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

Abstract

The invention provides a compound microbial agent for preventing and treating bacterial diseases of fruits and vegetables and a preparation method of the compound microbial agent. The compound microbial agent comprises brevibacillus brevis, pseudomonas chlororaphis and streptomyces. Wherein the ratio of the viable count of the brevibacillus brevis to the viable count of the pseudomonas chlororaphis to the viable count of the streptomyces is (1-3): (2-4): (1-2). According to the invention, brevibacillus brevis secretes antibacterial peptides for direct bacteriostasis, pseudomonas chlororaphis competes for ecological niche through siderophores, streptomycete generates antibiotics, and multiple strains cooperate to form multiple mechanisms of bacteriostasis-competition-immune activation, so that high-efficiency prevention and treatment of fruit and vegetable bacterial diseases are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial agent preparation, and in particular relates to a composite microbial agent for preventing and treating bacterial diseases of fruits and vegetables and its preparation method. Background Art

[0002] Bacterial diseases of fruits and vegetables (such as bacterial wilt, canker, and soft rot) are a major challenge hindering global agricultural production. According to FAO statistics, they cause economic losses exceeding US$30 billion annually. While traditional chemical control methods are effective, long-term use can lead to the development of serious drug resistance in pathogens and pose environmental risks such as excessive pesticide residues and damage to the soil microbiome.

[0003] Microbial control, as a green alternative, has attracted considerable attention in recent years. However, existing technologies still face several bottlenecks. First, microbial agents typically employ single strains, resulting in weak synergistic mechanisms. Currently available microbial agents often utilize only one or two strains, with modes of action limited to antibacterial or nutrient competition. Second, they suffer from poor stability and insufficient field adaptability, leading to rapid bacterial decay during storage and application. Furthermore, delivery efficiency is low, targeting is lacking, and existing microencapsulation technology has a low encapsulation rate and cannot distinguish between pathogens and symbiotic bacteria, easily harming beneficial microorganisms. Furthermore, the delivery of microbial agents within plants relies on passive diffusion, resulting in a less than 10% efficiency in reaching diseased areas. Furthermore, mechanisms for inducing resistance have not been effectively integrated. While plant-derived inducers (such as methyl salicylate) can activate systemic resistance, their compatibility with microbial agents is poor, and their direct addition inhibits bacterial growth. Conventional loading materials release the agent too quickly, resulting in a long-lasting effect of less than seven days.

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

[0005] The first objective of the present invention is to provide a composite microbial agent for preventing and controlling bacterial diseases of fruits and vegetables. The agent uses antimicrobial peptides secreted by Brevibacillus to directly inhibit bacteria, while Pseudomonas chlororaphis competes for ecological niches through siderophores, and Streptomyces produces antibiotics. These multiple strains synergistically form a multi-pronged mechanism of "bacteriostasis-competition-immune activation," thereby achieving highly efficient prevention and control of bacterial diseases of fruits and vegetables.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned composite microbial agent for preventing and controlling bacterial diseases of fruits and vegetables, which further achieves efficient synergy and improved stability of strains through three-stage stepped fermentation, addition of global induction inhibitors and pulsed electric field-assisted microencapsulation technology.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted: A composite microbial agent for preventing and controlling bacterial diseases of fruits and vegetables, comprising Bacillus brevis, Pseudomonas chlororaphis and Streptomyces; The ratio of the number of live bacteria of Brevibacillus, Pseudomonas chlororaphis and Streptomyces is (1-3): (2-4): (1-2).

[0008] In the present invention, synergistic enhancement is achieved by adopting a ternary synergistic system of Bacillus brevis, Pseudomonas chlororaphis and Streptomyces, thereby forming a complete "antibacterial-competitive-antibiotic" three-defense system. This is because the prevention and control effect of a single strain is often limited, because the pathogens may develop resistance to a single mechanism, or a single strain cannot cover multiple links of the disease. For example, although Bacillus brevis can directly inhibit bacteria when used alone, it cannot block the pathogen's acquisition of iron ions; although Pseudomonas chlororaphis can compete for iron ions when used alone, it has limited antibacterial effect on the established pathogen population; although Streptomyces can produce antibiotics, it lacks the ability to compete for other ecological niches. The mixed use of the three can achieve synergistic enhancement; Among them, Bacillus brevis can secrete lipopeptide antimicrobial substances (such as surfactant and iturin), whose molecular structure contains a hydrophilic head and a hydrophobic tail. This amphiphilic property enables it to insert into the phospholipid bilayer of the pathogen cell membrane through hydrophobic interactions, forming a transmembrane ion channel, resulting in an imbalance of ion gradients inside and outside the cell, ultimately causing leakage of cell contents and death of pathogens. Its action speed is significantly faster than traditional chemical fungicides; while Pseudomonas chlororaphis secretes high-affinity iron carriers (such as pseudomycin) with an extremely high binding constant (Kf), which enables Pseudomonas chlororaphis to efficiently chelate iron ions in the environment. Iron is an essential cofactor for pathogens to synthesize pathogenic factors. Therefore, Pseudomonas chlororaphis competes with pathogens for iron, reducing the iron ion concentration of pathogens to below the critical threshold, significantly inhibiting the expression of their pathogenic genes. At the same time, Pseudomonas chlororaphis can also form biofilms and physically occupy the rhizosphere niche of plants. , further blocking the colonization of pathogens; the actinomycin antibiotics (such as streptomycin) produced by Streptomyces can specifically bind to the 30S subunit of the pathogen ribosome, interfere with tRNA insertion 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, the present invention mainly uses these three strains to achieve the triple mechanism of "inhibition-competition-antibiotics", forming a complete defense system that can effectively prevent and control bacterial diseases of fruits and vegetables. Among them, Bacillus brevis can quickly destroy the cell membrane of pathogens within 0-24 hours, thereby achieving "instant inhibition", while Pseudomonas chlororaphis can continuously suppress the recovery of pathogens within 24-72 hours through iron carrier competition and biofilm formation, completing "niche blockade", and Streptomyces can use antibiotics to long-term inhibit the reproduction of pathogens after 48 hours, thereby providing "sustained protection".

[0009] Furthermore, the present invention also limits the ratio of the number of live bacteria among Bacillus brevis, Pseudomonas chlororaphis, and Streptomyces to ensure that the three strains can synergistically enhance their effectiveness. When the ratio of the number of live bacteria among Bacillus brevis, Pseudomonas chlororaphis, and Streptomyces is (1-3): (2-4): (1-2), the composite microbial agent prepared thereby has excellent effects. This is because when the ratio of live 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 ratio of live bacteria of Pseudomonas chlororaphis is greater than 4, excessive iron competition caused by Pseudomonas chlororaphis may easily lead to iron deficiency in plants. If the ratio of Streptomyces is too large, antibiotic accumulation may occur, thereby causing an imbalance in the microbial community in the soil.

[0010] Preferably, as a further specific embodiment, Bacillus amyloliquefaciens is further included, wherein the number of viable bacteria of the Bacillus amyloliquefaciens is 10-15% of the total number of viable bacteria of the Brevibacillus, Pseudomonas chlororaphis and Streptomyces.

[0011] In the present invention, the effect of the microbial agent is further improved by adding Bacillus amyloliquefaciens. It activates the systemic acquired resistance of the plant by secreting lipopeptides and plant hormones, upregulates the expression of PR proteins, and thereby enhances the degree of lignification of the plant cell walls. At the same time, the extracellular enzymes produced by it can degrade the biofilm matrix of pathogens, thereby enhancing the antibacterial effect of Bacillus brevis. At the same time, the present invention also limits the number of viable bacteria of Bacillus amyloliquefaciens. If the number of viable bacteria of Bacillus amyloliquefaciens is lower than the lower limit of 10%, the immune activation effect is insufficient due to the insufficient number of viable bacteria of Bacillus amyloliquefaciens, and it is difficult to cooperate with other strains to systematically defend fruits and vegetables. When the number of viable bacteria of Bacillus amyloliquefaciens exceeds 15% of the total number of viable bacteria, the Bacillus amyloliquefaciens exceeding this ratio may cause excessive stress response of the plant or compete for resources with other strains, thereby affecting the overall balance.

[0012] Preferably, as a further specific embodiment, the composite microbial agent further comprises Alteromonas of marine origin, and the ratio of live bacteria of the Alteromonas to the Streptomyces is 1:(1-1.5).

[0013] In the present invention, the performance of the composite microbial agent is further improved by adding marine-derived Alteromonas. As a marine-derived microorganism, Alteromonas can secrete brominated furanone compounds, thereby interfering with the quorum sensing system of pathogens and inhibiting the expression of virulence factors of pathogens. At the same time, its halophilic properties can give the microbial agent extremely high stability even in high-salt soils, and it can also synergize with Streptomyces to produce halogenated antibiotics, thereby further broadening the antibacterial spectrum of the composite microbial agent. Furthermore, the present invention also limits the number of viable bacteria of Alteromonas. When the ratio of viable bacteria of Alteromonas to Streptomyces is 1:1, the composite microbial agent prepared thereby has excellent effect. This is because when the ratio of viable bacteria of the two is 1:1, the metabolic products of Alteromonas and Streptomyces are optimally complementary, thereby avoiding excessive accumulation of antibiotics due to excessive Streptomyces, which inhibits the growth of beneficial microorganisms in the soil.

[0014] The present invention also provides a method for preparing the composite microbial agent for preventing and treating bacterial diseases of fruits and vegetables, comprising the following steps: Three-stage stepwise fermentation culture was carried out respectively, and the fermentation conditions of Bacillus brevis, Pseudomonas chlororaphis and Streptomyces were controlled at pH 6.8-7.2, pH 7.5-8.0 and pH 6.5-7.0 respectively. After mixing the bacterial solutions in proportion, 0.1% of the total volume of the quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone was added to prepare a preparative solution; The preparatory solution is prepared into a microbial agent by adopting a pulse electric field assisted microencapsulation technology, with an electric field strength of 15-20 kV / cm and a pulse frequency of 100 Hz.

[0015] In the present invention, the technology of three-stage stepped fermentation, addition of global sensing inhibitors and pulsed electric field-assisted microencapsulation is used to further achieve efficient synergy and stability improvement of strains. Compared with the existing technology, the preparation method of the present invention has significant progress in terms of strain activity maintenance, functional synergy, delivery efficiency and environmental adaptability. Among them, based on the fact that Pseudomonas chlororaphis has the highest iron carrier secretion efficiency in an alkaline environment and Bacillus brevis has the best antimicrobial peptide production at a neutral pH, the present invention adopts three-stage stepped fermentation to conduct independent fermentation for the optimal growth pH range of different strains, thereby avoiding cross-inhibition. At the same time, the stepped culture can also ensure the simultaneous optimization of bacterial density and metabolite accumulation by gradually amplifying the fermentation scale. The present invention competitively binds to the LuxR-type receptor of pathogens by adding quorum sensing inhibitors, blocks the acylhomoserine lactone signaling pathway, and inhibits the expression of virulence factors of pathogens, wherein the addition amount of quorum sensing inhibitor is 0.1%. It can significantly reduce the quorum sensing efficiency of pathogens while being non-toxic to beneficial bacteria. When the addition amount is less than 0.1%, quorum sensing can be inhibited. If the addition amount is too high, the synthesis of iron carriers of Pseudomonas chlororaphis will be inhibited. Subsequently, the present invention also adds a pulsed electric field to assist microencapsulation, thereby greatly improving the survival rate of the bacterial agent while also improving the embedding rate of the bacterial agent. When the electric field strength is between 15 and 20 kV / cm, the cell membrane can form reversible electroporation, thereby promoting the efficient embedding of the bacteria by the capsule material and improving the embedding rate. When the electric field parameter is lower than 15 kV / cm, the embedding rate of the bacteria is insufficient. When the electric field parameter is higher than 20 kV / cm, the excessively high electric field will cause irreversible damage to the cell membrane of the bacteria, thereby killing the bacteria. At the same time, the present invention further avoids thermal damage to the bacteria by using an auxiliary pulse frequency of 100 Hz. In addition, the nano-TiO2 in the capsule material can also be oriented in the electric field, thereby further enhancing the mechanical strength of the capsule material.

[0016] 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 capsule core to capsule material is 1:(0.3-0.5).

[0017] The present invention uses a sodium alginate-chitosan-nano-titanium dioxide composite as a composite capsule material to achieve bacterial encapsulation. Compared to a single capsule material, the capsule material provided by the present invention can ensure that the microbial agent can maintain a high survival rate under harsh conditions such as saline-alkali soil. The sodium alginate can form a three-dimensional gel network through calcium ion cross-linking, protecting the bacteria from the acidic environment of the root system. The positive charge of chitosan can effectively adsorb electropathogens, thereby enhancing the targeted antibacterial effect of the microbial agent. At the same time, the presence of nano-titanium dioxide can enable the bacteria to produce reactive oxygen species during subsequent operations, thereby directly oxidizing the cell membrane of the pathogens through the reactive oxygen species, and also improve the ultraviolet stability of the capsule material. In the present invention, the mass ratio of the capsule core to the capsule material is 1: (0.3-0.5), which can achieve excellent results. This is because when the mass ratio is below 0.3, the survival rate of the bacteria in the acidic environment is low due to the thin capsule material. When the mass ratio is above 0.5, the excessively thick capsule material hinders the diffusion of nutrients, resulting in delayed bacterial recovery.

[0018] Preferably, as a further specific embodiment, the surface of the microcapsule is grafted with targeting molecules galacturonic acid oligosaccharide, and the grafting density is 2-3 per μm².

[0019] In the present invention, the targeting molecule galacturonic acid oligosaccharide is grafted on the surface of the microcapsule to significantly improve the targeting and disease control efficiency of the microbial agent. 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 enables these pathogens to evolve a chemotactic recognition mechanism for galacturonic acid oligosaccharide to locate the infection site. The present invention grafts galacturonic acid oligosaccharide on the surface of the microcapsule to make the grafted oligosaccharide simulate plant pathogens. The chemical signals at the spots then attract pathogens to actively approach the microcapsules, achieving precise targeting. At the same time, oligosaccharides can bind to the active sites of pectinase of pathogens, partially inhibiting their enzyme activity and slowing down the degradation of plant cell walls. When the grafting density of galacturonic acid oligosaccharides grafted on the surface of the microcapsules is 2-3 / μm², the effect achieved is excellent. This is because when the grafting density is lower than 2 / μm², the pathogen recognition signal is insufficient, resulting in a significant decrease in targeting efficiency. When the grafting density is too high, the excessively high grafting density will over-activate the plant immune system and cause an allergic reaction.

[0020] Preferably, as a further specific embodiment, methyl salicylate, a plant-derived inducer supported by nano-hydroxyapatite, is added to the preparatory solution before microencapsulation, and the added amount is 0.05-0.1% of the total weight of the preparatory solution.

[0021] In the present invention, the plant-derived inducer methyl salicylate loaded by nanohydroxyapatite is added to the microbial agent before the microbial body is microencapsulated, thereby solving the problems of poor compatibility between the plant-derived inducer and the microbial agent and too fast release. The combination of the two can match the disease development cycle of the pathogen through sustained-release delivery and immune synergy, avoid the waste caused by the ineffective release of the microbial agent, and at the same time protect the activity of the microbial body, breaking through the application bottleneck of traditional inducers. Among them, nanohydroxyapatite is an inorganic material with excellent biocompatibility. Its layered structure and porous properties can Methyl salicylate is efficiently loaded through physical adsorption and ion exchange. Methyl salicylate is a key signaling molecule for systemic acquired resistance in plants. It can activate the salicylic acid signaling pathway, upregulate the expression of PR proteins, and thereby enhance cell wall lignification. It can also cooperate with Bacillus amyloliquefaciens to form a "microorganism-plant" dual defense network. The present invention has certain limitations on the amount of addition. When the amount of addition is too low, the expression of PR proteins is insufficient, making it difficult to effectively activate plant immunity. If the amount of addition is too high, the growth of Bacillus brevis will be slightly inhibited.

[0022] Preferably, as a further specific embodiment, the prepared composite microbial agent is further compounded with biochar, wherein the biochar is plasma modified, has a specific surface area of ≥800 m² / g, and a pore size distribution of 2-50 nm.

[0023] The present invention also combines the prepared microbial agent with plasma-modified biochar to significantly improve the soil colonization ability and environmental adaptability of the agent. The high specific area biochar can adsorb a large number of bacteria to prevent the agent from being washed away, and the pore size distribution of 2-50nm can take into account both the bacteria accommodation and the diffusion of nutrients. At the same time, the alkaline properties of biochar can neutralize acidic soil to provide a suitable living environment for microorganisms. In addition, the present invention also uses plasma to modify the biochar to introduce carboxyl and hydroxyl groups on the surface of the biochar, thereby enhancing its electrostatic adsorption with the bacterial cell wall, thereby increasing the loading rate of the agent; therefore, if the specific surface area is too small, the bacterial load is reduced and it is difficult to meet field needs. When the specific surface area is too large, it may adsorb too much water, thereby affecting the activity of the bacteria; and if the pore size is too small, it will hinder the transmission of nutrients, while if the pore size is too large, it will cause the loss of bacteria.

[0024] Preferably, as a further specific embodiment, the composite microbial agent is used in the prevention and treatment of bacterial diseases of fruits and vegetables by synergistic application through foliar spraying and root perfusion, wherein the concentration of the foliar spraying solution is 10 8 CFU / mL, the root infusion concentration was 10 6 CFU / mL.

[0025] Preferably, as a further specific embodiment, the drug is administered with an intermittent irradiation using an LED light source with a wavelength of 410 nm and a light intensity of 50-80 μmol / (m 2 ·s), irradiation 3 times a day, 10 minutes each time.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a composite microbial agent for preventing and controlling bacterial diseases of fruits and vegetables. The five strains synergistically form a multiple mechanism of "inhibition-competition-immune activation", thereby achieving high-efficiency prevention and control of bacterial diseases of fruits and vegetables. The antimicrobial peptides secreted by Bacillus brevis directly inhibit bacteria, Pseudomonas chlororaphis competes for ecological niches through siderophores, Streptomyces produces antibiotics, Bacillus amyloliquefaciens promotes plant immunity, and Alteromonas provides new active substances of marine origin.

[0027] (2) The present invention provides a method for preparing the composite microbial agent for preventing and controlling bacterial diseases of fruits and vegetables, which further achieves efficient synergy and improved stability of strains through three-stage step-by-step fermentation, addition of global induction inhibitors, and pulsed electric field-assisted microencapsulation technology. DETAILED DESCRIPTION

[0028] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, those skilled in the art will understand that the embodiments described below are part of the embodiments of the present invention, not all of them, 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 ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] In order to more clearly illustrate the technical solutions of the present invention, specific embodiments are provided below for illustration. Example 1

[0030] 1. Material Preparation 1.1 Bacterial preparation: Prepare Bacillus brevis, Pseudomonas chlororaphis, and Streptomyces separately; 1.2 Culture medium preparation: LB liquid medium (pH = 6.8 and pH = 7.5), Gao's medium No. 1 (pH = 6.5); 1.3 Reagents: quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone; Sodium alginate (food grade), chitosan (deacetylation degree ≥ 90%), nano-titanium dioxide (particle size 20nm); Nanohydroxyapatite (specific surface area ≥ 200m² / g), methyl salicylate (purity ≥ 98%); Plasma-modified biochar (surface area 850 m² / g, pore size 2 nm); 2. Bacteria Fermentation Culture 2.1 Cultivation of Bacillus brevis: Inoculate the bacteria into LB liquid medium (50 mL, pH = 6.8) and culture at 30°C and 180 rpm for 12 h. The strain was then transferred to a 5 L fermenter (pH = 7.0), aerated at 1.0 vvm, and stirred at 200 rpm for 24 h until OD 600 =4.5; Scale up to 50L fermentation tank (pH=7.2), control dissolved oxygen ≥30%, harvest bacterial solution (viable bacteria ≥10 9 CFU / mL); 2.2 Pseudomonas chlororaphis culture steps: Initial culture is the same as that of Bacillus brevis, pH 7.5; The bacteria were then transferred to a 5 L fermenter and the pH was adjusted to 7.8. 0.1 mM FeCl3 was added and cultured for 20 h until the OD 600 =5.0; Scale up to a 50 L tank, maintain pH = 8.0, and harvest the bacterial liquid; 2.3 Streptomyces culture steps: Streptomyces was inoculated into Gao's medium No. 1 (pH = 6.5) and cultured at 28°C for 48 h; 3. Bacterial liquid mixing and additive addition 3.1 Ratio adjustment: Adjust the ratio of live bacteria of Bacillus brevis, Pseudomonas chlororaphis and Streptomyces to 1:2:1; Additives: 0.1% quorum sensing inhibitor (final concentration) and 0.05% nanohydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent) were added; 4. Pulsed electric field assisted microencapsulation (1) Capsule material preparation Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized; Then microencapsulation was carried out according to the mass ratio of capsule core to capsule material: 1:0.3; (2) Microencapsulation parameters Electric field strength: 15kV / cm; Pulse frequency: 100Hz; Temperature control: <25℃.

[0031] Targeted modification: grafted galacturonic acid oligosaccharides (density 2 / μm²).

[0032] 5. Biochar compounding step: Mix the microencapsulated bacterial agent and plasma-modified biochar at a ratio of 1:5; Then, the mixture was shaken and adsorbed at 25°C for 2 h, and the composite bacterial agent was collected by centrifugation. Example 2

[0033] 1. Material Preparation 1.1 Bacterial preparation: Prepare Bacillus brevis, Pseudomonas chlororaphis, and Streptomyces separately; 1.2 Culture medium preparation: LB liquid medium (pH = 7.2 and pH = 7.5), Gao's medium No. 1 (pH = 6.5); 1.3 Reagents: quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone; Sodium alginate (food grade), chitosan (deacetylation degree ≥ 90%), nano-titanium dioxide (particle size 20nm); Nanohydroxyapatite (specific surface area ≥ 200m² / g), methyl salicylate (purity ≥ 98%); Plasma-modified biochar (surface area 850 m² / g, pore size 50 nm); 2. Bacteria Fermentation Culture 2.1 Cultivation of Bacillus brevis: Inoculate the bacteria into LB liquid medium (50 mL, pH = 6.8) and culture at 30°C and 180 rpm for 12 h. The strain was then transferred to a 5 L fermenter (pH = 7.0), aerated at 1.0 vvm, and stirred at 200 rpm for 24 h until OD 600 =4.5; Scale up to 50L fermentation tank (pH=7.2), control dissolved oxygen ≥30%, harvest bacterial solution (viable bacteria ≥10 9 CFU / mL); 2.2 Pseudomonas chlororaphis culture steps: Initial culture is the same as that of Bacillus brevis, pH 7.5; The bacteria were then transferred to a 5 L fermenter and the pH was adjusted to 7.8. 0.1 mM FeCl3 was added and cultured for 20 h until the OD 600 =5.0; Scale up to a 50 L tank, maintain pH = 8.0, and harvest the bacterial liquid; 2.3 Streptomyces culture steps: Streptomyces was inoculated into Gao's medium No. 1 (pH = 6.5) and cultured at 28°C for 48 h; 3. Bacterial liquid mixing and additive addition 3.1 Ratio adjustment: Adjust the ratio of live bacteria of Bacillus brevis, Pseudomonas chlororaphis and Streptomyces to 3:4:2; Additives: 0.1% quorum sensing inhibitor (final concentration) and 0.1% nanohydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent) were added; 4. Pulsed electric field assisted microencapsulation (1) Capsule material preparation Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized; Then microencapsulation was carried out according to the mass ratio of capsule core to capsule material: 1:0.5; (2) Microencapsulation parameters Electric field strength: 20 kV / cm; Pulse frequency: 100Hz; Temperature control: <25℃.

[0034] Targeted modification: grafted galacturonic acid oligosaccharides (density 3 / μm²).

[0035] 5. Biochar compounding step: Mix the microencapsulated bacterial agent and plasma-modified biochar at a ratio of 1:5; Then, the mixture was shaken and adsorbed at 25°C for 2 h, and the composite bacterial agent was collected by centrifugation. Example 3

[0036] 1. Material Preparation 1.1 Bacterial preparation: Prepare Bacillus brevis, Pseudomonas chlororaphis, and Streptomyces separately; 1.2 Culture medium preparation: LB liquid medium (pH = 7.2 and pH = 7.5), Gao's medium No. 1 (pH = 6.5); 1.3 Reagents: quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone; Sodium alginate (food grade), chitosan (deacetylation degree ≥ 90%), nano-titanium dioxide (particle size 20nm); Nanohydroxyapatite (specific surface area ≥ 200m² / g), methyl salicylate (purity ≥ 98%); Plasma-modified biochar (surface area 850 m² / g, pore size 30 nm); 2. Fermentation and culture of bacteria 2.1 Cultivation of Bacillus brevis: Inoculate the bacteria into LB liquid medium (50 mL, pH = 6.8) and culture at 30°C and 180 rpm for 12 h. The strain was then transferred to a 5 L fermenter (pH = 7.0), aerated at 1.0 vvm, and stirred at 200 rpm for 24 h until OD 600 =4.5; Scale up to 50L fermentation tank (pH=7.2), control dissolved oxygen ≥30%, harvest bacterial solution (viable bacteria ≥10 9 CFU / mL); 2.2 Pseudomonas chlororaphis culture steps: Initial culture is the same as that of Bacillus brevis, pH 7.5; The bacteria were then transferred to a 5 L fermenter and the pH was adjusted to 7.8. 0.1 mM FeCl3 was added and cultured for 20 h until the OD 600 =5.0; Scale up to a 50 L tank, maintain pH = 8.0, and harvest the bacterial liquid; 2.3 Streptomyces culture steps: Streptomyces was inoculated into Gao's medium No. 1 (pH = 6.5) and cultured at 28°C for 48 h; 3. Bacterial liquid mixing and additive addition 3.1 Ratio adjustment: Adjust the ratio of live bacteria of Bacillus brevis, Pseudomonas chlororaphis and Streptomyces to 2:3:1; Additives: 0.1% quorum sensing inhibitor (final concentration) and 0.06% nanohydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent) were added; 4. Pulsed electric field assisted microencapsulation (1) Capsule material preparation Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized; Then, microencapsulation was performed according to the mass ratio of capsule core to capsule material: 1:0.4; (2) Microencapsulation parameters Electric field strength: 16kV / cm; Pulse frequency: 100Hz; Temperature control: <25℃.

[0037] Targeted modification: grafted galacturonic acid oligosaccharides (density 2.5 / μm²).

[0038] 5. Biochar compounding step: Mix the microencapsulated bacterial agent and plasma-modified biochar at a ratio of 1:5; Then, the mixture was shaken and adsorbed at 25°C for 2 h, and the composite bacterial agent was collected by centrifugation. Example 4

[0039] 1. Material Preparation 1.1 Bacterial preparation: Prepare Bacillus brevis, Pseudomonas chlororaphis, Streptomyces, and Alteromonas separately; 1.2 Culture medium preparation: LB liquid medium (pH = 7.2 and pH = 7.5), Gao's medium No. 1 (pH = 6.5) and marine bacterial medium (pH = 7.5); 1.3 Reagents: quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone; Sodium alginate (food grade), chitosan (deacetylation degree ≥ 90%), nano-titanium dioxide (particle size 20nm); Nanohydroxyapatite (specific surface area ≥ 200m² / g), methyl salicylate (purity ≥ 98%); Plasma-modified biochar (surface area 850 m² / g, pore size 30 nm); 2. Bacteria Fermentation Culture 2.1 Cultivation of Bacillus brevis: Inoculate the bacteria into LB liquid medium (50 mL, pH = 6.8) and culture at 30°C and 180 rpm for 12 h. The strain was then transferred to a 5 L fermenter (pH = 7.0), aerated at 1.0 vvm, and stirred at 200 rpm for 24 h until OD 600 =4.5; Scale up to 50L fermentation tank (pH=7.2), control dissolved oxygen ≥30%, harvest bacterial solution (viable bacteria ≥10 9 CFU / mL); 2.2 Pseudomonas chlororaphis culture steps: Initial culture is the same as that of Bacillus brevis, pH 7.5; The bacteria were then transferred to a 5 L fermenter and the pH was adjusted to 7.8. 0.1 mM FeCl3 was added and cultured for 20 h until the OD 600 =5.0; Scale up to a 50 L tank, maintain pH = 8.0, and harvest the bacterial liquid; 2.3 Steps for co-fermentation of Streptomyces and Alteromonas: Streptomyces was inoculated into Gao's medium No. 1 (pH = 6.5) and cultured at 28°C for 48 h; Alteromonas was inoculated into marine culture medium containing 30% NaCl at pH 7.5 and cultured at 25°C for 36 h; Then, the live bacteria of Alteromonas and Streptomyces were mixed at a ratio of 1:1; 3. Bacterial liquid mixing and additive addition 3.1 Ratio adjustment: Adjust the ratio of live bacteria of Bacillus brevis, Pseudomonas chlororaphis and Streptomyces to 2:3:1; Additives: 0.1% quorum sensing inhibitor (final concentration) and 0.06% nanohydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent) were added; 4. Pulsed electric field assisted microencapsulation (1) Capsule material preparation Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized; Then, microencapsulation was performed according to the mass ratio of capsule core to capsule material: 1:0.4; (2) Microencapsulation parameters Electric field strength: 16kV / cm; Pulse frequency: 100Hz; Temperature control: <25℃.

[0040] Targeted modification: grafted galacturonic acid oligosaccharides (density 2.5 / μm²).

[0041] 5. Biochar compounding step: Mix the microencapsulated bacterial agent and plasma-modified biochar at a ratio of 1:5; Then, the mixture was shaken and adsorbed at 25°C for 2 h, and the composite bacterial agent was collected by centrifugation. Example 5

[0042] 1. Material Preparation 1.1 Bacterial preparation: Prepare Bacillus brevis, Pseudomonas chlororaphis, Streptomyces, Alteromonas, and Bacillus amyloliquefaciens respectively; 1.2 Culture medium preparation: LB liquid medium (pH = 7.2 and pH = 7.5), Gao's medium No. 1 (pH = 6.5) and marine bacterial medium (pH = 7.5); 1.3 Reagents: quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone; Sodium alginate (food grade), chitosan (deacetylation degree ≥ 90%), nano-titanium dioxide (particle size 20nm); Nanohydroxyapatite (specific surface area ≥ 200m² / g), methyl salicylate (purity ≥ 98%); Plasma-modified biochar (surface area 850 m² / g, pore size 30 nm); 2. Bacteria Fermentation Culture 2.1 Cultivation of Bacillus brevis: Inoculate the bacteria into LB liquid medium (50 mL, pH = 6.8) and culture at 30°C and 180 rpm for 12 h. The strain was then transferred to a 5 L fermenter (pH = 7.0), aerated at 1.0 vvm, and stirred at 200 rpm for 24 h until OD 600 =4.5; Scale up to 50L fermentation tank (pH=7.2), control dissolved oxygen ≥30%, harvest bacterial solution (viable bacteria ≥10 9 CFU / mL); 2.2 Pseudomonas chlororaphis culture steps: Initial culture is the same as that of Bacillus brevis, pH 7.5; The bacteria were then transferred to a 5 L fermenter and the pH was adjusted to 7.8. 0.1 mM FeCl3 was added and cultured for 20 h until the OD 600 =5.0; Scale up to a 50 L tank, maintain pH = 8.0, and harvest the bacterial liquid; 2.3 Steps for co-fermentation of Streptomyces and Alteromonas: Streptomyces was inoculated into Gao's medium No. 1 (pH = 6.5) and cultured at 28°C for 48 h; Alteromonas was inoculated into marine culture medium containing 30% NaCl at pH 7.5 and cultured at 25°C for 36 h; Then, the live bacteria of Alteromonas and Streptomyces were mixed at a ratio of 1:1.2; 3. Bacterial liquid mixing and additive addition 3.1 Ratio adjustment: Adjust the live bacterial count ratio of Bacillus brevis, Pseudomonas chlororaphis, and Streptomyces to 2:3:1, with Bacillus amyloliquefaciens accounting for 12% of the total live bacterial count; Additives: 0.1% quorum sensing inhibitor (final concentration) and 0.06% nanohydroxyapatite-loaded methyl salicylate (dry weight of bacterial agent) were added; 4. Pulsed electric field assisted microencapsulation (1) Capsule material preparation Sodium alginate (2%), chitosan (1%), and nano-TiO2 (0.5%) were dissolved in deionized water and homogenized; Then, microencapsulation was performed according to the mass ratio of capsule core to capsule material: 1:0.4; (2) Microencapsulation parameters Electric field strength: 16kV / cm; Pulse frequency: 100Hz; Temperature control: <25℃.

[0043] Targeted modification: grafted galacturonic acid oligosaccharides (density 2.5 / μm²).

[0044] 5. Biochar compounding step: Mix the microencapsulated bacterial agent and plasma-modified biochar at a ratio of 1:5; Then, the mixture was shaken and adsorbed at 25°C for 2 h, and the composite bacterial agent was collected by centrifugation. Example 6

[0045] The specific implementation steps are the same as those in Example 5, except that the ratio of the viable counts of Bacillus brevis, Pseudomonas chlororaphis, and Streptomyces is adjusted to 0.5:1:0.05. Example 7

[0046] The specific implementation steps are the same as those in Example 5, except that the ratio of the viable bacteria counts of Bacillus brevis, Pseudomonas chlororaphis, and Streptomyces is adjusted to 4:8:3. Example 8

[0047] The specific implementation steps are the same as those in Example 5, except that the number of viable bacteria of Bacillus amyloliquefaciens is adjusted to 2% of the total viable bacteria in the composite bacterial agent. Example 9

[0048] The specific implementation steps are the same as those in Example 5, except that the number of viable bacteria of Bacillus amyloliquefaciens is adjusted to 20% of the total viable bacteria in the composite bacterial agent. Example 10

[0049] The specific implementation steps are the same as those in Example 5, except that the live bacterial ratio of Alteromonas to Streptomyces is adjusted to 0.5:1. Example 11

[0050] The specific implementation steps are the same as those in Example 5, except that the ratio of live bacteria of Alteromonas to Streptomyces is adjusted to 1:3. Example 12

[0051] The specific implementation steps are the same as those in Example 5, except that the mass ratio of the capsule core to the capsule material is adjusted to 1:0.1. Example 13

[0052] The specific implementation steps are the same as those in Example 5, except that the mass ratio of the capsule core to the capsule material is adjusted to 1:1. Example 14

[0053] The specific implementation steps are the same as those in Example 5, except that the grafting density of the targeting molecule galacturonic acid oligosaccharide is adjusted to 1 per μm². Example 15

[0054] The specific implementation steps are the same as those in Example 5, except that the grafting density of the targeting molecule galacturonic acid oligosaccharide is adjusted to 5 per μm². Example 16

[0055] The specific implementation steps are consistent with those of Example 5, except that the addition amount of the plant-derived inducer methyl salicylate supported by nanohydroxyapatite is adjusted to 0.01% of the dry weight of the inoculum. Example 17

[0056] The specific implementation steps are consistent with those of Example 5, except that the addition amount of the plant-derived inducer methyl salicylate supported by nano-hydroxyapatite is adjusted to 1% of the dry weight of the inoculum. Comparative Example 1

[0057] The specific implementation steps are the same as those in Example 5, except that no quorum sensing inhibitor is added. Comparative Example 2

[0058] The specific implementation steps are the same as those in Example 5, except that only sodium alginate is used as the capsule material in the microencapsulation technology. Comparative Example 3

[0059] The specific implementation steps are the same as those in Example 5, except that the targeting molecule galacturonic acid oligosaccharide is not grafted onto the surface of the microcapsules. Comparative Example 4

[0060] The specific implementation steps are the same as those in Example 5, except that the plant-derived inducer methyl salicylate supported by nano-hydroxyapatite is not added to the bacterial agent before microencapsulation. Comparative Example 5

[0061] The specific implementation steps are the same as those in Example 5, except that the prepared bacterial agent is not compounded with biochar. Comparative Example 6

[0062] The specific implementation steps are the same as those in Example 5, except that the biochar is not subjected to plasma modification. Experimental Example 1: Testing the effectiveness of composite microbial agents in preventing and controlling bacterial diseases of fruits and vegetables

[0063] In this experimental example, the microbial composite agents obtained in Examples 1-17 and Comparative Examples 1-5 were used to verify their effectiveness in preventing and controlling bacterial diseases of fruits and vegetables. The specific steps are as follows: 1 Test agents: the composite microbial agents obtained in Examples 1-17 and Comparative Examples 1-6, control group 1: a commercially available single Bacillus brevis agent, and control group 2: a chemical fungicide; 2. Test pathogens and plants Pathogens: bacterial wilt, canker, and soft rot Plants: Tomato (bacterial wilt), citrus (canker), cabbage (soft rot) 3 Experimental design 3.1 Application method: Foliar spraying: bacterial solution concentration 10 8 CFU / mL, 10 mL per strain; Root irrigation: bacterial solution concentration 10 6 CFU / mL, 50 mL per strain; Light assist: 410 nm LED light source, 50 μmol / (m 2 ·s), 3 times a day, 10 minutes each time; 3.2 Pathogen inoculation: Inoculate pathogens 24 hours after application; 4 Experimental steps 4.1 Pathogen culture: The three pathogens were inoculated into LB medium respectively and cultured at 28℃ with shaking until OD 600 =0.8; Plant treatments: Tomato, citrus, and cabbage seedlings (5-leaf stage) were planted in sterilized soil; Treatments were performed according to the application method, and the control group was sprayed with sterile water; 4.2 Pathogen inoculation: Inoculate the pathogen into the leaves or stems using the needle puncture method.

[0064] Data Records: Day 7: Record the initial onset of disease; Day 14: Statistics on final incidence, control effects and plant growth indicators; Storage stability: The number of viable bacteria was measured after the bacterial agent was stored at 4°C for 30 days; The final results are shown in Table 1: Table 1 Test results

[0065] The above experimental data show that Examples 1-5 demonstrate the performance of the composite microbial agent under different optimized conditions, among which Example 5 has the best overall effect. This is because the mixture of five strains, namely Bacillus brevis, Pseudomonas chlororaphis, Streptomyces, Bacillus amyloliquefaciens, and Alteromonas, forms a multiple defense mechanism that covers all stages of disease development. In addition, the ratio of viable bacteria count and the ratio of Alteromonas to Streptomyces in Example 5 ensure the complementarity of metabolites, avoiding side effects caused by excessive proliferation of a single strain. By comparing Examples 6 and 7, it can be seen that the strain ratio in Example 6 is too low, resulting in insufficient viable bacteria of Brevibacillus and Streptomyces, resulting in insufficient secretion of antimicrobial peptides and antibiotics, and inability to effectively inhibit pathogens; while the strain ratio in Example 7 is too high, resulting in excessive iron competition caused by excessive Pseudomonas chlororaphis, which may cause iron deficiency in plants; and the antimicrobial peptides secreted by Brevibacillus inhibit the growth of other strains, disrupting the synergistic balance; and the unbalanced strain ratio in Examples 6 and 7 leads to intensified competition between strains, resulting in abnormal accumulation of metabolites between strains, which affects the survival of bacteria in the microcapsules. 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 of PR protein is low, and the disease resistance of the plant is not fully stimulated; and when the proportion of Bacillus amyloliquefaciens in Example 9 is too high, Bacillus amyloliquefaciens competes for resources with other strains, inhibiting the activity of Bacillus brevis; and excessive plant hormones may trigger a stress response in the plant, which is not conducive to growth. By comparing Examples 10 and 11, it can be seen that in Example 10, the ratio of Alteromonas to Streptomyces is too low, resulting in insufficient secretion of bromofuranone by Alteromonas, weakened quorum sensing inhibition effect, and insufficient blocking of pathogen virulence factor expression; and when the ratio of the two is too high in Example 11, excessive Streptomyces leads to antibiotic accumulation, which may inhibit beneficial soil microorganisms and disrupt the microecological balance; By comparing Examples 12 and 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 the acidic environment; when the capsule material in Example 13 is too thick, the diffusion of nutrients is easily blocked, the recovery of the bacteria is delayed, and the microcapsule particle size is too large, which affects the leaf surface adhesion. By comparing Examples 14 and 15, it can be seen that when the grafting density in Example 14 is low, the pathogen recognition signal is weak and the microcapsule cannot accurately locate the diseased site; and when the grafting density in Example 15 is too high, the galacturonic acid oligosaccharide overactivates the plant immune system, resulting in unnecessary energy consumption. By comparing Examples 16 and 17, it can be seen that when the loading amount of methyl salicylate in Example 16 is too low, the methyl salicylate is insufficient and the salicylic acid signaling pathway cannot be effectively activated; and when the loading rate of methyl salicylate in Example 17 is too high, the excessive amount of methyl salicylate causes the direct toxicity of the prepared microbial agent to affect the activity of the bacteria, and the sustained-release effect of the nano-hydroxyapatite is destroyed. It can be seen from Comparative Examples 1-6 that some key technologies of the preparation method of the present invention have a certain influence on the effect of the prepared microbial agent. When no sensing inhibitor is added in Comparative Example 1, the expression of virulence factors of pathogens is not inhibited due to the lack of sensing inhibitor, which greatly reduces the control effect; when the capsule material in Comparative Example 2 is single sodium alginate, the mechanical strength and ultraviolet stability of the capsule material in the prepared microcapsules are insufficient, which greatly reduces the survival rate of live bacteria; when no targeting molecule is added in Comparative Example 3, the agent relies on passive diffusion, which reduces the targeting efficiency; and in Comparative Example 4, methyl salicylate is not added, and the plant immune activation is insufficient, and the control effect and growth increase are significantly reduced; and in Comparative Example 5, biochar composite is not used, and the soil colonization ability of the agent is poor, which makes the agent easily washed away; and in Comparative Example 6, the biochar is not modified, and the specific surface area and adsorption capacity of the biochar are insufficient, which reduces the loading rate.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 composite microbial agent for preventing and treating bacterial diseases of fruits and vegetables, characterized in that: including Bacillus brevis, Pseudomonas chlororaphis, and Streptomyces; The ratio of the number of live bacteria of Brevibacillus, Pseudomonas chlororaphis and Streptomyces is (1-3): (2-4): (1-2).

2. The composite microbial agent according to claim 1, characterized in that Also included is Bacillus amyloliquefaciens, wherein the number of live bacteria of the Bacillus amyloliquefaciens is 10-15% of the total number of live bacteria of the Brevibacillus, Pseudomonas chlororaphis and Streptomyces.

3. The composite microbial agent according to claim 2, characterized in that The composite microbial agent also includes marine-derived Alteromonas, and the ratio of live bacteria of the Alteromonas to the live bacteria of the Streptomyces is 1:(1-1.5).

4. A method for preparing the composite microbial agent according to any one of claims 1 to 3, characterized in that: The following steps are involved: Three-stage stepwise fermentation culture was carried out respectively, and the fermentation conditions of Bacillus brevis, Pseudomonas chlororaphis and Streptomyces were controlled at pH 6.8-7.2, pH 7.5-8.0 and pH 6.5-7.0 respectively. After mixing the bacterial solutions in proportion, 0.1% of the total volume of the quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone was added to prepare a preparative solution; The preparatory solution is prepared into a microbial agent by adopting a pulse electric field assisted microencapsulation technology, with an electric field strength of 15-20 kV / cm and a pulse frequency of 100 Hz.

5. The preparation method according to claim 4, characterized in that 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).

6. The preparation method according to claim 4, characterized in that The surface of the microcapsule is grafted with targeting molecules galacturonic acid oligosaccharide, and the grafting density is 2-3 per μm².

7. The preparation method according to claim 6, characterized in that Before microencapsulation, methyl salicylate, a plant-derived inducer supported by nano-hydroxyapatite, is added to the preparatory solution in an amount of 0.05-0.1% of the total weight of the preparatory solution.

8. The preparation method according to claim 6, characterized in that The method also includes compounding the prepared composite microbial agent with biochar, wherein the biochar is plasma-modified, has a specific surface area of ≥800 m² / g, and a pore size distribution of 2-50 nm.

9. Use of the composite microbial agent according to any one of claims 1 to 3 in preventing and treating bacterial diseases of fruits and vegetables, characterized in that: The pesticide was applied by foliar spraying and root infusion, and the concentration of the foliar spray solution was 10 8 CFU / mL, the root infusion concentration was 10 6 CFU / mL.

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

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