Composite biocontrol agent for pepper phytophthora blight as well as preparation method and application of composite biocontrol agent

Abstract

The invention belongs to the field of biological agriculture, and particularly discloses a composite biocontrol preparation for pepper phytophthora blight as well as a preparation method and application of the composite biocontrol preparation. The preparation comprises the following components in parts by weight: 25-45 parts of freeze-dried powder of a complex microbial inoculant, 15-25 parts of a plant extract, 5-10 parts of seaweed oligosaccharide, 3-5 parts of a surfactant and 1-3 parts of a stabilizer, the freeze-dried powder of the complex microbial inoculant comprises bacillus velezensis, trichoderma harzianum and pseudomonas fluorescens, and the plant extract is composed of extracts of radix sophorae flavescentis, garlic bulbs and radix scutellariae roots. Under the synergistic effect of all the components, the prepared composite biocontrol agent can effectively inhibit the pepper phytophthora blight, whole-process prevention and control are achieved through seed treatment, root irrigation and foliage spraying, spreading of the pepper phytophthora blight is effectively prevented, the composite biocontrol agent is remarkably superior to a single fungicide and a chemical pesticide, and the composite biocontrol agent has the advantages of being efficient, environmentally friendly and low in cost and is wide in application prospect.

Description

Technical Field

[0001] This invention relates to the field of bio-agriculture, specifically to a compound biocontrol agent for pepper blight, its preparation method, and its application. Background Technology

[0002] Phytophthora capsici is one of the most destructive soil-borne diseases in global pepper cultivation. Its pathogen, Phytophthora capsici, can survive in the soil for years, infecting roots, stem bases, and fruits through zoospores. The long-term use of chemical pesticides (such as metalaxyl) has led to the widespread development of resistant strains and severe damage to the soil microbial community.

[0003] In recent years, biological control has become a research hotspot due to its environmentally friendly characteristics. However, existing technologies still have significant shortcomings. For example, while using single-species bacteria such as Bacillus subtilis can inhibit pathogens to some extent, it is difficult to cope with the complex infection cycle of Phytophthora, such as zoospore release, mycelial growth, and oospore survival. More seriously, some studies have found antagonistic phenomena when trying to combine different strains. For instance, chitinase secreted by Trichoderma can inhibit the germination of Bacillus spores, resulting in actual control efficacy lower than theoretical values. Plant-derived components, such as allicin and matrine, have been proven to have antifungal activity; however, the toxic effects of phenols and aldehydes in their crude extracts on biocontrol bacteria are often overlooked. Direct mixing of garlic extract with Trichoderma harzianum resulted in a decrease in viable bacteria count of over 70% within 6 months, indicating that existing formulations have not solved the compatibility problem of active ingredients. Conventional freeze-drying protectants such as skim milk and sucrose have limited effectiveness in protecting Gram-negative bacteria such as Pseudomonas fluorescens.

[0004] In application, existing products are mostly limited to a single application method such as foliar spraying, while Phytophthora can be spread through multiple routes such as soil, seeds, and irrigation water. For example, the mainstream product "Trichoderma granules" can only be used for soil treatment and cannot achieve simultaneous seed disinfection and protection during the growth period, resulting in a fluctuation in efficacy between 50% and 60%.

[0005] Therefore, there is an urgent need for a compound biocontrol agent that can effectively inhibit the spread of pepper blight while also comprehensively suppressing its transmission. Summary of the Invention

[0006] This invention prepares a compound biocontrol agent. When applied to pepper blight, it solves the problems of low efficacy, poor stability, and limited application scenarios of existing biocontrol agents through multi-strain synergy, optimized process compatibility, and innovative multi-pathway application. It provides an efficient, safe, and economical solution for the green control of pepper blight, which meets the needs of sustainable agricultural development.

[0007] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem:

[0008] On the one hand, the present invention provides a compound biocontrol agent for the prevention and control of pepper blight, comprising, by weight, 25-45 parts of compound microbial agent freeze-dried powder, 15-25 parts of plant extract, 5-10 parts of seaweed oligosaccharide, 3-5 parts of surfactant, and 1-3 parts of stabilizer.

[0009] The compound bacterial agent freeze-dried powder is made by freeze-drying and mixing the fermentation broth of Bacillus vesiculosus, Trichoderma harzianum and Pseudomonas fluorescens;

[0010] The plant extract consists of extracts from Sophora flavescens root, garlic bulb, and Scutellaria baicalensis root.

[0011] In some embodiments, the viable count of Bacillus berberis in the compound microbial agent is ≥1×10⁻⁶. 8 CFU / g, Trichoderma harzianum ≥1×10 7 CFU / g, ≥5×10⁻⁵ Pseudomonas fluorescens 7 CFU / g.

[0012] In some embodiments, the phycoolidone has a molecular weight of 800-1500 Da and a degree of deacetylation ≥90%.

[0013] In some embodiments, the method for preparing the plant extract includes the following steps:

[0014] (1) Grind the washed Sophora flavescens root, garlic bulb and Scutellaria baicalensis root into 50-200 mesh at a mass ratio of 3:(1-2):1;

[0015] (2) Use an ethanol-water mixture with a volume ratio of 7:(3-5) and ultrasonically extract 2-4 times at 50-60℃, each time for 1-2 hours;

[0016] (3) After combining the extracts, concentrate under reduced pressure to a relative density of 1.1-1.2, and freeze dry to obtain plant extract powder.

[0017] In some embodiments, the preparation of the compound microbial agent lyophilized powder includes:

[0018] S1. Inoculate Bacillus belye, Trichoderma harzianum, and Pseudomonas fluorescens into liquid culture medium for fermentation at a temperature of 28-32℃ and an aeration rate of 1.0-1.5 vvm.

[0019] S2. When the bacterial concentration reaches the end of the logarithmic growth phase, centrifuge to collect the bacterial sludge;

[0020] S3. Mix the bacterial sludge with the freeze-drying protectant at a mass ratio of 2:(1-3), pre-freeze for 2-4 hours, and then freeze-dry for 12-24 hours to obtain freeze-dried powder containing live bacteria;

[0021] The freeze-drying protectant contains 10-15% skim milk powder, 5-8% trehalose, 1-2% monosodium glutamate, and the remainder is glycerol.

[0022] In some embodiments, the fermentation medium of the Bacillus belye contains 20 g / L corn flour, 15 g / L soybean meal, and 2 g / L KH2PO4;

[0023] The fermentation medium for *Trichoderma harzianum* contains 30 g / L wheat bran, 10 g / L glucose, and 0.5 g / L MgSO4.

[0024] The fermentation medium for the fluorescent Pseudomonas bacteria contains: 10 g / L peptone, 5 g / L yeast extract, 8 g / L glycerol, and 1.5 g / L K2HPO4.

[0025] In some embodiments, the surfactant is a mixture of alkyl glycoside APG0810 and polyglycerol fatty acid ester PGFE in a weight ratio of 1:(2-4).

[0026] Secondly, the present invention also discloses a method for preparing the above-mentioned compound biocontrol agent, comprising the following steps: mixing the compound bacterial agent freeze-dried powder, plant extract freeze-dried powder, seaweed oligosaccharide, surfactant and stabilizer, mixing at 30-60 rpm for 40-60 min, passing through a 50-100 mesh sieve and then vacuum packaging.

[0027] Thirdly, this invention also discloses the application of the above-mentioned compound biocontrol agent for the control of Phytophthora capsici, wherein the application method includes any one or more of the following 1)-3):

[0028] 1) Chili seed treatment: Dilute the preparation 800-1200 times and soak the seeds for 1-2 hours;

[0029] 2) Transplanting root irrigation: Dilute 500-15000 times, and irrigate each plant with 200-500 mL;

[0030] 3) Foliar spraying at the early stage of disease: dilute 1000-2000 times and spray once every 5-7 days.

[0031] In some embodiments, when transplanting and irrigating chili peppers in continuously cropped soil, the diluted compound biocontrol agent is mixed with a 0.1% chitosan aqueous solution at a volume ratio of 1:1 and then applied.

[0032] The chili pepper variety is Lujiao No. 1, and the chitosan has a degree of deacetylation ≥85% and a molecular weight of 2000-5000 Da.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention prepares a compound biocontrol agent that utilizes the antimicrobial lipopeptide IturinA secreted by Bacillus belysus, the hyphae and oospores of the parasitic pathogen Trichoderma harzianum, and the induced systemic resistance in plants by Pseudomonas fluorescens, thereby achieving an antimicrobial spectrum covering the entire life cycle of Phytophthora capsici. Furthermore, when combined with plant extracts prepared from Sophora flavescens root, garlic bulb, and Scutellaria baicalensis root, it can effectively inhibit Phytophthora capsici.

[0035] The compound biocontrol agent of the present invention also includes low molecular weight seaweed oligosaccharides, which can induce a 3.2-fold increase in capsicum phenylalanine ammonia-lyase activity, and at the same time serve as a microbial carbon source to promote rhizosphere colonization, thus having the dual functions of enhancing disease resistance and promoting growth.

[0036] This invention utilizes a surfactant prepared by mixing trehalose and sodium carboxymethyl cellulose, making the compound biocontrol agent suitable for seed soaking, root irrigation, and foliar spraying. Compared with chemical pesticides, the compound biocontrol agent combined with chitosan root irrigation in continuously cropped soils increases the control efficacy by 10%-15% and increases yield by 23.6%, while reducing control costs and meeting the needs of green agriculture. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0039] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0040] In this invention, some of the raw materials and equipment designed are shown in Tables 1 and 2 below.

[0041] Table 1. Raw Material Sources

[0042]

[0043]

[0044] Table 2 Equipment List

[0045]

[0046]

[0047] Example 1

[0048] I. A compound biocontrol agent for pepper blight, comprising, by weight, 35 parts of compound bacterial agent freeze-dried powder, 20 parts of plant extract, 8 parts of seaweed oligosaccharide (molecular weight 1200 Da), 4 parts of surfactant, and 2 parts of stabilizer.

[0049] The compound bacterial agent freeze-dried powder is composed of Bacillus belyssus with a live bacteria ratio of 1:1:0.5 (roughly calculated based on the number of live bacteria) and a live bacteria count of 1×10⁻⁶. 8 CFU / g), Trichoderma harzianum (live count 1×10⁻⁶) 7 CFU / g), Pseudomonas fluorescens (viable count 5×10⁻⁶) 7 It is prepared by freeze-drying a mixture of mycelium sludge (CFU / g) and mycelium sludge.

[0050] The surfactant is a mixture of alkyl glycoside (APG0810) and polyglycerol fatty acid ester (PGFE) in a weight ratio of 1:2.

[0051] The stabilizer is a mixture of trehalose and sodium carboxymethyl cellulose (CMC-Na, viscosity 800-1200 mPa·s) in a weight ratio of 1:1.

[0052] II. A method for preparing a compound biocontrol agent for pepper blight, comprising the following steps:

[0053] 1. Preparation of compound microbial agent freeze-dried powder

[0054] (1) Bacillus vesiculosus fermentation:

[0055] Culture medium: 20 g / L corn flour, 15 g / L soybean meal, 2 g / L KH2PO4, pH 7.0.

[0056] Conditions: 5% inoculum, fermentation at 32℃ for 42 hours, stirring speed 200 rpm, aeration rate 1.2 vvm.

[0057] Collection: Centrifuge at 6000×g for 15 minutes, collect the bacterial sludge, and wash 3 times with physiological saline.

[0058] (2) Fermentation by Trichoderma harzianum:

[0059] Culture medium: 30 g / L wheat bran, 10 g / L glucose, 0.5 g / L MgSO4, pH 6.5.

[0060] Conditions: 10% inoculum, fermentation at 28℃ for 84 hours, stirring rate 150 rpm, aeration rate 1.0 vvm.

[0061] Collection: The mycelium was filtered through gauze, rinsed with distilled water, and then vacuum filtered.

[0062] (3) Fermentation by *Pseudomonas fluorescens*:

[0063] Culture medium: 10 g / L peptone, 5 g / L yeast extract, 8 g / L glycerol, 1.5 g / L K2HPO4, pH 7.2.

[0064] Conditions: 3% inoculum, fermentation at 30℃ for 30 hours, stirring speed 250 rpm, aeration rate 1.5 vvm.

[0065] Collection: Centrifuge at 8000×g for 10 minutes, and suspend the mycelium sludge in 0.85% NaCl solution.

[0066] (4) Freeze-drying protection and drying:

[0067] Protectant formulation: 10% skim milk powder, 5% trehalose, 2% monosodium glutamate, dissolved in sterile water.

[0068] Mixing: Mix the mixed sludge and the preservative at a ratio of 1:3 (w / v) and pre-freeze at -40℃ for 4 hours.

[0069] Freeze-drying: cold trap temperature -55℃, vacuum degree 10Pa, main drying for 20 hours (gradual temperature increase from -20℃ to 25℃).

[0070] 2. Preparation of plant extracts

[0071] (1) Wash the Sophora flavescens root, garlic bulb and Scutellaria baicalensis root separately and dry them at 50℃ until the moisture content is ≤8%, then crush them and pass them through a 60-mesh sieve.

[0072] (2) Ultrasonic extraction: Mixed raw materials (Sophora flavescens root, garlic bulb and Scutellaria baicalensis root mixed in a mass ratio of 3:2:1) were added to 70% ethanol solution (containing 0.1% ascorbic acid) at a material-to-liquid ratio of 1:15 (g / mL), and ultrasonically extracted twice at 50℃ (power 300W, frequency 40kHz, 1 hour each time).

[0073] (3) Concentration and drying: Combine the extracts, concentrate under reduced pressure at 50℃ to a relative density of 1.2 (measured at 25℃), freeze dry at -50℃ for 24 hours to obtain a brown powder (yield 18.5%).

[0074] 3. Formulation mixing

[0075] The compound microbial agent freeze-dried powder, plant extracts, seaweed oligosaccharides, surfactants, and stabilizers were mixed using a three-dimensional motion mixer (model SYH-1000) at 30 rpm for 40 minutes. The mixture was then passed through a 60-mesh sieve three times and vacuum-sealed in aluminum foil bags (residual oxygen content ≤0.5%), with each bag containing 500g.

[0076] Example 2

[0077] I. A compound biocontrol agent for pepper blight, comprising, by weight, 45 parts of compound bacterial agent freeze-dried powder, 25 parts of plant extract, 10 parts of seaweed oligosaccharide (molecular weight 1200 Da), 4 parts of surfactant, and 2 parts of stabilizer.

[0078] II. The preparation method of a compound biocontrol agent for pepper blight is the same as in Example 1.

[0079] Example 3

[0080] I. A compound biocontrol agent for pepper blight, comprising, by weight, 25 parts of compound bacterial agent freeze-dried powder, 15 parts of plant extract, 5 parts of seaweed oligosaccharide (molecular weight 800 Da), 4 parts of surfactant, and 2 parts of stabilizer.

[0081] II. The preparation method of a compound biocontrol agent for pepper blight is the same as in Example 1.

[0082] Example 4

[0083] The difference between Example 4 and Example 1 is that the weight of the compound bacterial agent freeze-dried powder in Example 4 is 35 parts, and the ratio of live bacteria is 1:0.8:0.6, while all other aspects are the same.

[0084] Example 5

[0085] The difference between Example 5 and Example 1 is that in the preparation of the plant extract in Example 5, the mass ratio of Sophora flavescens root, garlic bulb, and Scutellaria baicalensis root is 4:1:1, while all other aspects are the same.

[0086] Comparative Example 1

[0087] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain seaweed oligosaccharides and the weight of the stabilizer is 10 parts, while all other aspects are the same.

[0088] Comparative Example 2

[0089] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 used an equal amount of Bacillus belye lyophilized powder (viable count 1 × 10⁻⁶). 8 The CFU / g compound microbial agent freeze-dried powder is used as a substitute, and everything else is the same.

[0090] Bacillus vesiculosus fermentation:

[0091] Culture medium: 20 g / L corn flour, 15 g / L soybean meal, 2 g / L KH2PO4, pH 7.0.

[0092] Conditions: 5% inoculum, fermentation at 32℃ for 42 hours, stirring speed 200 rpm, aeration rate 1.2 vvm.

[0093] Collection: Centrifuge at 6000×g for 15 minutes, collect the bacterial sludge, and wash 3 times with physiological saline.

[0094] Mixing: Mix the bacterial sludge with the preservative (10% skim milk powder, 5% trehalose, 2% monosodium glutamate dissolved in sterile water) at a ratio of 1:3 (w / v) and pre-freeze at -40°C for 4 hours.

[0095] Freeze-drying: cold trap temperature -55℃, vacuum degree 10Pa, main drying for 20 hours (gradual temperature increase from -20℃ to 25℃).

[0096] Comparative Example 3

[0097] The difference between Comparative Example 3 and Example 1 is that the plant extract in Comparative Example 3 is different, but everything else is the same.

[0098] In the preparation of plant extracts, the mass ratio of Sophora flavescens root, garlic bulb, and Scutellaria baicalensis root is 1:1:1.

[0099] Test Example 1

[0100] Seed treatment efficacy test

[0101] Tested variety: 'Lu Jiao No. 1' chili pepper, with a seed germination rate of ≥95%;

[0102] Treatment groups: Examples 1-3, Comparative Examples 1-3, Blank control (sterile water), Chemical control (50% carbendazim WP diluted 800 times).

[0103] Test method:

[0104] 1. Dilute each compound biocontrol agent 800 times and soak seeds for 2 hours (25℃ constant temperature shaker, 100 rpm);

[0105] 2. Sow in seedling trays (substrate: peat moss: vermiculite = 2:1, inoculate with 1×10⁻⁶ Phytophthora spore suspension). 5 CFU / g);

[0106] 3. Cultivate in a greenhouse at 25℃ and 80% humidity, and count the incidence of sudden collapse disease after 30 days.

[0107] Data statistics: For every 100 seeds treated, the treatment was repeated 4 times. The formula for calculating the control efficacy is shown below:

[0108]

[0109] Table 3. Seed Treatment Efficacy Test Results

[0110] Group Seed disease incidence (%) Preventive efficacy (%) Example 1 8.2±1.3 81.5a Example 2 9.1±1.5 79.4ab Example 3 12.3±2.1 72.1c Comparative Example 1 15.7±1.8 64.3d Comparative Example 2 23.6±2.5 46.2e Comparative Example 3 18.9±2.0 57.1f Chemical comparison 10.5±1.2 76.4b Blank control 44.5±3.7 -

[0111] Note: Lowercase letters indicate Duncan's multiple comparisons (P<0.05), and there is no significant difference for the same letter.

[0112] As shown in Table 3, the disease incidence rate of seeds treated with the compound biocontrol agent containing seaweed oligosaccharides in Example 1 was significantly lower than that in Comparative Example 1. This is because seaweed oligosaccharides enhance the control efficacy by inducing seed resistance and promoting the colonization of biocontrol bacteria. Further comparison between Example 1 and Comparative Example 2 shows that Comparative Example 2 had the lowest control efficacy, indicating that Bacillus vesiculosus, Trichoderma, and Pseudomonas fluorescens can synergistically inhibit bacterial growth. Comparison between Example 1 and Comparative Example 3 shows that although Comparative Example 3 used the same plant extracts as Example 1, the proportions of Sophora flavescens root, garlic bulb, and Scutellaria baicalensis root were different, resulting in a much lower control efficacy than Example 1. This is because the reduced proportion of Sophora flavescens led to a decrease in the antibacterial fat-soluble component (matrine), which affected the inhibition of spore germination.

[0113] Test Example 2

[0114] Soil protection efficacy of transplanting, root irrigation, and continuous cropping

[0115] Experimental site: A 5-year continuous cropping chili pepper field (Lu Jiao No. 1) in Shouguang City, Shandong Province (Phytophthora spore count ≥ 1×10⁻⁶) 4 CFU / g).

[0116] Processing Group:

[0117] Example 1: Formulation diluted 500 times;

[0118] Example 1: Formulation diluted 500 times + 0.1% chitosan (mixing volume ratio 1:1);

[0119] Comparative Example 3 formulation diluted 500 times;

[0120] Chemical control (68% metalaxyl-mancozeb WP diluted 500 times);

[0121] Blank control (sterile water).

[0122] Test method:

[0123] At transplanting, drench each plant with 200 mL of solution. After 30 days, investigate the area of ​​lesions at the base of the stem (0-5 grades: 0 = no lesions, 5 = stem ring rot). Calculate the disease index as shown below:

[0124]

[0125] Table 4. Results of Soil Protection Test for Transplanted Irrigated Continuous Cropping

[0126]

[0127]

[0128] Note: Lowercase letters indicate Duncan's multiple comparisons (P<0.05), and there is no significant difference for the same letter.

[0129] As shown in Table 4, when the compound biocontrol agent of Example 1 was used together with chitosan to treat peppers, the control efficacy of peppers was higher than that of the group in Example 1 where the compound biocontrol agent was used alone. This is because chitosan enhances the colonization ability of biocontrol bacteria by chelating heavy metals in the soil and improving the rhizosphere microecology (the formation of biofilm of Pseudomonas fluorescens was increased by 35%). In contrast, the control efficacy of Comparative Example 3 was much lower than that of Example 1 because the proportion of Sophora flavescens was reduced, which led to a decrease in the activity of inhibiting mycelial growth.

[0130] Test Example 3

[0131] Foliar spraying to control fruit blight

[0132] 1. Test Sample

[0133] Treatment group: Example 1, Example 4, Example 5, Comparative Example 1.

[0134] 2. Testing Method:

[0135] Starting from the initial flowering stage of chili pepper (Lujiao No. 1), dilute the preparation 1000 times and spray once every 7 days (droplet diameter 150-200μm, coverage ≥90%).

[0136] Artificial inoculation with Phytophthora spores (1×10 5 Spores ( / mL) were applied to young fruit, and the disease rate was calculated after 15 days.

[0137] The activities of leaf defense enzymes (POD, PAL) were measured.

[0138] Table 5. Test Results of Foliar Spraying for Control of Fruit Disease

[0139] Group Diseased fruit rate (%) POD activity (U / g) PAL activity (U / g) Example 1 13.5±1.8 352±24a 28.5±2.1a Example 4 16.8±2.1 298±21b 23.7±1.9b Example 5 19.3±2.3 265±18c 20.3±1.5c Comparative Example 1 24.7±2.6 231±17d 17.8±1.2d Blank control 52.1±3.5 155±12e 12.4±0.9e

[0140] As shown in Table 5, the disease rate of the fruit in Example 1 was much lower than that in Examples 4 and 5 and Comparative Example 1. The higher POD and PAL activities indicate that the peppers were under stress and were resisting the adversity by enhancing their antioxidant capacity and cell wall reinforcement. This is because Pseudomonas fluorescens induced systemic resistance and enhanced cell wall lignification. At the same time, the high proportion of matrine provided by Sophora flavescens in Example 1 disrupted the permeability of the pathogen's cell membrane.

[0141] In summary, this invention has prepared a compound biocontrol agent for Phytophthora capsici. The compound bacterial agent freeze-dried powder, prepared by mixing Bacillus belye, Trichoderma harzianum, and Pseudomonas fluorescens, has an antibacterial spectrum covering the entire life cycle of Phytophthora capsici. The compound bacterial agent significantly increases the mycelial inhibition rate by 40%-60% compared to single species, and there is no antagonistic effect among the three species. When the compound bacterial agent freeze-dried powder is used in combination with plant extracts prepared by mixing Sophora flavescens root, garlic bulb, and Scutellaria baicalensis root within a certain range, it still has a high fungal infestation rate against Phytophthora capsici. This indicates that the present invention uses ultrasonic extraction with ethanol, which solves the problem of plant phenolic substances inhibiting the activity of bacterial agents in traditional processes.

[0142] Meanwhile, when applying the compound biocontrol agent, the present invention covers the entire cycle of Phytophthora infection through a three-dimensional application mode of seed treatment, root irrigation and foliar spraying, which not only reduces the amount of chemical pesticides by more than 70%, but also reduces the disease rate of peppers.

[0143] The raw materials of this invention are all commercially available bacterial agents and natural extracts, the production cost is much lower than that of similar biological agents, the shelf life is up to 18 months, it is compatible with existing agricultural equipment, and it has the value for large-scale promotion.

[0144] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A compound biocontrol agent for controlling blight in chili peppers, characterized in that, By weight, it includes 25-45 parts of compound microbial agent freeze-dried powder, 15-25 parts of plant extract, 5-10 parts of seaweed oligosaccharide, 3-5 parts of surfactant, and 1-3 parts of stabilizer. The compound bacterial agent freeze-dried powder is made by freeze-drying and mixing the fermentation broth of Bacillus vesiculosus, Trichoderma harzianum and Pseudomonas fluorescens; The plant extract consists of extracts from Sophora flavescens root, garlic bulb, and Scutellaria baicalensis root.

2. The compound biocontrol agent according to claim 1, characterized in that, The viable count of Bacillus vesiculosus in the compound microbial agent is ≥1×10⁻⁶. 8 CFU / g, Trichoderma harzianum ≥1×10 7 CF U / g, Fluorescent Pseudomonas ≥5×10 7 CFU / g.

3. The compound biocontrol agent according to claim 1, characterized in that, The sphagnum oligosaccharide has a molecular weight of 800-1500 Da and a degree of deacetylation ≥90%.

4. The compound biocontrol agent according to claim 1, characterized in that, The method for preparing the plant extract includes the following steps: (1) Grind the washed Sophora flavescens root, garlic bulb and Scutellaria baicalensis root into 50-200 mesh at a mass ratio of 3:(1-2):1; (2) Use an ethanol-water mixture with a volume ratio of 7:(3-5) and ultrasonically extract 2-4 times at 50-60℃, each time for 1-2 hours; (3) After combining the extracts, concentrate under reduced pressure to a relative density of 1.1-1.2, and freeze dry to obtain plant extract powder.

5. The compound biocontrol agent according to claim 1, characterized in that, The preparation of the compound microbial agent freeze-dried powder includes: S1. Inoculate Bacillus belye, Trichoderma harzianum, and Pseudomonas fluorescens into liquid culture medium for fermentation at a temperature of 28-32℃ and an aeration rate of 1.0-1.5 vvm. S2. When the bacterial concentration reaches the end of the logarithmic growth phase, centrifuge to collect the bacterial sludge; S3. Mix the bacterial sludge with the freeze-drying protectant at a mass ratio of 2:(1-3), pre-freeze for 2-4 hours, and then freeze-dry for 12-24 hours to obtain freeze-dried powder containing live bacteria; The freeze-drying protectant contains 10-15% skim milk powder, 5-8% trehalose, 1-2% monosodium glutamate, and the remainder is glycerol.

6. The compound biocontrol agent according to claim 5, characterized in that, The fermentation medium of the Bacillus belye contains 20 g / L corn flour, 15 g / L soybean meal flour, and 2 g / L KH2PO4; The fermentation medium for *Trichoderma harzianum* contains 30 g / L wheat bran, 10 g / L glucose, and 0.5 g / L MgSO4. The fermentation medium for the fluorescent Pseudomonas bacteria contains: 10 g / L peptone, 5 g / L yeast extract, 8 g / L glycerol, and 1.5 g / L K2HPO4.

7. The compound biocontrol agent according to claim 1, characterized in that, The surfactant is a mixture of alkyl glycoside APG0810 and polyglycerol fatty acid ester PGFE in a weight ratio of 1:(2-4).

8. The method for preparing the compound biocontrol agent according to any one of claims 1-7, characterized in that, Includes the following steps: After mixing the compound microbial agent freeze-dried powder, plant extract freeze-dried powder, seaweed oligosaccharide, surfactant and stabilizer, mix at 30-60 rpm for 40-60 min, pass through a 50-100 mesh sieve and then vacuum package.

9. The application of the compound biocontrol agent according to any one of claims 1-7, characterized in that, For the control of Phytophthora capsici, the application methods include any one or more of the following 1)-3): 1) Chili seed treatment: Dilute the preparation 800-1200 times and soak the seeds for 1-2 hours; 2) Transplanting root irrigation: Dilute 500-15000 times, and irrigate each plant with 200-500 mL; 3) Foliar spraying at the early stage of disease: dilute 1000-2000 times and spray once every 5-7 days.

10. The application according to claim 9, characterized in that, When transplanting and irrigating chili peppers in continuously cropped soil, the diluted compound biocontrol agent is mixed with 0.1% chitosan aqueous solution at a volume ratio of 1:1 before application.

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