Biocontrol composite microbial agent and application thereof

Abstract

The present application relates to the technical field of agricultural biological control, and discloses a kind of biological control composite microbial inoculant and its application, comprising: with natural biological base compound vanillin and vanillic acid as main raw material design and synthesis biodegradable tri-aldehyde monomer, and biodegradable tri-aldehyde monomer and p-phenylenediamine as organic building unit assembly form ordered porous crystal structure, obtain biodegradable COF material;Using impregnation adsorption method, the basic composite microbial inoculant that Bacillus thuringiensis, Bacillus subtilis and Streptomyces wareni are composed is loaded on biodegradable COF material, by tetraethylene pentamine and tannic acid copolymerization coating on its surface form degradable polytannic acid layer, prepared slow-release type composite microbial inoculant, its effective active component can be slowly released, can significantly improve the effective period and utilization rate of composite microbial inoculant, and to fusarium oxysporum and botrytis cinerea and other pathogenic fungi show obvious inhibitory effect, can be applied in the prevention and treatment of plant diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of agricultural biological control, in particular to a biological control composite microbial agent and application thereof. BACKGROUND

[0002] Biological control refers to the use of microorganisms to control various crop diseases, and microbial agent control is concerned due to its green, safe, efficient and no residue and other advantages. Common biological control mechanisms include microbial antagonism and competition, induced host resistance and plant growth promotion. At present, most biological control is based on the isolation, screening and application of single strains, but in field production, single-component biocontrol agents may have unstable control effect and narrow pathogen inhibition range, so composite microbial agents with better disease control effect have gradually become the development trend in the field of biological control.

[0003] Traditional biological control composite microbial agents have a too fast release rate of active ingredients in actual use, a large amount of active ingredients are released in a very short time, and then the release rate gradually decreases, and the subsequent release amount cannot meet the requirements of disease and pest control, and the loss phenomenon is easy to occur after application, which greatly limits the control effect on crop diseases.

[0004] Adsorptive sustained-release agents and coated sustained-release agents are the most commonly used sustained-release materials. Adsorptive sustained-release agents are composite microbial agents adsorbed in porous carriers (mesoporous silica, metal-organic frameworks, bentonite, calcium carbonate, covalent organic frameworks, etc.) with good biological safety and large specific surface area as storage bodies; coated sustained-release agents are composite microbial agents coated with biodegradable polymer compounds to achieve sustained-release function.

[0005] Research has found that covalent organic frameworks (COF) are a kind of crystalline porous framework material with adjustable pore size structure and high specific surface area, which can provide a larger space for the adsorption of composite microbial agents, and the ordered porous structure of covalent organic frameworks makes the adsorption of composite microbial agents regular, efficient and controllable, so as to have broad application space as a delivery carrier for biological control composite microbial agents. SUMMARY

[0006] The present application uses immersion adsorption technology and polymer coating technology to independently develop a sustained-release composite microbial agent with a core-shell coated structure, which can slowly release the effective active components, avoid the loss phenomenon caused by the too fast release rate of conventional biocontrol agents in actual use, and effectively improve the effective period and utilization rate of the composite microbial agent.

[0007] The biological control composite microbial agent is a core-shell coated structure, specifically, a degradable poly-tannin shell is coated on a biodegradable COF material loaded with the microbial agent.

[0008] The biodegradable COF material is prepared by Schiff base reaction of a biodegradable triformaldehyde-based monomer and p-phenylenediamine to form an imine bond for directional assembly.

[0009] Preferably, the microbial agent is a basic composite microbial agent composed of Bacillus thuringiensis liquid, Bacillus subtilis liquid and Streptomyces verticillus liquid.

[0010] Preferably, the volume ratio of the Bacillus thuringiensis liquid, the Bacillus subtilis liquid and the Streptomyces verticillus liquid in the basic composite microbial agent is 1:(0.5-2):(0.5-2), and the concentration of any of the liquid is 10 6 -10 9 CFU / mL.

[0011] Preferably, the preparation method of the biodegradable triformaldehyde-based monomer is as follows:

[0012] Vanillic acid is used as raw material, and through amidation reaction of 3.01-3.09 molar equivalents of carboxyl functional groups of vanillic acid and 1 molar equivalent of amino functional groups of melamine, a tris(amide phenolic hydroxyl) monomer is generated.

[0013] Vanillin is used as raw material, and through nucleophilic substitution reaction of 1 molar equivalent of phenolic hydroxyl functional groups of vanillin and 1.01-1.09 molar equivalents of chlorine functional groups of p-dichlorobenzene, a mono(chloro aldehyde) monomer is generated.

[0014] Through nucleophilic substitution reaction of 1 molar equivalent of phenolic hydroxyl functional groups of the tris(amide phenolic hydroxyl) monomer and 3.01-3.09 molar equivalents of chlorine functional groups of the mono(chloro aldehyde) monomer, a biodegradable triformaldehyde-based monomer is generated.

[0015] The biological control composite microbial agent is used for preventing and treating plant diseases caused by Fusarium oxysporum and / or Botrytis cinerea.

[0016] Preferably, the biological control composite microbial agent is used for preventing and treating cucumber fusarium wilt.

[0017] Preferably, the prevention method is that the biological control composite microbial agent is dissolved in sterile water and applied to cucumber seedlings by root irrigation.

[0018] Beneficial effects:

[0019] The application first designs and synthesizes a biodegradable triformal monomer containing an amide group by taking natural bio-based compounds vanillin and vanillic acid as main raw materials, and assembles the biodegradable triformal monomer and p-phenylenediamine as organic building units to form an ordered porous crystal structure, to obtain a biodegradable COF material;

[0020] Then, the complex microbial inoculant composed of Bacillus thuringiensis, Bacillus subtilis and Streptomyces verticillus is loaded on the biodegradable COF material by using the immersion adsorption method, and finally a degradable polytannin acid layer is formed on the surface by coating with tetraethylenepentamine and tannic acid, to obtain a slow-release complex microbial inoculant.

[0021] The active components of the slow-release complex microbial inoculant prepared in the application are gradually released from the biodegradable COF material along with the degradation of the polytannin acid coating layer, which can significantly improve the persistence period and utilization rate of the complex microbial inoculant, and the biodegradable COF material carrier can also be degraded in the soil, without causing environmental pollution due to residues.

[0022] The slow-release complex microbial inoculant prepared in the application shows obvious inhibitory effect on pathogenic fungi (taking Fusarium oxysporum and Botrytis cinerea as examples), and can effectively prevent and control crop diseases (taking cucumber fusarium wilt as an example). DETAILED DESCRIPTION

[0023] Example 1

[0024] The biodegradable triformal monomer is prepared by the following steps:

[0025] Step 1: taking natural bio-based compound vanillic acid as raw material, through the amidation reaction of 3.05 molar equivalents of carboxyl functional groups of vanillic acid and 1 molar equivalent of amino functional groups of melamine, a tris(amide phenolic hydroxyl) monomer is generated;

[0026] Step 2: taking natural bio-based compound vanillin as raw material, through the nucleophilic substitution reaction of 1 molar equivalent of phenolic hydroxyl functional groups of vanillin and 1.04 molar equivalents of chlorine functional groups of p-dichlorobenzene, a mono(chloro aldehyde) monomer is generated;

[0027] Step 3: through the nucleophilic substitution reaction of 1 molar equivalent of phenolic hydroxyl functional groups of the tris(amide phenolic hydroxyl) monomer and 3.08 molar equivalents of chlorine functional groups of the mono(chloro aldehyde) monomer, a biodegradable triformal monomer is generated, and the chemical structural formula is:

[0028]

[0029] The specific experimental steps for preparing the biodegradable triformal monomer are as follows:

[0030] Under the protection of nitrogen, 1.3 g of melamine, 5.2 g of vanillic acid and 50 mL of N,N-dimethylformamide were added to a three-necked flask with a water separator, stirred to dissolve at room temperature for 30 min, then 10 mL of N,N'-dicyclohexyl carbodiimide catalyst solution (prepared from 1.5 g of N,N'-dicyclohexyl carbodiimide catalyst and 10 mL of N,N-dimethylformamide) was added to the three-necked flask, and the reaction was stirred at 70°C for 6 h, then cooled to room temperature, and the solvent was removed by rotary evaporation, washed with dichloromethane, and dried in vacuum to obtain a tris(amide phenolic hydroxyl) monomer;

[0031] Under the protection of nitrogen, 1.5 g of p-dichlorobenzene and 20 mL of N,N-dimethylformamide were added to a three-necked flask, stirred to dissolve at room temperature, then 1.5 g of vanillin and 1.1 g of potassium carbonate were added to 30 mL of N,N-dimethylformamide, stirred to dissolve at room temperature for 30 min, then added dropwise to the three-necked flask, and the reaction was stirred at 60°C for 8 h, then cooled to room temperature, and the solvent was removed by rotary evaporation, washed with methanol, and dried in vacuum to obtain a mono(chloro aldehyde) monomer;

[0032] Under the protection of nitrogen, 1.9 g of tris(amide phenolic hydroxyl) monomer, 0.8 g of potassium carbonate and 30 mL of N,N-dimethylformamide were added to a three-necked flask, stirred to dissolve at room temperature for 30 min, then 30 mL of mono(chloro aldehyde) monomer solution (prepared from 2.6 g of mono(chloro aldehyde) monomer and 30 mL of N,N-dimethylformamide) was added to the three-necked flask, and the reaction was stirred at 70°C for 6 h, then cooled to room temperature, filtered, washed with methanol, and dried in vacuum to obtain a biodegradable trialdehyde monomer;

[0033] The molecular formula of the biodegradable trialdehyde monomer is C 69 H 54 O 18 N6, which was tested by a Vario EL III CHNSO element analyzer, and the experimental value (theoretical value, %) was C 65.95 (66.03), H 4.41 (4.34), and N 6.76 (6.70); the error range of the actual value and the theoretical value of C, H and N elements was within 0.3%.

[0034] Example Two:

[0035] Preparation of a biodegradable COF material: using biodegradable trialdehyde monomers and p-phenylenediamine as building blocks, a solvent thermal method was adopted to form imine bonds through Schiff base reaction to directionally assemble the building blocks into an ordered porous crystal structure, thereby obtaining a biodegradable COF material;

[0036] The specific experimental steps for preparing the biodegradable COF material are: 2.5 g of biodegradable triformaldehyde-based monomer, 0.4 g of p-phenylenediamine, 15 mL of mesitylene and 15 mL of 1,4-dioxane are added to a Schlenk reaction tube, ultrasonic dispersion is performed for 5 min, then 0.8 mL of glacial acetic acid is added, the temperature is increased to 90 DEG C, stirring is performed for 72 h, the temperature is cooled to room temperature, centrifugation is performed, methanol and tetrahydrofuran are used for washing in sequence, suction filtration is performed, and vacuum drying is performed, to obtain the biodegradable COF material.

[0037] Example three:

[0038] The preparation of the slow-release type composite microbial agent I includes the following steps:

[0039] Step one, the preparation of the basic composite microbial agent I, the preparation process is:

[0040] (1) Bacillus thuringiensis (model number CICC 21340) and Bacillus subtilis (model number CICC 20645) are inoculated in NA liquid medium, cultured at 30 DEG C and 180 rpm for 24 h, inoculated in fermentation medium at a inoculation amount of 1 vt%, and cultured at 30 DEG C and 180 rpm for 24 h, and the bacterial liquid concentration of Bacillus thuringiensis and Bacillus subtilis is adjusted to 1.0x10 8 CFU / mL by using sterile water;

[0041] (2) Streptomyces verticillus (model number CGMCC 4.1935) is inoculated in ISP2 liquid medium, cultured at 30 DEG C and 180 rpm for 3 days, inoculated in fermentation medium at a inoculation amount of 1 vt%, and cultured at 30 DEG C and 180 rpm for 5 days, and the bacterial liquid concentration of Streptomyces verticillus is adjusted to 1.0x10 8 CFU / mL by using sterile water;

[0042] (3) The bacterial liquid of Bacillus thuringiensis, Bacillus subtilis and Streptomyces verticillus is mixed in equal volume, to obtain the basic composite microbial agent I with a total bacterial liquid concentration of 1.0x10 8 CFU / mL;

[0043] The formula of the NA liquid medium is: beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, agar 20 g / L, distilled water to 1000 mL, and pH is 7.5;

[0044] The formula of the ISP2 liquid medium is: yeast powder 4 g / L, malt powder 10 g / L, glucose 4 g / L, agar 20 g / L, distilled water to 1000 mL, and pH is 7.3;

[0045] The fermentation medium formula is as follows: 20 g / L soybean flour, 10 g / L corn starch, 10 g / L glucose, 3 g / L beef extract, 2 g / L ammonium sulfate, 2 g / L sodium chloride, 2 g / L calcium carbonate, 0.04 g / L magnesium sulfate, 0.04 g / L potassium dihydrogen phosphate, 0.04 g / L dipotassium hydrogen phosphate, and distilled water to a final volume of 1000 mL, with a pH of 7.3.

[0046] Step two, prepare the slow-release compound microbial agent I, the preparation process is as follows:

[0047] (1) Loading of basic compound microbial agent I: The basic compound microbial agent I was loaded onto biodegradable COF material by impregnation and adsorption method. 5g of biodegradable COF material was completely impregnated in 40mL of basic compound microbial agent, left to stand for 24h to allow it to be fully adsorbed, and then freeze-dried to obtain loaded compound microbial agent I.

[0048] (2) Coating of basic compound bacterial agent I: 5g of loaded compound bacterial agent I was ultrasonically dispersed in 40mL Tris-HCl buffer (pH=7.4), 1.5g of tannic acid was added and stirred for 30min, then 0.8mL of tetraethylenepentamine was added, and the mixture was stirred at room temperature for 8h. After centrifugation, the mixture was ultrasonically dispersed again in 100mL Tris-HCl buffer (pH=7.4), washed with sterile water, and air-dried at room temperature to obtain sustained-release compound bacterial agent I.

[0049] Example 4:

[0050] The preparation of sustained-release compound microbial agent II includes the following steps:

[0051] Step 1: Prepare the basic compound microbial agent II. The preparation process is as follows:

[0052] (1) Bacillus thuringiensis (CICC 21340) and Bacillus subtilis (CICC 20645) were inoculated into NA liquid medium and cultured at 30℃ and 180 rpm for 24 h. Then, they were inoculated into fermentation medium at a 1 vt% inoculum and cultured at 30℃ and 180 rpm for 24 h. The bacterial concentrations of Bacillus thuringiensis and Bacillus subtilis were adjusted to 1.0 × 10⁻⁶ using sterile water. 7 CFU / mL;

[0053] (2) *Streptomyces vesicae* (model CGMCC 4.1935) was inoculated into ISP2 liquid medium and cultured at 30℃ and 180 rpm for 3 days. Then, it was inoculated into fermentation medium at a 1 vt% inoculum and cultured at 30℃ and 180 rpm for 5 days. The bacterial concentration of *Streptomyces vesicae* was adjusted to 1.0 × 10⁻⁶ using sterile water. 7 CFU / mL;

[0054] (3) The bacterial suspensions of Bacillus thuringiensis, Bacillus subtilis, and Streptomyces vesicatoria were mixed at a volume ratio of 1:0.5:1 to obtain a total bacterial suspension concentration of 1.0 × 10⁻⁶. 7 Basic compound bacterial agent II with CFU / mL;

[0055] The formula for NA liquid culture medium is as follows: 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L sodium chloride, 20 g / L agar, diluted to 1000 mL with distilled water, and pH 7.5.

[0056] The formulation of ISP2 liquid culture medium is as follows: 4 g / L yeast powder, 10 g / L malt powder, 4 g / L glucose, 20 g / L agar, diluted to 1000 mL with distilled water, and pH 7.3.

[0057] The fermentation medium formula is as follows: 20 g / L soybean flour, 10 g / L corn starch, 10 g / L glucose, 3 g / L beef extract, 2 g / L ammonium sulfate, 2 g / L sodium chloride, 2 g / L calcium carbonate, 0.04 g / L magnesium sulfate, 0.05 g / L potassium dihydrogen phosphate, 0.05 g / L dipotassium hydrogen phosphate, and distilled water to a final volume of 1000 mL, with a pH of 7.4.

[0058] Step two, prepare the slow-release compound bacterial agent II, the preparation process is as follows:

[0059] (1) Loading of basic compound microbial agent II: The basic compound microbial agent II was loaded onto the biodegradable COF material by the impregnation adsorption method. 5g of biodegradable COF material was completely impregnated in 40mL of basic compound microbial agent, and allowed to stand for 24h to allow it to be fully adsorbed. After freeze drying, the loaded compound microbial agent II was obtained.

[0060] (2) Coating of basic compound bacterial agent II: 5g of loaded compound bacterial agent II was ultrasonically dispersed in 40mL Tris-HCl buffer (pH=7.4), 1.5g of tannic acid was added and stirred for 30min, then 0.8mL of tetraethylenepentamine was added, and the mixture was stirred at room temperature for 8h. After centrifugation, it was ultrasonically dispersed again in 100mL Tris-HCl buffer (pH=7.4), washed with sterile water, and air-dried at room temperature to obtain sustained-release compound bacterial agent II.

[0061] Example 5:

[0062] The preparation of sustained-release compound microbial agent III includes the following steps:

[0063] Step 1: Prepare the basic compound microbial agent III. The preparation process is as follows:

[0064] (1) Bacillus thuringiensis (CICC 21340) and Bacillus subtilis (CICC 20645) were inoculated into NA liquid medium and cultured at 30℃ and 180 rpm for 24 h. Then, they were inoculated into fermentation medium at a 1 vt% inoculum and cultured at 30℃ and 180 rpm for 24 h. The bacterial concentrations of Bacillus thuringiensis and Bacillus subtilis were adjusted to 1.0 × 10⁻⁶ using sterile water. 9 CFU / mL;

[0065] (2) *Streptomyces vesicae* (model CGMCC 4.1935) was inoculated into ISP2 liquid medium and cultured at 30℃ and 180 rpm for 3 days. Then, it was inoculated into fermentation medium at a 1 vt% inoculum and cultured at 30℃ and 180 rpm for 5 days. The bacterial concentration of *Streptomyces vesicae* was adjusted to 1.0 × 10⁻⁶ using sterile water. 9 CFU / mL;

[0066] (3) Equal volumes of bacterial suspensions of Bacillus thuringiensis, Bacillus subtilis, and Streptomyces vesicularis were mixed to obtain a total bacterial suspension concentration of 1.0 × 10⁻⁶. 9 Basic compound bacterial agent III with CFU / mL;

[0067] The formula for NA liquid culture medium is as follows: beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, agar 22 g / L, distilled water to a final volume of 1000 mL, pH 7.6;

[0068] The formulation of ISP2 liquid culture medium is as follows: 4 g / L yeast powder, 10 g / L malt powder, 4 g / L glucose, 22 g / L agar, diluted to 1000 mL with distilled water, and pH 7.5.

[0069] The fermentation medium formula is as follows: 20 g / L soybean flour, 10 g / L corn starch, 10 g / L glucose, 3 g / L beef extract, 2 g / L ammonium sulfate, 2 g / L sodium chloride, 2 g / L calcium carbonate, 0.04 g / L magnesium sulfate, 0.05 g / L potassium dihydrogen phosphate, 0.05 g / L dipotassium hydrogen phosphate, and distilled water to a final volume of 1000 mL, with a pH of 7.4.

[0070] Step two, prepare the slow-release compound microbial agent III, the preparation process is as follows:

[0071] (1) Loading of basic compound microbial agent III: The basic compound microbial agent III was loaded onto biodegradable COF material by impregnation and adsorption method. 5g of biodegradable COF material was completely impregnated in 40mL of basic compound microbial agent, left to stand for 24h to allow it to fully adsorb, and then freeze-dried to obtain the loaded compound microbial agent III.

[0072] (2) Coating of basic compound bacterial agent III: 5g of loaded compound bacterial agent III was ultrasonically dispersed in 40mL Tris-HCl buffer (pH=7.4), 1.5g of tannic acid was added and stirred for 30min, then 0.8mL of tetraethylenepentamine was added, and the mixture was stirred at room temperature for 8h. After centrifugation, it was ultrasonically dispersed again in 100mL Tris-HCl buffer (pH=7.4), washed with sterile water, and air-dried at room temperature to obtain sustained-release compound bacterial agent III.

[0073] Performance testing:

[0074] I. Test on the inhibitory effect of slow-release compound microbial agents on pathogens

[0075] 7mm mycelial discs of *Fusarium oxysporum* (ATCC 16417) and *Botrytis cinerea* (ATCC 11542) were placed in the center of PDA medium. Holes were made 2.5cm apart from the center of each mycelial disc using a sterile punch. 3mL of a slow-release compound microbial agent solution (prepared from 5g of slow-release compound microbial agent and 40mL of sterile water) was added for an antagonistic experiment. A control group was prepared by inoculating only the pathogens onto the PDA medium. The medium was incubated at 28℃ for 7 days. Afterward, the colony diameter of the pathogens was measured, and the inhibition rate of the slow-release compound microbial agent against the pathogens was calculated. The specific method is as follows:

[0076] Inhibition rate = (Coronavirus colony diameter in control group 1 - Coronavirus colony diameter after treatment with slow-release compound bacterial agent) / Coronavirus colony diameter in control group 1 × 100%;

[0077] The formula for PDA culture medium is as follows: 200 g / L potato, 20 g / L glucose, 20 g / L agar, and 1000 mL distilled water, with a pH of 7.2.

[0078] The experimental results are shown in Table 1 below.

[0079] Table 1. Test results of the inhibitory effect of the slow-release compound microbial agent on Fusarium oxysporum and Botrytis cinerea.

[0080]

[0081] The experimental results in Table 1 show that the slow-release compound microbial agent prepared in this invention has a significant inhibitory effect on pathogens (Fusarium oxysporum and Botrytis cinerea);

[0082] II. Test of the sustained-release performance of the sustained-release compound microbial agent

[0083] The slow-release compound bacterial agent I solution prepared by 5g of slow-release compound bacterial agent I and 40mL of sterile water in performance test 1 was left to stand at room temperature for 5 days, filtered, and then ultrasonically dispersed in 40mL of sterile water to prepare the first slow-release compound bacterial agent I solution. The inhibition effect was tested according to the method in performance test 1.

[0084] The first slow-release compound bacterial agent I solution was left to stand at room temperature for 5 days, filtered, and then ultrasonically dispersed in 40 mL of sterile water to prepare the second slow-release compound bacterial agent I solution. The inhibition effect was tested according to the method in performance test one.

[0085] The second slow-release compound bacterial agent I solution was left to stand at room temperature for 10 days, filtered, and then ultrasonically dispersed again in 40 mL of sterile water to prepare the third slow-release compound bacterial agent I solution. The inhibition effect was tested according to the method in performance test one.

[0086] The experimental results are shown in Table 2 below.

[0087] Table 2. Results of sustained-release performance test of the sustained-release compound microbial agent.

[0088]

[0089] The experimental results in Table 2 show that the active components in the slow-release compound microbial agent prepared by this invention are released slowly. After 20 days, the slow-release compound microbial agent can still show a certain inhibitory effect on Fusarium oxysporum and Botrytis cinerea. In practical applications, this is beneficial to improving the utilization rate of the compound microbial agent.

[0090] III. Testing the Biocontrol Capacity of Slow-Release Compound Microbial Agents

[0091] Targeting cucumber wilt disease, a slow-release compound microbial agent solution was prepared by dispersing 10g of the solution in 80mL of sterile water. 50mL of this solution was then applied to the roots of 4-week-old cucumber seedlings (control group 2 was treated with plain water). After 24 hours of pretreatment, the seedlings were inoculated using a root drenching method with a bacterial solution concentration of 1×10⁻⁶. 5 The pathogen of cucumber wilt (Cucumber-specific Fusarium oxysporum, model BNCC 116160) was placed in a greenhouse for 28 days. The disease index and control effect were calculated according to the severity grading standard of cucumber wilt.

[0092] The severity level of cucumber wilt is as follows:

[0093] Grade 0: Healthy plant, no disease;

[0094] Grade 1: Cotyledons are yellowing, but not wilting;

[0095] Grade 2: Cotyledons wilting;

[0096] Grade 3: Cotyledons and true leaves wilting or plant stunting;

[0097] Level 4: Dead;

[0098] Disease index (%) = [∑(number of diseased plants at each level × representative value of disease severity at each level) / (representative value of highest disease severity × total number of plants surveyed)] × 100%;

[0099] Prevention and control effect (%) = [(disease index of control group 2 - disease index after treatment with slow-release compound bacterial agent) / disease index of control group 2] × 100%;

[0100] The experimental results are shown in Table 3 below.

[0101] Table 3. Results of the biocontrol capability test of the slow-release compound microbial agent.

[0102] Product type Disease index (%) Control effect (%) Cucumber growth after treatment with slow-release compound microbial agent I 12.8 83.9

[0103] Note: The disease index of control group 2 was 79.5%;

[0104] The experimental results in Table 3 show that the slow-release compound microbial agent prepared in this invention has a significant control effect on cucumber wilt disease.

Claims

1. A biological control compound microbial agent, characterized in that, The biological control compound microbial agent has a core-shell coated structure, specifically: a biodegradable polytannic acid shell is coated on a biodegradable COF material loaded with the basic compound microbial agent. The basic compound bacterial agent consists of Bacillus thuringiensis (Bt). Bacillus thuringiensis ) bacterial solution, Bacillus subtilis ( Bacillus subtilis ) bacterial suspension and Streptomyces vesicae ( Streptomyces violaceusniger The bacterial suspension has a volume ratio of 1:(0.5-2):(0.5-2), and the concentration of any bacterial suspension is 10. 6 -10 9 CFU / mL; Biodegradable COF materials are prepared by directional assembly of biodegradable trialdehyde monomers and p-phenylenediamine through a Schiff base reaction to form imine bonds; The chemical structural formula of the biodegradable trialdehyde monomer is: 。 2. The biological control compound microbial agent according to claim 1, characterized in that, The preparation method of the biodegradable trialdehyde monomer is as follows: Using vanillic acid as a raw material, an amidation reaction is carried out through 3.01-3.09 molar equivalents of the carboxyl functional group of vanillic acid and 1 molar equivalent of the amino functional group of melamine to generate tris(amidophenol hydroxy) monomers. Using vanillin as a raw material, a nucleophilic substitution reaction is carried out between the phenolic hydroxyl functional group of 1 molar equivalent of vanillin and the chlorine functional group of 1.01-1.09 molar equivalent of dichlorobenzene to generate a mono(chloroaldehyde) monomer. A biodegradable trialdehyde monomer is generated by a nucleophilic substitution reaction between the phenolic hydroxyl functional group of a 1 molar equivalent tri(amidophenol hydroxy) monomer and the chlorine functional group of a 3.01-3.09 molar equivalent mono(chloroaldehyde) monomer.

3. The application of the biological control compound microbial agent according to any one of claims 1-2, characterized in that, Biological control compound microbial agents are used to control plant diseases caused by Fusarium oxysporum and / or Botrytis cinerea.

4. The application of the biological control compound microbial agent according to claim 3, characterized in that, The aforementioned biological control compound microbial agent is used to control cucumber wilt disease.

5. The application of the biological control compound microbial agent according to claim 4, characterized in that, The control method is as follows: dissolve the biological control compound microbial agent in sterile water and apply it to cucumber seedlings by root irrigation.

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