol(+) / kg, or an Al content in the soil solution. 3+ The concentration is 20~200 µmol / L.

8. The application of the microbial agent according to claim 1 or 2 in improving the aluminum tolerance of soybeans in acidic soil soybean cultivation.

9. The application of the method according to any one of claims 3 to 7 in improving the aluminum resistance of soybeans in acidic soil soybean cultivation.

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