Experimental method for research on tomato bacterial wilt prevention and control performance of complex microbial inoculants

Abstract

The invention relates to the field of tomato bacterial wilt prevention and control, in particular to a research experiment method for the tomato bacterial wilt prevention and control performance of a complex microbial inoculant, which comprises the following steps: activation of bacterial strains, preparation of the complex microbial inoculant, inhibition of the complex microbial inoculant on GMI1000, colonization of the complex microbial inoculant at tomato roots, response of the complex microbial inoculant on tomato root exudates, and pot experiment. Measuring plant biomass and physicochemical properties; the method comprises the following steps: S1, measuring the content of plant rhizosphere soil nutrients, measuring the number of complex microbial inoculants and GMI1000 in soil, and performing high-throughput sequencing on tomato rhizosphere soil microorganisms, in the step S1, GMI1000 divides strains preserved in a glycerol tube at-80 DEG C into a sterilized BG solid culture medium through a plate streaking method, and the strains are inversely placed in a constant-temperature incubator at 28 DEG C to be cultured for 48 hours. Then, single colonies are selected and placed in a BG liquid culture medium; and regulating and controlling the community structure of rhizosphere fungi to repair or remodel tomato rhizosphere soil microbial communities infected by ralstonia solanacearum so as to indirectly prevent and treat tomato bacterial wilt.

Description

Technical Field

[0001] The invention relates to the field of tomato bacterial wilt prevention and treatment, and specifically is an experimental method for studying the prevention and treatment performance of a composite bacterial agent on tomato bacterial wilt. Background Art

[0002] As a bacterial pathogen, Ralstonia solanacearum can invade through wounds at the root or base of the plant and survive in the soil for a long time, causing the stems and leaves to wither without normal water supply. Bacterial wilt is a devastating soil-borne disease caused by Ralstonia solanacearum. It is one of the plant diseases with the most extensive distribution, extremely serious harm and the most serious losses in the world. Tomatoes play a key role in human diet and life, but tomato bacterial wilt seriously affects plant growth, often causing large-scale withering and death of tomato plants. Due to the complex pathogenic mechanism of Ralstonia solanacearum and its strong survival and transmission ability, there is currently no very effective method to completely control tomato bacterial wilt, so prevention is the main method to prevent Ralstonia solanacearum from invading and spreading plants. Although pesticides play an important role in the prevention and control of tomato bacterial wilt, their negative effects on soil, environment and food safety cannot be ignored.

[0003] Although there have been many studies on the use of PGPR to control tomato bacterial wilt, there are few studies that systematically explore the interaction between composite microbial agents and tomato rhizosphere to control tomato bacterial wilt.

[0004] Therefore, in response to the above problems, an experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt was proposed. Summary of the invention

[0005] In order to make up for the shortcomings of the existing technology, we systematically explored the migration, chemotaxis and colonization film formation of the compound bacterial agent in the rhizosphere of tomatoes; we clarified the direct and indirect synergistic mechanisms of the compound bacterial agent in preventing and controlling tomato bacterial wilt, that is, the compound bacterial agent directly prevents and controls tomato bacterial wilt by reducing the number of Ralstonia wilt in the rhizosphere soil, and indirectly prevents and controls tomato bacterial wilt by regulating the structure of soil microbial communities.

[0006] The technical solution adopted by the present invention to solve the technical problem is: an experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt, comprising the following steps:

[0007] S1. Activation of strains;

[0008] S2. Preparation of composite bacterial agent;

[0009] S3, inhibition of compound bacterial agent on GMI1000;

[0010] S4, colonization of the composite bacterial agent on tomato roots;

[0011] S5, Response of composite bacterial agent to tomato root exudates;

[0012] S6, potted plant experiment;

[0013] S7, plant biomass and physicochemical properties determination;

[0014] S8, determination of nutrient content in plant rhizosphere soil;

[0015] S9, determination of the amount of compound bacterial agent and GMI1000 in soil;

[0016] S10. High-throughput sequencing of tomato rhizosphere soil microorganisms.

[0017] Preferably, in step S1, GMI1000 streaks the strain stored in the glycerol tube at -80°C into the sterilized BG solid medium by the plate streaking method, and inverts it in a 28°C constant temperature incubator for 48 hours. Then, a single colony is picked and placed in a BG liquid medium, and cultured overnight at 28°C and 200rpm to complete activation.

[0018] Preferably, in step S2, the composite bacterial agent is compounded by Rs-5 and SL-44, and the culture medium used for Rs-5 and SL-44 is LB culture medium.

[0019] Preferably, in step S3, the inhibitory ability of the composite bacterial agent on GMI1000 is determined by the plate confrontation method. That is, 100 μL of activated GMI1000 is taken onto the BG solid culture medium and spread evenly, and then three sterilized circular filter papers (φ=0.5 cm) are placed on each BG solid culture medium, and then 10 μL of the composite bacterial agent bacterial solution is inoculated on the filter paper, cultured at 30°C for 48 hours, and then observed whether an inhibition zone appears. If an inhibition zone exists, it means that the composite bacterial agent can inhibit GMI1000, otherwise there is no inhibition. At the same time, strains Rs-5 and SL-44 are set as controls.

[0020] Preferably, in step S4, tomato seeds with full particles and uniform size are selected, and then they are disinfected by soaking in 75% alcohol and rinsed with sterile water for multiple times. The culture matrix (vermiculite and perlite mixed in a ratio of 3:2) is added to a tissue culture bottle (800mL) and sterilized at high temperature. The tomato seedlings grow to 4 true leaves in the culture matrix, and then the tomato seedlings are taken out and washed with sterile water to remove the vermiculite and perlite at the roots. The tomato seedlings are then placed in a centrifuge tube containing 20mL of a bacterial suspension of strains Rs-5, SL-44 or a composite bacterial agent. The centrifuge tube is then placed in a climate chamber and cultured for 48 hours before being taken out, the tomato roots are rinsed with sterile water, and then about 1 cm is cut from the meristem and elongation zones of the tomato roots. Finally, the tomato roots are fixed in a 2.5% glutaraldehyde solution and freeze-dried, and then observed by scanning electron microscopy.

[0021] Preferably, in step S5, considering that tomato root secretions are mainly sugars, amino acids and organic acids, the specific root secretion components selected in this chapter are: glucose, mannitol, sucrose, fructose, leucine, glutamic acid, aspartic acid, alanine, tartaric acid, malic acid, citric acid, and fumaric acid. The response of Rs-5, SL-44 and composite bacterial agents to tomato root secretions was determined by the swimming plate method. Different root secretions (10mM) were added to the sterilized semi-solid culture medium (agar content 0.2%), shaken and poured into a culture dish to cool. After 10mL of the cultured bacterial solution was centrifuged at 8000rpm and 4℃ for 10 minutes, it was resuspended in 1mL phosphate buffer (pH 7.3) to prepare a concentrated bacterial suspension, and then 20μL of the concentrated bacterial suspension was taken in the center of the semi-solid culture medium. The semi-solid culture medium without root secretions was used as a blank control. After the above culture medium was cultured overnight, the chemotactic radius was recorded. .

[0022] Preferably, in step S6, the tomato seeds are soaked in 70% ethanol, then soaked in 0.1% HgCl2 for 10 minutes, and finally rinsed with sterile water several times. The sterilized tomato seeds are sown in a seedling tray filled with sterilized soil, and the tomato seedlings are transplanted when they grow to three leaves and one heart. The soil used for the potted plant experiment was taken from the Lintong Campus of Xi'an Polytechnic University (34°21′N, 109°11′E). The organic matter, alkaline nitrogen, available phosphorus and available potassium contents of the original soil were 15.2 g·kg -1 , 50.62 mg kg -1 , 11.46 mg kg -1 and 180 mg kg -1 , the soil conductivity and pH were 7.90 and 480 μS·cm -1 . A total of 4 treatments were set up in the pot experiment: CK, no treatment (no compound bacterial agent and pathogen GMI1000 were added); T1, only compound bacterial agent was added; T2, only pathogen GMI1000 was added; T3, compound bacterial agent and pathogen GMI1000 were added. Six seedling pots (φ23.5cm×19.5cm) were set up for each treatment, each pot was filled with 3kg of non-sterile soil and transplanted with 3 tomato seedlings of the same growth. Before transplanting the tomato seedlings, the GMI1000 bacterial suspension was evenly sprayed on the soil for T2 and T3 treatments to control the pathogen concentration at 10 7 CFU·g -1 After transplanting the tomato seedlings, the compound bacterial agent was inoculated into the center of the seedling pot (the concentration of the compound bacterial agent was about 108 CFU·g -1 The seedling pots were placed in a constant temperature and light incubator at 26°C with a light exposure time of 14 h (350 μmol·m 2 S -1), relative humidity was 50%-60%. The pot experiment ended 30 days after the tomato seedlings were transplanted. After the pot experiment, the plant biomass (fresh weight, dry weight, plant height, root length, stem thickness), plant physical and chemical index content and plant rhizosphere soil nutrient content were measured. Collection of soil samples: Two sampling points, namely M site and R site, were set to study the migration of the composite bacterial agent in the soil and its effect on the soil microbial community. The M site is located between the tomato rhizosphere and the inoculation site of the composite bacterial agent, and the R site is located in the tomato rhizosphere. At 15 and 30 days after tomato transplantation, the soil at M and R of three pots was taken for each treatment. The soil at M and R of three seedlings was taken from each pot, and then the soil at M and R of each pot was mixed evenly. After the soil was sieved with a 2mm sieve to remove impurities such as plant roots, it was stored at -80℃. The incidence of bacterial wilt was counted on the 15th and 30th days after the tomato seedlings were transplanted. The incidence of tomato bacterial wilt is the percentage of the number of diseased plants to the total number of plants. Disease level statistics: Six disease levels are determined based on the proportion of the wilting degree of tomato seedlings to the whole tomato plant. n = 0, no symptoms; n = 1, 20% of the whole plant is wilted; 2, 40% of the whole plant is wilted; 3, 60% of the whole plant is wilted; 4, 80% of the whole plant is wilted; 5, 100% of the whole plant is wilted. The disease index (DI) and relative control efficacy (CE) are calculated:

[0023] DI = (100∑(disease level n×number of plants of that disease level)) / (5×total number of plants in that treatment)

[0024] CE (%) = (control disease index - treatment disease index) × 100 / control disease index.

[0025] Preferably, in step S7, the biomass of tomato and corn plants includes fresh weight, dry weight, plant height, root length and diameter. The fresh sample of the plant is washed with pure water and then dried with paper to measure its fresh weight. Then it is transferred to a drying oven and dried at 90°C to constant weight, and then taken out to measure the dry weight. The plant height is the distance from the base of the plant stem to the growth point, the root length is the distance from the longest root tip of the plant to the base of the stem, and the diameter of the plant is measured with a vernier caliper. Soluble protein: the determination of plant soluble protein refers to the Coomassie Brilliant Blue G-250 method

[102] . Weigh 0.2 g of fresh plant leaves and add them to 5 mL of distilled water. After grinding into a homogenate, centrifuge (10000 rpm, 30 min) and collect the supernatant as the test solution. Take 1.0 mL of the supernatant in a test tube, add 5 mL of Coomassie Brilliant Blue G-250 reagent, shake well and let stand for 2 minutes, measure the absorbance of each sample at a wavelength of 595 nm, and calculate the soluble protein content through the standard curve. Repeat for each sample three times; Soluble sugar: The soluble sugar content in the plant is determined by the sulfuric acid phenol method

[103] . Weigh 0.5 g of fresh plant leaves, cut them into pieces and add them to 5 mL of distilled water, seal them with a sealing film, extract them in boiling water for 30 minutes, collect the extract, filter it and transfer it to a 25 mL volumetric flask, cool it and make up the volume to 25 mL. This solution is the test solution. Take 0.5 mL of the test solution and mix it with 1.5 mL of distilled water, add 1 mL of 9% phenol solution, shake well and slowly drip 5 mL of concentrated sulfuric acid and shake well. Add distilled water to the colorimetric solution to make the total volume 8 mL, and let it stand at room temperature for half an hour. After the reaction is completed, the absorbance of each reaction solution is measured at a wavelength of 485 nm with the blank solution as the control, and the soluble sugar content is calculated from the standard curve. Each sample is repeated three times; Proline: The proline content of plants is determined by the sulfosalicylic acid method

[104] . Weigh 0.5 g of fresh plant leaves and place them in test tubes containing 5 mL of 3% sulfosalicylic acid solution. React in a boiling water bath for 10 min. After cooling, filter the extract into a clean test tube. The filtrate is the extract. Measure 2 mL of the extract into a clean test tube and mix it evenly with 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin reagent. Then place the test tube in a boiling water bath and heat for 30 min. After cooling, add 4 mL of toluene to the test tube, shake for 30 s, and let it stand for several minutes to allow all the proline in the leaves to be extracted into the toluene solution. Pipette the proline toluene extract solution on the upper layer of the test tube into a cuvette, and detect the absorbance at a wavelength of 520 nm with toluene as the blank control. The proline content in the plant was calculated based on the standard curve, and each sample was repeated three times; Chlorophyll and carotenoids: The chlorophyll and carotenoid content in the plant leaves was determined by ethanol extraction spectrophotometry

[105] . Weigh 0.2g of plant leaves, place them in a 10mL volumetric flask, add 10mL of 96% ethanol, shake well, and let stand in the dark overnight. After the plant leaves turn completely white, measure the absorbance of the extract at λ666, λ649 and λ470. The calculation formulas for each component are as follows:

[0026] Chlorophyll a (mg / g) = (13.95 × OD666 - 6.88 × OD649) × V / M;

[0027] Chlorophyll b (mg / g) = (24.96 × OD649 - 7.32 × OD666) × V / M;

[0028] Total chlorophyll (mg / g) = (20.29OD646 + 8.05OD661) × V / M;

[0029] Carotenoid C (mg / g) = 1000 × (OD470-2.05Chl a-114.8Chl b) / 245 × V / M

[0030] Note: In the above formula, V is the total volume of the extract (mL), and M is the fresh weight of pepper leaves (g).

[0031] Preferably, in step S8, the organic matter, available potassium, available phosphorus and alkaline nitrogen in the rhizosphere soil of the plants are determined by potassium dichromate bulk density method, flame photometry, molybdenum antimony colorimetry and alkaline diffusion method

[112] respectively; soil organic matter: accurately weigh 0.5 g of sieved air-dried soil sample and add it to a digestion tube, add 10.0 mL of potassium dichromate sulfuric acid solution (0.4 mol·L -1 ) and then cover the tube plug and place the digestion tube into the porous furnace. Control the temperature of the electric furnace and maintain the temperature inside the tube at 170℃~180℃. Start timing when the solution in the digestion tube begins to boil. Take out the digestion tube after 5 minutes and cool it for 30 minutes. Use water to transfer the digestion solution and soil residue to a triangular flask without damage. Rinse the test tube and the tube plug with water, and combine the washing solution into the triangular flask. The total volume of the solution in the triangular flask is controlled at 50mL~60mL. Add 3 drops of o-phenanthroline indicator and titrate with ferrous sulfate standard solution. The solution changes from orange to blue-green and finally to brown-red, which is the end point. At the same time, set up a blank test and calculate the content of organic matter by the formula; Soil available potassium: weigh 2.0g of sieved air-dried soil in a plastic bottle and add 20mL of neutral NH 4 OAc solution, plug the rubber stopper and shake for 30 minutes. Then filter the solution into a triangular flask with qualitative filter paper and measure it together with the potassium standard series solution on a flame photometer. The concentration of effective potassium is obtained from the standard curve. Soil available phosphorus: weigh 2.5g of sieved air-dried soil sample into a triangular flask, add 50mL NaHCO 3 (0.5mol·L -1 ) solution, add phosphorus-free activated carbon, plug the bottle tightly, shake for 30 minutes, filter with phosphorus-free filter paper into a 50mL plastic bottle, draw 10mL of the filtrate, and add 10mL of NaHCO 3(0.5mol·L-1) solution, add 5mL of molybdenum antimony reagent, shake well, remove carbon dioxide bubbles, and then dilute to 50mL with deionized water. After leaving for 30 minutes, perform colorimetry at 700nm. Use the absorption value of the blank solution as a reference, read the absorption value of the test solution, and calculate the effective phosphorus content from the standard curve; soil alkaline nitrogen: weigh 2.0g of sieved air-dried soil sample, place it in the outer chamber of a clean diffusion dish, and slowly rotate the diffusion dish to evenly spread the soil sample. Take 2mL of H3BO3 and add it to the inner chamber of the diffusion dish. Quickly add 10.0mL of NaOH solution and cover it tightly, slowly rotate the diffusion dish, and then place it in a constant temperature incubator at 38℃. Take it out after 24 hours of alkaline diffusion, and the NH3 in the inner chamber absorption liquid is diluted with 0.01mol·L - 1(1 / 2H2SO4) standard solution titration, and record the consumed 0.01mol·L -1 (1 / 2H 2 SO 4 ) volume. In addition, a blank test is set up to calibrate reagent and titration errors. The content of alkaline hydrolysis nitrogen is calculated by the formula.

[0032] Preferably, in step S9, the amount of composite bacteria and GMI1000 in the soil of the M and R sites is determined by absolute quantitative PCR (AQ-PCR), and 3 parallels are set for each treatment. Primers SL-44 (F) 5'-GCGTAGAGATGTGGAGGAA-3' and SL-44 (R) 5'-TAGGATTGTCAGAGGATGTCA-3' are used to detect the amount of SL-44 in the soil; primers Rs-5 (F) 5'-CACATCCGACTTGACAGACC-3' and Rs-5 (R) 5'-TTACCCGCAGAAGAAGCAC-3' are used to detect the amount of Rs-5 in the soil; primers GMI1000 (F) 5'-TAACCCAACATCTCACGACA-3' and GMI1000 (R) 5'-ATGCCACTAACGAAGCAGA-3' are used to detect the amount of GMI1000 in the soil. Take 0.5 g of soil from each sample and use Mag- Soil DNA was extracted using Soil DNA Kit M5635 (cat: M5635-02). Then, standard samples were prepared, standard curves were constructed, mixture A of SYBR and primers was prepared, 1 μL DNA was diluted several times and used as a template for the machine, and the machine system and Real-time PCR reaction were prepared; in step S10, the soil DNA that met the requirements was sent to the NovaSeq 6000 platform (PE250, Illumina, USA) of Wuhan Jinkairui Bioengineering Co., Ltd. for microbial high-throughput sequencing. Primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGAGT TT-3') were used to amplify the V4-V5 variable region of the bacterial 16S rRNA gene. The primers ITS5-1737F (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS2-2043R (5'-GCTGCGTTCTTCATCGATGC-3') were used to amplify the ITS1 region of fungi. The specific process of sequencing: genomic DNA extraction and sample detection, PCR product acquisition, PCR product mixing and purification, library construction and machine detection. Soil microbial high-throughput data analysis steps: using Flash (Version 0.23.1) 97% similarity and species annotation, and Qiime (Version 1.9.1) for α-diversity and β-diversity analysis.

[0033] The present invention is beneficial in that:

[0034] 1. The present invention provides an experimental method for studying the performance of a composite bacterial agent in preventing and controlling tomato bacterial wilt. Antagonistic experiments show that the composite bacterial agent can significantly inhibit bacterial wilt. Potted plant experiments show that bacterial wilt causes serious effects on the growth and physiological functions of tomato plants. After being treated with the composite bacterial agent, the incidence of bacterial wilt in tomato seedlings was reduced by 71.43% 30 days after transplanting, and the biological control effect on tomato bacterial wilt was as high as 77.78%. In addition, the physicochemical properties (soluble protein, soluble sugar, chlorophyll and carotenoids) content of tomato plants infected with bacterial wilt, the activity of plant antioxidant enzymes (SOD, POD and CAT), and the physicochemical properties of plant rhizosphere soil (organic matter, alkaline nitrogen, available phosphorus, and available potassium) content were significantly improved, all of which benefited from the direct inhibition of BE on bacterial wilt.

[0035] 2. The present invention can reveal through high-throughput sequencing and bioinformatics analysis that the composite bacterial agent inhibits Ralstonia solanacearum by increasing the relative abundance of beneficial bacteria such as Massilia, Pseudomonas, Bacillus and Enterobacter in the rhizosphere soil of tomatoes, and regulates the community structure of rhizosphere fungi to repair or reshape the microbial community of tomato rhizosphere soil after Ralstonia solanacearum infection, thereby indirectly preventing and controlling tomato bacterial wilt. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0037] Figure 1 This is a diagram showing the research idea of ​​the composite bacterial agent of the present invention on the control performance of tomato bacterial wilt. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Embodiment 1

[0040] An experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt comprises the following steps:

[0041] S1. Activation of strains; S2. Preparation of composite bacterial agents; S3. Inhibition of GMI1000 by composite bacterial agents; S4. Colonization of composite bacterial agents in tomato roots; S5. Response of composite bacterial agents to tomato root secretions; S6. Potted plant experiment; S7. Determination of plant biomass and physicochemical properties; S8. Determination of nutrient content in rhizosphere soil of plants; S9. Determination of the number of composite bacterial agents and GMI1000 in soil; S10. High-throughput sequencing of tomato rhizosphere soil microorganisms; In step S1, GMI1000 is streaked from a glycerol tube preserved at -80°C into a sterilized BG solid culture medium by the plate streak method, and then inverted in a constant temperature incubator at 28°C for 48 hours. Then, a single colony was picked and placed in a BG liquid culture medium, and cultured overnight at 28°C and 200rpm to complete activation; in the step S2, the composite bacterial agent was composed of Rs-5 and SL-44, and the culture medium used for Rs-5 and SL-44 was LB culture medium; in the step S3, the inhibition ability of the composite bacterial agent on GMI1000 was determined by the plate confrontation method. That is, 100 μL of activated GMI1000 was taken on the BG solid culture medium and spread evenly, and then three sterilized circular filter papers (φ=0.5 cm) were placed on each BG solid culture medium, and then 10 μL of the composite bacterial agent solution was inoculated on the filter paper, cultured at 30°C for 48 hours, and then observed whether an inhibition zone appeared. If an inhibition zone exists, it means that the composite bacterial agent can inhibit GMI1000, otherwise there is no inhibition. At the same time, strains Rs-5 and SL-44 were set as controls; in step S4, tomato seeds with plump particles and uniform size were selected, and then they were disinfected by soaking in 75% alcohol and rinsed with sterile water for multiple times. The culture matrix (vermiculite and perlite mixed in a ratio of 3:2) was added to the tissue culture bottle (800mL) and sterilized at high temperature. The tomato seedlings grew to 4 true leaves in the culture matrix, and then the tomato seedlings were taken out and washed with sterile water to remove the vermiculite and perlite at the roots. Then the tomato seedlings were placed in a centrifuge tube containing 20mL of bacterial suspension of strains Rs-5, SL-44 or composite bacterial agents. Then the centrifuge tube was placed in a climate box for 48 hours and then taken out, the tomato roots were rinsed with sterile water, and then about 1 cm was cut from the meristem and elongation zones of the tomato roots. Finally, the tomato roots were fixed in a 2.5% glutaraldehyde solution and freeze-dried, and then observed under a scanning electron microscope; in step S5, since the tomato root secretions are mainly sugars, amino acids and organic acids, the specific root secretion components selected in this chapter are: glucose, mannitol, sucrose, fructose, leucine, glutamic acid, aspartic acid, alanine, tartaric acid, malic acid, citric acid, and fumaric acid. The response of Rs-5, SL-44 and the composite bacterial agent to tomato root secretions was determined by the swimming plate method. Different root secretions (10mM) were added to the sterilized semi-solid culture medium (agar content 0.2%), shaken well and poured into a culture dish to cool.After 10 mL of the cultured bacterial solution was centrifuged at 8000 rpm and 4 °C for 10 minutes, it was resuspended with 1 mL of phosphate buffer (pH 7.3) to prepare a concentrated bacterial suspension, and then 20 μL of the concentrated bacterial suspension was taken in the center of the semi-solid culture medium. A semi-solid culture medium without root secretions was used as a blank control. After the above culture medium was cultured overnight, the chemotactic radius was recorded; in step S6, the tomato seeds were soaked in 70% ethanol, then soaked in 0.1% HgCl2 for 10 minutes, and finally rinsed with sterile water several times. The sterilized tomato seeds were sown in a seedling tray filled with sterilized soil, and the tomato seedlings were transplanted when they grew to three leaves and one heart. The soil used for the potted experiment was taken from the Lintong Campus of Xi'an Polytechnic University (34°21′N, 109°11′E), and the organic matter, alkaline nitrogen, available phosphorus and available potassium contents of the original soil were 15.2 g kg respectively. -1 , 50.62 mg kg -1 , 11.46 mg kg -1 and 180 mg kg -1 , the soil conductivity and pH were 7.90 and 480 μS·cm -1 . A total of 4 treatments were set up in the pot experiment: CK, no treatment (no compound bacterial agent and pathogen GMI1000 were added); T1, only compound bacterial agent was added; T2, only pathogen GMI1000 was added; T3, compound bacterial agent and pathogen GMI1000 were added. Six seedling pots (φ23.5cm×19.5cm) were set up for each treatment, each pot was filled with 3kg of non-sterile soil and transplanted with 3 tomato seedlings of the same growth. Before transplanting the tomato seedlings, the GMI1000 bacterial suspension was evenly sprayed on the soil for T2 and T3 treatments to control the pathogen concentration at 10 7 CFU·g -1 After transplanting the tomato seedlings, inoculate the compound bacterial agent into the center of the seedling pot (the concentration of the compound bacterial agent is about 10 8 CFU·g -1 The seedling pots were placed in a constant temperature and light incubator at 26°C with a light exposure time of 14 h (350 μmol·m 2 S -1), relative humidity was 50%-60%. The pot experiment ended 30 days after the tomato seedlings were transplanted. After the pot experiment, the plant biomass (fresh weight, dry weight, plant height, root length, stem thickness), plant physical and chemical index content and plant rhizosphere soil nutrient content were measured. Collection of soil samples: Two sampling points, namely M site and R site, were set to study the migration of the composite bacterial agent in the soil and its effect on the soil microbial community. The M site is located between the tomato rhizosphere and the inoculation site of the composite bacterial agent, and the R site is located in the tomato rhizosphere. At 15 and 30 days after tomato transplantation, the soil at M and R of three pots was taken for each treatment. The soil at M and R of three seedlings was taken from each pot, and then the soil at M and R of each pot was mixed evenly. After the soil was sieved with a 2mm sieve to remove impurities such as plant roots, it was stored at -80°C. The incidence of bacterial wilt was counted on the 15th and 30th days after the tomato seedlings were transplanted. The incidence of tomato bacterial wilt is the percentage of the number of diseased plants to the total number of plants. Disease level statistics: Six disease levels are determined based on the proportion of the wilting degree of tomato seedlings to the whole tomato plant. n = 0, no symptoms; n = 1, 20% of the whole plant is wilted; 2, 40% of the whole plant is wilted; 3, 60% of the whole plant is wilted; 4, 80% of the whole plant is wilted; 5, 100% of the whole plant is wilted. The disease index (DI) and relative control efficacy (CE) are calculated:

[0042] DI = (100∑(disease level n×number of plants of that disease level)) / (5×total number of plants in that treatment)

[0043] CE (%) = (control disease index - treatment disease index) × 100 / control disease index; In step S7, the biomass of tomato and corn plants includes fresh weight, dry weight, plant height, root length and diameter. The fresh sample of the plant is washed with pure water and then dried with paper, and its fresh weight is measured. Then it is transferred to a drying oven, dried at 90°C to constant weight, and taken out to measure the dry weight. The plant height is the distance from the base of the plant stem to the growth point, the root length is the distance from the longest root tip of the plant to the base of the stem, and the diameter of the plant is measured with a vernier caliper, and soluble protein: the determination of plant soluble protein refers to the Coomassie Brilliant Blue G-250 method. Weigh 0.2g of fresh plant leaves and add them to 5mL of distilled water. Grind them into a homogenate and centrifuge (10000rpm, 30min) to collect the supernatant as the test solution. Take 1.0 mL of the supernatant in a test tube, add 5 mL of Coomassie Brilliant Blue G-250 reagent, shake well and let stand for 2 minutes, measure the absorbance of each sample at a wavelength of 595 nm, and calculate the soluble protein content through the standard curve. Repeat for each sample three times; Soluble sugar: The soluble sugar content in the plant is determined by the sulfuric acid phenol method

[103] . Weigh 0.5 g of fresh plant leaves, cut them into pieces and add them to 5 mL of distilled water, seal them with a sealing film, extract them in boiling water for 30 minutes, collect the extract, filter it and transfer it to a 25 mL volumetric flask, cool it and make up the volume to 25 mL. This solution is the test solution. Take 0.5 mL of the test solution and mix it with 1.5 mL of distilled water, add 1 mL of 9% phenol solution, shake well and slowly drip 5 mL of concentrated sulfuric acid and shake well. Add distilled water to the colorimetric solution to make the total volume 8 mL, and let it stand at room temperature for half an hour. After the reaction is completed, the absorbance of each reaction solution is measured at a wavelength of 485 nm with the blank solution as the control, and the soluble sugar content is calculated from the standard curve. Each sample is repeated three times; Proline: The proline content of plants is determined by the sulfosalicylic acid method

[104] . Weigh 0.5 g of fresh plant leaves and place them in test tubes containing 5 mL of 3% sulfosalicylic acid solution. React in a boiling water bath for 10 min. After cooling, filter the extract into a clean test tube. The filtrate is the extract. Measure 2 mL of the extract into a clean test tube and mix it evenly with 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin reagent. Then place the test tube in a boiling water bath and heat for 30 min. After cooling, add 4 mL of toluene to the test tube, shake for 30 s, and let it stand for several minutes to allow all the proline in the leaves to be extracted into the toluene solution. Pipette the proline toluene extract solution on the upper layer of the test tube into a cuvette, and detect the absorbance at a wavelength of 520 nm with toluene as the blank control. The proline content in the plant was calculated based on the standard curve, and each sample was repeated three times; Chlorophyll and carotenoids: The chlorophyll and carotenoid content in the plant leaves was determined by ethanol extraction spectrophotometry

[105] . Weigh 0.2g of plant leaves, place them in a 10mL volumetric flask, add 10mL of 96% ethanol, shake well, and let stand in the dark overnight. After the plant leaves turn completely white, measure the absorbance of the extract at λ666, λ649 and λ470. The calculation formulas for each component are as follows:

[0044] Chlorophyll a (mg / g) = (13.95 × OD666 - 6.88 × OD649) × V / M;

[0045] Chlorophyll b (mg / g) = (24.96 × OD649 - 7.32 × OD666) × V / M;

[0046] Total chlorophyll (mg / g) = (20.29OD646 + 8.05OD661) × V / M;

[0047] Carotenoid C (mg / g) = 1000 × (OD470-2.05Chl a-114.8Chl b) / 245 × V / M

[0048] Note: In the above formula, V is the total volume of the extract (mL), and M is the fresh weight of the pepper leaves (g); in step S8, the organic matter, available potassium, available phosphorus and alkaline nitrogen in the rhizosphere soil of the plants were determined by potassium dichromate bulk density method, flame photometry, molybdenum antimony colorimetry and alkaline diffusion method

[112] respectively; Soil organic matter: Accurately weigh 0.5g of sieved air-dried soil sample and add it to the digestion tube, add 10.0mL of potassium dichromate sulfuric acid solution (0.4mol·L) and cover the tube with a stopper, and place the digestion tube in a porous furnace. Control the temperature of the electric furnace to maintain the temperature in the tube at 170℃~180℃. When the solution in the digestion tube starts to boil, start the timer. After 5 minutes, take out the digestion tube, cool it for 30 minutes, transfer the digestion solution and soil residue to a triangular flask without damage with water, rinse the test tube and the tube stopper with water, and combine the washing liquid into the triangular flask. The total volume of the solution in the triangular flask is controlled to be 50mL~60mL. Add 3 drops of o-phenanthroline indicator and titrate with ferrous sulfate standard solution. The solution changes from orange to blue-green and finally to brown-red, which is the end point. At the same time, set up a blank test and calculate the content of organic matter by the formula; Soil available potassium: weigh 2.0g of sieved air-dried soil in a plastic bottle, add 20mL of neutral NH4OAc solution, plug the rubber stopper, and shake for 30 minutes. Then filter the solution into a triangular flask with qualitative filter paper, and measure it on a flame photometer together with a potassium standard series solution. The concentration of available potassium is obtained from the standard curve; Soil available phosphorus: weigh 2.5g of sieved air-dried soil sample in a triangular flask, add 50mL of NaHCO 3 (0.5mol·L -1 ) solution, add phosphorus-free activated carbon, plug the bottle tightly, shake for 30 minutes, filter with phosphorus-free filter paper into a 50mL plastic bottle, draw 10mL of the filtrate, and add 10mL of NaHCO 3 (0.5mol·L -1) solution, add 5mL of molybdenum antimony reagent, shake well to remove carbon dioxide bubbles, and then dilute to 50mL with deionized water. After leaving for 30 minutes, perform colorimetry at 700nm. Use the absorption value of the blank solution as a reference, read the absorption value of the test solution, and calculate the effective phosphorus content from the standard curve; soil alkaline nitrogen: weigh 2.0g of sieved air-dried soil sample, place it in the outer chamber of a clean diffusion dish, and slowly rotate the diffusion dish to evenly spread the soil sample. Take 2mL H 3 BO 3 Add to the inner chamber of the diffusion dish. Quickly add 10.0mL of NaOH solution and cover tightly. Slowly rotate the diffusion dish and then place it in a 38℃ constant temperature incubator. Take it out after 24 hours of alkaline hydrolysis diffusion. Use 0.01mol·L NH3 in the absorption liquid in the inner chamber -1 (1 / 2H 2 SO 4 ) standard solution and record the consumed 0.01 mol·L-1(1 / 2H 2 SO 4 ) volume. In addition, a blank test was set to correct reagent and titration errors. The content of alkaline nitrogen was calculated by the formula; in step S9, the amount of composite bacterial agent and GMI1000 in the soil of sites M and R was determined by absolute quantitative PCR (AQ-PCR), and 3 parallels were set for each treatment. Primers SL-44 (F) 5'-GCGTAGAGATGTGGAGGAA-3' and SL-44 (R) 5'-TAGGATTGTCAGAGGATGTCA-3' were used to detect the amount of SL-44 in the soil; primers Rs-5 (F) 5'-CACATCCGACTTGACAGACC-3' and Rs-5 (R) 5'-TTACCCGCAGAAGAAGCAC-3' were used to detect the amount of Rs-5 in the soil; primers GMI1000 (F) 5'-TAACCCAACATCTCACGACA-3' and GMI1000 (R) 5'-ATGCCACTAACGAAGCAGA-3' were used to detect the amount of GMI1000 in the soil. 0.5 g of soil was taken from each sample and stained with Mag- Soil DNA was extracted using Soil DNA Kit M5635 (cat: M5635-02). Then, standard samples were prepared, standard curves were constructed, mixture A of SYBR and primers was prepared, 1 μL DNA was diluted several times and used as a template for the machine, and the machine system and Real-time PCR reaction were prepared; in step S10, the soil DNA that met the requirements was sent to the NovaSeq 6000 platform (PE250, Illumina, USA) of Wuhan Jinkairui Bioengineering Co., Ltd. for microbial high-throughput sequencing. Primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGAGT TT-3') were used to amplify the V4-V5 variable region of the bacterial 16S rRNA gene. The primers ITS5-1737F (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS2-2043R (5'-GCTGCGTTCTTCATCGATGC-3') were used to amplify the ITS1 region of fungi. The specific process of sequencing: genomic DNA extraction and sample detection, PCR product acquisition, PCR product mixing and purification, library construction and machine detection. Soil microbial high-throughput data analysis steps: 97% similarity and species annotation using Flash (Version 0.23.1), α-diversity and β-diversity analysis through Qiime (Version 1.9.1).

[0049] Working principle: First, the strain is activated. The strain stored in the glycerol tube at -80℃ is streaked into the sterilized BG solid medium by GMI1000 through the plate streaking method, and then inverted in a 28℃ constant temperature incubator for 48h. Then a single colony is picked in the BG liquid medium, and cultured overnight at 28℃ and 200rpm to complete the activation. After the strain activation is completed, the preparation of the composite bacterial agent begins. The composite bacterial agent is composed of Rs-5 and SL-44. Then the composite bacterial agent is colonized at the root of the tomato, and the tomato seeds with full particles and uniform size are selected, and then they are disinfected by soaking in 75% alcohol and rinsed with sterile water several times. The culture matrix (vermiculite and perlite mixed in a ratio of 3:2) is added to the tissue culture bottle (800mL) and sterilized at high temperature. The tomato seedlings grow to 4 true leaves in the culture matrix, and then the tomato seedlings are taken out and washed with sterile water to remove the vermiculite and perlite at the roots. Then, the tomato seedlings were placed in a centrifuge tube containing 20 mL of bacterial suspension of strain Rs-5, SL-44 or composite bacterial agent to colonize the composite bacterial agent in the tomato roots, and then the tomato seeds were soaked in 70% ethanol, then soaked in 0.1% HgCl2 for 10 minutes, and finally rinsed with sterile water several times. The sterilized tomato seeds were sown in a seedling tray filled with sterilized soil, and when the tomato seedlings grew to three leaves and one heart, they were transplanted into potted plants. Then, the plant biomass and physical and chemical properties, the nutrient content of the plant rhizosphere soil, and the number of composite bacterial agents and GMI1000 in the soil were determined at different times, and the results of the determination were analyzed and discussed.

[0050] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. An experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt, characterized in that: The following steps are involved: S1. Activation of strains; S2. Preparation of composite bacterial agent; S3, inhibition of compound bacterial agent on GMI1000; S4, colonization of the composite bacterial agent on tomato roots; S5, Response of composite bacterial agent to tomato root exudates; S6, potted plant experiment; S7, plant biomass and physicochemical properties determination; S8, determination of nutrient content in plant rhizosphere soil; S9, determination of the amount of compound bacterial agent and GMI1000 in soil; S10. High-throughput sequencing of tomato rhizosphere soil microorganisms. The step S4 is to select tomato seeds with plump grains and uniform size, and then culture them and then take samples for observation. The step S5 is to use different root secretions to determine the response of Rs-5, SL-44 and the composite bacterial agent to tomato root secretions through the swimming plate method. The step S9 is to set different treatment points at different points to detect the amount of GMI1000 in the soil.

2. The experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt according to claim 1, characterized in that: In step S1, GMI1000 streaks the strain stored in the glycerol tube at -80°C into the sterilized BG solid medium by the plate streaking method, and inverts it in a 28°C constant temperature incubator for 48 hours. Then, a single colony is picked and placed in the BG liquid medium, and cultured overnight at 28°C and 200rpm to complete activation.

3. The experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt according to claim 1, characterized in that: In step S2, the composite bacterial agent is compounded by Rs-5 and SL-44, and the culture medium used for Rs-5 and SL-44 is LB culture medium.

4. The experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt according to claim 1, characterized in that: In step S3, the inhibitory ability of the composite bacterial agent on GMI1000 is determined by the plate confrontation method. That is, 100 μL of activated GMI1000 is taken to the BG solid culture medium and spread evenly, and then three sterilized circular filter papers (φ=0.5 cm) are placed on each BG solid culture medium, and then 10 μL of the composite bacterial agent bacterial solution is inoculated on the filter paper, cultured at 30°C for 48 hours, and then observed whether an inhibition zone appears. If an inhibition zone exists, it means that the composite bacterial agent can inhibit GMI1000, otherwise there is no inhibition. At the same time, strains Rs-5 and SL-44 are set as controls.

5. The experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt according to claim 4, characterized in that: In step S4, tomato seeds with full particles and uniform size are selected, and then they are disinfected by soaking in 75% alcohol and rinsed with sterile water for multiple times. The culture matrix (vermiculite and perlite mixed in a ratio of 3:2) is added to a tissue culture bottle (800mL) and sterilized at high temperature. The tomato seedlings grow to 4 true leaves in the culture matrix, and then the tomato seedlings are taken out and washed with sterile water to remove the vermiculite and perlite at the roots. Then the tomato seedlings are placed in a centrifuge tube containing 20mL of a bacterial suspension of strains Rs-5, SL-44 or a composite bacterial agent. Then the centrifuge tube is placed in a climate box and cultured for 48 hours before being taken out, the tomato roots are rinsed with sterile water, and then about 1 cm is cut from the meristem and elongation zones of the tomato roots. Finally, the tomato roots are fixed in a 2.5% glutaraldehyde solution and freeze-dried, and then observed under a scanning electron microscope.

6. The experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt according to claim 1, characterized in that: In step S5, considering that the root secretions of tomatoes are mainly sugars, amino acids and organic acids, the specific root secretion components selected in this chapter are: glucose, mannitol, sucrose, fructose, leucine, glutamic acid, aspartic acid, alanine, tartaric acid, malic acid, citric acid, and fumaric acid. The response of Rs-5, SL-44 and composite bacterial agents to tomato root secretions was determined by the swimming plate method. Different root secretions (10mM) were added to the sterilized semi-solid culture medium (agar content 0.2%), shaken and poured into a culture dish to cool. After 10mL of the cultured bacterial solution was centrifuged at 8000rpm and 4℃ for 10 minutes, it was resuspended in 1mL phosphate buffer (pH 7.3) to prepare a concentrated bacterial suspension, and then 20μL of the concentrated bacterial suspension was taken in the center of the semi-solid culture medium. The semi-solid culture medium without root secretions was used as a blank control. After the above culture medium was cultured overnight, the chemotactic radius was recorded.

7. The experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt according to claim 6, characterized in that: In step S6, the tomato seeds are soaked in 70% ethanol, then soaked in 0.1% HgCl2 for 10 minutes, and finally rinsed with sterile water several times. The sterilized tomato seeds are sown in a seedling tray filled with sterilized soil, and the tomato seedlings are transplanted when they grow to three leaves and one heart. The soil used for the potted plant experiment was taken from the Lintong Campus of Xi'an Polytechnic University (34°21′N, 109°11′E). The organic matter, alkaline nitrogen, available phosphorus and available potassium contents of the original soil were 15.2 g·kg -1 , 50.62 mg kg -1 , 11.46 mg kg -1 and 180 mg kg -1 , the soil conductivity and pH were 7.90 and 480 μS·cm -1 The potted plant experiment was set up with 4 treatment groups: CK, no treatment (no compound bacterial agent and pathogen GMI1000 were added); T1, only the compound bacterial agent was added; T2, only the pathogen GMI1000 was added; T3, the compound bacterial agent and the pathogen GMI1000 were added. Six seedling pots (φ23.5cm×19.5cm) were set up for each treatment, each pot was filled with 3kg of non-sterile soil and transplanted with 3 tomato seedlings of the same growth. Before transplanting the tomato seedlings, the GMI1000 bacterial suspension was evenly sprayed on the soil in the T2 and T3 treatments to control the pathogen concentration at 10 7 CFU·g -1 After transplanting the tomato seedlings, inoculate the compound bacterial agent into the center of the seedling pot (the concentration of the compound bacterial agent is about 10 8 CFU·g -1 The seedling pots were placed in a constant temperature and light incubator at 26°C with a light exposure time of 14 h (350 μmol·m 2 S -1 ), relative humidity was 50%-60%. The pot experiment ended 30 days after the tomato seedlings were transplanted. After the pot experiment, the plant biomass (fresh weight, dry weight, plant height, root length, stem thickness), plant physical and chemical index content and plant rhizosphere soil nutrient content were measured. Collection of soil samples: Two sampling points, namely site M and site R, were set up to study the migration of the composite bacterial agent in the soil and its effect on the soil microbial community. Site M is located between the tomato rhizosphere and the site of the composite bacterial agent inoculation, and site R is located in the tomato rhizosphere. At 15 and 30 days after tomato transplantation, soils at M and R of three pots were taken for each treatment. Soils at M and R of three seedlings were taken from each pot, and then the soils at M and R of each pot were mixed evenly. The soil was passed through a 2mm sieve to remove impurities such as plant roots and stored at -80°C. The incidence of bacterial wilt was counted on the 15th and 30th days after the tomato seedlings were transplanted. The incidence of tomato bacterial wilt is the percentage of diseased plants to the total number of plants. Disease level statistics: Six disease levels are determined based on the proportion of the wilting degree of tomato seedlings to the whole tomato plant. n = level 0, no symptoms; n = level 1, 20% of the whole plant is wilted; level 2, 40% of the whole plant is wilted; level 3, 60% of the whole plant is wilted; level 4, 80% of the whole plant is wilted; level 5, 100% of the whole plant is wilted. And calculate the disease index (DI) and relative control efficacy (CE): DI = (100∑(disease level n×number of plants of that disease level)) / (5×total number of plants in that treatment) CE (%) = (control disease index - treatment disease index) × 100 / control disease index.

8. The experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt according to claim 1, characterized in that: In step S7, the biomass of tomato and corn plants includes fresh weight, dry weight, plant height, root length and diameter. The fresh sample of the plant was washed with pure water and then dried with paper to measure its fresh weight. Then it was transferred to a drying oven and dried at 90°C to constant weight, and then taken out to measure the dry weight. The plant height is the distance from the base of the plant stem to the growth point, the root length is the distance from the longest root tip of the plant to the base of the stem, and the diameter of the plant was measured with a vernier caliper. Soluble protein: the determination of plant soluble protein refers to the Coomassie Brilliant Blue G-250 method [102]. Weigh 0.2g of fresh plant leaves and add them to 5mL of distilled water. After grinding into a homogenate, centrifuge (10000rpm, 30min) and collect the supernatant as the test solution. Take 1.0 mL of the supernatant in a test tube, add 5 mL of Coomassie Brilliant Blue G-250 reagent, shake well and let stand for 2 minutes, measure the absorbance of each sample at a wavelength of 595 nm, and calculate the soluble protein content through the standard curve. Repeat for each sample three times; Soluble sugar: The soluble sugar content in the plant is determined by the sulfuric acid phenol method [103]. Weigh 0.5 g of fresh plant leaves, cut them into pieces and add them to 5 mL of distilled water, seal them with a sealing film, extract them in boiling water for 30 minutes, collect the extract, filter it and transfer it to a 25 mL volumetric flask, cool it and make up the volume to 25 mL. This solution is the test solution. Take 0.5 mL of the test solution and mix it with 1.5 mL of distilled water, add 1 mL of 9% phenol solution, shake well and slowly drip 5 mL of concentrated sulfuric acid and shake well. Add distilled water to the colorimetric solution to make the total volume 8 mL, and let it stand at room temperature for half an hour. After the reaction is completed, the absorbance of each reaction solution is measured at a wavelength of 485 nm with the blank solution as the control, and the soluble sugar content is calculated from the standard curve. Each sample is repeated three times; Proline: The proline content of plants is determined using the sulfosalicylic acid method [104]. Weigh 0.5 g of fresh plant leaves and place them in test tubes containing 5 mL of 3% sulfosalicylic acid solution. React in a boiling water bath for 10 minutes. After cooling, filter the extract into a clean test tube. The filtrate is the extract. Measure 2 mL of the extract into a clean test tube and mix it with 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin reagent. Then place the test tube in a boiling water bath and heat it for 30 minutes. After cooling, add 4 mL of toluene to the test tube, shake it for 30 seconds, and then let it stand for several minutes to allow all the proline in the leaves to be extracted into the toluene solution. Pipette the proline toluene extract solution on the upper layer of the test tube into a cuvette, and use toluene as a blank control to detect the absorbance at a wavelength of 520 nm. Calculate the proline content in the plant according to the standard curve, and repeat each sample three times; Chlorophyll and carotenoids: The chlorophyll and carotenoid content in the plant leaves was determined by ethanol extraction spectrophotometry [105]. Weigh 0.2 g of plant leaves, place them in a 10 mL volumetric flask, add 10 mL of 96% ethanol, shake well, and let it stand overnight in the dark. After the plant leaves turn completely white, measure the absorbance values ​​of the extract at λ666, λ649, and λ470. The calculation formulas for each component are as follows: Chlorophyll a (mg / g) = (13.95 × OD666 - 6.88 × OD649) × V / M; Chlorophyll b (mg / g) = (24.96 × OD649 - 7.32 × OD666) × V / M; Total chlorophyll (mg / g) = (20.29OD646 + 8.05OD661) × V / M; Carotenoid C (mg / g) = 1000 × (OD470-2.05Chl a-114.8Chl b) / 245 × V / M Note: In the above formula, V is the total volume of the extract (mL), and M is the fresh weight of pepper leaves (g).

9. The experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt according to claim 8, characterized in that: In step S8, the organic matter, available potassium, available phosphorus and alkaline nitrogen in the rhizosphere soil of the plants were determined by potassium dichromate bulk density method, flame photometry, molybdenum antimony colorimetry and alkaline diffusion method [112] respectively; Soil organic matter: 0.5 g of sieved air-dried soil sample was accurately weighed and added to a digestion tube, and 10.0 mL of potassium dichromate sulfuric acid solution (0.4 mol·L -1 ) and then cover the tube plug and put the digestion tube into the porous furnace. Control the temperature of the electric furnace and keep the temperature inside the tube at 170℃~180℃. Start timing when the solution in the digestion tube starts to boil. Take out the digestion tube after 5 minutes and cool it for 30 minutes. Use water to transfer the digestion solution and soil residue to the triangular flask without damage. Rinse the test tube and the tube plug with water, and combine the washing liquid into the triangular flask. The total volume of the solution in the triangular flask is controlled at 50mL~60mL. Add 3 drops of o-phenanthroline indicator and titrate with ferrous sulfate standard solution. The solution changes from orange to blue-green and finally to brown-red, which is the end point. At the same time, set up a blank test and calculate the content of organic matter by the formula; Soil available potassium: weigh 2.0g of sieved air-dried soil in a plastic bottle, add 20mL of neutral NH4OAc solution, plug the rubber stopper, and shake for 30 minutes. Then filter the solution into a conical flask with qualitative filter paper and measure it together with the potassium standard series solution on a flame photometer. The concentration of effective potassium can be obtained from the standard curve. Soil available phosphorus: weigh 2.5g of sieved air-dried soil sample into a conical flask, add 50mL NaHCO3 (0.5mol·L -1 ) solution, add phosphorus-free activated carbon, plug the bottle tightly, shake for 30 minutes, filter with phosphorus-free filter paper into a 50mL plastic bottle, draw 10mL of the filtrate, and add 10mL NaHCO3 (0.5mol·L -1 ) solution, add 5mL of molybdenum antimony reagent, shake well, remove carbon dioxide bubbles, then dilute to 50mL with deionized water, let it stand for 30 minutes, and perform colorimetry at 700nm. Use the absorption value of the blank solution as a reference, read the absorption value of the test solution, and calculate the effective phosphorus content from the standard curve; soil alkaline nitrogen: weigh 2.0g of sieved air-dried soil sample, place it in the outer chamber of a clean diffusion dish, and slowly rotate the diffusion dish to evenly spread the soil sample. Take 2mLH3BO3 and add it to the inner chamber of the diffusion dish. Quickly add 10.0mL NaOH solution and cover it tightly, slowly rotate the diffusion dish, and then place it in a 38℃ constant temperature incubator. Take it out after 24 hours of alkaline diffusion, and the NH3 in the inner chamber absorption liquid is diluted with 0.01mol·L -1 (1 / 2H2SO4) standard solution titration, and record the consumption of 0.01mol·L -1 (1 / 2H2SO4) volume. In addition, set up a blank test to calibrate reagent and titration errors. Calculate the content of alkaline nitrogen by the formula.

10. The experimental method for studying the control performance of a composite bacterial agent on tomato bacterial wilt according to claim 1, characterized in that: In step S9, the amount of composite bacteria and GMI1000 in the soil of sites M and R was determined by absolute quantitative PCR (AQ-PCR), with three parallels for each treatment. Primers SL-44 (F) 5'-GCGTAGAGATGTGGAGGAA-3' and SL-44 (R) 5'-TAGGATTGTCAGAGGATGTCA-3' were used to detect the amount of SL-44 in the soil; primers Rs-5 (F) 5'-CACATCCGACTTGACAGACC-3' and Rs-5 (R) 5'-TTACCCGCAGAAGAAGCAC-3' were used to detect the amount of Rs-5 in the soil; primers GMI1000 (F) 5'-TAACCCAACATCTCACGACA-3' and GMI1000 (R) 5'-ATGCCACTAACGAAGCAGA-3' were used to detect the amount of GMI1000 in the soil. Take 0.5 g of soil from each sample for Soil DNA was extracted using Soil DNA Kit M5635 (cat: M5635-02). Then, standard samples were prepared, standard curves were constructed, mixture A of SYBR and primers was prepared, 1 μL DNA was diluted several times and used as a template for the machine, and the machine system and Real-time PCR reaction were prepared; in step S10, the soil DNA that met the requirements was sent to the NovaSeq 6000 platform (PE250, Illumina, USA) of Wuhan Jinkairui Bioengineering Co., Ltd. for microbial high-throughput sequencing. Primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGAGT TT-3') were used to amplify the V4-V5 variable region of the bacterial 16S rRNA gene. The primers ITS5-1737F (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS2-2043R (5'-GCTGCGTTCTTCATCGATGC-3') were used to amplify the ITS1 region of fungi. The specific process of sequencing: genomic DNA extraction and sample detection, PCR product acquisition, PCR product mixing and purification, library construction and machine detection. Soil microbial high-throughput data analysis steps: using Flash (Version 0.23.1) 97% similarity and species annotation, and Qiime (Version 1.9.1) for α-diversity and β-diversity analysis.

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