Compound microbial agent for preventing and treating bacterial diseases of fruits and vegetables and preparation method thereof

Abstract

The application provides a compound microbial agent for preventing and treating bacterial diseases of fruits and vegetables and a preparation method thereof, wherein the compound microbial agent comprises Brevibacillus brevis, Pseudomonas chlororaphis and Streptomyces; wherein the ratio of the viable bacterial count of the Brevibacillus brevis, the Pseudomonas chlororaphis and the Streptomyces is (1-3):(2-4):(1-2). The Brevibacillus brevis directly inhibits bacteria by secreting antibacterial peptides, the Pseudomonas chlororaphis competes for ecological niches through iron carriers, and the Streptomyces produces antibiotics, so that multiple strains form a "bacteriostasis-competition-immune activation" multiple mechanism, thereby achieving efficient prevention and treatment of bacterial diseases of fruits and vegetables.

Description

Technical Field

[0001] This invention belongs to the field of microbial agent preparation technology, specifically, it relates to a compound microbial agent for preventing and controlling bacterial diseases of fruits and vegetables and its preparation. Background Technology

[0002] Bacterial diseases of fruits and vegetables (such as bacterial wilt, bacterial canker, and soft rot) are a major challenge to global agricultural production, causing economic losses exceeding US$30 billion annually, according to FAO statistics. While traditional chemical control methods are fast-acting, long-term use leads to severe drug resistance in pathogens and causes environmental problems such as excessive pesticide residues and damage to the soil microecology.

[0003] Microbial control, as a green alternative, has received considerable attention in recent years. However, existing technologies still face several bottlenecks. First, microbial agents often employ single-strain functionalities and have weak synergistic mechanisms. Furthermore, most commercially available microbial agents use only one or two strains, limiting their action to inhibition or nutrient competition. Second, agents suffer from poor stability and insufficient field adaptability, with conventional agents experiencing rapid cell death during storage and application. Additionally, delivery efficiency is low, targeting is lacking, and current microencapsulation technology has low encapsulation rates and cannot distinguish between pathogens and symbiotic bacteria, easily harming beneficial microorganisms. Moreover, agent transport within plants relies on passive diffusion, reaching diseased sites with an efficiency of less than 10%. Resistance induction mechanisms are not effectively integrated. While plant-derived inducers (such as methyl salicylate) can activate systemic resistance, their compatibility with microbial agents is poor; direct addition inhibits cell growth, and conventional loading materials release too quickly, resulting in a duration of action of less than 7 days for microbial agents.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this invention is to provide a compound microbial agent for the prevention and control of bacterial diseases in fruits and vegetables. This agent utilizes Bacillus shortbread to secrete antimicrobial peptides for direct bacterial inhibition, Pseudomonas aeruginosa to compete for ecological niches via siderophores, and Streptomyces to produce antibiotics. These multiple strains work synergistically to form a multi-mechanism of "bacterial inhibition-competition-immune activation," thereby achieving highly efficient prevention and control of bacterial diseases in fruits and vegetables.

[0006] The second objective of this invention is to provide a method for preparing the above-mentioned compound microbial agent for preventing and controlling bacterial diseases of fruits and vegetables. Through three-stage step fermentation, addition of whole-body sensing inhibitors, and pulsed electric field-assisted microencapsulation technology, the efficient synergy and stability of the strains are further improved.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0008] A compound microbial agent for preventing bacterial diseases in fruits and vegetables, comprising Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces;

[0009] The ratio of viable Bacillus shortbread, Pseudomonas aeruginosa, and Streptomyces is (1-3):(2-4):(1-2).

[0010] This invention employs a ternary synergistic system of Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces to achieve synergistic effects, thus forming a complete "antibacterial-competitive-antibiotic" three-pronged defense system. This is because the control effect of a single strain is often limited, as pathogens may develop resistance to a single mechanism, or a single strain may not cover multiple stages of the disease. For example, while Bacillus brevis alone can directly inhibit bacteria, it cannot block the pathogen's acquisition of iron ions; while Pseudomonas aeruginosa alone can compete for iron ions, its inhibitory effect on established pathogen populations is limited; and while Streptomyces can produce antibiotics, it lacks the ability to compete for other ecological niches. The combined use of these three strains achieves synergistic effects.

[0011] Bacillus brevis can secrete lipopeptide antibacterial substances (such as surfactants and iturobrine), whose molecular structure includes a hydrophilic head and a hydrophobic tail. This amphiphilic property allows it to insert into the phospholipid bilayer of the pathogen cell membrane through hydrophobic interactions, forming a transmembrane ion channel. This leads to an imbalance of the intracellular and extracellular ion gradient, ultimately causing leakage of cell contents and death of the pathogen. Its action speed is significantly faster than that of traditional chemical fungicides. Meanwhile, *Pseudomonas aeruginosa* secretes high-affinity siderophores (such as pseudomonobrine) with extremely high binding constants (Kf). This allows *P. aeruginosa* to efficiently chelate iron ions in the environment. Iron is an essential cofactor for the synthesis of pathogenic factors in pathogens. Therefore, *P. aeruginosa* competes with pathogens for iron, reducing the iron ion concentration of pathogens below the critical threshold and significantly inhibiting the expression of pathogenic genes. Simultaneously, *P. aeruginosa* can also form biofilms, physically occupying the plant rhizosphere ecological niche. This further blocks pathogen colonization; the actinomycin antibiotics (such as streptomycin) produced by Streptomyces can specifically bind to the 30S subunit of the pathogen's ribosome, interfering with tRNA translocation and peptide bond formation, thereby blocking protein synthesis. Compared with chemical antibiotics, the antibiotic complex secreted by Streptomyces can reduce the risk of resistance caused by single-point mutations in pathogens. Therefore, this invention mainly achieves a triple mechanism of "antibacterial inhibition-competition-antibiotics" through these three strains, forming a complete defense system that can effectively prevent and control bacterial diseases of fruits and vegetables. Among them, Bacillus shortbread can quickly destroy the cell membrane of pathogens within 0-24 hours, thereby achieving "immediate antibacterial action," while Pseudomonas aeruginosa can continuously suppress pathogen resuscitation within 24-72 hours through siderophore competition and biofilm formation, completing "niche blockade." Streptomyces can provide "continuous protection" by using antibiotics to inhibit pathogen reproduction for a long time after 48 hours.

[0012] Furthermore, this invention also ensures that the three strains can work synergistically to achieve better results by limiting the ratio of viable bacteria among Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces. When the ratio of viable bacteria of Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces is (1-3):(2-4):(1-2), the prepared compound microbial agent has excellent effects. This is because when the proportion of viable bacteria of Bacillus brevis is greater than 3, the excessive secretion of antimicrobial peptides by Bacillus brevis may inhibit the growth of other strains. If the proportion of viable bacteria of Pseudomonas aeruginosa is greater than 4, the excessive iron competition caused by Pseudomonas aeruginosa may easily lead to iron deficiency in plants. If the proportion of Streptomyces is too large, it will lead to antibiotic accumulation and cause an imbalance in the microbial community in the soil.

[0013] Preferably, as a further specific embodiment, it also includes Bacillus amyloliquefaciens, wherein the viable count of Bacillus amyloliquefaciens is 10-15% of the total viable count of Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces.

[0014] In this invention, the effect of the microbial agent is further enhanced by adding Bacillus amyloliquefaciens. Bacillus amyloliquefaciens activates the plant's systemic acquired resistance by secreting lipopeptides and plant hormones, upregulates PR protein expression, and thus enhances the lignification of plant cell walls. Simultaneously, its extracellular enzymes can degrade the pathogenic bacteria biofilm matrix, thereby improving the antibacterial effect of Bacillus amyloliquefaciens. Furthermore, this invention limits the viable count of Bacillus amyloliquefaciens. If the viable count is below 10%, the immune activation effect is insufficient, making it difficult to simultaneously coordinate with other strains for systemic defense against fruits and vegetables. Conversely, if the viable count exceeds 15% of the total viable count, this may lead to excessive stress in the plant or competition for resources with other strains, thus affecting the overall balance.

[0015] Preferably, as a further specific embodiment, the compound microbial agent also includes marine-derived Alternaria alterniflora, wherein the live bacteria ratio of Alternaria alterniflora to Streptomyces is 1:(1-1.5).

[0016] In this invention, the performance of the compound microbial agent is further improved by adding marine-derived Alternaria alternifolia. As a marine-derived microorganism, Alternaria alternifolia can secrete bromofuranone compounds, thereby interfering with the quorum sensing system of pathogens and inhibiting the expression of pathogen virulence factors. Its halophilic properties also endow the microbial agent with extremely high stability in high-salt soils. Furthermore, it can synergistically produce halogenated antibiotics with Streptomyces, further broadening the antibacterial spectrum of the compound microbial agent. Moreover, this invention limits the viable count of Alternaria alternifolia. When the viable count ratio of Alternaria alternifolia to Streptomyces is 1:1, the prepared compound microbial agent exhibits excellent effects. This is because when the viable count ratio is 1:1, the complementarity of the metabolites of Alternaria alternifolia and Streptomyces is optimal, thus avoiding excessive accumulation of antibiotics due to excessive Streptomyces, which inhibits the growth of beneficial microorganisms in the soil.

[0017] This invention also provides a method for preparing the above-mentioned compound microbial agent for preventing and controlling bacterial diseases of fruits and vegetables, comprising the following steps:

[0018] Three-stage stepwise fermentation culture was carried out, with Bacillus brevis fermenting at pH 6.8-7.2, Pseudomonas aeruginosa at pH 7.5-8.0, and Streptomyces at pH 6.5-7.0.

[0019] After mixing the bacterial cultures in proportion, add 0.1% of the total volume of the quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone to obtain the preparative solution;

[0020] The preparative solution was prepared into a microbial agent using pulsed electric field-assisted microencapsulation technology, with an electric field strength of 15-20 kV / cm and a pulse frequency of 100 Hz.

[0021] In this invention, a three-stage stepwise fermentation, the addition of quorum sensing inhibitors, and pulsed electric field-assisted microencapsulation techniques are used to further achieve efficient synergy and improved stability of bacterial strains. Compared with existing technologies, the preparation method of this invention has significant improvements in terms of strain activity maintenance, functional synergy, delivery efficiency, and environmental adaptability. Specifically, *Pseudomonas aeruginosa* exhibits the highest siderophore secretion efficiency under alkaline conditions, while *Bacillus brevis* shows the optimal antimicrobial peptide yield at neutral pH. This invention employs a three-stage stepwise fermentation process to independently ferment different strains within their optimal growth pH range, avoiding cross-inhibition. Furthermore, the stepwise culture allows for gradual scaling up of the fermentation scale, ensuring simultaneous optimization of cell density and metabolite accumulation. This invention competitively binds to the LuxR receptor of pathogens by adding quorum sensing inhibitors, blocking the acyl-homoserine lactone signaling pathway and inhibiting the expression of pathogen virulence factors. The addition of 0.1% quorum sensing inhibitors is... It can significantly reduce the quorum sensing efficiency of pathogenic bacteria while being non-toxic to beneficial bacteria. The inhibition of quorum sensing is achieved when the addition amount is below 0.1%, while excessive addition will inhibit the synthesis of siderophores in *Pseudomonas aeruginosa*. Furthermore, this invention significantly improves the survival rate of the bacterial agent and its encapsulation rate by adding a pulsed electric field to assist microencapsulation. When the electric field strength is between 15-20 kV / cm, the cell membrane can form reversible electroporation, promoting efficient encapsulation of bacteria and increasing the encapsulation rate. When the electric field parameter is below 15 kV / cm, the encapsulation rate is insufficient, and when the electric field parameter is above 20 kV / cm, the excessively high electric field will cause irreversible damage to the cell membrane, leading to bacterial death. Simultaneously, this invention further avoids thermal damage to the bacteria by using an auxiliary pulse frequency of 100 Hz. Additionally, the nano-TiO2 in the encapsulation material can achieve directional alignment in this electric field, further enhancing the mechanical strength of the encapsulation material.

[0022] Preferably, as a further specific embodiment, the capsule material in the microcapsule technology is a sodium alginate-chitosan-nano titanium dioxide composite, and the mass ratio of the capsule core to the capsule material is 1:(0.3-0.5).

[0023] This invention employs a sodium alginate-chitosan-nano titanium dioxide composite as a composite encapsulation material to achieve bacterial cell encapsulation. Compared to a single encapsulation material, the encapsulation material provided by this invention ensures that the microbial agent maintains a high survival rate even under harsh conditions such as saline-alkali soil. Sodium alginate forms a three-dimensional gel network through calcium ion cross-linking, protecting the bacteria from the acidic environment of the root system. Chitosan, with its positive charge, effectively adsorbs electropathogenic bacteria, thereby enhancing the targeted antibacterial effect of the microbial agent. Simultaneously, the presence of nano titanium dioxide allows the bacteria to generate reactive oxygen species during subsequent operations, directly oxidizing the cell membrane of pathogens and improving the UV stability of the encapsulation material. For this invention, the optimal effect is achieved when the core-to-encapsulation material mass ratio is 1:(0.3-0.5). This is because when the mass ratio is below 0.3, the thin encapsulation material results in a low survival rate of the bacteria in an acidic environment, while when the mass ratio is above 0.5, the thick encapsulation material hinders nutrient diffusion, delaying bacterial recovery.

[0024] Preferably, as a further specific embodiment, the surface of the microcapsule is grafted with the targeting molecule galacturonic acid oligosaccharide, and the grafting density is 2-3 molecules / μm².

[0025] This invention further enhances the targeting and disease control efficiency of the microbial agent by grafting the targeting molecule galacturonic acid oligosaccharide onto the surface of the microcapsules. Galacturonic acid oligosaccharide is a degradation product of plant cell walls, and many fruit and vegetable bacterial pathogens can secrete polygalacturonase, which specifically decomposes pectin in plant cell walls to produce oligosaccharide fragments. This mechanism has led these pathogens to evolve a chemotactic recognition mechanism for galacturonic acid oligosaccharide, thereby locating the infection site. This invention, by grafting galacturonic acid oligosaccharide onto the surface of the microcapsules, allows the grafted oligosaccharide to mimic plant diseases. The chemical signals at the spots attract pathogens to actively approach the microcapsules, achieving precise targeting. At the same time, oligosaccharides can bind to the active sites of pectinase in pathogens, partially inhibiting their enzyme activity and slowing down the degradation of plant cell walls. The effect is excellent when the grafting density of galacturonic acid oligosaccharides grafted on the surface of the microcapsules is 2-3 per μm². This is because when the grafting density is less than 2 per μm², the pathogen recognition signal is insufficient, resulting in a significant reduction in targeting efficiency. When the grafting density is too high, it will overactivate the plant's immune system, leading to an allergic reaction.

[0026] Preferably, as a further specific embodiment, methyl salicylate, a plant-derived inducer loaded with nano-hydroxyapatite, is added to the preparative solution before microencapsulation, and the amount added is 0.05-0.1% of the total weight of the preparative solution.

[0027] This invention also addresses the problems of poor compatibility and rapid release between plant-derived inducers and microbial agents by adding methyl salicylate, a plant-derived inducer loaded with nano-hydroxyapatite, to the inoculant before microencapsulation of the bacterial cells. The combination of the two, through slow-release delivery and immune synergy, matches the disease development cycle of the pathogen, avoiding waste caused by ineffective release of the microbial agent, while also protecting bacterial cell activity. This overcomes the application bottleneck of traditional inducers. Nano-hydroxyapatite, as an inorganic material with excellent biocompatibility, possesses a layered structure and porous properties that enable… Methyl salicylate is efficiently loaded through physical adsorption and ion exchange. Methyl salicylate is a key signaling molecule for acquired resistance in plants. It can activate the salicylic acid signaling pathway, upregulate PR protein expression, and thus enhance cell wall lignification. It can also synergize with Bacillus amyloliquefaciens to form a dual defense network of "microbe-plant". However, the present invention has certain limitations on the amount of methyl salicylate added. When the amount added is too low, the expression of PR protein is insufficient, making it difficult to effectively activate plant immunity. On the other hand, if the amount added is too high, it will slightly inhibit the growth of Bacillus brevis.

[0028] Preferably, as a further specific embodiment, the prepared composite microbial agent is further combined with biochar, wherein the biochar is plasma modified and has a specific surface area ≥800m² / g and a pore size distribution of 2-50nm.

[0029] This invention further enhances the soil colonization capacity and environmental adaptability of the prepared microbial agent by combining it with plasma-modified biochar. The high specific surface area of ​​the biochar allows for the adsorption of a large number of microorganisms, preventing the agent from being washed away. The pore size distribution of 2-50 nm balances microbial capacity with nutrient diffusion. The alkaline properties of the biochar neutralize acidic soil, providing a suitable environment for microorganisms. Furthermore, the invention employs plasma modification to introduce carboxyl and hydroxyl groups onto the surface of the biochar, enhancing its electrostatic adsorption to the cell walls of microorganisms and thus increasing the agent's loading rate. Therefore, if the specific surface area is too small, the microbial loading rate is reduced, making it difficult to meet field requirements; if the specific surface area is too large, it may adsorb excessive water, affecting microbial activity. Conversely, if the pore size is too small, it hinders nutrient transport; and if the pore size is too large, it leads to microbial loss.

[0030] Preferably, as a further specific embodiment, the compound microbial agent is applied to control bacterial diseases of fruits and vegetables through synergistic application of foliar spraying and root irrigation, wherein the concentration of the foliar spray solution is 10. 8 CFU / mL, root irrigation concentration was 10 6 CFU / mL.

[0031] Preferably, as a further specific embodiment, the application of the pesticide is combined with intermittent irradiation using an LED light source with a wavelength of 410 nm, and the light intensity is 50-80 μmol / (m²). 2 •s), irradiate 3 times a day, 10 minutes each time.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) This invention provides a compound microbial agent for preventing and controlling bacterial diseases of fruits and vegetables. It uses Bacillus shortness of breath to secrete antimicrobial peptides to directly inhibit bacteria, Pseudomonas aeruginosa to compete for ecological niches through side carriers, Streptomyces to produce antibiotics, Bacillus amyloliquefaciens to promote plant immunity, and Alternaria alternata to provide novel active substances from the ocean. The five strains work together to form a multi-mechanism of "antibacterial inhibition-competition-immune activation", thereby achieving high-efficiency prevention and control of bacterial diseases of fruits and vegetables.

[0034] (2) The present invention provides a method for preparing the above-mentioned compound microbial agent for preventing and controlling bacterial diseases of fruits and vegetables. Through three-stage step fermentation, addition of whole-body sensing inhibitors and pulsed electric field-assisted microencapsulation technology, the efficient synergy and stability of the strains are further improved. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] To more clearly illustrate the technical solutions in this invention, specific embodiments are described below. Example 1

[0037] 1. Material preparation

[0038] 1.1 Preparation of bacterial strains: Prepare Bacillus shortbread, Pseudomonas aeruginosa, and Streptomyces respectively;

[0039] 1.2 Culture medium preparation: LB liquid medium (pH=6.8 and pH=7.5), Gao's No. 1 medium (pH=6.5);

[0040] 1.3 Reagents:

[0041] Quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone;

[0042] Sodium alginate (food grade), chitosan (degree of deacetylation ≥90%), nano titanium dioxide (particle size 20nm);

[0043] Nano-hydroxyapatite (specific surface area ≥200m² / g), methyl salicylate (purity ≥98%);

[0044] Plasma-modified biochar (specific surface area 850 m² / g, pore size 2 nm);

[0045] 2. Fermentation culture of microbial strains

[0046] 2.1 Bacillus brevis culture procedure: Inoculate the bacterial strain into LB liquid medium (50 mL, pH=6.8) and culture with shaking at 30℃ and 180 rpm for 12 h;

[0047] The culture was then transferred to a 5L fermenter (pH=7.0), with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm, and cultured for 24 hours until OD. 600 =4.5;

[0048] Scale up to a 50L fermenter (pH=7.2), control dissolved oxygen ≥30%, and harvest the bacterial culture (live bacteria count ≥10). 9 (CFU / mL)

[0049] 2.2 Pseudomonas aeruginosa culture procedure: Initial culture is the same as for Bacillus brevis, pH 7.5;

[0050] The culture was then transferred to a 5L fermenter and the pH was adjusted to 7.8. 0.1mM FeCl3 was added, and the culture was incubated for 20 hours until the OD reached its maximum. 600 =5.0;

[0051] Scale up to a 50L tank, maintain pH=8.0, and harvest the bacterial culture;

[0052] 2.3 Streptomyces culture steps:

[0053] Streptomyces was inoculated into Gao's No. 1 medium (pH=6.5) and cultured at 28°C for 48 h;

[0054] 3. Mixing of bacterial solution and addition of additives

[0055] 3.1 Ratio Adjustment: The ratio of live bacteria of Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces was adjusted to 1:2:1;

[0056] Additives: Add 0.1% quorum sensing inhibitor (final concentration) and 0.05% nano hydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent).

[0057] 4. Pulsed electric field-assisted microencapsulation

[0058] (1) Preparation of capsule material

[0059] Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized.

[0060] Subsequently, microencapsulation was carried out according to a core-to-encapsulation material mass ratio of 1:0.3;

[0061] (2) Microencapsulation parameters

[0062] Electric field strength: 15 kV / cm;

[0063] Pulse frequency: 100Hz;

[0064] Temperature control: <25℃.

[0065] Targeted modification: grafting galacturonic acid oligosaccharides (density 2 units / μm²).

[0066] 5. Biochar compounding step: Mix the microencapsulated bacterial agent with plasma-modified biochar at a ratio of 1:5;

[0067] The compound bacterial agent was then shaken and adsorbed at 25°C for 2 hours, and then collected by centrifugation. Example 2

[0068] 1. Material preparation

[0069] 1.1 Preparation of bacterial strains: Prepare Bacillus shortbread, Pseudomonas aeruginosa, and Streptomyces respectively;

[0070] 1.2 Culture medium preparation: LB liquid medium (pH=7.2 and pH=7.5), Gao's No. 1 medium (pH=6.5);

[0071] 1.3 Reagents:

[0072] Quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone;

[0073] Sodium alginate (food grade), chitosan (degree of deacetylation ≥90%), nano titanium dioxide (particle size 20nm);

[0074] Nano-hydroxyapatite (specific surface area ≥200m² / g), methyl salicylate (purity ≥98%);

[0075] Plasma-modified biochar (specific surface area 850 m² / g, pore size 50 nm);

[0076] 2. Fermentation culture of microbial strains

[0077] 2.1 Bacillus brevis culture procedure: Inoculate the bacterial strain into LB liquid medium (50 mL, pH=6.8) and culture with shaking at 30℃ and 180 rpm for 12 h;

[0078] The culture was then transferred to a 5L fermenter (pH=7.0), with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm, and cultured for 24 hours until OD. 600 =4.5;

[0079] Scale up to a 50L fermenter (pH=7.2), control dissolved oxygen ≥30%, and harvest the bacterial culture (live bacteria count ≥10). 9 (CFU / mL)

[0080] 2.2 Pseudomonas aeruginosa culture procedure: Initial culture is the same as for Bacillus brevis, pH 7.5;

[0081] The culture was then transferred to a 5L fermenter and the pH was adjusted to 7.8. 0.1mM FeCl3 was added, and the culture was incubated for 20 hours until the OD reached its maximum. 600 =5.0;

[0082] Scale up to a 50L tank, maintain pH=8.0, and harvest the bacterial culture;

[0083] 2.3 Streptomyces culture steps:

[0084] Streptomyces was inoculated into Gao's No. 1 medium (pH=6.5) and cultured at 28°C for 48 h;

[0085] 3. Mixing of bacterial solution and addition of additives

[0086] 3.1 Ratio Adjustment: The ratio of live bacteria of Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces was adjusted to 3:4:2;

[0087] Additives: Add 0.1% quorum sensing inhibitor (final concentration) and 0.1% nano hydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent).

[0088] 4. Pulsed electric field-assisted microencapsulation

[0089] (1) Preparation of capsule material

[0090] Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized.

[0091] Subsequently, microencapsulation was carried out according to a core-to-encapsulation material mass ratio of 1:0.5;

[0092] (2) Microencapsulation parameters

[0093] Electric field strength: 20 kV / cm;

[0094] Pulse frequency: 100Hz;

[0095] Temperature control: <25℃.

[0096] Targeted modification: grafting galacturonic acid oligosaccharides (density 3 units / μm²).

[0097] 5. Biochar compounding step: Mix the microencapsulated bacterial agent with plasma-modified biochar at a ratio of 1:5;

[0098] The compound bacterial agent was then shaken and adsorbed at 25°C for 2 hours, and then collected by centrifugation. Example 3

[0099] 1. Material preparation

[0100] 1.1 Preparation of bacterial strains: Prepare Bacillus shortbread, Pseudomonas aeruginosa, and Streptomyces respectively;

[0101] 1.2 Culture medium preparation: LB liquid medium (pH=7.2 and pH=7.5), Gao's No. 1 medium (pH=6.5);

[0102] 1.3 Reagents:

[0103] Quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone;

[0104] Sodium alginate (food grade), chitosan (degree of deacetylation ≥90%), nano titanium dioxide (particle size 20nm);

[0105] Nano-hydroxyapatite (specific surface area ≥200m² / g), methyl salicylate (purity ≥98%);

[0106] Plasma-modified biochar (specific surface area 850 m² / g, pore size 30 nm);

[0107] 2. Fermentation culture of microbial strains

[0108] 2.1 Bacillus brevis culture procedure: Inoculate the bacterial strain into LB liquid medium (50 mL, pH=6.8) and culture with shaking at 30℃ and 180 rpm for 12 h;

[0109] The culture was then transferred to a 5L fermenter (pH=7.0), with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm, and cultured for 24 hours until OD. 600 =4.5;

[0110] Scale up to a 50L fermenter (pH=7.2), control dissolved oxygen ≥30%, and harvest the bacterial culture (live bacteria count ≥10). 9 (CFU / mL)

[0111] 2.2 Pseudomonas aeruginosa culture procedure: Initial culture is the same as for Bacillus brevis, pH 7.5;

[0112] The culture was then transferred to a 5L fermenter and the pH was adjusted to 7.8. 0.1mM FeCl3 was added, and the culture was incubated for 20 hours until the OD reached its maximum. 600 =5.0;

[0113] Scale up to a 50L tank, maintain pH=8.0, and harvest the bacterial culture;

[0114] 2.3 Streptomyces culture steps:

[0115] Streptomyces was inoculated into Gao's No. 1 medium (pH=6.5) and cultured at 28°C for 48 h;

[0116] 3. Mixing of bacterial solution and addition of additives

[0117] 3.1 Ratio Adjustment: The ratio of live bacteria of Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces was adjusted to 2:3:1;

[0118] Additives: Add 0.1% quorum sensing inhibitor (final concentration) and 0.06% nano hydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent).

[0119] 4. Pulsed electric field-assisted microencapsulation

[0120] (1) Preparation of capsule material

[0121] Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized.

[0122] Subsequently, microencapsulation was carried out according to a core-to-encapsulation material mass ratio of 1:0.4;

[0123] (2) Microencapsulation parameters

[0124] Electric field strength: 16 kV / cm;

[0125] Pulse frequency: 100Hz;

[0126] Temperature control: <25℃.

[0127] Targeted modification: grafting galacturonic acid oligosaccharides (density 2.5 units / μm²).

[0128] 5. Biochar compounding step: Mix the microencapsulated bacterial agent with plasma-modified biochar at a ratio of 1:5;

[0129] The compound bacterial agent was then shaken and adsorbed at 25°C for 2 hours, and then collected by centrifugation. Example 4

[0130] 1. Material preparation

[0131] 1.1 Preparation of bacterial strains: Prepare Bacillus shortbread, Pseudomonas aeruginosa, Streptomyces and Alternaria alterniflora respectively;

[0132] 1.2 Culture medium preparation: LB liquid medium (pH=7.2 and pH=7.5), Gao's No. 1 medium (pH=6.5) and marine bacteria medium (pH=7.5);

[0133] 1.3 Reagents:

[0134] Quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone;

[0135] Sodium alginate (food grade), chitosan (degree of deacetylation ≥90%), nano titanium dioxide (particle size 20nm);

[0136] Nano-hydroxyapatite (specific surface area ≥200m² / g), methyl salicylate (purity ≥98%);

[0137] Plasma-modified biochar (specific surface area 850 m² / g, pore size 30 nm);

[0138] 2. Fermentation culture of microbial strains

[0139] 2.1 Bacillus brevis culture procedure: Inoculate the bacterial strain into LB liquid medium (50 mL, pH=6.8) and culture with shaking at 30℃ and 180 rpm for 12 h;

[0140] The culture was then transferred to a 5L fermenter (pH=7.0), with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm, and cultured for 24 hours until OD. 600 =4.5;

[0141] Scale up to a 50L fermenter (pH=7.2), control dissolved oxygen ≥30%, and harvest the bacterial culture (live bacteria count ≥10). 9 (CFU / mL)

[0142] 2.2 Pseudomonas aeruginosa culture procedure: Initial culture is the same as for Bacillus brevis, pH 7.5;

[0143] The culture was then transferred to a 5L fermenter and the pH was adjusted to 7.8. 0.1mM FeCl3 was added, and the culture was incubated for 20 hours until the OD reached its maximum. 600 =5.0;

[0144] Scale up to a 50L tank, maintain pH=8.0, and harvest the bacterial culture;

[0145] 2.3 Steps for co-fermentation culture of Streptomyces and Alternaria:

[0146] Streptomyces was inoculated into Gao's No. 1 medium (pH=6.5) and cultured at 28°C for 48 h;

[0147] Alternating monoclonal bacteria were inoculated into a marine culture medium containing 30% NaCl at pH 7.5 and cultured at 25°C for 36 hours.

[0148] Subsequently, the bacteria were mixed at a ratio of 1:1 between Alternaria alternifolia and Streptomyces.

[0149] 3. Mixing of bacterial solution and addition of additives

[0150] 3.1 Ratio Adjustment: The ratio of live bacteria of Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces was adjusted to 2:3:1;

[0151] Additives: Add 0.1% quorum sensing inhibitor (final concentration) and 0.06% nano hydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent).

[0152] 4. Pulsed electric field-assisted microencapsulation

[0153] (1) Preparation of capsule material

[0154] Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized.

[0155] Subsequently, microencapsulation was carried out according to a core-to-encapsulation material mass ratio of 1:0.4;

[0156] (2) Microencapsulation parameters

[0157] Electric field strength: 16 kV / cm;

[0158] Pulse frequency: 100Hz;

[0159] Temperature control: <25℃.

[0160] Targeted modification: grafting galacturonic acid oligosaccharides (density 2.5 units / μm²).

[0161] 5. Biochar compounding step: Mix the microencapsulated bacterial agent with plasma-modified biochar at a ratio of 1:5;

[0162] The compound bacterial agent was then shaken and adsorbed at 25°C for 2 hours, and then collected by centrifugation. Example 5

[0163] 1. Material preparation

[0164] 1.1 Preparation of bacterial strains: Prepare Bacillus shortbread, Pseudomonas aeruginosa, Streptomyces, Alternaria alterniflora and Bacillus amyloliquefaciens respectively;

[0165] 1.2 Culture medium preparation: LB liquid medium (pH=7.2 and pH=7.5), Gao's No. 1 medium (pH=6.5) and marine bacteria medium (pH=7.5);

[0166] 1.3 Reagents:

[0167] Quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone;

[0168] Sodium alginate (food grade), chitosan (degree of deacetylation ≥90%), nano titanium dioxide (particle size 20nm);

[0169] Nano-hydroxyapatite (specific surface area ≥200m² / g), methyl salicylate (purity ≥98%);

[0170] Plasma-modified biochar (specific surface area 850 m² / g, pore size 30 nm);

[0171] 2. Fermentation culture of microbial strains

[0172] 2.1 Bacillus brevis culture procedure: Inoculate the bacterial strain into LB liquid medium (50 mL, pH=6.8) and culture with shaking at 30℃ and 180 rpm for 12 h;

[0173] The culture was then transferred to a 5L fermenter (pH=7.0), with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm, and cultured for 24 hours until OD. 600 =4.5;

[0174] Scale up to a 50L fermenter (pH=7.2), control dissolved oxygen ≥30%, and harvest the bacterial culture (live bacteria count ≥10). 9 (CFU / mL)

[0175] 2.2 Pseudomonas aeruginosa culture procedure: Initial culture is the same as for Bacillus brevis, pH 7.5;

[0176] The culture was then transferred to a 5L fermenter and the pH was adjusted to 7.8. 0.1mM FeCl3 was added, and the culture was incubated for 20 hours until the OD reached its maximum. 600 =5.0;

[0177] Scale up to a 50L tank, maintain pH=8.0, and harvest the bacterial culture;

[0178] 2.3 Steps for co-fermentation culture of Streptomyces and Alternaria:

[0179] Streptomyces was inoculated into Gao's No. 1 medium (pH=6.5) and cultured at 28°C for 48 h;

[0180] Alternating monoclonal bacteria were inoculated into a marine culture medium containing 30% NaCl at pH 7.5 and cultured at 25°C for 36 hours.

[0181] The mixture was then prepared by mixing Alternaria alternatae with Streptomyces live bacteria at a ratio of 1:1.2.

[0182] 3. Mixing of bacterial solution and addition of additives

[0183] 3.1 Ratio Adjustment: The ratio of viable Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces was adjusted to 2:3:1, with Bacillus amyloliquefaciens accounting for 12% of the total viable count;

[0184] Additives: Add 0.1% quorum sensing inhibitor (final concentration) and 0.06% nano hydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent).

[0185] 4. Pulsed electric field-assisted microencapsulation

[0186] (1) Preparation of capsule material

[0187] Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized.

[0188] Subsequently, microencapsulation was carried out according to a core-to-encapsulation material mass ratio of 1:0.4;

[0189] (2) Microencapsulation parameters

[0190] Electric field strength: 16 kV / cm;

[0191] Pulse frequency: 100Hz;

[0192] Temperature control: <25℃.

[0193] Targeted modification: grafting galacturonic acid oligosaccharides (density 2.5 units / μm²).

[0194] 5. Biochar compounding step: Mix the microencapsulated bacterial agent with plasma-modified biochar at a ratio of 1:5;

[0195] The compound bacterial agent was then shaken and adsorbed at 25°C for 2 hours, and then collected by centrifugation. Example 6

[0196] The specific implementation steps are the same as in Example 5, except that the ratio of viable Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces is adjusted to 0.5:1:05. Example 7

[0197] The specific implementation steps are the same as in Example 5, except that the ratio of viable bacteria of Bacillus brevis, Pseudomonas aeruginosa, and Streptomyces is adjusted to 4:8:3. Example 8

[0198] The specific implementation steps are the same as in Example 5, except that the number of viable Bacillus amyloliquefaciens is adjusted to 2% of the total viable bacteria in the compound bacterial agent. Example 9

[0199] The specific implementation steps are the same as in Example 5, except that the number of viable Bacillus amyloliquefaciens is adjusted to 20% of the total viable bacteria in the compound bacterial agent. Example 10

[0200] The specific implementation steps are the same as in Example 5, except that the ratio of live bacteria of Alternaria alternifolia to Streptomyces is adjusted to 0.5:1. Example 11

[0201] The specific implementation steps are the same as in Example 5, except that the ratio of live bacteria of Alternaria alternifolia to Streptomyces is adjusted to 1:3. Example 12

[0202] The specific implementation steps are the same as in Example 5, except that the mass ratio of the core to the material is adjusted to 1:0.1. Example 13

[0203] The specific implementation steps are the same as in Example 5, except that the mass ratio of the core to the material is adjusted to 1:1. Example 14

[0204] The specific implementation steps are the same as in Example 5, except that the grafting density of the target molecule galacturonic acid oligosaccharide is adjusted to 1 per μm². Example 15

[0205] The specific implementation steps are the same as in Example 5, except that the grafting density of the target molecule galacturonic acid oligosaccharide is adjusted to 5 molecules / μm². Example 16

[0206] The specific implementation steps are the same as in Example 5, except that the amount of plant-derived inducer methyl salicylate loaded with nano hydroxyapatite is adjusted to 0.01% of the dry weight of the inoculant. Example 17

[0207] The specific implementation steps are the same as in Example 5, except that the amount of plant-derived inducer methyl salicylate loaded with nano hydroxyapatite is adjusted to 1% of the dry weight of the inoculant. Comparative Example 1

[0208] The specific implementation steps are the same as in Example 5, except that no quorum sensing inhibitor is added. Comparative Example 2

[0209] The specific implementation steps are the same as in Example 5, except that the capsule material used in the microcapsule technology is only sodium alginate. Comparative Example 3

[0210] The specific implementation steps are the same as in Example 5, except that the target molecule galacturonic acid oligosaccharide is not grafted onto the surface of the microcapsule. Comparative Example 4

[0211] The specific implementation steps are the same as in Example 5, except that methyl salicylate, a plant-derived inducer loaded with nano-hydroxyapatite, is not added to the bacterial agent before microencapsulation. Comparative Example 5

[0212] The specific implementation steps are the same as in Example 5, except that the prepared bacterial agent is not combined with biochar. Comparative Example 6

[0213] The specific implementation steps are the same as in Example 5, except that the biochar is not modified by plasma.

[0214] Experiment Example 1: Efficacy Test of Compound Microbial Inoculants in Controlling Bacterial Diseases of Fruits and Vegetables

[0215] In this experimental example, the microbial compound inoculants obtained in Examples 1-17 and Comparative Examples 1-5 were used to verify their control effect on bacterial diseases of fruits and vegetables. The specific steps are as follows:

[0216] 1. Test bacterial agents: Microbial compound bacterial agents obtained in Examples 1-17 and Comparative Examples 1-6, Control Group 1 was a commercially available single Bacillus shortbacterium bacterial agent, and Control Group 2 was a chemical bactericide;

[0217] 2. Tested pathogens and plants

[0218] Pathogens: Bacterium wilt, Bacterium canker, and Bacterium soft rot

[0219] Plants: Tomato (bacterial wilt), citrus (canker), cabbage (soft rot)

[0220] 3 Experimental Design

[0221] 3.1 Application method:

[0222] Foliar spray: bacterial solution concentration 10 8 CFU / mL, 10 mL per plant;

[0223] Root irrigation: bacterial solution concentration 10 6 CFU / mL, 50 mL per plant;

[0224] Illumination assistance: 410 nm LED light source, 50 μmol / (m 2•s), 3 times a day, 10 minutes each time;

[0225] 3.2 Pathogen inoculation: Inoculate with pathogens 24 hours after application of the drug;

[0226] 4 Experimental Steps

[0227] 4.1 Pathogen culture: The three pathogens were inoculated separately onto LB medium and cultured at 28°C with shaking until OD. 600 =0.8;

[0228] Plant treatment: Tomato, citrus, and cabbage seedlings (5-leaf stage) were planted separately in sterilized soil;

[0229] Treatment was based on the method of application; the control group was sprayed with sterile water.

[0230] 4.2 Pathogen inoculation: Inoculate the leaves or stems with pathogens using the needle prick method.

[0231] Data Records:

[0232] Day 7: Record the initial symptoms of the illness;

[0233] Day 14: Statistical analysis of final morbidity, control effectiveness, and plant growth indicators;

[0234] Storage stability: The viable cell count was determined after the bacterial agent was stored at 4℃ for 30 days.

[0235] The final results are shown in Table 1:

[0236] Table 1 Test Results

[0237]

[0238] The experimental data above show that the compound microbial agent demonstrated in Examples 1-5 under different optimized conditions. Among them, Example 5 has the best overall effect. This is because it forms a multi-layered defense mechanism by mixing five strains: Bacillus brevis, Pseudomonas aeruginosa, Streptomyces, Bacillus amyloliquefaciens, and Alternaria alterniflora, which covers all stages of disease development. In addition, the ratio of viable bacteria and the ratio of Alternaria alterniflora to Streptomyces in Example 5 ensure the complementarity of metabolites and avoid the side effects caused by the excessive proliferation of a single strain.

[0239] Comparing Examples 6 and 7, it is evident that the low proportion of strains in Example 6 resulted in insufficient viable counts of Bacillus brevis and Streptomyces, leading to insufficient secretion of antimicrobial peptides and antibiotics, thus failing to effectively inhibit pathogens. Conversely, the high proportion of strains in Example 7 resulted in an excessive amount of Pseudomonas aeruginosa, causing excessive iron competition and potentially leading to iron deficiency in plants. Furthermore, the antimicrobial peptides secreted by Bacillus brevis inhibited the growth of other strains, disrupting the synergistic balance. Moreover, the imbalance in the proportion of strains in Examples 6 and 7 intensified competition among strains, resulting in abnormal accumulation of metabolites among strains, which affected the survival of bacteria within the microcapsules.

[0240] By comparing Examples 8 and 9, it can be seen that when the proportion of Bacillus amyloliquefaciens in Example 8 is too low, the immune activation of the microbial agent is insufficient, the expression level of PR protein is low, and the plant's disease resistance is not fully stimulated; while when the proportion of Bacillus amyloliquefaciens in Example 9 is too high, Bacillus amyloliquefaciens competes with other strains for resources and inhibits the activity of Bacillus brevis; moreover, excessive plant hormones may trigger plant stress response, which is not conducive to growth.

[0241] Comparing Examples 10-11, it can be seen that in Example 10, the ratio of Alternaria alternifolia to Streptomyces was too low, resulting in insufficient secretion of bromofuranone by Alternaria alternifolia, weakened quorum sensing inhibition effect, and insufficient blocking of pathogen virulence factor expression; while in Example 11, when the ratio of the two was too high, the excessive Streptomyces led to antibiotic accumulation, which may inhibit beneficial soil microorganisms and disrupt the microecological balance.

[0242] By comparing Examples 12-13, it can be seen that when the capsule material in Example 12 is too thin, the mechanical strength of the sodium alginate-chitosan composite layer is insufficient and cannot effectively protect the bacteria from acidic environment; while when the capsule material in Example 13 is too thick, it is easy to hinder the diffusion of nutrients, delay the recovery of bacteria, and the microcapsule particle size is too large, affecting the adhesion to the leaf surface.

[0243] Comparing Examples 14-15, it can be seen that when the grafting density is low in Example 14, the pathogen recognition signal is weak and the microcapsules cannot accurately locate the diseased parts; while when the grafting density is too high in Example 15, the galacturonic acid oligosaccharide over-activates the plant immune system, resulting in unnecessary energy consumption.

[0244] Comparing Examples 16-17, it can be seen that when the loading of methyl salicylate in Example 16 is too low, there is insufficient methyl salicylate, which cannot effectively activate the salicylic acid signaling pathway; while when the loading of methyl salicylate in Example 17 is too high, the excess of methyl salicylate causes the prepared microbial agent to have direct toxicity affecting the cell activity, and the sustained-release effect of nano hydroxyapatite is destroyed.

[0245] As can be seen from Comparative Examples 1-6, some key technologies of the preparation method of this invention have a certain impact on the effectiveness of the prepared microbial agents. In Comparative Example 1, without the addition of a sensing inhibitor, the expression of pathogenic virulence factors is not suppressed due to the lack of a sensing inhibitor, resulting in a significant decrease in the control effect. In Comparative Example 2, when sodium alginate is used as the encapsulation material, the mechanical strength and UV stability of the encapsulation material in the prepared microcapsules are insufficient, resulting in a significant decrease in the survival rate of live bacteria. In Comparative Example 3, without the addition of a targeting molecule, the agent relies on passive diffusion, resulting in a decrease in targeting efficiency. In Comparative Example 4, without the addition of methyl salicylate, the plant immune activation is insufficient, resulting in a significant decrease in both the control effect and growth increase. In Comparative Example 5, without the use of biochar composites, the agent has poor soil colonization ability, making it easy for the agent to be washed away. In Comparative Example 6, without modification of the biochar, the specific surface area and adsorption capacity of the biochar are insufficient, resulting in a decrease in the loading rate.

[0246] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a compound microbial agent for preventing and controlling bacterial diseases in fruits and vegetables, characterized in that, Includes the following steps: Three-stage stepwise fermentation culture was carried out, with Bacillus brevis fermenting at pH 6.8-7.2, Pseudomonas aeruginosa at pH 7.5-8.0, and Streptomyces at pH 6.5-7.

0. After mixing the bacterial cultures in proportion, add 0.1% of the total volume of the quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone to obtain the preparative solution; The preparative solution was prepared into a microbial agent using pulsed electric field-assisted microencapsulation technology, with an electric field strength of 15-20 kV / cm and a pulse frequency of 100 Hz. The microcapsule technology uses a sodium alginate-chitosan-nano titanium dioxide composite as the capsule material, with a core-to-material mass ratio of 1:(0.3-0.5).

2. The preparation method according to claim 1, characterized in that, The surface of the microcapsule is grafted with the target molecule galacturonic acid oligosaccharide, with a grafting density of 2-3 molecules / μm².

3. The preparation method according to claim 2, characterized in that, Before microencapsulation, methyl salicylate, a plant-derived inducer loaded with nano-hydroxyapatite, is added to the preparative solution at an amount of 0.05-0.1% of the total weight of the preparative solution.

4. The preparation method according to claim 2, characterized in that, It also includes combining the prepared composite microbial agent with biochar, wherein the biochar is plasma modified and has a specific surface area ≥800m² / g and a pore size distribution of 2-50nm.

5. A compound microbial agent for preventing and controlling bacterial diseases of fruits and vegetables, prepared by the method according to any one of claims 1-4, characterized in that, The ratio of viable Bacillus shortbread, Pseudomonas aeruginosa, and Streptomyces is (1-3):(2-4):(1-2). The composite microbial agent is a microcapsule, wherein the capsule material is a sodium alginate-chitosan-nano titanium dioxide composite, and the mass ratio of the capsule core to the capsule material is 1:(0.3-0.5).

6. The compound microbial agent for preventing and controlling bacterial diseases of fruits and vegetables according to claim 5, characterized in that, It also includes Bacillus amyloliquefaciens, wherein the viable count of Bacillus amyloliquefaciens is 10-15% of the total viable count of Bacillus shortbread, Pseudomonas aeruginosa, and Streptomyces.

7. The compound microbial agent for preventing and controlling bacterial diseases of fruits and vegetables according to claim 6, characterized in that, The compound microbial agent also includes marine-derived Alternaria alterniflora, and the live count ratio of Alternaria alterniflora to Streptomyces is 1:(1-1.5).

8. The application of the compound microbial agent according to any one of claims 5-7 in the prevention and control of bacterial diseases in fruits and vegetables, characterized in that, The pesticide was applied through a combination of foliar spraying and root irrigation, with the foliar spraying solution concentration being 10. 8 CFU / mL, root irrigation concentration was 10 6 CFU / mL.

9. The application according to claim 8, characterized in that, When applying the pesticide, use an LED light source with a wavelength of 410nm for intermittent irradiation. The light intensity should be 50-80μmol / (m²·s), and the irradiation should be performed 3 times a day for 10 minutes each time.

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