Bacillus velezensis biological activity enhancing method and application

Abstract

The invention discloses a bacillus velezensis biological activity enhancing method and application, and aims to solve the technical problems that in the prior art, a co-culture synergistic mechanism of bacillus velezensis and spiropolyspora pink is unknown, and the rhizosphere colonization ability of bacillus velezensis and spiropolyspora pink is difficult to directionally enhance. According to the method disclosed by the invention, the coculture of the spiropolyspora roseus and the bacillus velezensis is adopted, and the L-proline secreted by the spiropolyspora roseus and the bacillus velezensis is used as a key technical means for specifically up-regulating the expression of a biomembrane matrix gene epsC / tasA by using a signal molecule, so that the directional enhancement of the biomembrane forming ability is realized; the colonization durability and the stress tolerance of the strain in rhizosphere and the prevention and control and plant growth promoting effects on root-knot nematode are remarkably improved, and meanwhile, the co-culture process has the controllability based on metabolic targets and the product batch stability.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a method and application for enhancing the bioactivity of Bacillus belyssus. Background Technology

[0002] Bacillus belesiensis ( Bacillus velezensis As an important class of rhizosphere growth-promoting bacteria, Bacillus subtilis exhibits broad application potential in biological control and green agriculture by secreting antibacterial substances, inducing systemic resistance in plants, and promoting nutrient absorption. However, its field application effectiveness is often limited by bottlenecks such as weak rhizosphere colonization capacity and insufficient tolerance to environmental stresses. In particular, when facing persistent soil-borne diseases (such as root-knot nematode disease), the stability and persistence of a single strain are insufficient to meet actual production needs. Related studies have shown that the formation of a structurally complete and matrix-rich biofilm on the plant root surface is a key prerequisite for Bacillus subtilis to successfully colonize and exert its biocontrol function; while insufficient biofilm formation capacity has become a core factor restricting its application effectiveness.

[0003] To enhance the function of biocontrol bacteria, microbial co-culture strategies have been widely explored, which involve activating silent metabolic pathways or inducing new functional phenotypes through interspecific interactions. Although some bacterial-fungal co-culture systems have been reported to enhance antibacterial activity, significant limitations remain: First, most co-culture studies focus on macroscopic phenotypic observations (such as expanded inhibition zones and increased plant fresh weight), lacking mechanistic analysis of key signaling molecules and molecular regulatory pathways driving synergistic effects. Second, due to the lack of mechanistic understanding, co-culture processes often rely on empirical mixing of strains or fermentation at fixed ratios, failing to implement targeted regulation of key rhizosphere adaptive traits of biocontrol bacteria (such as biofilm formation). This results in poor batch-to-batch stability of the prepared inoculants, large fluctuations in field colonization efficiency, and difficulty in maximizing and reproducible synergistic effects.

[0004] Therefore, there is an urgent need in this field for a technical solution with a clear mechanism and precise controllability that can analyze and directionally enhance the biofilm formation ability of Bacillus belye at the molecular interaction level, thereby systematically improving its rhizosphere colonization efficiency and environmental adaptability, and providing theoretical basis and technical support for the development of efficient and stable microbial biocontrol agents.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] This invention provides a method and application for enhancing the bioactivity of Bacillus belyssus, aiming to solve the technical problem in the prior art that the synergistic mechanism of co-culturing Bacillus belyssus and Polyspora pinkis is unclear and it is difficult to directionally enhance their rhizosphere colonization ability.

[0007] The inventors discovered that pink spiral polyspora ( Clonostachys rosea NF-06 and Bacillus belesiensis ( Bacillus velezensis The combined application of YB-1652 showed superior synergistic effects compared to single-strain application in controlling root-knot nematodes and promoting plant growth, demonstrating the potential of this combination in biocontrol applications. Based on this, metabolomics analysis revealed significant changes in the metabolic profile of fungi after co-culturing *Polyspora rosenbergii* and *Bacillus belyssum*. Further targeted quantitative analysis identified L-proline as a key differential metabolite, with a clear gradient distribution: highest in *Polyspora rosenbergii* monoculture, intermediate in co-culture, and lowest in *Bacillus belyssum* monoculture. This gradient distribution indicates that metabolites such as proline produced by *Polyspora rosenbergii* can be utilized by *Bacillus belyssum*. Functional experiments confirmed that proline can enhance bacterial biofilm formation and upregulate matrix genes. epsC and tasA The results indicate that the enhanced biocontrol effect achieved through co-culture can be attributed to the synergistic effect of proline-mediated enhanced bacterial rhizosphere adaptability and increased production of other bioactive metabolites. Based on these findings, this invention establishes a method for directionally enhancing the biofilm formation ability of Bacillus by adding proline or regulating co-culture conditions.

[0008] The first aspect disclosed in this application relates to the use of L-proline in the preparation of Bacillus belye (…). Bacillus velezensis Application in formulations with biofilm-forming capacity and / or biological activity.

[0009] The second aspect disclosed in this application relates to *Polyspora pinkis* (… Clonostachys rosea The application of Bacillus berberis or its fermentation products in enhancing the biofilm formation ability of Bacillus berberis.

[0010] In some embodiments of this disclosure, the enhanced biofilm-forming ability of *Bacillus belyi* is manifested in any one or more of the following: (1) Enhanced colonization ability and / or stress tolerance of Bacillus belye in plant rhizosphere; (2) Genes related to biofilm matrix synthesis in Bacillus belyssus epsC and / or tasA The level of expression was increased; (3) Bacillus belye has enhanced inhibitory activity against plant parasitic nematodes or improved growth-promoting effects on plants.

[0011] In some embodiments of this disclosure, the *Polyspora pinkis* is *Polyspora pinkis* with accession number CGMCC No. 16262. Clonostachys roseaNF-06; The Bacillus belyssus mentioned is Bacillus belyssus with accession number CGMCC No. 36152 (NF-06); Bacillus velezensis YB-1652.

[0012] The third aspect disclosed in this application provides a method for enhancing the bioactivity of Bacillus belyssus, which involves simultaneously inoculating Bacillus belyssus and Polyspora pinkis in a liquid culture medium and then co-culturing them.

[0013] In some embodiments of this disclosure, the liquid culture medium is a semi-synthetic culture medium with soybean meal as the main nitrogen source, and each liter of culture medium contains: 16-24 g of corn flour, 8-12 g of soybean meal, 0.3-0.7 g of MgSO4, 0.04-0.06 g of FeSO4·7H2O, and 0.04-0.06 g of ZnSO4·7H2O.

[0014] In some embodiments of this disclosure, the *Bacillus belye* is inoculated in seed liquid form with an inoculation volume percentage of 1-5%; the *Polyspora pinkis* is inoculated in spore suspension form with an inoculation volume percentage of 1-5%; the co-culture conditions are: temperature 25-30°C, shaker speed 150-200 rpm, and culture time 48-72 h.

[0015] In some embodiments of this disclosure, the initial concentration or ratio of arginine and / or proline is adjusted by adding arginine and / or proline to the liquid culture medium, thereby enhancing the biofilm-forming ability or nematode-inhibiting activity of the co-culture product.

[0016] The fourth aspect of this application discloses a highly active Bacillus belye product, prepared by any of the methods described herein, in the form of fermentation broth, concentrate, lyophilized powder, or a formulation made with an agriculturally acceptable carrier.

[0017] The fifth aspect disclosed in this application relates to the use of the aforementioned highly active Bacillus belyssus product in any of the following: (1) Control of plant parasitic nematodes; (2) Promote plant growth or enhance plant resistance to adverse conditions; (3) Enhance the colonization ability of Bacillus belye in the plant rhizosphere.

[0018] This invention is based on the study of *Polyspora pinkis* (… Clonostachys rosea A thorough analysis of the interspecies metabolic interactions between L-proline and Bacillus belyssum reveals that L-proline, as a key signaling molecule, can specifically upregulate the biofilm matrix synthesis genes of Bacillus belyssum. epsC and tasAThe expression of this mechanism can be used to target and enhance the biofilm formation capacity. Based on this understanding, this invention constructs a co-culture system using soybean meal as the nitrogen source, and the biofilm properties can be precisely controlled by adding exogenous *Polyspora pinkis* fermentation product.

[0019] 1. Significantly enhances the biofilm formation ability of Bacillus belyssus: Co-culturing Bacillus belyssus with Polyporus pulveratum pinkis, or directly adding Polyporus pulveratum pinkis ferment broth and L-proline standard, can specifically upregulate key genes of biofilm matrix. epsC and tasA The transcriptional level significantly increased the biomass of Bacillus belyss biofilm compared to culture alone.

[0020] 2. Enhanced colonization ability and tolerance to environmental stress: Thanks to the enhanced biofilm structure, the initial attachment efficiency of Bacillus belye treated by the method of this invention on the plant root surface can be greatly improved, and its survival rate under abiotic stress conditions such as drought and high salt is significantly better than that of single-strain cultures.

[0021] 3. Synergistic Effect of Nematode Control and Plant Growth Promotion: Based on the aforementioned enhanced rhizosphere adaptability, the highly active Bacillus belyceae product prepared in this invention achieves a mortality rate of over 95% against second-instar larvae of the southern root-knot nematode, and a growth promotion effect (fresh weight increase) of 70%–80% on crops such as cucumber, both significantly superior to single-strain treatment and physical mixture control. This synergistic effect is not a simple superposition of strain functions, but rather stems from the enhanced persistence of rhizosphere colonization mediated by the biofilm, enabling stable and continuous biocontrol function.

[0022] 4. Achieving precise control of the co-cultivation process and ensuring product stability: This invention identifies the key signaling molecule (L-proline) and its target site ( epsC / tasA A mechanism-oriented co-culture optimization pathway was established: proline can be provided endogenously by specific fungal partners, or the biofilm formation intensity can be directly regulated by adding exogenous *Polyspora pinkis* fermentation product or proline standards. This strategy overcomes the shortcomings of traditional co-culture, which relies on experience and has unpredictable results, and provides technical support for the standardized production and stable field application of microbial preparations.

[0023] In summary, this invention forms a complete technology chain from mechanism analysis (proline signaling) → targeted enhancement of traits (biofilm) → functional system improvement (colonization / prevention efficacy / growth promotion) → precise and controllable process (adjustable co-culture), realizing a leap from empirical bacterial agent compounding to mechanism-driven precision design, and providing a systematic solution for developing a new generation of efficient, stable, and controllable microbial biocontrol products. Attached Figure Description

[0024] Figure 1 This is a comparison of total ion chromatograms of the filtrates from the individual cultures of *Polyspora pinkis* NF-06 and *Bacillus belyssus* YB-1652, and the co-culture of both, in positive ion mode (A, C, E) and negative ion mode (B, D, F) of one embodiment of this application.

[0025] Figure 2 This is a non-targeted metabolomics analysis result of the co-culture system of *Polyspora pinkis* and *Bacillus belyssae* in one embodiment of this application; where A is the principal component analysis (PCA) score plot; B is the Venn diagram of the number of differential metabolic features between the co-culture and the two single cultures; C and D are volcano plots of differential metabolic features between the co-culture and *Polyspora pinkis* single culture (C) and *Bacillus belyssae* single culture (D), respectively (significantly upregulated and downregulated features are highlighted in red and green, respectively); E and F are the KEGG pathway enrichment analysis results of differential metabolites between the co-culture and *Polyspora pinkis* single culture (E) or *Bacillus belyssae* single culture (F), respectively (x-axis is the enrichment factor, y-axis is the pathway name, and the size of the point corresponds to the number of enriched metabolites).

[0026] Figure 3 This embodiment of the present application demonstrates the targeted validation and absolute quantitative analysis of proline; wherein, A is a full-scan mass spectrum of proline standard; B is a tandem mass spectrum of proline standard; C is a tandem mass spectrum of the target analyte in the co-culture sample; D is an ion chromatogram of the extracted target ion from the co-culture sample; and E is an external standard calibration curve for proline quantification. R 2 >0.999); F represents the absolute proline content measured in the three culture methods (different lowercase letters indicate statistically significant differences). p <0.05).

[0027] Figure 4 This paper describes the effect of L-proline on biofilm formation and matrix gene expression in *Bacillus belyssiensis* YB-1652 in one embodiment of this application. A shows the macroscopic phenotype of *Bacillus belyssiensis* biofilm formation under different treatments, including monoculture (Ba), simultaneous co-culture (CoSim), addition of *Polyspora pinkis* fermentation filtrate (Ba+Cl), and addition of exogenous L-proline (Ba+Pro). B shows the biofilm image stained with crystal violet (left) and the quantitative analysis results (right). C shows key genes for biofilm matrix synthesis. epsC and tasA The relative expression level; D is the scanning electron microscope image of the microstructure of the biofilm under each treatment, scale bar = 2 μm. Detailed Implementation

[0028] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0029] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and materials involved are all commercially available conventional products; unless otherwise specified, the test and detection methods involved are all conventional methods.

[0030] Example 1: Culture of the test strain and preparation of fermentation samples 1. Strains

[0031] Pink Spiral Polyporus ( Clonostachys rosea , hereinafter referred to as C. rosea NF-06, originally named "Pink Broom Mold NF-06", has the accession number CGMCC No.16262 (for the source and isolation process, please refer to the applicant's earlier patent document CN109762743A).

[0032] Bacillus belesiensis ( Bacillus velezensis , hereinafter referred to as B. velezensis YB-1652, with accession number CGMCC No.36152 (for the source and separation process, please refer to the applicant's previous patent application CN202511673699.2).

[0033] 2. Preparation of seed cultures

[0034] Seed cultures were prepared by separate fermentation: A single colony of *Polyspora pinkis* NF-06 was picked from a potato dextrose agar (PDA; containing 200 g / L potato extract, 20 g / L glucose, and 20 g / L agar) plate and inoculated into 100 mL of potato dextrose broth (PDB; 200 g / L potato extract and 20 g / L glucose). The broth was incubated at 25°C and 150 rpm for 48 h with shaking.

[0035] Similarly, a single colony of Bacillus belyssus YB-1652 was picked from an LA (L; 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar) plate and inoculated into 100 mL LB broth (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and cultured at 30°C and 180 rpm for 48 h.

[0036] Fungal conidial concentrations were normalized to 1.0 × 10⁻⁶ using a hemocytometer. 7Spores / mL, while bacterial cell density was normalized to 1.0 × 10⁻⁶ by serial dilution plate counting. 8 CFU / mL. These standardized cultures were used as inoculum for subsequent experiments.

[0037] 3. Preparation of cell-free fermentation filtrate

[0038] Culture medium composition: 20 g / L corn flour, 0.5 g / L MgSO4, 0.05 g / L FeSO4·7H2O, 0.05 g / L ZnSO4·7H2O, 10 g / L soybean meal. PDB was used as a control for the conventional culture medium.

[0039] Based on the above culture medium, four treatments were prepared in parallel: (1) Synchronous inoculation and co-culture (2% v / v) C. rosea + 2% v / v B. velezensis ); (2) C. rosea Single culture (2%, v / v); (3) B. velezensis Single culture (2%, v / v); and (4) A 1:1 (v / v) physical mixture of two single-culture broths, with the same inoculum amount for each single-culture broth as the 2% (v / v) inoculum amount used in co-culture.

[0040] The preparation method of the above-mentioned physical mixture control was consistently used in all relevant experiments of this invention, to distinguish the effect of active microbial interactions from the effect of simply doubling the total microbial biomass or metabolites. All cultures were cultured in 250 mL Erlenmeyer flasks at 28°C and 180 rpm for 48 h, and autoclaved at 121°C for 20 min before inoculation. After culturing, the broth was centrifuged at 10,000 g and 4°C for 5 min. The supernatant was filtered through a 0.22 μm filter membrane for sterilization to obtain cell-free fermentation filtrate.

[0041] Example 2: Verification of nematicidal activity of co-cultured products

[0042] Collect Southern Root-Knot Nematodes ( Meloidogyne incognita Second-instar larvae (J2) were used to prepare a suspension (approximately 2000 larvae / mL). 100 μL of the cell-free fermentation filtrate from each treatment group obtained in Example 1 was mixed with an equal volume of the J2 suspension in a 96-well plate. Uninoculated culture medium was used as a control. After treatment at 25°C for 48 h, larval mortality was calculated by microscopic examination. The results are shown in Table 1.

[0043] Table 1. Lethality of different fermented products against Southern Root-knot Nematode J2

[0044] The above results indicate that the nematicidal activity of the co-culture product was significantly synergistically enhanced, with effects far exceeding those of single bacteria and their physical mixtures.

[0045] Example 3: Verification of the plant growth-promoting effect of co-culture products

[0046] Cucumber seeds (variety: Chunqiu Huangguan) were selected and soaked in the cell-free fermentation filtrate of each group in Example 1 for 4 hours before sowing. Seedlings were cultured under greenhouse conditions for 20 days, and their growth indicators were measured. The results are shown in Table 2.

[0047] Table 2 Effects of different fermentation product treatments on cucumber seedling growth

[0048] The results showed that the co-culture product also exhibited a synergistic effect on promoting plant growth, especially with a fresh weight increase rate of 77.0%, demonstrating its powerful plant health enhancement function.

[0049] Example 4: Metabolomics Analysis of Co-culture System

[0050] To elucidate the molecular mechanism by which *Polyspora pinkis* and *Bacillus belye* produce a synergistic effect through simultaneous co-culture, this embodiment employs a non-targeted metabolomics strategy to systematically compare the differences in metabolomics profiles between simultaneous co-culture, two single cultures, and a physical mixed control.

[0051] 1. Sample Preparation

[0052] Cell-free fermentation filtrates were obtained from single cultures of *Polyspora pinkis* and *Bacillus belye*, as well as their simultaneous co-cultures, following the method described in Example 1. For metabolite extraction, 50 μL of thawed fermentation filtrate was mixed with 150 μL of pre-chilled extraction solvent (methanol:acetonitrile = 4:1, v / v) containing an internal standard mixture. The mixture was vortexed vigorously for 3 min and then centrifuged at 12,000 g for 10 min at 4 °C. 150 μL of the supernatant was transferred to a new tube and incubated at -20 °C for 30 min to precipitate residual proteins, followed by centrifugation again for 3 min under the same conditions. Finally, 120 μL of the supernatant was collected and transferred to a glass-lined tube for LC-MS analysis.

[0053] 2. LC-MS Analysis

[0054] Metabolomics analysis was performed using a Vanquish UHPLC system coupled with a Q Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Chromatographic separations were performed on a Waters ACQUITY Premier HSS T3 column (1.8 μm, 2.1 × 100 mm) at a temperature maintained at 40 °C. Mass spectrometry detection was performed in electrospray ionization (ESI) mode. Throughout the analytical sequence, a mixed quality control (QC) sample, generated by mixing equal volumes of all experimental samples, was periodically injected to monitor system stability and ensure data quality.

[0055] Metabolomics analysis of single cultures and co-cultures was performed using liquid chromatography-mass spectrometry (LC-MS) in both positive and negative ion modes. Total ion chromatogram (TIC) analysis showed ( Figure 1 All three groups of samples exhibited unique and reproducible metabolic profiles. The metabolic profile of the co-culture was generally closer to that of the Bacillus belye monoculture, while it differed significantly from that of the Polyspora pinkis monoculture, suggesting that co-culture mainly induces fungal metabolic reprogramming.

[0056] 3. Data processing and multivariate statistical analysis

[0057] After format conversion, the raw data were processed using the XCMS package (v3.18.0) in R software (v4.2.0) for metabolic feature extraction, retention time correction, and peak area integration. To ensure data reliability, metabolic features with a relative standard deviation (RSD) > 30% in the QC samples were removed. A total of 6,159 metabolic features were ultimately obtained, defined by precise mass-to-charge ratio (m / z) and retention time (RT).

[0058] Principal component analysis (PCA) was performed using the ropls package (v1.30.0) to visualize the overall distribution of metabolites and inter-group differences. Differential metabolites were screened using univariate statistical analysis, with features having a variable importance projection (VIP) score >1.0 and a p-value <0.05 considered significant. The results are presented as a volcano plot. Additionally, Venn diagrams were used to show the overlap of differential features when comparing the co-culture group with each of the two single-culture groups.

[0059] PCA results showed significant segregation among the groups, and the co-culture group was spatially closer to the Bacillus belyss monoculture group. Figure 2 A). Differential analysis showed that, compared to the *Polyspora pinkis* monoculture, 3,192 characteristics changed significantly in the co-culture group; compared to the *Bacillus belyssae* monoculture, 1,713 characteristics changed (…). Figure 2(C, D). Among them, 1,124 features are common to both classes and constitute the core metabolic response specifically triggered by co-culture (C, D). Figure 2 B). This result indicates that co-culture primarily produces a synergistic effect by remodeling the metabolic network of *Polyspora pinkis*.

[0060] 4. Metabolite annotation and pathway analysis

[0061] A stratified strategy was employed to annotate significantly altered metabolic features. All differentially expressed features were queried in the following databases: (1) an internal database of real chemical standards, (2) public metabolite libraries including HMDB and KEGG, and (3) computer-predicted and mass spectrometry libraries within the GNPS platform. High-confidence features were assigned to those matching real standards in the internal database and further supported by either (i) an MS / MS mass spectrometry similarity score >0.8 or (ii) corroborating evidence from MetDNA. This high-confidence metabolite selection was then subjected to KEGG pathway enrichment analysis using MetaboAnalyst 5.0 to identify significantly altered biological pathways in the co-culture system. A total of 1,204 high-confidence annotated metabolites were obtained.

[0062] KEGG pathway enrichment analysis showed that ( Figure 2 E, F: Compared with monoculture of *Polyspora pinkis*, the coculture was significantly enriched in amino acid metabolic pathways, including arginine and proline metabolism (ko00330), tryptophan metabolism (ko00380), phenylalanine metabolism (ko00360), and amino acid biosynthesis (ko01230), while secondary metabolite biosynthesis (ko01110) was also significantly activated. Compared with Bacillus belyi monoculture, the coculture mainly enriched lipid metabolism pathways, such as glycerophospholipid metabolism (ko00564), α-linolenic acid metabolism (ko00592) and linoleic acid metabolism (ko00591), while the changes in amino acid metabolism pathways were relatively limited.

[0063] Notably, arginine and proline metabolism (ko00330) were significantly enriched in both groups, suggesting a central role in interspecies interactions. Within this pathway, L-proline was identified as a key differentially regulated metabolite. Quantitative analyses consistently confirmed that proline content in *Polyspora pinkis* monocultures was significantly higher than in *Bacillus belyssiensis* monocultures, and although proline levels decreased in cocultures, they remained significantly higher than in *Bacillus belyssiensis* monocultures.

[0064] Example 5: Targeted LC-MS / MS Validation and Quantification of Proline

[0065] Based on the preliminary identification of proline as a key differential metabolite using non-targeted analysis, a targeted LC-MS / MS method based on real standards was further employed to confirm and quantify it.

[0066] 1. Sample Preparation

[0067] Fermentation filtrates from simultaneous co-cultures and corresponding single cultures of *C. rosea* and *B. velezensis* were used for targeted analysis. Frozen aliquots were thawed at room temperature and vortexed. For extraction, 200 μL of sample was mixed with 800 μL of ice-cold methanol, vortexed for 5 min, and centrifuged at 12,000 g for 5 min at 4 °C. 80 μL of the supernatant was filtered through a 0.22 μm PTFE syringe filter into glass autosampler vials for subsequent LC-MS / MS analysis.

[0068] 2. Chromatographic and mass spectrometric conditions

[0069] Analysis was performed using an ultra-high performance liquid chromatography (UHPLC) system coupled with an AB Sciex Triple Quad™ 4500 mass spectrometer (Flemingham, Massachusetts, USA). Chromatographic conditions: Separation was achieved on a Thermo Scientific™ HYPERSIL GOLD C18 column (3 μm, 2.1 × 100 mm) at a temperature maintained at 35 °C. The mobile phase was (A) 0.1% (v / v) formic acid aqueous solution and (B) acetonitrile, with a flow rate of 0.3 mL / min. The injection volume was 3 μL. The gradient program was: 0–3 min, 10% B; 3–6 min, 10–90% B; 6–6.5 min, 90% B; 6.5–6.6 min, 90–10% B; 6.6–10 min, 10% B. Detection was performed using a Turbo Spray® ionization source in positive electrospray ionization mode. The relevant parameters were set as follows: ion spray voltage, +5500 V; source temperature, 550℃; curtain gas, 30 psi; collision gas, 9 psi. Proline was quantified in multiple reaction monitoring mode by monitoring the transition from m / z 116.1 to 70.1.

[0070] Targeted LC-MS / MS analysis based on real standards was used to validate and quantify proline—a key metabolite derived from non-targeted screening. The protonated ion of proline [M+H] was also analyzed. + (Theoretical m / z 116.0707) m / z 115.9 was detected in the standard and m / z 116.0 was detected in the sample. Figure 3A). These measurements were all within the acceptable mass accuracy tolerance (±0.5 Da) specified for targeted quantification using the AB Sciex 4500 triple quadrupole system at unit resolution. More importantly, the tandem mass spectrometry (MS / MS) fragmentation mode of the analyte (precursor ion m / z 116.0) was completely consistent with that of the true standard (precursor ion m / z 115.9), conclusively confirming the presence of proline. Figure 3 B, C). Extraction ion chromatogram (XIC) at m / z 116.0 further validated the detection of proline and demonstrated its efficient chromatographic separation from other matrix components. Figure 3 D).

[0071] 3. Quantitative and Quality Control

[0072] Calibration curves were constructed using a dilution series of real L-proline standards (Sigma-Aldrich, USA), with each concentration analyzed three times. A linear regression of the average peak area against the standard concentration (μg / mL) yielded the calibration equation. The proline concentration of the sample extract was determined by interpolating the peak area into this curve. The final concentration in the original fermentation filtrate was calculated using the formula: Cfinal (μg / mL) = c × Vtotal / vsample, where c is the interpolated concentration, Vtotal is the total volume of the extraction mixture, and vsample is the volume of the original fermentation filtrate used for extraction. Method reliability was monitored by periodically analyzing the mixed QC sample and by interspersing low, medium, and high concentrations of calibration standards throughout the analytical sequence.

[0073] Quantitative analysis was performed using an external standard calibration curve, which exhibited excellent linearity (R0) across the tested concentration range. 2 >0.999), ensuring the accuracy and precision of the measurement. Figure 3 E). Consistent with the trend of non-targeted metabolomics identification, absolute quantification confirmed a significantly elevated proline concentration in *Polyspora pulvinata* monocultures. Proline levels in cocultures were significantly lower than in *Polyspora pulvinata* monocultures, but still much higher than the concentration detected in *Bacillus belyssiensis* monocultures. Figure 3 F), forming a concentration gradient, indicates that proline is transferred from fungi (Pink Spiral Polyporus) to bacteria (Bacillus belye) and utilized.

[0074] Example 6: Validation of the mechanism by which co-culture enhances biofilm formation of Bacillus belyssus.

[0075] Previous studies have confirmed that L-proline is a key metabolite capable of regulating bacterial biofilm structure and stability. In vitro experiments were conducted to assess the role of L-proline in bacterial biofilm formation in co-culture systems.

[0076] 1. Sample Preparation

[0077] Prepared according to the method described in Example 1 C. rosea and B. velezensis Seed culture. Subsequently, 2 mL of culture medium with soybean meal as the nitrogen source was dispensed into each well of a 12-well cell culture plate. Four treatment groups were set up, with each group being repeated three times: (1) B. velezensis (1) Single culture (2.0%, v / v); (2) Simultaneous co-culture (2.0%, v / v for each); (3) Supplement with 0.1% (v / v) filtration sterilization C. rosea Fermentation filtrate B. velezensis (2.0%, v / v); (4) Supplement with 15 mM exogenous L-proline B. velezensis (2.0%, v / v). Contains only C. rosea Wells containing NF-06 (2.0%, v / v) served as an additional control. All culture plates were statically incubated at 37°C for 48 h.

[0078] 2. Quantification of biofilm biomass

[0079] After culturing, planktonic cells and supernatant were carefully removed. Adhering biofilm was gently washed twice with sterile distilled water and then air-dried. The biofilm was stained with 2 mL of 0.1% (w / v) crystal violet at room temperature for 10 min, then washed three times with distilled water to remove unbound dye. Bound dye was eluted with 1 mL of 33% acetic acid for 30 min. The absorbance of the eluent was measured at 595 nm using a UV-Vis spectrophotometer (Thermo Fisher Scientific, USA) to quantify the relative biofilm biomass.

[0080] 3. Observation of biomembrane morphology

[0081] Biofilm samples were fixed with 1% (v / v) osmium tetroxide at 25 °C for 2 h, and then dehydrated stepwise in an ethanol gradient (30%, 50%, 70%, 80%, 90%, and 100%). The samples were critically dried using standard biofilm protocols. The dried samples were sputter-coated with gold and imaged using a Hitachi SU8100 scanning electron microscope (Tokyo Hitachi, Japan) at an accelerating voltage of 3.0 kV.

[0082] 4. Gene expression analysis

[0083] Total RNA was extracted from biofilm samples using RNAiso Plus (Takara, Dalian, China). For each treatment, RNA was extracted from three independent biofilm cultures. Genomic DNA was removed using the PrimeScript RT Reagent Kit with gDNAEraser (Takara). First-strand cDNA was synthesized from 1 μg of total RNA from each sample, following the manufacturer's protocol. Quantitative PCR was performed on a QuantStudio 7500 Fast Real-Time PCR System (Applied Biosystems) using TB Green Premix Ex Taq II (Tli RNaseH Plus, Takara). −∆∆Ct Methods for analyzing biomembrane matrix genes ( epsC , tasA The expression level of qPCR was measured. The 16S rRNA gene was normalized as an endogenous reference. Primer sequences are listed in Table 3. Each qPCR reaction was performed in triplicate. The entire experiment was independently repeated three times.

[0084] Table 3 Primer Sequences

[0085] Macroscopic observation and quantitative analysis with crystal violet showed that, compared with single bacterial culture, *Bacillus belye* exhibited significantly enhanced biofilm biomass under three conditions: co-culture with *Polyspora pinkis*, supplementation with filtered and sterilized *Polyspora pinkis* fermentation filtrate, or supplementation with pure L-proline (… Figure 4 A, B). No biofilm formation was detected in single fungal cultures.

[0086] At the molecular level, under the same three induction conditions, the expression of key biofilm matrix genes epsC (extracellular polysaccharide biosynthesis) and tasA (major matrix protein) in Bacillus belyi was significantly upregulated. Figure 4 C). Scanning electron microscopy visually confirmed these findings, showing that in co-culture and in the presence of fungal fermentation filtrate or L-proline, dense, matrix-encapsulated bacterial aggregates formed, while in monoculture they appeared as dispersed cells. Figure 4 D).

[0087] The above experiments collectively demonstrate that, in the co-culture system, fungi activate the biofilm formation program of Bacillus belyssus by providing L-proline, which is one of the key intrinsic mechanisms for its enhanced activity and improved rhizosphere adaptability.

[0088] Example 6: Formulation preparation of highly active products

[0089] The co-culture fermentation broth obtained in Example 1 was centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant (i.e., cell-free filtrate) was collected, which can be used directly as a liquid dosage form. Alternatively, a lyophilization protectant (such as 5% trehalose) was added to the supernatant, and after pre-freezing, it was lyophilized in a freeze dryer to obtain a lyophilized powder formulation. When using, the lyophilized powder can be reconstituted with sterile water, or directly mixed with carriers such as diatomaceous earth and clay to prepare powder or granules.

[0090] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0091] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this application and their equivalents, this invention is also intended to include these modifications and variations.

Claims

1. L-proline in the preparation of Bacillus belye ( Bacillus velezensis Application in formulations with biofilm-forming capacity and / or biological activity.

2. *Acetobacter pylori* (Pink Spiral Polyspora) Clonostachys rosea The application of Bacillus berberis or its fermentation products in enhancing the biofilm formation ability of Bacillus berberis.

3. The application according to claim 1 or 2, characterized in that, The enhanced biofilm-forming ability of Bacillus belesi is manifested in any one or more of the following: (1) Enhanced colonization ability and / or stress tolerance of Bacillus belye in plant rhizosphere; (2) Genes related to biofilm matrix synthesis in Bacillus belyssus epsC and / or tasA The level of expression was increased; (3) Bacillus belye has enhanced inhibitory activity against plant parasitic nematodes or improved growth-promoting effects on plants.

4. The application according to claim 3, characterized in that, The *Polyspora pinkis* species is *Polyspora pinkis* with accession number CGMCC No. 16262. Clonostachys rosea NF-06; The Bacillus belyssus mentioned is Bacillus belyssus with accession number CGMCC No. 36152 (NF-06); Bacillus velezensis YB-1652.

5. A method for enhancing the bioactivity of Bacillus belye, characterized in that, Bacillus belye and Polyspora pinkis were co-cultured after being simultaneously inoculated in liquid culture medium.

6. The method according to claim 5, characterized in that, The liquid culture medium is a semi-synthetic culture medium with soybean meal as the main nitrogen source. Each liter of culture medium contains: 16-24 g corn flour, 8-12 g soybean meal, 0.3-0.7 g MgSO4, 0.04-0.06 g FeSO4·7H2O, and 0.04-0.06 g ZnSO4·7H2O.

7. The method according to claim 6, characterized in that, The *Bacillus belye* was inoculated in seed liquid form at a volume percentage of 1-5%; the *Polyspora pinkis* was inoculated in spore suspension form at a volume percentage of 1-5%; the co-culture conditions were: temperature 25-30℃, shaker speed 150-200 rpm, and culture time 48-72 h.

8. The method according to claim 7, characterized in that, The initial concentration or ratio of the co-culture product can be adjusted by adding *Polyspora pinkis* ferment or proline standard to the liquid culture medium, thereby improving the biofilm formation ability or nematode inhibitory activity of the product.

9. A highly active Bacillus belyceae product, characterized in that, The product is prepared by the method described in any one of claims 5-8 and is in the form of fermentation broth, concentrate, freeze-dried powder, or formulation made with an agriculturally acceptable carrier.

10. The use of the highly active Bacillus belyceae product according to claim 9 in any of the following: (1) Control of plant parasitic nematodes; (2) Promote plant growth or enhance plant resistance to adverse conditions; (3) Enhance the colonization ability of Bacillus belye in the plant rhizosphere.

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