A composite biocontrol agent, a preparation method, an application and an application method

Abstract

The present application provides a kind of compound biocontrol agent and its preparation method and application technology, and it is composed of 1000ml of fermentation mixed liquor of multiple strains of biocontrol bacteria mixed in equal volume ratio and 0.5%-1.5% stabilizer with complementary mechanism and target.The fermentation concentrated solution of multiple strains of bacteria is prepared in fermentation medium to obtain the present application, which is used for preventing and controlling fusarium, pythium, phytophthora, rhizoctonia solani, ralstonia solanacearum, erwinia carotovora, streptomyces scabiei, xanthomonas campestris and other plant pathogenic bacteria and nematodes, which can inhabit in soil for a long time and spread through soil, and one or several of them can cause plant soil-borne diseases.The preparation process of the present application is not affected by too many chemical and physical factors, the activity of live bacteria and metabolites is high, the average control effect is 78%-85%, the application effect is good;The production process is simple, energy and auxiliary materials are saved, and the production cost is low.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticides, specifically relating to a compound biocontrol bacterial liquid formulation with complementary biocontrol mechanisms and targets and their metabolites as the main active components, its application, and application method. Background Technology

[0002] Soil-borne plant diseases are a collective term for diseases that occur in the roots or neck of plants and are transmitted through the soil. Soil-borne pathogens are numerous and can cause significant economic losses. They mainly include diseases caused by pathogens such as *Fusarium oxysporum*, *Verticillium dahliae* Kleb., *Ralstonia solanacearum*, *Phytophthora parasitica var. nicotianae*, nematodes, *Phytophthora cactus*, and *Pythium aphandidermatum*, such as wilt, verticillium wilt, bacterial wilt, black shank, and various root rots. These pathogens can reside in the soil, crop residues, and other humus for extended periods, primarily active in the soil. When suitable conditions arise, the pathogens germinate and infect the rhizosphere, neck, branches, leaves, and fruits of plants, causing crop diseases. In recent years, in pursuit of higher profits, people have continuously expanded the cultivation area of ​​cash crops, resulting in continuous planting of single crops. Unreasonable variety structures, inappropriate cultivation models, and management techniques have led to severe continuous cropping obstacles in the soil, and soil-borne plant diseases are becoming increasingly frequent and serious. Furthermore, the growing scale of facility agriculture, with its dense planting and enclosed environments, easily creates conditions conducive to the growth and spread of soil-borne pathogens, causing severe outbreaks of soil-borne diseases. The incidence rate of Fusarium wilt reaches 70%–80%, Phytophthora blight exceeds 60%, bacterial wilt reaches as high as 80%, and the incidence rates of seedling damping-off and seedling blight also exceed 50%. These factors severely restrict the sustainable development of cash crop production in my country and have become urgent problems that need to be solved.

[0003] Currently, the main control technologies for soil-borne plant diseases include disease-resistant plant variety breeding, grafting and crop rotation, chemical pesticide control, and biological control. These technologies can effectively suppress the occurrence and damage of soil-borne diseases to varying degrees. Chemical pesticide control, in particular, is widely used due to its rapid, efficient, and low-cost nature. Biological control was initially proposed to address the shortcomings of chemical pesticide control. It involves isolating beneficial or antagonistic microorganisms from the rhizosphere soil or plant tissues and applying them to the soil to inhibit the reproduction and invasion of pathogens while promoting plant growth—a novel control technology aligned with the concept of ecological balance. Currently, the functional microorganisms used for biological control mainly include bacteria, fungi, and non-pathogenic microorganisms. Bacterial microorganisms mainly include Bacillus, Pseudomonas, and Acinetobacter; fungal microorganisms include Trichoderma and Streptomyces; and among non-pathogenic microorganisms, the non-pathogenic bacterium *Bacillus polymyxa* has been extensively studied for its biocontrol effect on pathogens. Other non-pathogenic microorganisms mainly have inhibitory effects on their corresponding pathogens. The mechanisms by which these biological control methods control soil-borne diseases mainly include the production of antagonistic substances for sterilization, induction of systemic resistance in plants, and competition with pathogens for rhizosphere nutrients and ecological niches. Biological control's unique advantages—being environmentally friendly, pollution-free, resource-rich, and possessing diverse control mechanisms—make it a more popular new technology for controlling soil-borne plant diseases in modern green agricultural production. While these technologies for controlling soil-borne plant diseases each have their advantages, they also have problems. Disease-resistant variety breeding technology results in a limited number of resistant varieties with narrow control spectra; some new varieties have low yields and poor quality, making widespread adoption difficult. Grafting and crop rotation cultivation techniques are time-consuming, labor-intensive, and costly; furthermore, due to the wide host range of pathogens, coupled with limitations in land resources and economic interests, widespread adoption is difficult. Chemical pesticide control leaves harmful residues that reduce fruit quality, damage the ecological environment, disrupt the soil ecosystem, and easily induce vicious cycles; the pathogens are diverse, have high genetic variability, and are prone to inducing pesticide resistance. Chemical control overcomes the shortcomings of agricultural control techniques such as disease-resistant varieties and cultivation methods, improving control efficacy and significantly reducing control costs. Biological control, to some extent, overcomes the drawbacks of chemical control, and extensive research has shown that this method is economically effective against most soil-borne diseases. Therefore, it has become a new direction for controlling soil-borne plant diseases and has received considerable attention and development. To date, more than 80 kinds of biological pesticides, including *Trichoderma*, *Trichoderma*, *Cladosporium*, *Bacillus*, *Streptomyces*, and numerous strains producing agricultural antibiotics, are used domestically and internationally to control soil-borne plant diseases, achieving good control results. However, these biological pesticides still have the following shortcomings: a predominance of single-strain formulations, weak strain resistance, application efficacy needs improvement, formulations are mostly wettable powders, and production processes and costs need further simplification and reduction. Summary of the Invention

[0004] This invention provides a compound biocontrol agent that achieves a better and faster biocontrol effect than a single bacterium through the complementary action of multiple biocontrol strains of different genera and their metabolites with complementary mechanisms of action and targets.

[0005] Another objective of this invention is to provide a method for preparing a compound biocontrol agent, using an auxiliary stabilizer to form a liquid biocontrol agent, which has relatively low cost, high activity, and is suitable for integrated water and fertilizer application.

[0006] The third objective of this invention is to provide an application technology for a compound biocontrol agent for controlling soil-borne plant diseases caused by one or more of the following: Fusarium, Phytophthora, Pythium, Rhizoctonia solani, Raulella, Erwinia carotenoides pathogenic type, Streptomyces pathogenicus, Xanthomonas dahliae, and other plant pathogens that can reside in the soil for a long time and spread through the soil, as well as nematodes.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A compound biocontrol agent, which is a liquid preparation composed of a fermentation mixture of multiple biocontrol bacteria and a stabilizer; specifically

[0009] The formulation is as follows: 1000 mL of a fermentation mixture of multiple biocontrol bacteria strains in equal volume ratio, and 0.5%–1.5% stabilizer, all in weight-volume percentage. The fermentation mixture of multiple biocontrol bacteria strains is a fermentation mixture of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus thuringiensis, and Streptomyces tarda. The Bacillus subtilis strain is Bacillus subtilis (CGMCC No. 24484); the Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens (CGMCC No. 24481); the Bacillus cereus strain is Bacillus cereus (CGMCC No. 24485); and the Bacillus thuringiensis strain is Bacillus thuringiensis (CGMCC No. 24485). No. 24483; The aforementioned Streptomyces atroolivaceus is Streptomyces atroolivaceus, with accession number CGMCC No. 24482.

[0010] Further:

[0011] The stabilizer is preferably one or a mixture of several of the following in any proportion: skim milk, dipotassium hydrogen phosphate, sodium alginate, mannitol, vitamin C, poloxamer, molasses, bentonite, polyvinyl alcohol, aesculin, folic acid, urea, colloidal silica, polysorbate, chlorogenic acid, mercaptoethanol, chitosan, amino acids, inositol, ferrous sulfate, superphosphate, ethylene glycol, glycerol, and polyoxyethylene.

[0012] A method for preparing a compound biocontrol agent involves using *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus cereus*, and *Bacillus thuringiensis* in a fermentation broth containing 0.5%–2.0% beef extract and 1.0%–3.0% glucose, with a sample loading of 50%–70%, an aeration rate of 3.0 L / ml–6.0 L / ml, an inoculum size of 5%–10%, a temperature of 28–31°C, an rpm of 100–200 rpm, an initial pH of 7.0, a dissolved oxygen content of 20%–35%, and the direct addition of 0.05%–0.3% GP-330 antifoaming agent. The culture is then incubated for 30–48 days. h for later use; the aforementioned *Streptomyces olfleurone* strain was fermented with millet (1.5%–4.0%), glucose (0.5%–2.5%), and dipotassium hydrogen phosphate (0.01%–0.3%) as the fermentation broth, with a sample volume of 50%–70%, an aeration rate of 2.0–4.5 L / ml, an inoculum size of 7%–12%, a temperature of 26–29°C, an rpm of 200–300 rpm, and 0.1%–0.5% GP-330 defoaming added continuously. The initial pH was 7.2. After 72 hours of fermentation, 16%–23% of the fermentation broth was added at one time to continue fermentation, with a dissolved oxygen content of 20%–26%, and the culture time was 132–168 hours. All fermentation broths were mixed, and 0.5%–1.5% stabilizer was added. The mixture was thoroughly mixed to obtain the final product.

[0013] The application method of a compound biocontrol agent is as follows:

[0014] 1) Prevention: When planting plants of the same family in succession, apply the compound biocontrol agent provided by this invention at a concentration of 50 mL / m² before planting. 2 ~200mL / m 2 Spray on the soil or apply with irrigation water to the soil or treat the planting substrate. Apply 1-2 times consecutively. Plant 3-5 days after application.

[0015] 2) Prevention and control: When soil-borne plant diseases such as wilt, root rot, damping-off, seedling blight, bacterial wilt, soft rot, and Verticillium wilt caused by the above-mentioned pathogens occur in the previous crop, treat the soil with bio-organic fertilizer and bio-pesticides after harvest, or use the compound biocontrol agent provided by this invention at 100mL / m³. 2 ~350mL / m 2 Apply the compound biocontrol agent provided by this invention to the soil along with irrigation water, then cover with a thin layer of soil; after sowing this crop, when the temperature is above 30℃ and the humidity is above 75%, apply 80mL / m² of the compound biocontrol agent provided by this invention. 2 ~250mL / m2 Spray on the soil or apply with irrigation water, 2 to 5 times consecutively. The first 2 to 3 times should be spaced 7 to 10 days apart, and the next 4 to 5 times should be spaced 15 to 20 days apart. It is especially suitable for fertigation.

[0016] Based on the characteristics of soil-borne diseases—including their diverse types, tendency to recur, and need for repeated control—this invention provides a multi-effect compound biocontrol bacterial liquid formulation and its highly efficient application technology. This opens up new avenues for efficient and green control of soil-borne plant diseases and provides a highly effective and applicable biocontrol agent for fertigation technology. Its specific effects are as follows:

[0017] 1. Broad-spectrum control through microbial community action. The compound biocontrol agent provided by this invention is composed of five different genera of biocontrol strains and their metabolites with complementary mechanisms of action and target effects. The microbial community action leverages the strengths of each strain to effectively broaden the control spectrum of the formulation. It has a stronger control effect on soil-borne plant diseases caused by soil-borne pathogenic fungi, bacteria, actinomycetes, and nematodes than single-strain agents, thereby effectively controlling the spread and damage of the corresponding soil-borne plant diseases.

[0018] 2. Liquid formulation with good application effect. The compound biocontrol liquid formulation provided by this invention is not affected by chemical or physical factors during the formulation process. The live bacteria and metabolites have high activity. The live bacteria do not undergo a dormancy period when applied to the soil and colonize quickly. Moreover, the five biocontrol bacteria in this formulation effectively improve the microecological environment of the pathogenic soil through reproduction, growth and metabolites, increase soil enzyme activity, enhance soil available nutrients, induce plant disease resistance, enhance their disease resistance, and build a good plant ecosystem, resulting in good application effect.

[0019] 3. Simple production process and low cost. The compound biocontrol agent provided by this invention is a liquid formulation of biocontrol bacteria. The key production process only includes fermentation, followed by the addition of a fermentation broth stabilizer. The preparation process is simple, saves energy and excipients, and has low production costs.

[0020] 4. Wide applicability, safe and environmentally friendly. The compound biocontrol agent provided by this invention can be used for spraying, watering, root irrigation, and mixing. It is suitable for the control of soil-borne diseases in protected cultivation and open-field planting, and is especially suitable for fertigation. There is no dust drift during use. The active biocontrol bacteria are derived from nature and have been verified to be safe for humans and animals through acute toxicity tests. It belongs to the category of non-toxic pesticides and is safe for natural enemies and the environment. Detailed Implementation

[0021] The present invention will be further described in detail below through principles and specific implementations, so that those skilled in the art can have a more comprehensive understanding of the present invention, but it is not intended to limit the present invention in any way.

[0022] The principle of this invention is explained as follows:

[0023] The main active ingredients of this invention are composed of five different genera of biocontrol bacteria and their metabolites. Because they have different mechanisms of action and targets, they complement each other and enhance each other in application, thereby achieving a broad-spectrum, high-efficiency, and wider-ranging composite target.

[0024] Streptomyces atroolivaceus is rich in metabolically active substances, including compounds such as strychnine, androstenone monosulfate, salvia miltiorrhizin, furanyltetrahydropyrrole, aminotrimethylimidazoquinoline, methylquinoline, ethoxymethylcoumarin, arecoline, pyrimethanil, isodesin, isopiclin, isonicotinic acid, protodiosgenin, and myristic acid. These compounds inhibit the biosynthesis of ergosterol in the cell membrane of pathogenic fungi, disrupting cell membrane structure and stability. Simultaneously, they inhibit the activity of protective enzymes in pathogenic fungi, exacerbating lipid peroxidation and causing rapid cell death. This results in the inhibition of pathogenic Fusarium, Rhizoctonia solani, Phytophthora, Pythium spp., Pseudomonas aeruginosa, Cladosporium, and pathogenic monotyphi. Furthermore, its metabolically active substances also include amino acids and plant hormones, which, in synergistic with these active compounds, activate the resistance system of protected plants, enhancing their stress resistance and synergistically improving the antibacterial effect.

[0025] Bacillus amyloliquefaciens is rich in metabolically active substances, including organic acids, alkaloids, and bacteriostatic agents such as acrylic acid, rhein, pyridylic acid, quinic acid, isocobacterium, isochoryl glycoside, ephemeralin, isopyroxin, piracetamine, demethoxycurcumin, and anestheticin. These fermentation broth active substances increase the lipid peroxidation level of pathogens (leading to a decrease in intracellular malondialdehyde content), disrupting the cell membrane stability of pathogens and strongly inhibiting fungal spore germination and hyphal growth. This achieves the goal of inhibiting plant pathogenic fungi diseases caused by Fusarium oxysporum, Erwinia carotenoides soft rot type, Verticillium dahliae, Rhizoctonia solani, Pseudomonas syringae, and Pseudomonas xanthoides, as well as diseases caused by Cyclosporium and Peronospora. In addition, metabolically active substances include amino acids and melatonin, which enhance plant photosynthesis and carbon assimilation, increase chlorophyll content, and activate the plant's stress resistance system, significantly increasing the activity of its disease-fighting and defense enzymes. This improves the protected plant's ability to resist pathogen infection and stress, and synergistically enhances the antibacterial effect.

[0026] Bacillus subtilis contains metabolically active substances such as indole acids, cytokinins, plant hormones, sugars, and various amino acids, which promote plant growth and development and have a significant growth-promoting effect on protected plants. It also contains antibacterial substances such as epoxyoctadecanoic acid, itaconic acid, styracidin, benapralli, hydroxyphenylcarvedilol, hydroxyfurandan, furanone, methylfuranaldehyde, and nitrosothiazolidinecarboxylic acid. It inhibits the growth of pathogenic fungal cell wall chitinases and related protective enzymes, promotes lipid peroxidation in pathogenic cells, and disrupts the stability of the cell wall and cell membrane, leading to protoplasmic leakage and cell death. Simultaneously, it inhibits the activity of cell wall degrading enzymes in pathogenic fungi, reducing their infectivity to host plants, thus achieving the goal of inhibiting plant pathogens such as Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Pseudomonas pathogenica, Rhizoctonia solani, and Pseudomonas nigra.

[0027] Bacillus cereus contains bacteriocinoids and organic acids such as isotetracycline, ethylcarboxamide, methylmalonic acid, methoxyphenol, and ricinoleic acid in its metabolically active substances, acting as an antibacterial agent. Bacillus cereus has high reproductive capacity and spreads rapidly. It inhibits plant pathogens by competing with pathogens for space and suppressing their cell membrane protein synthesis, thus severely damaging the cell membrane. These pathogens include Streptomyces scabica, Ralstonia solanacearum, Erwinia carotenoidea (soft rot type), Erwinia carotenoidea (black rot type), Corynebacterium circumferentiale, Pseudomonas syringae (pathogenic type), Pseudomonas aeruginosa, Fusarium oxysporum, Fusarium sulfide, and Alternaria solanacearum. In addition, its fermentation metabolites include plant growth regulators such as indoleacetic acid, cytokinins, and amino acids, promoting the growth and development of protected plants.

[0028] Bacillus thuringiensis contains a variety of insecticidal and bacteriostatic compounds in its metabolically active substances, including dihydroxycinnamic acid, homovanillic acid, aminobutyraldehyde, ethyl dimethylpyrazine, methyldeoxyrhezin, pyridine bases, peptides, complex amino acids, rotenone, carbapenem dehydropiperazine, and tetrahydropyrrole. These compounds exert their killing or inhibitory effects on lepidopteran pests such as armyworms and diamondback moths, homopteran pests such as aphids, and crop nematodes such as root-knot nematodes and root-rot nematodes through stomach poisoning, contact killing, and growth inhibition. Furthermore, its metabolites also contain monoamino acid groups and phytokinins, which promote robust growth in protected plants and enhance their resistance to diseases and pests.

[0029] Of the five biocontrol bacteria mentioned above, four synergistically attack the cell membranes or cell walls of different or the same target pathogens through different mechanisms, disrupting their structure and stability, or inhibiting the activity of cell wall degrading enzymes. This leads to the cessation of mycelial growth, spore germination, and infection capacity of the target pathogens, achieving a broad-spectrum and highly effective bactericidal effect through combined application. Bacillus thuringiensis, primarily effective against nematodes and other underground pests, compensates for the shortcomings of the other four antibacterial strains, achieving a broad-spectrum target of controlling both diseases and pests. Furthermore, their metabolites contain specific amino acids, phytokinins, and plant growth regulators, which can effectively increase the chlorophyll content of protected plants, activate the plant's stress resistance system, and promote the growth and development of protected plants, synergistically enhancing their antibacterial and insecticidal effects, thereby achieving highly effective control of the spread and damage of corresponding plant diseases and pests.

[0030] The present invention and its beneficial effects are further illustrated below through examples and test cases.

[0031] I. Examples 1-9: The proportions of each component in a compound biocontrol agent are shown in Table 1.

[0032] Table 1. Component ratio of the compound biocontrol agent

[0033]

[0034] Note: Prepare 1000mL of compound biocontrol agent in advance.

[0035] II. A method for preparing a composite biocontrol agent

[0036] Example 10: A method for preparing a compound biocontrol agent. The *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus cereus*, and *Bacillus thuringiensis* strains were fermented in a culture medium containing 0.5% beef extract and 1.0% glucose, with a 50% sample volume, an aeration rate of 3.0 L / mL, a 5% inoculum, at 31°C, 100 rpm, an initial pH of 7.0, and a dissolved oxygen level of 35%. 0.05% GP-330 defoamer was added directly at once, and each strain was cultured for 30 hours. The *Streptomyces urophylla* strain was fermented in a culture medium containing 1.5% millet, 2.5% glucose, and 0.3% dipotassium hydrogen phosphate, with a 50% sample volume, an aeration rate of 2.0 L / mL, a 7% inoculum, at 29°C, 200 rpm, with 0.1% GP-330 added as defoamer, an initial pH of 7.2, and 16% fermentation medium added at 72 hours of fermentation for further fermentation. The dissolved oxygen level was approximately 20%, and the fermentation was continued for 132 hours. Mix all the fermentation broth, add 0.5% stabilizer, and mix thoroughly to obtain the finished product.

[0037] Example 11: A method for preparing a compound biocontrol agent. The *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus cereus*, and *Bacillus thuringiensis* strains were fermented in a culture medium containing 1.0% beef extract and 2.0% glucose, with a 60% loading volume, an aeration rate of 5.0 L / ml, an 8% inoculum, at 30°C, 150 rpm, an initial pH of 7.0, and a dissolved oxygen level of 30%. 0.2% GP-330 defoaming agent was added directly at once, and each strain was cultured for 38 h. The *Streptomyces urophylla* strain was fermented in a culture medium containing 2.0% millet, 1.5% glucose, and 0.1% dipotassium hydrogen phosphate, with a 60% loading volume, an aeration rate of 3.5 L / ml, a 9.0% inoculum, at 28°C, 260 rpm, with 0.4% GP-330 added as defoaming agent, an initial pH of 7.2, and 21% fermentation medium added at 72 h of fermentation for continued fermentation. The dissolved oxygen level was approximately 23%, and the culture was cultured for 155 h. Mix all the fermentation broth, add 1.0% stabilizer, and mix thoroughly to obtain the finished product.

[0038] Example 12: A method for preparing a compound biocontrol agent. The *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus cereus*, and *Bacillus thuringiensis* strains were fermented in a culture medium containing 2.0% beef extract and 3.0% glucose, with a 70% loading volume, an aeration rate of 6.0 L / ml, a 10% inoculum, at 28°C, 200 rpm, an initial pH of 7.0, and a dissolved oxygen level of 20%. 0.3% GP-330 defoamer was added directly at once, and each strain was cultured for 48 h. The *Streptomyces urophylla* strain was fermented in a culture medium containing 4.0% millet, 0.5% glucose, and 0.01% dipotassium hydrogen phosphate, with a 70% loading volume, an aeration rate of 4.5 L / ml, a 12% inoculum, at 26°C, 300 rpm, with 0.5% GP-330 added as defoamer, an initial pH of 7.2, and 23% fermentation medium added at 72 h of fermentation for continued fermentation. The dissolved oxygen level was approximately 26%, and the fermentation was continued for 168 h. Mix all the fermentation broth, add 1.5% stabilizer, and mix thoroughly to obtain the finished product.

[0039] III. Application Technology of Compound Biocontrol Agents

[0040] 1. Application Objectives: For the control of soil-borne plant diseases caused by one or more of the following fungi: Fusarium, Phytophthora, Pythium, Rhizoctonia solani, Raulella, Erwinia carotenoides pathogenic type, Streptomyces pathogenicus, Xanthoceras dahliae, and other soil-transmittable plant pathogens, as well as nematodes.

[0041] 2. Application method:

[0042] 1) Prevention: When planting plants of the same family in succession, apply the compound biocontrol agent provided by this invention at a concentration of 50 mL / m² before planting. 2 ~

[0043] 200mL / m 2Spray on the soil or apply with irrigation water to the soil or treat the planting substrate. Apply 1-2 times consecutively. Plant 3-5 days after application.

[0044] 2) Prevention and control: When soil-borne plant diseases such as wilt, root rot, damping-off, seedling blight, bacterial wilt, soft rot, and Verticillium wilt caused by the above-mentioned pathogens occur in the previous crop, treat the soil with bio-organic fertilizer and bio-pesticides after harvest, or use the compound biocontrol agent provided by this invention at 100mL / m³. 2 ~350mL / m 2 Apply the solution to the soil with irrigation water, then cover with a thin layer of soil. After sowing this crop, when the temperature is above 30℃ and the humidity is above 75%, use 80mL / m² of the compound biocontrol agent provided by this invention. 2 ~250mL / m 2 Spray on the soil or apply with irrigation water, 2 to 5 times. The first 2 to 3 times should be spaced 5 to 10 days apart, and the next 4 to 5 times should be spaced 15 to 20 days apart.

[0045] Specific application examples are as follows:

[0046] Example 13: When planting plants of the same family in succession, apply 200 mL / m² of the compound biocontrol agent provided by this invention before planting. 2 Spray the soil once, and plant two days after application.

[0047] Example 14: When planting plants of the same family in succession, apply 100 mL / m² of the compound biocontrol agent provided by this invention before planting. 2 Apply the pesticide to the soil with irrigation water, twice, and plant 5 days after application.

[0048] Example 15: When planting plants of the same family in succession, apply 50 mL / m² of the compound biocontrol agent provided by this invention before planting. 2 Treat the planting substrate twice, and plant 3 days after application.

[0049] Example 16: When one or more soil-borne plant diseases such as wilt, root rot, damping-off, seedling blight, bacterial wilt, soft rot, and Verticillium wilt occurred in the previous crop, the soil was treated with bio-organic fertilizer and bio-pesticide after harvest. After sowing in the current crop, when the temperature is above 30℃ and the humidity is above 75%, the compound biocontrol agent provided by this invention was applied at a rate of 80 mL / m². 2 Spray on the soil, apply 5 times consecutively, with an interval of 5 days between the first 3 applications and an interval of 10 days between the last 2 applications.

[0050] Example 17: When one or more of the following soil-borne plant diseases occurred in the previous crop: Fusarium wilt, root rot, damping-off, seedling blight, bacterial wilt, soft rot, Verticillium wilt, etc., apply the compound biocontrol agent provided by this invention at a concentration of 100 mL / m² after harvest. 2Apply the solution to the soil with irrigation water, then cover with a thin layer of soil. After sowing this crop, when the temperature is above 30℃ and the humidity is above 75%, apply 200mL / m² of the compound biocontrol agent provided by this invention. 2 Apply to the soil with irrigation water, and repeat 3 times. The first 2 applications should be 7 days apart, and the last application should be 15 days apart.

[0051] Example 18: When one or more of the following soil-borne plant diseases occurred in the previous crop: Fusarium wilt, root rot, damping-off, seedling blight, bacterial wilt, soft rot, Verticillium wilt, etc., apply the compound biocontrol agent provided by this invention at a rate of 250 mL / m² after harvest. 2 Apply the solution to the soil with irrigation water, then cover with a thin layer of soil. After sowing this crop, when the temperature is above 30℃ and the humidity is above 75%, apply 250mL / m² of the compound biocontrol agent provided by this invention. 2 Spray on the soil or apply with irrigation water, twice consecutively, with an interval of 10 days between each application.

[0052] Example 19: When the previous crop suffered from one or more soil-borne plant diseases such as wilt, root rot, damping-off, seedling blight, bacterial wilt, soft rot, and Verticillium wilt, the soil was treated with bio-organic fertilizer and bio-pesticide after harvest. After sowing the current crop, when the temperature is above 30℃ and the humidity is above 75%, the compound biocontrol agent provided by this invention was applied at a rate of 250 mL / m². 2 Spray on the soil or apply with irrigation water, repeat 3 times, with an interval of 7 days between each application.

[0053] Example 20: When one or more of the following soil-borne plant diseases occurred in the previous crop: Fusarium wilt, root rot, damping-off, seedling blight, bacterial wilt, soft rot, Verticillium wilt, etc., apply the compound biocontrol agent provided by this invention at 350 mL / m² after harvest. 2 Apply the solution to the soil with irrigation water, then cover with a thin layer of soil. After sowing this crop, when the temperature is above 30℃ and the humidity is above 75%, use the compound biocontrol agent provided by this invention at a concentration of 150 mL / m². 2 Spray on the soil and apply 4 times. The first 3 times should be 10 days apart, and the last time should be 20 days apart.

[0054] IV. Experimental Verification of the Invention

[0055] Experimental Example 1: The control effect of this invention on damping-off and wilt diseases in cabbage seedlings.

[0056] Test reagents: The compound biocontrol agent and each single bacterial agent of this invention are the test reagents, 64% oxamyl manganese zinc wettable powder is the positive control, and water treatment is the blank control.

[0057] Sporotypes with a spore content greater than 1×10 8Inoculate 5 ml / g of each of the *Fusarium oxysporum* cuf / mL cuf / mL *Rhizoctonia solani* suspensions into a mixed substrate of coconut coir, black soil, and vermiculite (3:4:1), keep it moist, cover with a thin film, and incubate at (26±1) ℃ for 7 days. Mix well, fill 14 seedling trays of 50 cells each, with 2 trays per treatment, and label them. Measure 15 ml of each test agent per gram of substrate, dilute appropriately, and spray evenly into the corresponding labeled trays. Weigh the control agent according to the standard (1:500) and dilute it in the same amount as the test agents before spraying. Spray the blank control with the same amount of water. Incubate all treatments at (26±1) ℃ for 3 days, then treat again with the same amount and method of the test agent for later use (see Example 12 for this technique). Select plump cabbage seeds, disinfect them with 70% alcohol for 1 min, then with 0.5% sodium hypochlorite for 1 min, rinse 5-6 times with sterile water, and then soak them in sterile water at 40 ℃ for 2 h. Germinate them at (27±1) ℃ in the dark. After most seeds have germinated, select seeds with consistent germination and sow them in seedling trays containing the treatment substrate, 100 seeds per treatment. Culture under a 10h / 14h photoperiod and normal management. Observe and record seedling survival, plant growth, and disease incidence daily. After the number of diseased plants in the blank control exceeds 10%, count the number of diseased plants and their disease status, calculate the disease incidence rate and control effect. The experimental results are shown in Table 2.

[0058] Calculation formula:

[0059] Disease incidence rate % = (Number of diseased plants / Total number of plants surveyed) × 100

[0060] Control efficacy % = (Control disease incidence rate - Treatment disease incidence rate) / Control disease incidence rate × 100

[0061] Table 2. Control efficacy of the present invention against Fusarium wilt and damping-off in cabbage seedlings.

[0062]

[0063] Notes: 1. BAS, BAC, BAT, BSS, and STRA formulations are single-strain formulations prepared using the same formula (Example 6) and preparation method for Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus amyloliquefaciens, and Streptomyces urophylla in this invention with the compound biocontrol agent; 2. The positive control is 64% mancozeb wettable powder; 3. Different lowercase letters in the same row in the table indicate a significant difference at the 0.05 level (p≤0.05).

[0064] Experimental Example 2: The control effect of the compound biocontrol agent of the present invention on cucumber wilt disease in the seedling stage.

[0065] Test reagents: compound biocontrol agent and each single bacterial agent were used as test reagents, 50% carbendazim wettable powder was used as a positive control, and water treatment was used as a blank control.

[0066] Each test agent was mixed at a rate of 10 ml / g with a mixed substrate of coconut coir, black soil, and vermiculite (2:2:1). The control agent was diluted with the same amount of water at a ratio of 1:500 and mixed with the above mixed substrate. A blank control was sprayed with the same amount of water as the test agent and mixed with the above mixed substrate. The mixtures were kept moist and placed at a constant temperature and humidity of (28±1) ℃ for 3 days. Two 50-cell seedling trays were prepared for each test treatment, and the mixed substrate was labeled. On the 4th day, the spore content was greater than 1×10⁻⁶. 8 A 6 ml / g inoculation of *Fusarium oxysporum* spore suspension (cuf / mL) specifically for cucumber was applied to the mixed substrate of each experimental treatment. The inoculated substrate was mixed thoroughly, placed in trays, and labeled. (Refer to Example 10) Plump cucumber seeds were selected, disinfected with 70% alcohol for 1 min, then with 0.5% sodium hypochlorite for 1 min, rinsed 5-6 times with sterile water, and then soaked in sterile water at 40 ℃ for 2 h. Germination was carried out at (30±1) ℃ in the dark. After most seeds germinated, seeds with consistent germination were selected and sown in seed trays containing the treatment substrate, 100 seeds per treatment. The plants were cultured under a 9h / 15h photoperiod and managed normally. Seedling emergence, plant growth, and disease incidence were observed and recorded daily. When the number of diseased plants in the blank control exceeded 10%, the number of diseased plants and their disease severity were counted, and the disease incidence rate and control effect were calculated using the same formula as in Example 1. The experimental results are shown in Table 3.

[0067] Table 3. Control efficacy of the present invention against cucumber wilt disease in the seedling stage.

[0068]

[0069] Notes: 1. The present invention and each single agent in the table are prepared according to the formulation ratio of Example 4, and the other notes are the same as those in Table 2; 2. The positive control is 50% carbendazim wettable powder; 3. Different lowercase letters in the same row in the table indicate that there is a significant difference at the 0.05 level (p≤0.05).

[0070] Experimental Example 3: The control effect of this invention on various soil-borne diseases of potato

[0071] Test reagents: The compound biocontrol agent of this invention is the test reagent, 50% carbendazim wettable powder is the positive control, and water treatment is the blank control.

[0072] An appropriate amount of original soil was collected from a dryland potato field that had been continuously cropped for 6-7 years. Testing revealed that the original soil contained a large amount (greater than 10...) of... 4The pathogenic fungi and nematodes (infected tubers, which were inoculated into each treatment substrate in this experiment) were collected (cfu / g). The diseased soil was mixed evenly with the treated coconut coir (4:1) and kept warm (26±1℃) for 7 days for later use. The test agent was mixed evenly with the treated soil substrate at a rate of 20 ml / g. The control agent was diluted with the same amount of water at a mass ratio of 1:500 and mixed evenly with the substrate. The blank control was sprayed with the same amount of water as the test agent and mixed evenly with the substrate. All treatments were kept warm (30±1℃) for 3 days for later use. The same amount and method were then used to treat twice more, with an interval of 3 days between each treatment. The tubers were then placed in pots (7 pots per treatment), labeled, and kept for later use (refer to Example 14). Select sufficient quantities of pre-sprouted potato seed stock (Longshu 10) that are roughly the same size and clean on the surface. Cut them into pieces (disinfect the cutting knife promptly with 0.2% potassium permanganate solution), mix with wood ash, and sow 6 seeds per pot into the substrates of each treatment. Each treatment contains 42 seeds. Perform routine management. Observe and record seedling emergence, plant growth, and disease incidence (including those that have not yet emerged; investigate the cause of disease in those that have not yet emerged). When the incidence rate in the blank control is greater than 10%, count the number of diseased plants and their disease severity, and calculate the control effect using the same formula as in Experiment 1. The experimental results are shown in Table 4.

[0073] Table 4. Control efficacy of this invention against various soil-borne diseases of potato

[0074]

[0075] Notes: 1. The present invention and each single agent in the table are prepared according to the formulation ratio of Example 1, and the other notes are the same as in Table 2; 2. The pathogens of bacterial wilt, rot, wilt, root rot and damping-off are respectively Laurella, Erwinia carotenoidea, Fusarium, nematode and Rhizoctonia solani. Since Streptomyces scabica mainly damages tubers, it was not included in the statistics at the time of the survey because tubers had not yet formed; 3. Different lowercase letters in the same column in the table indicate that there is a significant difference at the 0.05 level (p≤0.05).

[0076] Experimental Example 4: The effect of this invention on the control of soil-borne diseases of continuously cropped cabbage.

[0077] The experimental plot was farmland that had been continuously cropped with cruciferous vegetables for three years. Rainfall and temperature were normal in the experimental year. The cabbage variety used in the experiment was Zhonggan 21. This experiment was a summer crop, with baby bok choy planted in the spring. There was a slight outbreak of wilt disease. At harvest, the main pathogens found in the soil were Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Erwinia carotenoides, Sclerotinia sclerotiorum, and Pseudomonas aeruginosa.

[0078] The experiment consisted of three treatments: one test agent (the present invention), one positive control (64% cymoxanil·mancozeb wettable powder), and one blank control (no treatment). Each treatment was replicated three times, for a total of nine experimental plots, each plot being 30 m². 2 Randomized block design. A flat, unmulched planting method was adopted. During the seedling stage, 60 mL / m² of the compound biocontrol agent provided by this invention was used. 2 Treat the soil with fungicide and spray twice with the same amount to ensure germination rate and seedling quality. After the previous crop is harvested, apply 80 kg / 667 m² of fungicide. 2 Biological organic fertilizer and 8kg / 667 m 2 The compound biocontrol granules are broadcast, deeply tilled, and the land is prepared for soil treatment. After transplanting this crop, when the temperature is above 30℃, apply the compound biocontrol granules provided by this invention at a concentration of 80 mL / m². 2 Apply once with irrigation water, then spray twice more after a 5-day interval, followed by two more applications with irrigation water, each 10 days apart. A positive control was treated with a 600-fold dilution using the same method. Other routine management practices were followed. Throughout the entire growth period and at harvest, seedling emergence, plant growth, and disease occurrence were observed and recorded. Disease types, disease incidence, and control effectiveness were statistically analyzed. The experimental results are shown in Table 5.

[0079] Calculation formula: Disease incidence rate (%) = Number of diseased plants / Total number of plants surveyed × 100

[0080] Control efficacy (%) = (Disease rate in control area - Disease rate in treatment area) / Disease rate in control area × 100

[0081] Table 5. Effect of this invention on the control of soil-borne diseases in continuously cropped cabbage.

[0082]

[0083] Notes: 1. This invention is prepared according to the formulation of Example 8, and the application technique is applied according to Example 16; 2. The pathogens of Fusarium wilt, damping-off, soft rot, black shank, and sclerotinia rot are Fusarium, Rhizoctonia solani, Erwinia carotenoides, Pseudomonas sclerotiorum, and Sclerotinia sclerotiorum, respectively; 3. Different lowercase letters in the same column of the table indicate significant differences at the 0.05 level (p≤0.05).

[0084] Experimental Example 5: The effect of this invention on the control of soil-borne diseases in greenhouse eggplant

[0085] The previous crop in the experimental greenhouse was eggplant. Soil surveys at harvest time revealed the presence of several pathogenic fungi, including *Fusarium oxysporum*, *Fusarium solanum*, *Cladosporium dahliae*, *Sclerotinia sclerotiorum*, and *Pythium spp.* All of these pathogens were present in the previous crop but were controlled with timely pesticide application. After the previous crop was harvested, the compound biocontrol agent provided by this invention was applied at a concentration of 350 mL / m². 2 Apply the solution to the soil along with irrigation water, then till and prepare the land. In the summer heat, seal the soil in a greenhouse for 20 days, then let it air dry for 15 days.

[0086] The experiment consisted of three treatments: one test agent (compound biocontrol agent), one positive control (50% carbendazim wettable powder), and one blank control (no treatment). Each treatment was replicated three times, resulting in a total of nine experimental plots, each with an area of ​​30 m². 2 The planting was arranged in a randomized block design. A raised-ridge and mulch planting pattern was adopted. Before ridging, the compound biocontrol agent provided by this invention was applied at a rate of 250 mL / m² during land preparation. 2 Apply the compound biocontrol agent provided by this invention to the soil along with irrigation water, and prepare the land. After transplanting, when the temperature remains above 30℃ and the humidity is greater than 75%, apply 250mL / m² of the compound biocontrol agent provided by this invention. 2 Apply by spraying or watering the soil twice, with a 10-day interval between applications. After a 20-day interval, water the soil once more with the same amount. The positive control is diluted 500 times and treated in the same manner.

[0087] Cultivation and management were carried out according to standard procedures. Throughout the entire growth period and at harvest, observations and records of seedling emergence, plant growth, and disease occurrence were kept. Disease types, disease incidence, and control effectiveness were statistically analyzed using the same formula as in Experiment 4. The experimental results are shown in Table 6.

[0088] Table 6. Control efficacy of this invention against soil-borne diseases in greenhouse eggplant.

[0089]

[0090] Notes: 1. This invention is prepared according to the formulation of Example 9, and the application technique is applied according to Example 18; 2. The pathogens of Verticillium wilt, damping-off, Fusarium wilt, root rot and sclerotinia rot are Cladosporium dahliae, Pythium cucurbita, Fusarium oxysporum, Fusarium solani, and Sclerotinia sclerotiorum, respectively; 3. Different lowercase letters in the same column of the table indicate significant differences at the 0.05 level (p≤0.05).

[0091] Data Analysis: The results of Experiments 1-5 (Tables 2, 3, 4, 5, 6) show that this invention can effectively control soil-borne plant diseases caused by pathogens such as Fusarium, Phytophthora, Pythium, Rhizoctonia solani, Raulella, Erwinia carotenoides pathogenic type, Streptomyces pathogenicus, Cistanche deserticola, Blackleg mold, and nematodes, which can reside in the soil for a long time and spread through the soil. Moreover, its control effect is good, and there is no significant difference with the positive control at the 0.05 level (p>0.05). The control effect is even higher than that of the positive control for some of the diseases. The pot experiment results of Examples 1 and 2 (Tables 2 and 3) show that the present invention has a control effect of 83.90% to 85.91% on Fusarium wilt and Rhizoctonia solani, which are common, highly damaging and difficult to control, and caused by Fusarium and Rhizoctonia solani. The control effect is much stronger than that of each single fungicide preparation, with a significant difference (p≤0.05). The control effect is comparable to that of the positive control (50% carbendazim wettable powder and 64% cymoxanil-mancozeb wettable powder), with no significant difference (p>0.05). The results of the pot experiment in Example 3 (Table 4) showed that the present invention had a control effect of 80.27%–85.20% on soil-borne diseases such as bacterial wilt, rot, wilt, root rot, and damping-off caused by pathogens commonly found in potato soils after continuous cropping for many years, including *Raphanus latifolius*, *Erwinia carotenoides* pathogenic type, *Fusarium*, nematodes, and *Rhizoctonia solani*. This effect was significantly stronger than that of individual spore preparations (p≤0.05), and most of the control effects were comparable to the positive control (50% carbendazim wettable powder), with no significant difference (p>0.05). However, the control effect on potato root rot, which is mainly caused by nematodes (80.27%), was significantly different from that of 50% carbendazim wettable powder (38.61%), which does not have a nematode-killing effect (p≤0.05). The field trial results of Experiments 5 and 6 (Tables 5 and 6) show that the present invention has a control effect of 79.73% to 85.46% on soil-borne diseases such as Fusarium wilt, root rot, damping-off, soft rot, black shank, sclerotinia soft rot, black shank, stem rot, sclerotiorum, dahlia spores, and Pythium moniliforme caused by pathogens that are common and serious in the soil of continuously cropped cabbage and greenhouse eggplant. Most of these diseases are comparable to the positive control (50% carbendazim wettable powder and 64% cymoxanil-mancozeb wettable powder), with no significant difference (p>0.05). However, the control effect on eggplant damping-off and root rot is better than that of 50% carbendazim wettable powder (positive control), and the difference is significant (p≤0.05).

Claims

1. A compound biocontrol agent, characterized in that, It is a liquid preparation composed of a fermentation mixture of multiple biocontrol bacteria and a stabilizer; the specific ratio is: 1000 mL of a fermentation mixture of multiple biocontrol bacteria mixed in equal volumes, and 0.5%–1.5% stabilizer, the unit being weight-volume percentage; the fermentation mixture of the multiple biocontrol bacteria is a fermentation mixture of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus thuringiensis, and Streptomyces urophylla; the Bacillus subtilis is Bacillus subtilis, preservation number CGMCC No. 24484; the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens, preservation number CGMCC No. 24481; the Bacillus cereus is Bacillus cereus, preservation number CGMCC No. 24485; the Bacillus thuringiensis is Bacillus thuringiensis. The species *Streptomyces thuringiensis* has the accession number CGMCC No. 24483; the species *Streptomyces atroolivaceus* has the accession number CGMCC No. 24482. The preparation steps of the fermentation broth of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, and Bacillus thuringiensis include: using 0.5%–2.0% beef extract and 1.0%–3.0% glucose as the fermentation culture medium, with a sample loading of 50%–70%, an aeration rate of 3.0 L / ml–6.0 L / ml, an inoculum size of 5%–10%, a temperature of 28–31°C, an rpm of 100–200 rpm, an initial pH of 7.0, a dissolved oxygen content of 20%–35%, and the direct addition of 0.05%–0.3% GP-330 antifoaming agent, and culturing for 30–48 hours for later use; The preparation steps of the fermentation broth of *Streptomyces urophylla* include: using millet (1.5%–4.0%), glucose (0.5%–2.5%), and dipotassium hydrogen phosphate (0.01%–0.3%) as the fermentation broth, with a sample volume of 50%–70%, an aeration rate of 2.0–4.5 L / ml, an inoculum size of 7%–12%, a temperature of 26–29°C, an operating speed of 200–300 rpm, and the addition of 0.1%–0.5% GP-330 for defoaming. The initial pH value is 7.

2. After 72 hours of fermentation, 16%–23% of the fermentation broth is added once to continue fermentation. The dissolved oxygen content is 20%–26%, and the culture time is 132–168 hours. The stabilizer is one or a mixture of several of the following in any proportion: skim milk, dipotassium hydrogen phosphate, sodium alginate, mannitol, vitamin C, poloxamer, molasses, bentonite, polyvinyl alcohol, aesculin, folic acid, urea, colloidal silica, polysorbate, chlorogenic acid, mercaptoethanol, chitosan, amino acids, inositol, ferrous sulfate, superphosphate, ethylene glycol, glycerol, and polyoxyethylene.

2. The preparation method of the composite biocontrol agent as described in claim 1, characterized in that: The *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus cereus*, and *Bacillus thuringiensis* strains were fermented in a medium of 0.5%–2.0% beef extract and 1.0%–3.0% glucose, with a loading volume of 50%–70%, an aeration rate of 3.0 L / ml–6.0 L / ml, an inoculum size of 5%–10%, a temperature of 28–31°C, an rpm of 100–200 rpm, an initial pH of 7.0, a dissolved oxygen content of 20%–35%, and the addition of 0.05%–0.3% GP-330 antifoaming agent in a single step. The culture was carried out for 30–48 hours for later use. The *Streptomyces urophylla* strain was fermented in a medium of 1.5%–4% millet extract. The fermentation broth consists of 0% glucose, 0.5%–2.5% dipotassium hydrogen phosphate, and 0.01%–0.3% glucose. The sample loading volume is 50%–70%, the aeration rate is 2.0–4.5 L / ml, the inoculum size is 7%–12%, the temperature is 26–29℃, the rpm is 200–300 rpm, and 0.1%–0.5% GP-330 is added for defoaming. The initial pH is 7.

2. After 72 hours of fermentation, 16%–23% of the fermentation broth is added to continue fermentation. The dissolved oxygen content is 20%–26%, and the culture time is 132–168 hours. All fermentation broths are mixed, and 0.5%–1.5% stabilizer is added. The mixture is thoroughly mixed to obtain the final product.

3. The application of the composite biocontrol agent as described in claim 1, characterized in that, It is used to control the following diseases: Fusarium wilt, damping-off, black shank, soft rot, sclerotinia stem rot, cucumber wilt, bacterial wilt, potato rot, potato root rot, damping-off, eggplant damping-off, eggplant wilt, verticillium wilt, eggplant root rot, and eggplant sclerotinia stem rot.

4. The application of the composite biocontrol agent as described in claim 3, characterized in that, Specifically as follows: 1) Prevention: When planting plants of the same family in succession, apply the aforementioned compound biocontrol agent at a concentration of 50 mL / m² before planting. 2 ~200mL / m 2 Spray on the soil or apply with irrigation water to the soil or treat the planting substrate. Apply 1-2 times consecutively. Plant 3-5 days after application. 2) Prevention and control: If diseases occurred in the previous crop, treat the soil with bio-organic fertilizer and bio-pesticides after harvest, or use the aforementioned compound biocontrol agent at a concentration of 100 mL / m³. 2 ~350mL / m 2 Apply the compound biocontrol agent to the soil with irrigation water, then cover with a thin layer of soil; after sowing this crop, when the temperature is above 30℃ and the humidity is above 75%, apply the compound biocontrol agent at a rate of 80mL / m². 2 ~250mL / m 2 Spray on the soil or apply with irrigation water, 2 to 5 times consecutively. The first 2 to 3 times should be spaced 7 to 10 days apart, and the next 4 to 5 times should be spaced 15 to 20 days apart.

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