Method for promoting root rot resistance by analyzing soybean core endophyte flora based on fluorescence labeling

Abstract

The invention relates to the technical field of biomedical engineering, and discloses a method for promoting root rot resistance by analyzing soybean core endophyte flora based on fluorescence labeling, and the method comprises the following steps: S1, respectively separating microbial communities from root systems of healthy soybean plants and susceptible soybean plants to obtain a healthy group microbial suspension and a susceptible group microbial suspension; according to the method for promoting root rot resistance by analyzing soybean core endophyte flora based on fluorescence labeling, phytophthora sojae and associated microorganisms are specifically recognized through an immunofluorescence labeling technology, fluorescence-negative flora in healthy soybean root systems is precisely enriched in combination with flow cytometry sorting, screening leakage of non-culturable microorganisms is avoided from the source, and the yield of root rot resistance is improved. Meanwhile, the invalid workload in the screening process is greatly reduced, the mining period of the core beneficial endophytic flora is shortened, and the pertinence and efficiency of the screening process are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a method for promoting resistance to root rot disease by analyzing the core endophytic flora of soybeans based on fluorescent labeling. Background Technology

[0002] In the field of biomedical engineering, soybean root rot, a devastating soil-borne disease affecting global soybean production, faces key challenges in its control due to the lack of precise pathogen localization and targeted discovery of beneficial functional microorganisms. In recent years, utilizing beneficial plant endophytic microorganisms for biological control has become an important strategy for green and sustainable plant protection. Plant endophytes are microorganisms that live within plant tissues without causing obvious disease symptoms. They help plants resist pathogen infection through various mechanisms, including nutrient competition, antimicrobial production, and induction of systemic resistance. However, the following technical bottlenecks still exist in the practical operation of discovering beneficial endophytes.

[0003] Traditional screening of functional strains mainly relies on blind plate isolation and in vitro confrontation culture. This method is labor-intensive, time-consuming, and most environmental microorganisms are unculturable, resulting in extremely low positive screening rates. Secondly, while modern molecular ecology techniques such as high-throughput sequencing can comprehensively analyze the structure of microbial communities, they cannot directly identify functional strains, making it difficult to translate research results into practical applications. Furthermore, existing pathogen detection techniques such as enzyme-linked immunosorbent assays (ELISA) and polymerase chain reactions (PCR) can only determine the infection status of plants, which is separate from the process of discovering beneficial functional bacteria.

[0004] Therefore, there is an urgent need in this field for a method to promote resistance to root rot by analyzing the core endophytic flora of soybeans based on fluorescent labeling, in order to solve the above problems. Summary of the Invention

[0005] The technical problem this invention aims to solve is that traditional methods have the disadvantage of being unable to efficiently and accurately identify functional strains directly related to plant disease resistance from complex microbial communities. To address this, we propose a method based on fluorescent labeling to analyze the core endophytic flora of soybeans to promote resistance to root rot.

[0006] To achieve the above objectives, this application adopts the following technical solution: a method for promoting resistance to root rot based on fluorescent labeling analysis of soybean core endophytic flora, comprising the following steps:

[0007] S1: Microbial communities were isolated from the roots of healthy soybean plants and diseased soybean plants, respectively, to obtain microbial suspensions of the healthy group and the diseased group.

[0008] S2: Using specific antibodies against Phytophthora soybeanis, immunofluorescence labeling was performed on microorganisms in the healthy group and the diseased group microbial suspensions, respectively.

[0009] S3: Use flow cytometry to sort the labeled healthy group microbial suspension and collect the microbial population with fluorescence signal intensity below a preset threshold, which is recorded as the healthy group fluorescence negative population;

[0010] S4: Nucleic acid extraction and high-throughput sequencing were performed on the healthy fluorescent negative population, and the core beneficial endophytic bacteria related to healthy soybean growth were identified based on the sequencing data;

[0011] S5: Perform functional verification on the identified core beneficial endophytic flora.

[0012] Preferably, in S1, an optimized extraction buffer is used to isolate the microbial community, the optimized extraction buffer containing polysaccharide hydrolase and protease inhibitor.

[0013] Preferably, the polysaccharide hydrolase includes cellulase and pectinase.

[0014] Preferably, the specific antibody in S2 is a combination of two antibodies, including a monoclonal antibody against the cell wall structural proteins of Phytophthora soybean and a polyclonal antibody against the secretory effector proteins of Phytophthora soybean.

[0015] Preferably, the preset threshold in S3 is determined by using a healthy microbial suspension without labeled specific antibodies as a negative control, and using the mean fluorescence signal intensity plus three times the standard deviation as the preset threshold.

[0016] Preferably, the determination system for identifying the core beneficial endophytic flora described in S4 includes abundance correlation, intragroup-specific enrichment, universality, and functional indicative indicators.

[0017] Preferably, the functional verification described in S5 includes in vitro antibacterial experiments and in vivo efficacy evaluation.

[0018] Preferably, the concentrations of the healthy group microbial suspension and the infected group microbial suspension are adjusted to... When adjusting the concentration, use sterile phosphate buffer for gradient dilution, and observe the integrity of the microbial morphology under a microscope after dilution.

[0019] Preferably, the high-throughput sequencing includes dual-region sequencing of the 16S rRNA gene V3-V4 region and V4-V5 region.

[0020] Preferably, the functional verification also includes the determination of the growth-promoting characteristics and stress resistance characteristics of the strain.

[0021] The technical effects and advantages of this invention are as follows:

[0022] In this invention, immunofluorescence labeling technology is used to specifically identify Phytophthora soybeanis and related microorganisms. Combined with flow cytometry, fluorescent negative bacteria in healthy soybean roots are accurately enriched. This avoids missing unculturable microorganisms from the source, while significantly reducing the amount of ineffective work in the screening process, shortening the discovery cycle of core beneficial endophytic bacteria, and improving the targeting and efficiency of the screening process. Attached Figure Description

[0023] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts:

[0024] Figure 1 This is an overall flowchart of the present invention;

[0025] Figure 2 This is a schematic diagram of the sample preparation and processing flow of the present invention. Detailed Implementation

[0026] It is readily understood that, based on the technical solution of this invention, those skilled in the art can propose various interchangeable structural methods and implementations without altering the essential spirit of the invention. Therefore, the following detailed embodiments and accompanying drawings are merely illustrative examples of the technical solution of this invention and should not be considered as the entirety of the invention or as limitations or restrictions on the technical solution of this invention.

[0027] Reference Figure 1-2 As shown, the present invention provides a technical solution: a method for promoting resistance to root rot disease based on fluorescent labeling analysis of soybean core endophytic flora, comprising the following steps:

[0028] S1: First, microbial communities were isolated from the root systems of healthy and diseased soybean plants, respectively, to obtain microbial suspensions for the healthy and diseased groups. To ensure the accuracy and comparability of the experiment, the concentrations of the microbial suspensions for the healthy and diseased groups were adjusted. During concentration adjustment, serial dilutions were performed using sterile phosphate buffer to ensure the diluted suspension concentration met experimental requirements. After dilution, the morphological integrity of the microorganisms was carefully observed under a microscope to ensure they were not damaged during the dilution process. Furthermore, to more effectively isolate the microbial community, an optimized extraction buffer containing various enzymes and inhibitors, including polysaccharide hydrolases and protease inhibitors, was used. Specifically, the polysaccharide hydrolases consisted of cellulase and pectinase, which effectively break down polysaccharide components in plant cell walls, thereby promoting the isolation of the microbial community. This series of meticulous procedures ensured the reliability of the experimental data and the accuracy of the results.

[0029] When conducting the experiment, samples of soybean plants in the pod-setting stage were selected. Specifically, two types of plants were chosen: one type consisted of healthy plants without any symptoms of root rot, characterized by bright green leaves and clean, white roots without any signs of rot; the other type consisted of plants infected with root rot, with localized or complete root rot, yellowing leaves, and wilting. Ten to fifteen plants from each type were selected to ensure the reliability and representativeness of the experimental data.

[0030] Next, treat each plant. Cut a root segment of about 0.5 grams from 5 to 10 centimeters away from the root. To preserve as many microorganisms as possible on the root surface, gently rinse the root segment with sterile water three times, each time for 30 seconds. During rinsing, avoid damaging the microorganisms on the root surface. After rinsing, gently blot the surface of the root segment dry with sterile filter paper.

[0031] Place the treated root segments into a sterile grinding tube, and then add 2 mL of pre-cooled optimized extraction buffer. To prepare the extraction buffer, take 100 mL of sterile phosphate buffer and add 1.0 g of polyvinylpyrrolidone, 50 μL of β-mercaptoethanol, 0.1 mL of polyethylene glycol octylphenyl ether, 0.5% cellulase, and 0.3% pectinase. Simultaneously add an appropriate amount of protease inhibitor mixture to ensure extraction efficiency.

[0032] Finally, the root segments were ground using a high-speed tissue grinder under ice bath conditions. The grinding was performed twice, for 30 seconds each time, with a 1-minute interval. The purpose was to fully break down the cells while minimizing the rupture of microbial cells, thereby ensuring the accuracy and validity of subsequent experimental results.

[0033] The prepared homogenate was carefully transferred to sterile centrifuge tubes, which were then centrifuged at 4°C for 5 minutes to effectively remove any plant tissue fragments. After the initial centrifugation, the supernatant was gently aspirated with a pipette and centrifuged again for 10 minutes to collect the microorganisms that had precipitated at the bottom of the tube. After ensuring complete separation of the microbial precipitate, the supernatant was carefully discarded, and the precipitate was resuspended in sterile phosphate buffer for subsequent experiments.

[0034] Next, 10 μL of the resuspended microbial suspension was added to a hemocytometer, and detailed cell counting was performed using an optical microscope. To ensure the accuracy and comparability of the experimental data, serial dilutions were performed using sterile phosphate buffer to uniformly adjust the concentration of the microbial suspension in both the healthy and infected groups. Meanwhile, another 5 μL of the diluted suspension was placed under a microscope for observation to carefully examine the integrity of the microbial cells, in order to avoid affecting the results of subsequent immunolabeling experiments due to cell damage.

[0035] The optimized extraction buffer formulation included the addition of cellulase and pectinase, two enzymes that specifically degrade polysaccharide components in root cell walls, thereby improving the release efficiency of microorganisms. Furthermore, the addition of polyvinylpyrrolidone K30 and β-mercaptoethanol effectively removed phenolic substances from the sample and inhibited oxidation reactions, significantly reducing non-specific interference from plant-derived impurities in subsequent immunofluorescence labeling experiments. These optimization measures greatly enhanced the binding efficiency of antibodies to pathogen antigens, providing a foundation for subsequent immunoassay.

[0036] S2: By using specific antibodies against Phytophthora soybeanis, precise immunofluorescence labeling is performed on microorganisms in both healthy and infected microbial suspensions. The specific antibody is composed of two antibodies: a monoclonal antibody against the cell wall structural proteins of Phytophthora soybeanis and a polyclonal antibody against the secretory effector proteins of Phytophthora soybeanis. This dual-antibody combination can more effectively identify and label Phytophthora soybeanis microorganisms.

[0037] Monoclonal antibodies against cell wall structural proteins of *Phytophthora sojae* were mixed with polyclonal antibodies against secretory effector proteins at a volume ratio of 2:1, and then diluted to a concentration of 0.8 μg / ml using 0.05 M carbonate buffer. Next, 100 μL of the diluted double-antibody mixture was added to each well of a 96-well microplate, and the plate was incubated at 4°C for 12 hours to ensure uniform and sufficient antibody adsorption onto the plate surface. After incubation, the coating solution in the wells was carefully discarded, and each well was washed three times with phosphate buffer containing polysorbate-20, immersing the plate for 5 minutes each time to ensure thorough removal of residual liquid and excess liquid from the wells. Then, 200 μL of a composite blocking solution was added to each well, and the plate was incubated at 37°C for 2 hours to block unbound antibody sites on the plate surface, effectively reducing non-specific binding. After incubation, the blocking solution was discarded, and each well was washed three times again with phosphate buffer containing polysorbate-20. Next, 100 μL of microbial suspension from either the healthy or infected group was added to each well, along with negative and blank control wells to ensure the accuracy and reliability of the experimental results. Finally, the ELISA plate was incubated at a constant temperature of 37°C for 1 hour to allow the dual antibody combination to fully bind with the Phytophthora soybean antigen in the microbial suspension, providing a solid foundation for subsequent detection and analysis.

[0038] After discarding the original liquid in the wells, the wells were carefully washed twice with phosphate buffer containing polysorbate-20. Then, a more thorough washing was performed using the same phosphate buffer containing polysorbate-20, repeated three times, with each wash lasting strictly controlled to 5 minutes. After washing, 150 μL of sterile phosphate buffer was accurately added to each well, and then the bottom of the plate was repeatedly and evenly pipetted to fully resuspend the bound microbial community in the buffer. Finally, these resuspended microbial communities were collected into sterile centrifuge tubes.

[0039] S3: Flow cytometry was used to sort the specifically labeled healthy microbial suspension. During this process, microbial populations with fluorescence signal intensity below a pre-set threshold were collected and defined as the fluorescent negative population of the healthy group. The specific procedure for determining the pre-set threshold is as follows:

[0040] First, a healthy microbial suspension without specific antibody labeling was selected as a negative control group. Next, the fluorescence signal intensity of the negative control group was measured and its average value was calculated. Finally, the average value was added to three times the standard deviation, and this result was used as a preset threshold to ensure the accuracy and reliability of the sorting process.

[0041] The labeled healthy microbial suspension was resuspended and then filtered through a sterile cell sieve with a pore size of 35 micrometers to ensure that the obtained sample was in a single-cell suspension state, thereby effectively avoiding the adverse effects of cell aggregation and other factors on the subsequent sorting accuracy.

[0042] During the sorting process, flow cytometry was used as the core equipment. Before formal sorting, the instrument channels were precisely calibrated using fluorescent microspheres to ensure that the coefficient of variation of fluorescence intensity detection was strictly controlled within 3%, thus guaranteeing the accuracy and reliability of the detection data. To prevent cross-contamination between different samples, the tubing was first thoroughly rinsed with 75% ethanol solution for 10 minutes, followed by a second rinse with sterile phosphate buffer for 15 minutes to ensure that the tubing environment met sterility standards.

[0043] In the first step of the sorting process, the bacteria, fungi, and debris regions are divided by using two parameters of forward scattered light (FSC) and side scattered light (SSC). The proportion of debris is strictly controlled to ensure that it does not exceed 5% in order to eliminate the interference of debris on the sorting results.

[0044] In the second step, the fluorescence intensity of the microbial suspension in the negative control wells was first detected, and the mean and standard deviation of the fluorescence intensity were recorded in detail. Based on this data, a scientifically preset threshold was set, namely M+3SD (mean plus three times the standard deviation). The population below this threshold was defined as the fluorescence-negative population. Subsequently, the microbial suspension of the healthy group was precisely sorted. After sorting, 10 microliters of the sorted sample were taken and observed under a fluorescence microscope. At the same time, the contamination rate of the sample was detected by plate counting method to ensure that the final sorted population has a high purity.

[0045] S4: For the fluorescently negative population in the healthy group, high-throughput sequencing analysis was conducted. This high-throughput sequencing not only covered the V3-V4 region of the 16S rRNA gene but also extended to dual-region sequencing of the V4-V5 region to ensure data comprehensiveness and accuracy. Based on the obtained sequencing data, core beneficial endophytic bacteria closely related to soybean healthy growth were detected. Key indicators included: first, the abundance correlation of the bacteria, i.e., the correlation between the relative abundance of the bacteria in the sample and the soybean health status; second, the intragroup-specific enrichment, examining the specificity and enrichment degree of these bacteria in the healthy group; third, universality indicators to assess the prevalence of these bacteria in different samples; and finally, functional indicators to analyze the functional impact of these bacteria on soybean healthy growth.

[0046] S5: Perform functional verification on the identified core beneficial endophytic flora. Functional verification includes in vitro antibacterial experiments and in vivo efficacy evaluation. Functional verification also includes determination of the growth-promoting characteristics and stress resistance characteristics of the strains.

[0047] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0048] Example 1:

[0049] Selecting reagents:

[0050] Soybean Phytophthora specific monoclonal antibody;

[0051] Polyclonal antibodies against secretory effector proteins of Phytophthora soybean;

[0052] Streptavidin labeled with fluorescein isothiocyanate;

[0053] Streptavidin labeled with AF488 fluorescent dye;

[0054] Casein, gelatin, plant-derived protein sealing agents;

[0055] Polyvinylpyrrolidone (PVP), β-mercaptoethanol;

[0056] Cellulase (activity ≥10U / mg), pectinase (activity ≥5U / mg);

[0057] Protease inhibitor cocktail;

[0058] Microbial genomic DNA extraction kit;

[0059] Primers for amplification of the V3-V4 and V4-V5 regions of the 16S rRNA gene;

[0060] Biotinylated anti-mouse immunoglobulin;

[0061] In the preparation of the buffer solution, take 100 ml of phosphate buffer, add 0.1 ml of polyethylene glycol octylphenyl ether, 1.0 g of polyvinylpyrrolidone, 50 μ L of β-mercaptoethanol, 0.5 g of cellulase, 0.3 g of pectinase, and 1 × working concentration of protease inhibitor mixture, mix thoroughly and store at 4 °C.

[0062] Next, weigh out 1.5g casein, 1g gelatin, and 0.5g plant-derived protein blocking agent. Dissolve the above components in 100ml phosphate buffer, add 0.1ml polysorbate-200, and stir thoroughly until completely dissolved.

[0063] Add 1% casein and 20% glycerol to sterile phosphate buffer, mix thoroughly, aliquot and store at 4°C.

[0064] Main equipment:

[0065] High-speed tissue homogenizer, flow cytometer, laser confocal microscope, high-throughput sequencing platform, enzyme-linked immunosorbent assay (ELISA) reader, and PCR instrument.

[0066] First, in the experimental area of ​​the soybean field, two groups of plant samples were selected: one group consisted of healthy plants with good growth and no obvious symptoms, and the other group consisted of susceptible plants showing typical symptoms of root rot. Fifteen plants were selected from each group to ensure the reliability and representativeness of the experimental data. Next, root samples were collected from both groups of plants. The collected root samples were thoroughly washed with sterile water to remove surface soil and impurities, and then gently blotted dry with absorbent paper to ensure the purity and dryness of the samples.

[0067] Subsequently, 0.5 g of root sample was weighed and placed in 2 ml of pre-cooled optimized extraction buffer. To ensure the stability and consistency of experimental conditions, the entire process was conducted under ice bath conditions. The sample was mechanically homogenized using a high-speed tissue homogenizer to fully disrupt the cells and release the biomolecules. Specific operating parameters were set as follows: 3 mm diameter grinding beads, two homogenization cycles, each lasting 30 seconds, with a 1-minute interval between cycles to ensure optimal homogenization of the sample.

[0068] To efficiently purify the homogenate, we employed a gradient centrifugation method. First, the homogenate was placed in a centrifuge and centrifuged at 500×g for 5 minutes at 4°C. This step aimed to remove debris from the plant tissue, ensuring smooth subsequent operations. After centrifugation, the supernatant was collected, avoiding disturbance of the precipitate. Next, the collected supernatant was returned to the centrifuge and, under the same 4°C conditions, the centrifugation force was increased to 8000×g, and the centrifugation time was extended to 10 minutes. This step aimed to effectively precipitate the microorganisms. After centrifugation, the microbial precipitate formed at the bottom was collected. To further process these precipitates, they were resuspended in sterile phosphate buffer and adjusted to a suitable concentration. Finally, precise quantitative analysis was performed using a hemocytometer to ensure the accuracy and reliability of the experimental data.

[0069] For samples requiring long-term preservation, 20% (v / v) glycerol was added to the resuspension as an effective preservative to ensure stability and activity during storage. These treated samples were then cryopreserved at -80°C to minimize biomolecular degradation. Simultaneously, to ensure sample quality met experimental requirements, firstly, the protein concentration was precisely measured using a micro-spectrophotometer to ensure it remained within the ideal target range of 0.5 to 2.0 mg / mL. Secondly, the purity of DNA was assessed by measuring the absorbance ratio (A260 / A280) at 260 nm and 280 nm wavelengths, ensuring this ratio was between 1.8 and 2.0, thus confirming DNA purity met standards. Furthermore, to further verify the microbial state of the samples, microbial morphology was observed under a microscope to ensure normal morphology and no abnormal changes.

[0070] Next, using 0.05 mol / L carbonate buffer, the specific monoclonal antibody against Phytophthora soybeanis and the helper antibody were mixed at a volume ratio of 2:1. The mixture was then diluted to a total concentration of 0.8 μg / mL. 100 μL of the diluted mixture was added to each well of a 96-well plate and incubated overnight at 4°C to ensure sufficient antibody binding. After incubation, the coating solution in the wells was discarded. The plate was then washed three times with phosphate buffer containing polysorbate-20 to remove unbound antibodies and other impurities. After washing, 200 μL of composite blocking buffer was added to each well, and the plate was incubated at 37°C for 2 hours to block non-specific binding sites. After incubation, the plate was washed again, and then 100 μL of pre-prepared microbial suspension from either the healthy or infected group was added to each well and incubated at 37°C for 1 hour to allow the microorganisms to fully react with the antibody. After incubation, continue with the washing step, then add 100 μL of fluorescein isothiocyanate or streptavidin labeled with AF488 fluorescent dye diluted 1:1000 with phosphate buffer to each well. Incubate at 37°C in the dark for 45 minutes to ensure sufficient binding of the fluorescent label to the antibody-microbial complex. After incubation, discard the liquid in the wells, gently wash the plate twice with phosphate buffer containing polysorbate-20, and then wash three more times with the same buffer, each wash lasting 5 minutes. After each wash, centrifuge at 5000×g. Finally, add 150 μL of sterile phosphate buffer to each well, and repeatedly pipette to the bottom of the plate to ensure thorough resuspension of the bound microbial community. Collect the resuspension in a sterile centrifuge tube, and use flow cytometry to detect the coefficient of variation of fluorescence intensity to verify whether the difference between the proportion of positive signals and the negative control is 10-fold or greater.

[0071] To ensure the accuracy and reliability of the experiment, the microbial suspension collected in the above steps was carefully filtered through a cell sieve with a pore size of 35 micrometers to ensure the smooth progress of the subsequent sorting process. Next, a flow cytometer was used to accurately sort the filtered suspension. Before the sorting process officially began, the fluorescence intensity of the FITC channel was calibrated using standard fluorescent microspheres to ensure that its coefficient of variation (CV value) was controlled within 3%, thus ensuring the accuracy and consistency of the sorting results. In addition, to prevent cross-contamination and ensure aseptic operation, the tubing system of the sorter was first rinsed with 75% ethanol solution for 10 minutes to thoroughly disinfect and remove any possible residual contaminants; then, it was rinsed with sterile phosphate buffer for 15 minutes to further ensure the cleanliness and sterility of the tubing system.

[0072] Using forward scattering (FSC) and side scattering (SSC) gating techniques, microorganisms are meticulously segmented based on their morphological characteristics to accurately identify and classify target microbial communities while effectively eliminating various fragment interferences. In this process, unlabeled healthy samples are selected as negative controls. A scientifically reasonable threshold is set based on the mean fluorescence intensity of the fluorescein isothiocyanate channel, plus three times the standard deviation. Next, the labeled healthy group microbial suspensions are precisely sorted, collecting those microbial populations with fluorescence signal intensities below the aforementioned threshold and recording them as the healthy group's fluorescent negative population. During the sorting process, flexible selection is made based on the actual concentration of the samples: for high-concentration samples... A purity-priority sorting mode is adopted to ensure that the purity of the sorted samples reaches or exceeds 98%; while for low-concentration samples... A yield-priority sorting mode is adopted to ensure a sorting purity of at least 95%. After sorting, a 10 μL sample is taken and carefully observed using a fluorescence microscope, and purity is verified by plate counting. This step aims to ensure that the proportion of positive cells in the sorted sample is not less than 95%, while controlling the contamination rate of other microorganisms to within 3%, thereby ensuring the accuracy and reliability of the experimental results.

[0073] Total DNA was extracted from the sorted healthy fluorescent negative population using a soil microbial genomic DNA extraction kit. A host DNA removal step was added. Using this DNA as a template, 16S rRNA gene dual-region sequencing was performed, and the V3-V4 region (primer 341F / 806R) and V4-V5 region (primer 515F / 907R) were amplified simultaneously. Metagenomic sequencing was performed on the core microbial community sample, with a sequencing depth ≥10Gbp.

[0074] The raw sequencing data underwent adapter removal via Trimmomatic and primer removal via Cutadapt. Data quality was validated using FastQC (Q30 ≥ 90%). OTU clustering was performed using a 97% similarity threshold, and singleton OTUs were removed. Species annotation was based on the SILVA (v138) and NCBInr databases. A multi-indicator judgment system was established to screen core beneficial endophytic communities. Specific indicators and standards are shown in Figure 1 below.

[0075] Judgment Indicators Weight Judgment criteria Calculation method Abundance correlation 40% The abundance was significantly higher in the healthy group than in the infected group. The difference fold is ≥1.8, and the significance level of the T-test is p<0.05. Intragroup specificity 30% Enriched in fluorescent negative populations Relative abundance ≥ 1.5 times the total community universality 20% Stable in healthy individuals Detection rate ≥80% Functional indication 10% Negatively correlated with pathogen load The correlation coefficient r ≤ 0.6 and the significance level < 0.05.

[0076] Chart 1

[0077] During the experiment, the bacterial communities detected in the blank control group were excluded. In addition, known plant pathogens were also excluded to avoid these harmful bacteria interfering with the experimental results. At the same time, low-abundance bacterial communities with a relative abundance of less than 0.1% were also excluded.

[0078] To isolate the core bacterial strain from healthy soybean roots, the traditional streak plating method was used for isolation and purification. This method effectively separates different strains, facilitating subsequent identification. For those difficult-to-culture bacterial groups, further optimization was performed on the R2A medium, as follows:

[0079] Adding 5% soybean root extract to simulate the natural growth environment of soybean roots promotes the growth of difficult-to-culture bacteria. At the same time, adding 0.1% humic acid and vitamin mixture provides additional nutritional support, further optimizing the composition of the culture medium and thus improving the isolation and culture efficiency of difficult-to-culture bacteria.

[0080] In the antibacterial experiment, a double-layer culture medium method was used, and the specific operation was as follows: First, the bottom layer was potato dextrose agar medium, which provided basic nutritional support for the experiment. Then, the top layer was potato dextrose agar medium containing 10% soybean root extract. The diameter of the pathogenic bacterial cake was 5 mm. The test strain was then inoculated... The quantity was precisely controlled by colony formation units / point to ensure the reliability of experimental data. All petri dishes were cultured at a constant temperature of 28℃ for 5 days. The antibacterial rate was calculated by comparing the growth of the experimental group and the control group.

[0081] In another experiment, potato dextrose liquid medium was used as the culture medium, and the test strain and *Phytophthora indicum* were co-cultured at a mycelial mass ratio of 10:1. After 7 days of co-culture, the dry weight inhibition rate of *Phytophthora indicum* hyphae and the spore germination inhibition rate were measured to evaluate the inhibitory effect of the test strain on *Phytophthora indicum*. Furthermore, to further verify the effect of the test strain on soybean growth, soybean seedlings were treated with its fermentation broth. After 14 days of growth observation, the growth rates of plant height, root length, fresh weight, and chlorophyll content of soybean seedlings were measured to comprehensively evaluate the promoting effect of the test strain on soybean growth.

[0082] In the pot experiment, two different substrates, sterilized soil and natural soil, were selected for comparison. Soybean seeds were sown after surface sterilization to ensure consistency of experimental conditions. Seven days after emergence, the test strain was inoculated, followed by inoculation with Phytophthora spore suspension three days later to simulate the natural infection process. After 30 days of growth observation, the disease index and control efficacy were measured to evaluate the practical application effect of the test strain. Samples were taken at 7, 15, and 30 days after inoculation, and the colonization location of the test strain in soybean roots was observed using a laser confocal microscope. The number of strains in the roots was determined by plate counting to understand the colonization status. In addition, the activities of superoxide dismutase, peroxidase, and phenylalanine ammonia-lyase in soybean roots were measured at 1, 3, 5, and 7 days after inoculation to evaluate the effect of the test strain on the physiological activities of soybean roots.

[0083] To further validate the findings, experimental fields were set up in three major soybean producing areas using a multi-variety experimental design. Each experimental site had three treatment groups: a single-strain treatment group, a core microbial community mixed treatment group, and a blank control group. Each group was replicated three times, with each plot measuring 15 square meters. Disease indices were recorded at the seedling, flowering, and grain-filling stages to comprehensively evaluate the control efficacy of different treatment groups against soybean diseases.

[0084] Example 2:

[0085] This embodiment selected three soybean varieties with different resistance levels: Suinong 42 (a disease-resistant variety), Hefeng 50 (a moderately susceptible variety), and Jiyu 86 (a susceptible variety). Twenty healthy and twenty susceptible plants were collected from each variety. Three replicate groups were set up, and parallel experiments were conducted using the standardized method of Example 1. The focus was on comparing the differences in the composition and functional characteristics of the core beneficial endophytic flora among the different varieties.

[0086] During the sample collection phase, root samples were collected from each variety at the same growth stage to ensure consistency in physiological state.

[0087] The monoclonal antibody and helper antibody specific to Phytophthora soybeanis were mixed at a volume ratio of 2:1 using 0.05M carbonate buffer and diluted to a total concentration of 0.8 μg / mL. However, the antibody concentration was adjusted to a gradient of 0.5-1.2 μg / mL to determine the optimal labeling conditions for each variety. Variety-specific fluorescence compensation was added to the flow cytometry sorting process to eliminate autofluorescence interference. The differences in microbial community structure among different varieties were compared by principal coordinate analysis and similarity analysis.

[0088] The functional verification results are shown in Figure 2 below:

[0089] Evaluation indicators Suinong 42 Hefeng 50 Jiyu 86 Antibacterial rate 82.3% 75.6% 68.9% Colonization capacity (CFU / g) Growth-promoting effect Plant height +18.5% Plant height +15.2% Plant height +12.8% Field efficacy 78.9% 65.3% 52.1%

[0090] Chart 2

[0091] As shown in Table 2, the coefficient of variation of the relative abundance of the core microbial community is ≤15% in different varieties, the similarity of microbial community composition between greenhouse and field environments is ≥80%, and the functional stability retention rate after three generations of subculture is ≥90%. Due to differences in root structure and pathogen content, the optimal antibody concentration varies among different varieties, thus avoiding insufficient labeling efficiency or non-specific interference caused by uniform concentration.

[0092] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. A method for promoting resistance to root rot in soybeans based on fluorescent labeling analysis of core endophytic microbiota, characterized in that, Includes the following steps: S1: Microbial communities were isolated from the roots of healthy soybean plants and diseased soybean plants, respectively, to obtain microbial suspensions of the healthy group and the diseased group. S2: Using specific antibodies against Phytophthora soybeanis, immunofluorescence labeling was performed on microorganisms in the healthy group and the diseased group microbial suspensions, respectively. S3: Use flow cytometry to sort the labeled healthy group microbial suspension and collect the microbial population with fluorescence signal intensity below a preset threshold, which is recorded as the healthy group fluorescence negative population; S4: Nucleic acid extraction and high-throughput sequencing were performed on the healthy fluorescent negative population, and the core beneficial endophytic bacteria related to healthy soybean growth were identified based on the sequencing data; S5: Perform functional verification on the identified core beneficial endophytic flora.

2. The method for promoting resistance to root rot based on fluorescent labeling analysis of soybean core endophytic flora according to claim 1, characterized in that: In S1, an optimized extraction buffer containing polysaccharide hydrolase and protease inhibitor is used to isolate the microbial community.

3. The method for promoting resistance to root rot based on fluorescent labeling analysis of soybean core endophytic flora according to claim 2, characterized in that: The polysaccharide hydrolases include cellulase and pectinase.

4. The method for promoting resistance to root rot based on fluorescent labeling of soybean core endophytic flora according to claim 1, characterized in that: The specific antibody described in S2 is a combination of two antibodies, including a monoclonal antibody against structural proteins of the cell wall of Phytophthora soybean and a polyclonal antibody against secretory effector proteins of Phytophthora soybean.

5. The method for promoting resistance to root rot based on fluorescent labeling of soybean core endophytic flora according to claim 1, characterized in that: The preset threshold mentioned in S3 is determined by using a healthy microbial suspension without labeled specific antibodies as a negative control, and using the mean fluorescence signal intensity plus three times the standard deviation as the preset threshold.

6. The method for promoting resistance to root rot based on fluorescent labeling of soybean core endophytic flora according to claim 1, characterized in that: The identification system for core beneficial endophytic flora described in S4 includes abundance correlation, intragroup-specific enrichment, universality, and functional indicative indicators.

7. The method for promoting resistance to root rot based on fluorescent labeling of soybean core endophytic flora according to claim 1, characterized in that: The functional verification described in S5 includes in vitro antibacterial experiments and in vivo efficacy evaluation.

8. The method for promoting resistance to root rot based on fluorescent labeling analysis of soybean core endophytic flora according to claim 1, characterized in that: The concentrations of the microbial suspensions from the healthy group and the infected group were adjusted to: When adjusting the concentration, use sterile phosphate buffer for gradient dilution, and observe the integrity of the microbial morphology under a microscope after dilution.

9. The method for promoting resistance to root rot based on fluorescent labeling analysis of soybean core endophytic flora according to claim 1, characterized in that: The high-throughput sequencing includes dual-region sequencing of the 16S rRNA gene V3-V4 and V4-V5 regions.

10. The method for promoting resistance to root rot based on fluorescent labeling analysis of soybean core endophytic flora according to claim 1, characterized in that: The functional verification also includes the determination of the growth-promoting characteristics and stress resistance characteristics of the strain.

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