Microenvironment modification composition for inducing pseudomonas and serratia biofilm formation and use thereof

Abstract

The application discloses a microenvironment reconstruction composition for inducing Pseudomonas and Serratia to form a film and application thereof. The microenvironment reconstruction composition comprises core film-forming bacteria and a film-forming core guide signal molecule, wherein the core film-forming bacteria include Pseudomonas and Serratia, and the signal molecule comprises osthole. Based on multi-omics analysis of the aerial root mucilage microecosystem under low nitrogen stress of corn, it is found for the first time that osthole can specifically induce the core film-forming bacteria to secrete extracellular polymers and assemble to form a biofilm. Osthole and N-phenylacetyl-L-glutamine have a synergistic effect when used together, and high-efficiency film formation can be realized at a low dose. The biofilm covers the surface of the aerial root of crops, can effectively block the diffusion of oxygen, forms a low-oxygen microenvironment in the film, thereby protecting the nitrogen fixation enzyme activity of indigenous nitrogen-fixing bacteria in situ, and improving the nitrogen fixation efficiency of crops.

Description

Technical Field

[0001] This application relates to the fields of agricultural microbiology and biofertilizer technology, specifically to a microenvironment modification composition that specifically induces Pseudomonas or Serratia to form biofilms, and the application of this composition in constructing a low-oxygen microenvironment, protecting native nitrogen-fixing bacteria in the field, and promoting nitrogen fixation in crops. Background Technology

[0002] Nitrogen is a critical limiting nutrient essential for crop growth. For a long time, agricultural production has heavily relied on the application of chemical nitrogen fertilizers, which has not only significantly increased production costs but also led to a series of serious ecological and environmental problems, including soil acidification and compaction, eutrophication of water bodies, and increased greenhouse gas emissions. Therefore, fully exploring and utilizing the potential of biological nitrogen fixation, while ensuring crop yields, to reduce the input of chemical nitrogen fertilizers, is one of the core pathways to achieving green and sustainable agricultural development. Gramineous crops such as maize produce numerous aerial roots at the base of the stem nodes after the jointing stage. The mucus secreted by the surface of these aerial roots is rich in carbohydrates and secondary metabolites, providing a unique carbon source and habitat for various microorganisms. However, nitrogenase, the core enzyme in biological nitrogen fixation, is extremely sensitive to oxygen. Oxygen can cause irreversible oxidative inactivation of nitrogenase. Maize aerial roots are directly exposed to air, and their mucus microenvironment is in a typical high-oxygen-tension state. This high-oxygen condition severely inhibits the nitrogenase activity of indigenous nitrogen-fixing bacteria in the mucus, greatly limiting the efficiency of in-situ biological nitrogen fixation. How to construct a stable "micro-oxygen / hypo-oxygen sanctuary" for indigenous nitrogen-fixing bacteria in an open agricultural niche without relying on genetic modification or exogenous inoculation for large-scale colonization has become a core challenge in breaking through the bottleneck of efficient biological nitrogen fixation technology for non-leguminous crops.

[0003] In existing technologies, a common strategy is to directly inoculate crop rhizospheres or leaves with highly efficient nitrogen-fixing bacteria. However, due to the lack of effective physical protective barriers, exogenously inoculated nitrogen-fixing bacteria exhibit extremely low survival and colonization rates when facing multiple abiotic stresses in the open field environment, such as high oxygen stress, water fluctuations, ultraviolet radiation, and competition from indigenous microorganisms. This results in highly unstable nitrogen fixation efficiency, falling far short of the expected effects for industrial applications. Although some studies in recent years have attempted to enhance the colonization ability of strains by adding film-forming promoters or biofilm inducers, these strategies are mostly limited to single-strain systems under laboratory conditions. They have neither revealed the intrinsic mechanisms by which plant metabolites drive microenvironmental remodeling in natural ecosystems nor provided a microecological modification solution that can simulate the laws of natural co-evolution and has universal applicability and field operability.

[0004] Therefore, how to discover and simulate the signaling mechanisms by which plants regulate their own microbial communities from natural ecosystems, and how to construct stable, low-oxygen biofilm barriers on crop surfaces in situ through artificial intervention, thereby providing a functional protective microenvironment for indigenous nitrogen-fixing bacteria, while taking into account production costs, formulation stability, and ease of field application, is a practical technical problem that urgently needs to be solved in the field of agricultural microbiology. Summary of the Invention

[0005] The inventors of this application discovered in long-term research on farmland ecosystems under low-available nitrogen treatment that the micro-ecosystem of maize's aerial root mucilage undergoes specific reorganization under low nitrogen stress, manifested as a significant increase in overall nitrogenase activity. Further metagenomic and metabolomics analysis revealed that this increase in nitrogenase activity was not dependent on an increase in the abundance of nitrogen-fixing bacteria, but rather accompanied by the abundant secretion of specific metabolites (such as phenylacetylglutamine and osthol) and a significant enrichment of non-nitrogen-fixing bacteria (such as Pseudomonas and Serratia). This suggests the existence of a cross-species synergistic mechanism driven by plant metabolites in nature. Therefore, this application extracts key signaling molecules and core film-forming microorganisms from the maize aerial root mucilage system and reconstructs this hypoxic microenvironment through artificial intervention, thereby providing a universally applicable strategy for microecological modification.

[0006] Based on this, the purpose of this application is to provide a microenvironment modification composition for inducing film formation by Pseudomonas sp. and Serratia sp., and its application. The microenvironment modification composition comprises Pseudomonas sp. and Serratia sp., and a core film-forming guiding signal molecule, wherein the signal molecule is osthol or a mixture of osthol and phenylacetylglutamine. The core scheme involves using the aforementioned specific metabolites as signal molecules to target and stimulate the core film-forming bacteria to secrete a rapid and large amount of extracellular polymers under conditions that do not significantly increase bacterial biomass, and to synergistically assemble them into a physically dense composite biofilm. This biofilm can construct a physical barrier on the crop surface, effectively blocking the diffusion of oxygen into the internal microregions, thereby forming a microaerobic / hypoxic microenvironment within the membrane. This provides enzyme activity protection for the indigenous nitrogen-fixing bacteria in situ, ultimately achieving the technical effects of improving the efficiency of in-situ nitrogen fixation in crops and reducing the application of chemical nitrogen fertilizers.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] In a first aspect, this application provides a microenvironment modification composition, comprising:

[0009] Core film-forming bacteria include Pseudomonas sp. and Serratia sp.; and

[0010] The film-forming core guides the signaling molecule, which includes osthol.

[0011] In some embodiments, the Pseudomonas is Pseudomonas putida and the Serratia is Serratia marcescens.

[0012] In some preferred embodiments, the *Pseudomonas putida* is derived from the American Center for Type Culture Collection (ATCC) with accession number ATCC49128; and the *Serratia marcescens* is derived from the China Center for Medical Bacteriology Collection (CMCC) with accession number CMCC(B)41002.

[0013] In some preferred embodiments, the film-forming core guiding signal molecule is a mixture of phenylacetylglutamine and osthol.

[0014] In some embodiments, the final concentration of the film-forming core guiding signal molecule in the composition is 50 μM to 100 μM.

[0015] Secondly, this application provides the use of the microenvironment modification composition described in the first aspect in the preparation of formulations for constructing a hypoxic microenvironment.

[0016] Thirdly, this application provides the use of the microenvironment modification composition described in the first aspect in the preparation of formulations for protecting indigenous nitrogen-fixing bacteria or promoting nitrogen fixation in crops.

[0017] Fourthly, this application provides a microenvironment modification method, which includes applying the microenvironment modification composition described in the first aspect to the surface of crop aerial roots.

[0018] In some preferred embodiments, the crop is corn.

[0019] Fifthly, this application provides a complete product for microenvironment modification, comprising individually packaged Agent A and Agent B:

[0020] Agent A contains core film-forming bacteria, including *Pseudomonas putida* and *Serratia marcescens*.

[0021] The agent B contains a film-forming core guiding signal molecule, which is osthol or a mixture of osthol and phenylacetylglutamine;

[0022] Agent A and Agent B are mixed before application.

[0023] Compared with the prior art, this application has at least the following beneficial effects:

[0024] 1. Based on long-term in-situ observations of farmland ecosystems treated with low-efficiency nitrogen, this application, through metagenomic and metabolomics network analysis, for the first time precisely identified phenylacetylglutamine and osthol as specific film-forming signaling molecules from the mucilage of maize aerial roots, and confirmed that *Pseudomonas putida* and *Serratia marcescens* are the core functional strains responsible for film-forming assembly. In vitro validation showed that these film-forming signaling molecules did not significantly increase bacterial biomass (OD). 600 Under the premise of increased activity, it can target and stimulate the core film-forming bacteria to rapidly secrete extracellular polymers and assemble to form a biofilm (OD). 530 (Significantly improved). This application is completely different from the traditional approach of directly adding nitrogen-fixing bacteria to the environment. Instead, it indirectly constructs a physical protective barrier for indigenous nitrogen-fixing bacteria by activating the film-forming microbial community that is widely present in the environment, resulting in higher field adaptability and ecological safety.

[0025] 2. After application, the composition provided in this application forms a composite biofilm actively assembled by the core film-forming bacteria under the guidance of metabolic signaling molecules. This biofilm tightly covers the surface of the crop's aerial roots, effectively blocking the inward diffusion of free oxygen from the air. This creates a stable microaerophilic / hypoxic microenvironment within the biofilm and the surrounding mucus layer. This physical barrier directly protects the indigenous nitrogen-fixing bacteria present in situ in the mucus from irreversible oxidative inactivation of nitrogenase by oxygen, thereby enhancing the crop's ability to capture atmospheric free nitrogen in situ and reducing the need for chemical nitrogen fertilizers.

[0026] 3. This application, through systematic in vitro screening and synergistic verification, discovered that osthol alone can specifically induce biofilm formation in *Pseudomonas putida* or *Serratia marcescens*. Furthermore, when osthol and phenylacetylglutamine are applied in combination, both exhibit a synergistic promoting effect on early bacterial proliferation at doses as low as 50 μM. The biofilm formation in the co-culture system is significantly higher than that in either single-strain system, confirming a synergistic assembly effect between the two metabolites and the two bacterial strains. Crucially, the complete mixture containing all five differential metabolites did not show superior biofilm formation; instead, it confirmed the irreplaceable specificity of the simplified osthol and phenylacetylglutamine combination. This discovery simplifies chemical components and reduces costs, while also providing a flexible option for single or combined use in different field applications.

[0027] 4. The composition of this application is easy to use in the field. It can be evenly sprayed onto the surface of the aerial roots using a conventional agricultural sprayer during the corn's jointing to tasseling stage. Simultaneously, this application provides a highly commercially valuable split-packaging solution—the bacterial fermentation concentrate or freeze-dried powder serves as Agent A, and the signaling molecule composition serves as Agent B, which is mixed and used immediately upon application in the field. This formulation design completely solves the industry pain points of short shelf life and easy inactivation of live bacteria preparations in fertilizer commercial distribution, meeting the actual needs of large-scale agricultural production and possessing extremely high industrial promotion value and market competitiveness. Attached Figure Description

[0028] Figure 1 Figure 1 shows the changes in nitrogen fixation function of maize aerial root mucus and the results of differential microbial abundance analysis under low available nitrogen treatment in Example 1 of this application. Among them, A is a comparison of the content of inorganic nitrogen (ammonium nitrogen and nitrate nitrogen) in rhizosphere soil under normal nitrogen and low available nitrogen treatment; B is a comparison of the content of inorganic nitrogen (ammonium nitrogen and nitrate nitrogen) in non-rhizosphere soil under normal nitrogen and low nitrogen treatment; C is a comparison of nitrogenase activity in aerial root mucus and total nitrogen content in leaves under normal nitrogen and low nitrogen treatment; D is an analysis of the module species proportion of the microbial co-occurrence network in aerial root mucus under low nitrogen conditions; E is an analysis of the abundance difference of Pseudomonas and Serratia at the species level; and F is a microbial source analysis of nitrogen cycle functional genes based on metagenomic data.

[0029] Figure 2 This is a network analysis diagram of the differential metabolites, differential microorganisms, and microbial functional genes in maize aerial root mucus under normal nitrogen and low-efficiency nitrogen treatments in Example 1 of this application; wherein, A is the network of differential metabolites, differential microorganisms, and microbial functional genes in aerial root mucus, B is the heatmap of screening and association of key differential metabolites and differential genes, and C is the correlation between differential metabolites, differential genes, and the abundance of Pseudomonas and Serratia.

[0030] Figure 3 The graph shows the results of biofilm measurement of *Pseudomonas putida* and *Serratia marcescens* under the action of different metabolites in Example 3 of this application; where A represents the biofilm amount of *Pseudomonas putida* under the action of different metabolites, and B represents the biofilm amount of *Serratia marcescens* under the action of different metabolites.

[0031] Figure 4 Figure 4 shows the results of measuring the growth of core film-forming bacteria under the action of compound metabolites and dual-bacterial system in Example 4 of this application. In Figure A, Pseudomonas putida grows under different metabolite treatments, with final metabolite concentrations of 100 μM (left) and 50 μM (right). Figure B shows the growth of Serratia marcescens under different metabolite treatments, with final metabolite concentrations of 100 μM (left) and 50 μM (right).

[0032] Figure 5Figure 4 shows the results of measuring the biofilm amount of the core film-forming bacteria under the action of the compound metabolite and the dual-bacterial system in Example 4 of this application. In Figure 4, Figure A shows the biofilm amount of *Pseudomonas putida* under different metabolite treatments, with final metabolite concentrations of 100 μM (left) and 50 μM (right), respectively. Figure B shows the biofilm amount of *Serratia marcescens* under different metabolite treatments, with final metabolite concentrations of 100 μM (left) and 50 μM (right), respectively. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0034] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0035] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0036] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may be adjusted according to the desired performance. At a minimum, each numerical parameter should be interpreted based on the reported significant figures and by applying common rounding techniques.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following explanations of terminology are provided to better understand this application.

[0038] 1. Core biofilm-forming bacteria: In this application, it refers to functional microorganisms that, under the induction of core guiding signal molecules, secrete extracellular polymers and actively assemble to form biofilms, specifically including *Pseudomonas putida* and *Serratia marcescens*. These core biofilm-forming bacteria do not possess core nitrogen-fixing genes; their main function is to provide a low-oxygen protective microenvironment for indigenous nitrogen-fixing bacteria in the environment by forming a biofilm barrier.

[0039] 2. Film-forming core guiding signal molecule: In this application, it refers to a substance derived from the mucilage metabolites of maize aerial roots that can significantly increase bacterial biomass (OD). 600 Under the premise of ), specific stimulation of core film-forming bacteria to initiate the expression of biofilm-forming related genes and secrete extracellular polymers, and finally assemble small molecule organic compounds to form biofilms, specifically including osthol or a combination of phenylacetylglutamine and osthol.

[0040] The following are specific examples:

[0041] Example 1: Preliminary analysis and identification of key factors regarding nitrogen fixation efficiency and microbial community structure changes in maize aerial root mucus under low-available nitrogen conditions.

[0042] This embodiment aims to identify key microbial species and metabolites that mediate the microecological reorganization of maize aerial root mucus under low-available nitrogen stress by systematically analyzing samples of maize aerial root mucus, rhizosphere soil, and non-rhizosphere soil under low-available nitrogen treatment and conventional nitrogen application in a long-term field, thus providing a data foundation for the construction of subsequent compositions.

[0043] 1.1 Experimental Design

[0044] Experimental location: Henan Modern Agricultural Experimental Demonstration Base, Yuanyang County, Xinxiang City, Henan Province.

[0045] This study was conducted at the Yuanyang Experimental Base of the Henan Academy of Agricultural Sciences, using a long-term, fixed-location experimental field for wheat-maize rotation (starting in 2015). The experimental plots were arranged in a randomized block design, with a plot area of ​​18.24 m². 2 The specific management measures for the experiment are as follows: Wheat season management: Fertilizer application rate: Pure nitrogen (N) application rate was 14 kg / mu. Phosphate fertilizer (P2O5) and potassium fertilizer (K2O) application rates were both 6.0 kg / mu. Application method: Phosphate and potassium fertilizers were applied as base fertilizer in one application. Nitrogen fertilizer was applied in multiple applications according to the ratio of "base fertilizer: jointing stage: grain filling stage = 5:3:2". Irrigation management: Irrigation was carried out at the jointing stage and the grain filling stage, with each irrigation volume being 20 m³. 3 / mu. Maize season management: Fertilizer application rate: Pure nitrogen (N) application rate is 18 kg / mu. Phosphate fertilizer (P2O5) application rate is 6.0 kg / mu, and potassium fertilizer application rate is 6.0 kg / mu. Management method: Nitrogen fertilizer is applied in multiple applications; ordinary urea is applied as topdressing at the jointing and grain-filling stages. Irrigation management: Irrigation is carried out at the jointing and grain-filling stages, with each irrigation volume being 20 m³. 3 / mu.

[0046] Experimental Treatments: During the maize season, three different treatments were selected: conventional nitrogen application (NPK) and low-available nitrogen (LRF) treatment. The LRF treatment included two methods: 20% nitrogen reduction + organic fertilizer (NPK+M) and 20% nitrogen reduction + slow-release fertilizer (NPK+SRF). Four plots with the same treatment were selected as replicates. Sample Collection: At the maize tasseling stage, a five-point sampling method was used to select five plants from each treatment's corresponding plot. The mucus on the surface of the aerial roots of the five plants was collected using sterile collection tubes and mixed to form one replicate. Four replicates were collected for each treatment, resulting in three samples per treatment. Two samples were flash-frozen in liquid nitrogen and stored at -80°C for DNA extraction and metabolomics assays, respectively; the other sample was used for nitrogenase activity assays. Non-rhizosphere soil samples were collected from the five corresponding plants using conventional soil sampling methods. Rhizosphere soil samples were collected from the root surface of the corresponding plants using a sterile fine brush and wrapped in sterile aluminum foil. Four replicates were collected for each treatment. A portion of the rhizosphere and non-rhizosphere soil samples were flash-frozen in liquid nitrogen and stored at -80°C for multi-omics analysis, while the remaining portion was used for physicochemical index analysis. Leaves from the corresponding plants were also collected simultaneously, with four replicates collected for each treatment.

[0047] 1.2 Determination of soil physicochemical properties and nitrogenase activity

[0048] The soil physicochemical properties and nitrogenase activity in aerial root mucilage were determined using the following methods:

[0049] Soil ammonia nitrogen and nitrate nitrogen were determined using the potassium chloride solution extraction-spectrophotometric method (HJ 634—2012), and plant nitrogen content was determined using the NY / T 2017-2011 standard method. For the determination of nitrogenase activity, approximately 10 mL of collected aerial root mucilage was placed in a 20 mL headspace vial and sealed. 10% of the air was removed, and an equal volume of high-purity acetylene gas was injected. The vial was incubated at 28 ℃ in the dark for 2–4 hours. Headspace gas was then extracted, and the amount of ethylene produced was detected using gas chromatography (GC). Nitrogenase activity was calculated using a no-acetylene control and a no-mucilage control as corrections. For each treatment, three replicates were performed for the determination of physicochemical indicators and enzyme activity.

[0050] Results: Under low nitrogen treatment, the available nitrogen content in both rhizosphere and non-rhizosphere soils was significantly lower than that under conventional nitrogen application, confirming the successful establishment of a low-nitrogen environment. Simultaneously, the activity of nitrogenase in aerial root mucilage and the total nitrogen content of plants were significantly increased under low nitrogen treatment compared to conventional nitrogen application. Figure 1 (AC). This indicates that the mucilage micro-ecosystem of maize aerial roots exhibits a strong in-situ nitrogen fixation compensation effect under low nitrogen conditions.

[0051] 1.3 Microbiome and metagenomic sequencing analysis

[0052] Total DNA was extracted from samples using the FastDNA Spin Kit for Soil (MP Biomedicals). PCR amplification was performed targeting the V5-V7 region of the bacterial 16S rRNA gene (primers 799F / 1193R) and the ITS1 region of the fungus (primers ITS1F / ITS2). The purified products were then subjected to paired-end sequencing (2 × 300 bp) on the Illumina MiSeq platform. Raw data were processed using the QIIME2 workflow, and amplicon sequence variant (ASV) table was obtained through DADA2 noise reduction. Species annotation was performed based on the SILVA and UNITE databases, respectively. Alpha diversity was assessed using Chao1 and Shannon indices calculated based on ASV levels, and Beta diversity was assessed using principal coordinate analysis (PCoA) based on Bray-Curtis distance. LEfSe analysis was used to screen for differentially expressed species among groups. Microbiome analysis was performed in quadruplicate for each treatment.

[0053] Based on amplicon analysis results, metagenomic sequencing was performed on core samples. Three replicates were performed for each treatment. Paired-end sequencing (150 bp) was performed using the NovaSeq 6000 platform, with each sample yielding at least 10 Gb of data. High-quality reads were assembled using Megahit software, and open reading frames were predicted using MetaGeneMark to construct a non-redundant gene set. The gene set was aligned with the KEGG and NCBI-nr databases for functional annotation, and HMMER was used to search for key genes involved in nitrogen cycling, chemotaxis, and biofilm formation, calculating their total nitrogen per mille (TPM) abundance. Functional differential analysis was performed using STAMP software.

[0054] Clean reads were aligned to the assembled contigs using BWA-MEM (v.0.7.17) and SAMtools (v1.546) to generate a sorted BAM file. Based on this BAM file, the sequencing depth of each contig was calculated using the script "jgi_summarize_bam_contig_depths" in the MetaBAT2 (v.2.12.1) package. Subsequently, the contigs were binning using MetaBAT2 (default parameters, minimum contig length ≥ 1500 bp) to obtain metagenomic assembled genomes (MAGs). The quality (integrity and contamination) of the MAGs was evaluated using the lineage_wf workflow of CheckM2 (v.1.0.2), and assembly information was obtained. Redundancy was removed from the MAGs using dRep (ANI > 95%), and the MAG with the highest QualityScore (defined as integrity - 5 × contamination) was selected as the representative MAG. Finally, GTDB-Tk (version r214) was used to perform taxonomic annotations on each MAG.

[0055] Results: Under low nitrogen conditions, the relative abundance of Pseudomonadota microorganisms significantly increased in aerial root mucus, becoming a core node in the microecological network. Figure 1 D). Further analysis of species-level differential microorganisms revealed that the abundance of *Pseudomonas putida* and *Serratia marcescens* significantly increased under low-nitrogen treatment. Figure 1 E). Functional gene tracing analysis showed that neither of these microorganisms possessed a core nitrogen-fixing gene (E). Figure 1 F).

[0056] 1.4 Non-targeted metabolomics analysis

[0057] 100 μL of mucus sample was placed in an EP tube, and 400 μL of 80% methanol aqueous solution was added. Each treatment was measured in quadruplicate. The sample was vortexed, incubated on ice for 5 min, and centrifuged at 15000 g, 4℃ for 20 min. An appropriate amount of supernatant (volume determined according to preliminary experiments) was taken and diluted with mass spectrometry-grade water to a final methanol concentration of 53%. The sample was centrifuged at 15000 g, 4℃ for 20 min, and the supernatant was collected and analyzed by LC-MS. UHPLC-MS / MS analysis was performed at BIOZERON Co., Ltd. in Shanghai, using a Vanquish UHPLC system equipped with an Orbitrap Q Exactive™ HF or Orbitrap Q Exactive™ HF-X mass spectrometer (both purchased from Thermo Fisher Scientific, Germany). Samples were injected into a Hypersil Gold column (100 × 2.1 mm, 1.9 μm) for separation at a flow rate of 0.2 mL / min using a 12-minute linear gradient elution. The mobile phases for both positive and negative ion modes were phase A (water + 0.1% formic acid) and phase B (methanol). The solvent gradient settings were as follows: 0–1.5 min, 2% B; 1.5–3 min, 2%–85% B; 3–10 min, 85%–100% B; 10–10.1 min, 100%–2% B; 10.1–12 min, 2% B. The mass spectrometer was operated in positive / negative ion switching mode, with the following main parameters: spray voltage 3.5 kV, capillary temperature 320°C, sheath gas pressure 35 psi, auxiliary gas flow rate 10 L / min, S-lens RF level 60, and auxiliary gas heating temperature 350°C. The raw UHPLC-MS / MS data were processed using Compound Discoverer 3.3 (CD3.3, ThermoFisher) software for peak alignment, peak extraction, and metabolite quantification. Metabolites were annotated using the KEGG, HMDB, and LIPIDMaps databases. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed using MetaX software. Statistical significance (P-value) was calculated using univariate analysis (t-test). Metabolites with a VIP > 1 and P < 0.05, and a fold change (FC) ≥ 2 or ≤ 0.5, were selected as differential metabolites.

[0058] Correlation network analysis was performed on the mucus differential metabolites, microbial abundance, functional gene abundance, and nitrogenase activity of different treatment groups. Combined with FC values ​​and the degree of association with core microorganisms, core differential metabolites were screened.

[0059] Results: Preliminary analysis of non-targeted metabolomics showed that the composition of mucus metabolites differed significantly between low-nitrogen and normal-nitrogen environments. Correlation network analysis of differentially metabolites, differentially abundant microorganisms, and functional genes revealed that the relative contents of specific metabolites such as isoliquiritin, osthol, sorbitol, phenylacetylglutamine, and N-acetyl-L-asparagine were not only significantly upregulated under low-nitrogen conditions, but also showed highly significant positive correlations with the abundance of *Pseudomonas putida* and *Serratia marcescens*, as well as biofilm formation-related genes. Figure 2 AC).

[0060] Based on the above multi-omics in situ data, the metabolites isoglycyrrhizin, osthol, sorbitol, phenylacetylglutamine, and N-acetyl-L-asparagine were identified as key coupling factors for modifying the microecological environment, along with *Pseudomonas putida* and *Serratia marcescens*. Since *Pseudomonas* is a widely known microbial community in nature, to verify the universality of this mechanism, subsequent validation experiments used commercially available *Pseudomonas putida* (ATCC49128) and *Serratia marcescens* (CMCC(B)41002).

[0061] Example 2: Preparation of Microenvironment Modification Composition

[0062] This embodiment provides a method for preparing a microenvironment modification composition, the composition comprising a core film-forming bacterial solution and a film-forming core guiding signal molecule.

[0063] 2.1 Strain activation and bacterial culture preparation

[0064] (1) Source of strains: Pseudomonas putida, purchased from Shenzhen Baoan District Xins Biotechnology Business Department, accession number ATCC49128. Serratia marcescens, purchased from Shenzhen Baoan District Xins Biotechnology Business Department, accession number CMCC(B)41002.

[0065] (2) Activation of bacterial strains: Pseudomonas putida (ATCC49128) and Serratia marcescens (CMCC(B)41002) preserved in glycerol were streaked onto LB agar plates (10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride) and incubated upside down at 28 °C for 24 h. Single colonies were picked and inoculated into test tubes containing 5 mL of LB liquid medium (excluding agar powder, the rest of the formula is the same as above) and incubated at 28 °C and 180 rpm for 12 h until the logarithmic growth phase.

[0066] (3) Adjustment of bacterial concentration: The bacterial culture that has been cultured to the logarithmic growth phase is serially diluted with sterile LB liquid medium or sterile physiological saline to adjust the bacterial concentration to 1×10⁻⁶. 6 CFU / mL.

[0067] 2.2 Preparation of the mother liquor for guiding signal molecules in film formation

[0068] (1) Metabolite information and source: Purchased from Aladdin Reagents (Shanghai) Co., Ltd.

[0069] Osthol: 7-methoxy-8-8-isopentenylcoumarin, molecular formula C 15 H 16 O3, CAS: 484-12-8; O408868-1ml.

[0070] Phenylacetylglutamine: Molecular formula C 13 H 16 N2O4, CAS: 28047-15-6; S422982-1ml.

[0071] Isoliquiritin: (E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one, molecular formula C 15 H 12 O4, CAS: 961-29-5; I407952-1ml.

[0072] Sagolacin: (S)-7-hydroxy-5-methoxy-2-phenylchroman-4-one, molecular formula C 16 H 14 O4, CAS: 3853-83-6; A423623-1ml.

[0073] N-Acetyl-L-Asparagine: Molecular formula C6H 10 N2O4, CAS: 1492-63-3; N580468-1ml.

[0074] (2) Preparation of stock solutions: Osthol and phenylacetylglutamine were mixed in an equimolar ratio and added to a sterile aqueous solution to prepare a 10 mM single stock solution or a mixed stock solution. At the same time, isoliquiritigenin, sorbitol, and N-acetyl-L-asparagine were each prepared into 10 mM single stock solutions using the same method for use in subsequent experiments.

[0075] 2.3 Formulation of Microenvironment Modification Composition

[0076] The Pseudomonas putida bacterial suspension or Serratia marcescens bacterial suspension (or a mixture of both) prepared in step 2.1 is mixed with the metabolite mother liquor prepared in step 2.2 at an appropriate volume ratio so that the final concentration of metabolites in the mixed system reaches 50 μM or 100 μM, thus obtaining the microenvironment modification composition.

[0077] 2.4 Quality Control

[0078] Each batch of prepared compositions should be sampled and tested: the viable count should be determined using the plate coating method to ensure that the bacterial concentration reaches 1×10⁻⁶. 6 CFU / mL; the actual concentration of metabolites is detected by high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) to ensure that the preparation accuracy is within ±10% of the labeled concentration.

[0079] Example 3: Inducing effects of different metabolites on the growth of core biofilm-forming bacteria and biofilm formation

[0080] This embodiment aims to systematically screen the induction effects of five differential metabolites (isoglycyrrhizin, osthol, sorbitol, phenylacetylglutamine, and N-acetyl-L-asparagine) identified in Example 1 on biofilm formation of core film-forming bacteria (Pseudomonas putida and Serratia marcescens) using a 96-well plate crystal violet staining method, in order to screen for signaling molecules with specific film-forming induction activity.

[0081] 3.1 Experimental Materials

[0082] Test strains: The source of the strains and the method of preparing the bacterial solution are the same as in Example 2.

[0083] Test metabolites: a single stock solution (10 mM) of isoliquiritigenin, osthol, sorbitol, phenylacetylglutamine, and N-acetyl-L-asparagine, and a mixed stock solution of the five metabolites (each component was mixed in equimolar ratio, with a total concentration of 10 mM), prepared in the same manner as in Example 2.

[0084] 3.2 Experimental Design

[0085] A total of 16 treatment groups were set up, with each treatment repeated three times (n=3). The specific groupings are as follows:

[0086] Control group 1: *Pseudomonas putida* bacterial suspension + sterile water;

[0087] Control group 2: Serratia marcescens bacterial suspension + sterile water;

[0088] Isoliquiritin group 1: Pseudomonas putida bacterial suspension + isoliquiritin;

[0089] Group 2: Serratia marcescens bacterial solution + isoliquiritigenin;

[0090] Osthol group 1: *Pseudomonas putida* bacterial suspension + osthol;

[0091] Group 2: Serratia marcescens bacterial solution + osthol;

[0092] Alpinia sorbitol group 1: *Pseudomonas putida* bacterial culture + alkaloids;

[0093] Group 2: Serratia marcescens bacterial solution + galangin;

[0094] Phenylacetylglutamine group 1: *Pseudomonas putida* bacterial culture + phenylacetylglutamine;

[0095] Group 2: Serratia marcescens bacterial culture + phenylacetylglutamine;

[0096] Group 1: Pseudomonas putida bacterial suspension + N-acetyl-L-asparagine;

[0097] Group 2: Serratia marcescens bacterial culture + N-acetyl-L-asparagine;

[0098] Mixed metabolite group 1: *Pseudomonas putida* bacterial culture + isoliquiritigenin + phenylacetylglutamine + N-acetyl-L-asparagine;

[0099] Mixed metabolite group 2: Serratia marcescens bacterial culture + isoliquiritigenin + phenylacetylglutamine + N-acetyl-L-asparagine;

[0100] Mixed metabolite group 3: *Pseudomonas putida* bacterial culture + mixture of five metabolites;

[0101] Mixed metabolite group 4: Serratia marcescens bacterial culture + mixture of five metabolites.

[0102] 3.3 Biofilm assay methods

[0103] The bacterial culture, having reached the logarithmic growth phase, was inoculated into 96-well plates containing sterile LB liquid medium. The inoculum size was adjusted to achieve the desired optical density (OD) at 600 nm. 600The initial concentration of the metabolite was 0.1. Then, 2 μL of the metabolite or a mixture of metabolites (initial concentration 10 mM) was added to the mixing system, bringing the total volume of the bacterial culture, metabolites, and culture medium to 200 μL. Therefore, the final concentration of the metabolite in each well reached 100 μM. The 96-well plate was then incubated at 28°C and 150 rpm with shaking. After 48 h of incubation, the liquid contents were removed, and the wells were washed with sterile double-distilled water. The wells were then stained with 0.1% crystal violet for 20 min and washed three times with sterile double-distilled water. The biofilm structure was removed from the wells by adding 2 mL of a 4:1 (v / v) solution of ethanol and acetic acid and gently shaking until completely dissolved. The optical density of the resulting solution was measured at 530 nm using a microplate reader to quantify the amount of biofilm formed. Each treatment was repeated three times.

[0104] 3.4 Experimental Results

[0105] The results are as follows Figure 3 As shown:

[0106] Single metabolite treatment effects: Osthol alone significantly increased the biofilm mass of *Pseudomonas putida* and *Serratia marcescens*. However, phenylacetylglutamine alone did not significantly increase the biofilm mass of either strain. This result suggests that phenylacetylglutamine may not directly act as the dominant catalyst for film formation in the construction of the microecological environment, but rather plays a potential role in community initiation or compatibility signal regulation.

[0107] Effects of mixed metabolite treatments: The mixed treatment groups containing isoliquiritigenin, phenylacetylglutamine, and N-acetyl-L-asparagine (mixed metabolite groups 1 and 2) showed no significant promoting effect on biofilm formation of the two bacteria. The full mixture treatment groups containing all five metabolites (mixed metabolite groups 3 and 4) did not show superior film-forming effects, and their OD... 530 The values ​​were not significantly higher than those of the control group, and were even lower than those of the group that received osthol alone.

[0108] The above results indicate that osthol, a metabolite significantly enriched in the mucilage of maize aerial roots under low-nitrogen conditions, can specifically stimulate *Pseudomonas* and *Serratia*, bacteria capable of biofilm formation, to secrete extracellular polymers, enabling them to actively assemble into physically dense biofilm structures within a short period. A complete mixture containing all five metabolites did not exhibit superior film-forming effects; instead, it confirmed the specificity and irreplaceable nature of osthol as a core film-forming signaling molecule. This discovery successfully simplifies the chemical composition, significantly reducing the raw material costs for future industrial production and compounding. This finding provides extremely strong evidence for subsequent simulation of natural conditions to construct low-oxygen microenvironments and protect indigenous nitrogen-fixing bacterial communities.

[0109] Example 4: Synergistic effect of compound metabolites and co-culture of two bacteria on the growth of core biofilm-forming bacteria and biofilm formation

[0110] Based on the preliminary screening results of Example 3, osthol alone exhibits film-forming specificity, while phenylacetylglutamine alone has no significant effect. To further investigate whether there is a synergistic effect when these two core metabolites are used in combination, and to examine the spatial community compatibility of the two core film-forming bacteria (Pseudomonas putida and Serratia marcescens), this example conducted in-depth validation of the compound metabolites and co-culture of the two bacteria.

[0111] 4.1 Experimental Materials

[0112] Test strains: Same as in Example 3. In the co-culture group, *Pseudomonas putida* and *Serratia marcescens* were mixed at a 1:1 (volume ratio).

[0113] Test metabolites: osthol single-component stock solution (10 mM), phenylacetylglutamine single-component stock solution (10 mM), and osthol and phenylacetylglutamine mixed stock solution in equimolar ratio (5 mM each, total concentration 10 mM), prepared in the same way as in Example 2.

[0114] 4.2 Experimental Design

[0115] This embodiment included 11 treatment groups, with each treatment repeated three times (n=3). Two levels of final metabolite concentration were set: 100 μM and 50 μM. The specific groupings are as follows:

[0116] Control group 1: *Pseudomonas putida* bacterial suspension + sterile water;

[0117] Control group 2: Serratia marcescens bacterial suspension + sterile water;

[0118] Phenylacetylglutamine group 1: *Pseudomonas putida* bacterial culture + phenylacetylglutamine;

[0119] Group 2: Serratia marcescens bacterial culture + phenylacetylglutamine;

[0120] Osthol group 1: *Pseudomonas putida* bacterial suspension + osthol;

[0121] Group 2: Serratia marcescens bacterial solution + osthol;

[0122] Mixed metabolite group 1: *Pseudomonas putida* bacterial culture + phenylacetylglutamine + osthol;

[0123] Mixed metabolite group 2: Serratia marcescens bacterial culture + phenylacetylglutamine + osthol;

[0124] Bis-bacterial metabolite group 1: *Pseudomonas putida* bacterial culture + *Serratia marcescens* bacterial culture + phenylacetylglutamine;

[0125] Bistrophic metabolite group 2: *Pseudomonas putida* bacterial suspension + *Serratia marcescens* bacterial suspension + osthol;

[0126] The compound metabolite dual-bacterial treatment group consisted of *Pseudomonas putida* bacterial suspension + *Serratia marcescens* bacterial suspension + phenylacetylglutamine + osthol.

[0127] 4.3 Methods for determining bacterial cell growth and biofilm quantity

[0128] The specific procedures for bacterial growth and biofilm measurement were the same as in Example 3, except that the metabolites were set at 100 μM and 50 μM, respectively. After 12 h of culture, the 96-well plate was removed. The absorbance (OD600) of the bacterial cells was measured at 600 nm using a microplate reader to assess the real-time proliferation (growth rate). After 48 h of culture, the biofilm amount was measured using the same method as in Example 3. Each treatment was repeated three times.

[0129] 4.4 Experimental Results

[0130] 4.4.1 Effects of complex metabolites and dual-strain system on bacterial growth (OD) 600 The impact of

[0131] The results are as follows Figure 4 As shown. After 12 hours of incubation:

[0132] Single-strain culture system: whether it is *Pseudomonas putida* (… Figure 4 A) or Serratia marcescens ( Figure 4 B), the addition of either osthol or phenylacetylglutamine alone showed a promoting effect on bacterial growth. When osthol and phenylacetylglutamine were applied in combination (100 μM or 50 μM), the growth (OD) of the two strains was significantly different. 600 The levels of both were significantly higher than those of the blank control group, and the growth-promoting effect was still maintained at a low concentration of 50 μM.

[0133] Co-culture system: In a co-culture system of *Pseudomonas putida* and *Serratia marcescens*, the total proliferation (OD) after application of a compound metabolite (osthol + phenylacetylglutamine) or a single metabolite was measured. 600 No inhibition due to niche competition was observed; OD 600 The values ​​remained within a stable and reasonable range at both 100 μM and 50 μM.

[0134] The above results indicate that osthol and phenylacetylglutamine metabolites have excellent synergistic or complementary effects on the early proliferation of core film-forming bacteria. Furthermore, the dual-bacterial compound system constructed in this application exhibits excellent community compatibility and synergistic proliferation activity mediated by the compound metabolites, laying a good biomass foundation for subsequent synergistic film formation.

[0135] 4.5.2 Effects of complex metabolites and dual-strain system on biofilm quantity (OD) 530 The impact of

[0136] The results are as follows Figure 5 As shown. After 48 h of static culture:

[0137] Single-strain system: For *Pseudomonas putida* (… Figure 5 A) with Serratia marcescens ( Figure 5 In the single-strain system of B), at a concentration of 100 μM, osthol significantly promoted biofilm formation in both bacteria. Phenylacetylglutamine alone showed no significant promoting effect at concentrations of 100 μM and 50 μM, consistent with the results of Example 3.

[0138] Two-strain co-culture system: When two core strains are co-cultured, the biofilm formation is significantly higher than in either single-strain system. At a low dose of 50 μM, the biofilm formation is significantly increased in both the two-strain and two-metabolite co-culture systems. Figure 5 A and 5B are shown on the right.

[0139] The above results indicate that phenylacetylglutamine, which has a limited effect when used alone, can produce a synergistic and complementary effect when combined with osthol. Pseudomonas and Serratia exhibit good spatial assembly synergy in a two-strain co-culture system. The complex metabolite (osthol + phenylacetylglutamine) at low doses (50 μM) can specifically activate the two-strain complex community to secrete large amounts of extracellular polymers, actively assembling into a dense and highly resistant complex biofilm barrier.

[0140] 4.6 Conclusion

[0141] This embodiment, through systematic in vitro synergistic verification, ultimately revealed a strong metabolic compatibility and spatial assembly synergistic effect between the dual signaling molecules osthol and phenylacetylglutamine and the dual bacterial communities of *Pseudomonas putida* and *Serratia marcescens*: osthol alone can efficiently induce single-bacterial film formation, while phenylacetylglutamine, although showing no significant film-forming activity when used alone, can synergistically promote the early proliferation of both bacteria at low doses (50 μM) when used in combination with osthol. Figure 4 Furthermore, in a dual-bacterial co-culture system, it can specifically activate *Pseudomonas putida* and *Serratia marcescens* to secrete large amounts of extracellular polymers, actively assembling them into a dense, highly resistant composite biofilm barrier. Figure 5 ).

[0142] Example 5: Application method and prospects of the composition of this application in field crops.

[0143] This embodiment aims to provide a specific method for applying the microenvironment modification composition described in this application to maize crops in the field, and to illustrate its application prospects in constructing a low-oxygen microenvironment, protecting indigenous nitrogen-fixing bacteria, and promoting crop nitrogen fixation.

[0144] 5.1 Field preparation of the composition

[0145] The microenvironment modification composition prepared in Example 2 was diluted in the field to achieve a bacterial concentration of 1×10⁻⁶ in the final spray solution. 6 CFU / mL (i.e., the total number of viable Pseudomonas putida and Serratia marcescens in the composition), with the final metabolite concentration adjusted to 50 μM–100 μM. Considering application costs, a 50 μM mixture of metabolites can be added directly.

[0146] Preparation method: Take the bacterial stock solution from Example 2 (containing *Pseudomonas putida* and *Serratia marcescens*, concentration 1×10⁻⁶) 6 Add the CFU / mL solution of osthol and phenylacetylglutamine metabolites to field water or irrigation water in an appropriate ratio. Add a conventional agricultural surfactant (such as 0.01% Tween-20 or an equivalent agricultural organosilicon adjuvant; specific brand and concentration to be added later), and stir thoroughly to prepare an aerial root targeted spray solution. Prepare and use immediately; do not store for extended periods.

[0147] 5.2 Field application method

[0148] Application period: From the jointing stage to the large trumpet stage of corn, which is the stage when a large number of aerial roots occur.

[0149] Application time: Spray in the evening (around sunset) to avoid the adverse effects of high temperature and strong ultraviolet radiation on the activity of the strain.

[0150] Application method: Use a conventional agricultural sprayer (such as a backpack sprayer or a towed sprayer; specific models and nozzle types will be added later) to evenly spray the prepared spray solution onto the surface of the corn aerial roots and their secreted mucus. The amount of spray should be enough to completely wet the surface of the aerial roots without causing runoff.

[0151] 5.3 Mechanisms and Effects of Microenvironment Modification

[0152] After application, *Pseudomonas putida* and *Serratia marcescens* secrete extracellular polymers and form a biofilm under the stimulation of metabolites. This biofilm effectively blocks the penetration of free oxygen from the air, providing a low-oxygen microenvironment for the indigenous free-living nitrogen-fixing microbial community encased in the mucus of the aerial roots. This eliminates the inhibition of nitrogenase activity by high oxygen levels and indirectly enhances the maize plant's ability to capture atmospheric free nitrogen in situ.

[0153] 5.4 Dosage Form Design and Industrial Application

[0154] In practical industrial applications of this application, the composition can be commercialized using separate packaging:

[0155] Agent A (bacterial agent): is a fermentation concentrate or freeze-dried powder of core film-forming bacteria (containing Pseudomonas putida and Serratia marcescens), which can be packaged in aseptic aluminum foil bags or high-barrier plastic bottles.

[0156] Agent B (Signaling molecule agent): A film-forming core guiding signaling molecule composition, comprising a high-concentration water-based emulsion or wettable powder of osthol or a mixture of osthol and phenylacetylglutamyl, with accompanying cosolvents (alkyl glycosides or ethanol / glycerol system) and surfactants.

[0157] Pre-application procedures: Before applying in the field, mix Agent A and Agent B in a sprayer or mixing tank and use immediately. Add an appropriate amount of water to dilute to the target concentration and spray immediately.

[0158] This formulation design completely solves the industry pain points of short shelf life and easy inactivation of live bacteria preparations in the commercial distribution of field fertilizers. The live bacteria (A agent) and the signaling molecule (B agent) are isolated from each other during storage. The signaling molecule does not prematurely trigger the film-forming metabolic energy consumption of the strains, allowing the strains to remain dormant or in a low-metabolic state. Only when mixed and used immediately in the field do the two functionally meet and initiate the film-forming process. This design has significant commercialization and industrialization potential.

[0159] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A microenvironment modification composition comprising: Core film-forming bacteria, including Pseudomonas and Serratia; and The film-forming core guides the signaling molecule, which includes osthol.

2. The microenvironment modification composition according to claim 1, wherein the Pseudomonas is Pseudomonas putida and the Serratia is Serratia marcescens.

3. The microenvironment modification composition according to claim 2, wherein the *Pseudomonas putida* is derived from the American Center for Type Culture Collection (ATCC49128); and the *Serratia marcescens* is derived from the China Center for Medical Bacteriology Collection (CMCC(B)41002).

4. The microenvironment modification composition according to claim 1, wherein the film-forming core guiding signal molecule is a mixture of phenylacetylglutamine and osthol.

5. The microenvironment modification composition according to claim 1, wherein the final concentration of the film-forming core guiding signal molecule is 50 μM to 100 μM.

6. The use of the microenvironment modification composition according to any one of claims 1-5 in the preparation of formulations for constructing a hypoxic microenvironment.

7. The use of the microenvironment modification composition according to any one of claims 1-5 in the preparation of formulations for protecting indigenous nitrogen-fixing bacteria or promoting nitrogen fixation in crops.

8. A method for modifying a microenvironment, comprising applying the microenvironment modification composition of any one of claims 1-5 to the surface of crop aerial roots.

9. The microenvironment modification method according to claim 8, wherein the crop is corn.

10. A complete product for microenvironment modification, comprising individually packaged Agent A and Agent B, wherein: Agent A contains core film-forming bacteria, including *Pseudomonas putida* and *Serratia marcescens*. The agent B contains a film-forming core guiding signal molecule, which is osthol or a mixture of osthol and phenylacetylglutamine; Agent A and Agent B are mixed before application.

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