Environment-friendly composite pesticide based on marine microorganism and preparation method thereof

Abstract

This invention relates to the field of pesticide technology, and more particularly to an environmentally friendly compound pesticide based on marine microorganisms and its preparation method. The pesticide comprises the following raw materials by weight percentage: 40-52% functionalized microspheres, 5-6% matrine, 2-3% rotenone, 3-4% rhamnolipid, 1-1.5% sodium alginate, 30-35% diatomaceous earth, 1-2% zinc sulfate, and 2-4% hydroxyapatite, with the balance being water. This invention uses natural marine materials such as alginate, sodium alginate, and diatomaceous earth as carriers, combined with plant-derived active ingredients and biosurfactants. Matrine acts as a cholinesterase inhibitor, and rotenone acts as a mitochondrial complex I inhibitor, forming a dual mechanism of action while avoiding chemical pesticide residue pollution.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, and in particular to an environmentally friendly compound pesticide based on marine microorganisms and its preparation method. Background Technology

[0002] With the intensive development of agriculture, the overuse of chemical pesticides has led to a series of ecological problems, including soil degradation, water pollution, loss of biodiversity, and increased pesticide resistance in pests and diseases. Traditional pesticides are mostly chemically synthesized, and their high residue and persistent degradation characteristics pose a long-term threat to the environment and human health. In recent years, biopesticides have attracted much attention due to their advantages such as natural origin, strong targeting, and good environmental compatibility. The marine environment, with its extreme characteristics of high salinity, high pressure, and low oxygen, has prompted microorganisms to evolve metabolic pathways distinct from terrestrial strains, producing novel antibacterial, insecticidal, or antiviral active substances (such as polyketides, peptides, and terpenoids). Studies have shown that the secondary metabolites of some marine actinomycetes, Bacillus, and fungi have significant inhibitory effects on crop pathogens (such as Fusarium and Botrytis cinerea) and pests (such as aphids and nematodes), and are easily degraded by the natural environment, meeting the development requirements of green pesticides.

[0003] Chinese patent CN106577774A discloses a method for producing a marine animal-derived pesticide. Using starfish as raw material, the method employs a gradient alcohol extraction, salting-out precipitation combined with immobilized proteolysis, chromatographic purification, and compounding processes to prepare an environmentally friendly animal-derived pesticide. This pesticide's control effect on pests such as leek maggots and cabbage caterpillars is comparable to that of natural pyrethroids. However, secondary metabolites of marine microorganisms (such as saponins and polyketides) are easily degraded in the field environment by ultraviolet radiation, high temperatures, or pH fluctuations, resulting in poor efficacy and requiring frequent application. Furthermore, compatibility issues between microbial active ingredients and chemical adjuvants (such as emulsifiers) can easily lead to stratification and precipitation, resulting in uneven spraying or clogging of application equipment. Summary of the Invention

[0004] To address the problems mentioned in the background section, this invention provides an environmentally friendly compound pesticide based on marine microorganisms and its preparation method.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] An environmentally friendly compound pesticide based on marine microorganisms comprises the following raw materials by weight percentage: 40-52% functionalized microspheres, 5-6% matrine, 2-3% rotenone, 3-4% rhamnolipid, 1-1.5% sodium alginate, 30-35% diatomaceous earth, 1-2% zinc sulfate, and 2-4% hydroxyapatite, with the balance being water.

[0007] Furthermore, the functionalized microspheres are prepared by the following steps:

[0008] S1. Dissolve alginate in 0.85% sterile saline, stir until completely dissolved, cool to 30°C, add chitosan oligosaccharide, fucoxanthin and concentrated bacterial solution, control the temperature and stir magnetically for 30-60 min, then add 0.05M calcium chloride solution dropwise at 4°C for 15-20 min, centrifuge, discard the supernatant, wash twice with PBS solution, and resuspend in cryoprotectant containing 5% trehalose and 1% glycerol to obtain alginate microsphere solution;

[0009] S2. Polyaspartic acid and octyl glycidyl ether were added to dimethyl sulfoxide, and triethylamine was added. Under nitrogen protection, the mixture was stirred and reacted at a controlled temperature for 24-48 hours. The reaction solution was poured into anhydrous ethanol, filtered, and the precipitate was collected and dried to constant weight to obtain modified polyaspartic acid.

[0010] S3. Add the alginate microsphere solution and modified polyaspartic acid to a 0.1M phosphate buffer solution with a pH of 6.5, sonicate for 5-10 min, add genipin, stir and react for 4 h while controlling the temperature, collect the precipitate by centrifugation, freeze dry at 0℃ for 24 h to obtain functionalized microspheres.

[0011] Furthermore, in step S1, the temperature is controlled at 30-35℃, and the magnetic stirring speed is 100-200 rpm.

[0012] Further, in step S1, the mass ratio of alginate, physiological saline, chitosan oligosaccharide, fucoxanthin, concentrated bacterial solution and calcium chloride solution is (3-4):100:(2-3):(0.5-0.6):(2-2.5):(100-110).

[0013] Furthermore, the concentrated bacterial solution in step S1 includes Bacillus amyloliquefaciens and Pseudomonas alterniflora, with the concentration of Bacillus amyloliquefaciens being 1 × 10⁻⁶. 7 CFU / mL, the concentration of *Pseudomonas aeruginosa* is 1×10⁻⁶. 6 CFU / mL.

[0014] The concentrated bacterial culture was prepared by the following steps: Bacillus amyloliquefaciens glycerol-preserved strain was streaked onto LB solid medium (containing 1.5% agar) and incubated at 30°C for 24 hours. Single colonies were then picked and inoculated into LB liquid medium and incubated at 30°C and 200 rpm on a shaker until OD reached [value missing]. 600 ≈0.8, centrifuge and discard the supernatant, wash the bacterial cells twice with sterile physiological saline to obtain a suspension of Bacillus amyloliquefaciens. Streak the glycerol-preserved *Pseudomonas alterniflora* strain onto 2216E marine solid medium (containing 1.5% agar) and incubate at 25°C for 24 hours. Pick a single colony and inoculate it into 2216E liquid medium, incubating at 25°C and 150 rpm on a shaker until OD reaches 0.5%. 600≈0.8, centrifuge and discard the supernatant, wash the bacterial cells twice with sterile physiological saline to obtain a *Pseudomonas amyloliquefaciens* suspension. Mix the *Bacillus amyloliquefaciens* suspension and the *Pseudomonas amyloliquefaciens* suspension at a volume ratio of 10:1, add sterile physiological saline to adjust the final concentration, and centrifuge to concentrate to a *Bacillus amyloliquefaciens* concentration of 1×10⁻⁸. 7 CFU / mL, Pseudomonas alterniflora concentration 1×10 6 CFU / mL was stored at 4℃ to obtain concentrated bacterial culture. The LB liquid medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl, with a pH of 7.0. The 2216E liquid medium consisted of 5 g / L peptone, 1 g / L yeast extract, and sterilized by filtration of aged seawater, with a pH of 7.5.

[0015] Further, in step S2, the mass ratio of polyaspartic acid, octyl glycidyl ether, dimethyl sulfoxide and triethylamine is (10-12):(0.16-0.2):(110-115):(0.01-0.02).

[0016] Furthermore, in step S2, the temperature is controlled at 60-65℃ and the stirring speed is 200-300 rpm.

[0017] Further, in step S3, the mass ratio of alginate microsphere solution, modified polyaspartic acid, phosphate buffer and genipin is (1-1.1):(0.5-0.7):(100-110):(0.15-0.18).

[0018] Furthermore, in step S3, the ultrasonic treatment temperature is 4-7℃, the ultrasonic treatment power is 40-50W, the controlled temperature is 30-35℃, and the stirring speed is 200-300rpm.

[0019] According to another aspect of the present invention, a method for preparing the above-mentioned environmentally friendly compound pesticide is provided, comprising the following steps:

[0020] Functionalized microspheres and diatomaceous earth were dry-mixed and ball-milled until the particle size was ≤50μm. Matrine, rotenone, and rhamnolipid were added sequentially, and the mixture was stirred at 35-37℃ for 2-3 hours to obtain material A. Sodium alginate was dissolved in hot water at 50-55℃, and zinc sulfate and hydroxyapatite were added. The mixture was homogenized and emulsified at 5000rpm for 5 minutes to obtain material B. Material A and material B were mixed, dried to constant weight, and passed through a 100-mesh sieve to obtain an environmentally friendly compound pesticide.

[0021] The beneficial effects of this invention are:

[0022] 1. In the technical solution of this invention, functionalized microspheres form a core through alginate-Ca²⁺ cross-linking, and a shell is formed by secondary cross-linking of modified polyaspartic acid with octyl hydrophobic chains and genipin. A dynamic loading system is formed through the ionic cross-linking network of alginate and the positive charge modification of chitosan oligosaccharides. Its internal multi-level porous structure regulates the diffusion path of pesticide molecules through gradient pore size, and combined with the synergistic effect of the hydrophobic-hydrophilic interface, it achieves directional adsorption and long-term sustained release of active ingredients, significantly improving pesticide utilization efficiency and reducing environmental degradation.

[0023] 2. In the technical solution of the present invention, the surface of the microspheres can effectively block the damage of external light, heat and oxidizing factors to the core microorganisms and pesticides, create a stable microenvironment for microorganisms, significantly extend the cell activity preservation period, and delay the oxidative degradation of active ingredients, thus ensuring the long-term stability of pesticides.

[0024] 3. In this invention, natural marine materials such as alginate, sodium alginate, and diatomaceous earth are used as carriers, combined with plant-derived active ingredients and biosurfactants. Matrine acts as a cholinesterase inhibitor, and rotenone acts as a mitochondrial complex I inhibitor, forming a dual mechanism of action while avoiding chemical pesticide residue pollution. The Bacillus amyloliquefaciens and pseudoalternating monoamine bacteria loaded in the functionalized microspheres can target and inhibit soil-borne pathogens while promoting soil microecological balance. The microsphere framework materials and excipients are all biodegradable, naturally decomposing into small molecules in the environment, avoiding secondary pollution. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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.

[0026] Unless otherwise specified, the raw materials used in this invention are all from commercially available conventional products.

[0027] Preparation Example 1

[0028] Functionalized microspheres are prepared by the following steps:

[0029] S1. Dissolve 30g of alginate in 1000g of 0.85% sterile physiological saline, stir until completely dissolved, cool to 30℃, add 20g of chitosan oligosaccharide, 20g of fucoxanthin, and 20g of concentrated bacterial solution, wherein the concentration of Bacillus amyloliquefaciens is 1×10⁻⁶. 7 CFU / mL, the concentration of *Pseudomonas aeruginosa* is 1×10⁻⁶. 6After stirring magnetically at 100 rpm for 30 min at 30 °C, 1000 g of 0.05 M calcium chloride solution was added dropwise at 4 °C for 15 min. After centrifugation, the supernatant was discarded, the solution was washed twice with PBS solution, and then resuspended in a cryoprotectant containing 5% trehalose and 1% glycerol to obtain alginate microsphere solution.

[0030] S2. Add 100g of polyaspartic acid and 1.6g of octyl glycidyl ether to 1100g of dimethyl sulfoxide, add 0.1g of triethylamine, and stir at 200rpm for 24h at 60℃ under nitrogen protection. Pour the reaction solution into anhydrous ethanol, filter to collect the precipitate, and dry to constant weight to obtain modified polyaspartic acid.

[0031] S3. Add 10g of alginate microsphere solution and 5g of modified polyaspartic acid to 1000g of 0.1M phosphate buffer solution with pH 6.5, sonicate at 4℃ for 5min with a sonication power of 40W, add 1.5g of genipin, stir at 200rpm at 30℃ for 4h, collect the precipitate by centrifugation, freeze-dry at 0℃ for 24h to obtain functionalized microspheres.

[0032] Preparation Example 2

[0033] Functionalized microspheres are prepared by the following steps:

[0034] S1. Dissolve 35g of alginate in 1000g of 0.85% sterile physiological saline, stir until completely dissolved, cool to 30℃, add 25g of chitosan oligosaccharide, 23g of fucoxanthin, and 22g of concentrated bacterial solution, wherein the concentration of Bacillus amyloliquefaciens is 1×10⁻⁶. 7 CFU / mL, the concentration of *Pseudomonas aeruginosa* is 1×10⁻⁶. 6 After stirring magnetically at 150 rpm for 45 min at 32 °C, 1050 g of 0.05 M calcium chloride solution was added dropwise at 4 °C for 18 min. After centrifugation, the supernatant was discarded, the solution was washed twice with PBS solution, and then resuspended in a cryoprotectant containing 5% trehalose and 1% glycerol to obtain alginate microsphere solution.

[0035] S2. Add 110g of polyaspartic acid and 1.8g of octyl glycidyl ether to 1120g of dimethyl sulfoxide, add 0.15g of triethylamine, and stir at 250rpm for 36h at 62℃ under nitrogen protection. Pour the reaction solution into anhydrous ethanol, filter to collect the precipitate, and dry to constant weight to obtain modified polyaspartic acid.

[0036] S3. 10.8g of alginate microsphere solution and 6g of modified polyaspartic acid were added to 1050g of 0.1M phosphate buffer solution with a pH of 6.5. The mixture was sonicated at 5℃ for 8min with a sonication power of 45W. 1.7g of genipin was added, and the mixture was stirred at 250rpm at 32℃ for 4h. The precipitate was collected by centrifugation and freeze-dried at 0℃ for 24h to obtain functionalized microspheres.

[0037] Preparation Example 3

[0038] Functionalized microspheres are prepared by the following steps:

[0039] S1. Dissolve 40g of alginate in 1000g of 0.85% sterile physiological saline, stir until completely dissolved, cool to 30℃, add 30g of chitosan oligosaccharide, 25g of fucoxanthin, and 25g of concentrated bacterial solution, wherein the concentration of Bacillus amyloliquefaciens is 1×10⁻⁶. 7 CFU / mL, the concentration of *Pseudomonas aeruginosa* is 1×10⁻⁶. 6 After stirring magnetically at 200 rpm for 60 min at 35 °C, 1100 g of 0.05 M calcium chloride solution was added dropwise at 4 °C for 20 min. After centrifugation, the supernatant was discarded, the solution was washed twice with PBS solution, and then resuspended in a cryoprotectant containing 5% trehalose and 1% glycerol to obtain alginate microsphere solution.

[0040] S2. Add 120g of polyaspartic acid and 2g of octyl glycidyl ether to 1150g of dimethyl sulfoxide, add 0.2g of triethylamine, and stir at 300rpm for 48h at 65℃ under nitrogen protection. Pour the reaction solution into anhydrous ethanol, filter to collect the precipitate, and dry to constant weight to obtain modified polyaspartic acid.

[0041] S3. 11g of alginate microsphere solution and 7g of modified polyaspartic acid were added to 1100g of 0.1M phosphate buffer solution with a pH of 6.5. The mixture was sonicated at 7℃ for 10min with a sonication power of 50W. 1.8g of genipin was added, and the mixture was stirred at 300rpm at 35℃ for 4h. The precipitate was collected by centrifugation and freeze-dried at 0℃ for 24h to obtain functionalized microspheres.

[0042] Example 1

[0043] A method for preparing an environmentally friendly compound pesticide includes the following steps:

[0044] 4.5g of the functionalized microspheres prepared in Example 1 were dry-mixed with 3.5g of diatomaceous earth and ball-milled to a particle size of 50μm. Then, 0.55g of matrine, 0.25g of rotenone, and 0.35g of rhamnolipid were added sequentially and stirred at 35℃ for 2h to obtain material A. 0.12g of sodium alginate was dissolved in 0.18g of hot water at 50℃, and 0.15g of zinc sulfate and 0.3g of hydroxyapatite were added. The mixture was homogenized and emulsified at 5000rpm for 5min to obtain material B. Material A and material B were mixed, dried to constant weight, and passed through a 100-mesh sieve to obtain an environmentally friendly compound pesticide.

[0045] Example 2

[0046] A method for preparing an environmentally friendly compound pesticide includes the following steps:

[0047] 5g of the functionalized microspheres prepared in Example 2 were dry-mixed with 3.2g of diatomaceous earth and ball-milled to a particle size of 50μm. 0.6g of matrine, 0.2g of rotenone, and 0.4g of rhamnolipid were added sequentially, and the mixture was stirred at 36℃ for 2.5h to obtain material A. 0.1g of sodium alginate was dissolved in 0.1g of hot water at 52℃, and 0.18g of zinc sulfate and 0.22g of hydroxyapatite were added. The mixture was homogenized and emulsified at 5000rpm for 5min to obtain material B. Material A and material B were mixed, dried to constant weight, and passed through a 100-mesh sieve to obtain an environmentally friendly compound pesticide.

[0048] Example 3

[0049] A method for preparing an environmentally friendly compound pesticide includes the following steps:

[0050] 5.2g of the functionalized microspheres prepared in Example 3 were dry-mixed with 3.3g of diatomaceous earth and ball-milled to a particle size of 40μm. Then, 0.52g of matrine, 0.28g of rotenone, and 0.32g of rhamnolipid were added sequentially and stirred at 37℃ for 3h to obtain material A. 0.14g of sodium alginate was dissolved in 0.18g of hot water at 55℃, and 0.13g of zinc sulfate and 0.31g of hydroxyapatite were added. The mixture was homogenized and emulsified at 5000rpm for 5min to obtain material B. Material A and material B were mixed, dried to constant weight, and passed through a 100-mesh sieve to obtain an environmentally friendly compound pesticide.

[0051] Comparative Example 1

[0052] The difference between this comparative example and Example 1 is that alginate is used instead of functionalized microspheres; the remaining steps are the same as in Example 1.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 2 is that polyaspartic acid is used instead of functionalized microspheres; the remaining steps are the same as in Example 2.

[0055] Comparative Example 3

[0056] The difference between this comparative example and Example 3 is that chitosan oligosaccharide is used instead of functionalized microspheres; the remaining steps are the same as in Example 3.

[0057] Third-instar diamondback moth larvae were selected, with 30 larvae per group and three replicates. 10 mg pesticide samples from Examples 1-3 and Comparative Examples 1-3 were diluted to 0.1% (w / v) with deionized water. The diluted solution was evenly sprayed into 10 cm diameter insect rearing containers using a sprayer, with 1 mL of pesticide per container. A control group was also included, sprayed with an equal volume of water. The treated insects were placed in an artificial climate chamber at a temperature of 25±1℃, humidity of 70%, and a photoperiod of 16L:8D. Mortality was recorded at 24 h and 48 h after treatment. Complete immobility was considered the mortality criterion. The corrected mortality rate (%) was calculated as: Corrected mortality rate (%) = (Treatment group mortality rate - Control group mortality rate) / (1 - Control group mortality rate) × 100. The results are shown in Table 1.

[0058] Table 1. Corrected mortality rates for Examples 1-3 and Comparative Examples 1-3

[0059]

[0060] Prepare standard methanol solutions of matrine at concentration gradients of 0.1, 0.5, 1.0, 5.0, and 10.0 μg / mL. Take 10 mg of pesticide samples from Examples 1-3 and Comparative Examples 1-3 and add them to 50 mL of phosphate buffer solution with a pH of 6.5. Place the solution in a constant temperature shaker at 37℃ and 100 rpm. Take samples at 0.5 h, 2 h, 6 h, 12 h, 24 h, and 48 h to determine the matrine concentration. Take 1 mL of solution each time, filter it through a 0.22 μm filter membrane, and then add an equal volume of fresh buffer solution. Calculate the cumulative release rate over 48 h. The cumulative release rate over 48 h (%) = Where Ct represents the matrine concentration (μg / mL) measured at each time point (0.5h, 2h, 6h, 12h, 24h, 48h); V t M represents the volume of each sample taken (1 mL); 总 The total loading of matrine in the pesticide sample

[0061] (μg). The results are shown in Table 2:

[0062] Table 2. Cumulative release rates over 48 hours for Examples 1-3 and Comparative Examples 1-3

[0063]

[0064] As shown in Table 1, the 48-hour corrected mortality rate (92.9-93.5%) of Examples 1-3 was significantly higher than that of the comparative examples (47.2-60.3%). The functionalized microspheres, through the cross-linking structure of alginate and modified polyaspartic acid, may form a porous network, effectively loading and protecting active ingredients such as matrine and rotenone, as well as the bacterial culture. The synergistic effect of the bacterial community and the plant-derived pesticide enhances the insecticidal activity. Genipin-crosslinked microspheres may release the drug through pH or enzyme responses, allowing for the slow release of active ingredients within the pest's body and prolonging the duration of action. In contrast, the single materials such as alginate and polyaspartic acid in Comparative Examples 1-3 may lack controlled-release capabilities, leading to sudden drug release or premature degradation.

[0065] As shown in Table 2, the cumulative release rate of the example after 48 hours reached over 95%, while the comparative example only reached 86-90%. The cross-linked structure of the functionalized microspheres balanced drug encapsulation and release rate, avoiding excessive release in the initial stage while ensuring long-term effective concentration. The hydrophobic groups of the modified polyaspartic acid may enhance the hydrophobicity of the microspheres, delay the diffusion of water-soluble drugs, and promote complete release in the later stages.

[0066] In summary, the functionalized microspheres prepared in Examples 1-3 and the process optimizations in Examples 1-3 achieved efficient loading, targeted controlled release, and synergistic effects of active ingredients and bacterial solutions, thereby improving the insecticidal effect and release stability of pesticides.

[0067] In the description of this specification, the terms "preparation example," "example," "various examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that example or preparation example, which are included in at least one example or preparation example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same example or preparation example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more examples or preparation examples.

[0068] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An environmentally friendly compound pesticide based on marine microorganisms, characterized in that, The ingredients, by weight percentage, include: 40-55% functionalized microspheres, 5-6% matrine, 2-3% rotenone, 3-4% rhamnolipid, 1-1.5% sodium alginate, 30-40% diatomaceous earth, 1-2% zinc sulfate, and 2-4% hydroxyapatite, with the balance being water. The functionalized microspheres are prepared by the following steps: S1. Dissolve alginate in physiological saline, stir until completely dissolved, cool to 30°C, add chitosan oligosaccharide, fucoxanthin and concentrated bacterial solution, control the temperature and stir magnetically for 30-60 min, then add calcium chloride solution dropwise at 4°C for 15-20 min, centrifuge, wash with PBS solution, and resuspend in cryoprotectant to obtain alginate microsphere solution. S2. Polyaspartic acid and octyl glycidyl ether were added to dimethyl sulfoxide, and triethylamine was added. The mixture was stirred and reacted under nitrogen protection for 24-48 hours with controlled temperature. The mixture was then filtered and dried to obtain modified polyaspartic acid. S3. Add the alginate microsphere solution and modified polyaspartic acid to phosphate buffer, sonicate for 5-10 min, add genipin, stir and react for 4 h while controlling the temperature, centrifuge to collect the precipitate, freeze dry to obtain functionalized microspheres. The concentrated bacterial solution in step S1 includes Bacillus amyloliquefaciens and Pseudomonas alterniflora.

2. The environmentally friendly compound pesticide based on marine microorganisms according to claim 1, characterized in that, In step S1, the temperature is controlled at 30-35℃ and the magnetic stirring speed is 100-200rpm.

3. The environmentally friendly compound pesticide based on marine microorganisms according to claim 1, characterized in that, In step S1, the mass ratio of alginate, physiological saline, chitosan oligosaccharide, fucoxanthin, concentrated bacterial solution and calcium chloride solution is (3-4):100:(2-3):(0.5-0.6):(2-2.5):(100-110).

4. The environmentally friendly compound pesticide based on marine microorganisms according to claim 1, characterized in that, The concentration of Bacillus amyloliquefaciens was 1×10⁻⁶. 7 CFU / mL, the concentration of *Pseudomonas aeruginosa* is 1×10⁻⁶. 6 CFU / mL.

5. The environmentally friendly compound pesticide based on marine microorganisms according to claim 1, characterized in that, In step S2, the mass ratio of polyaspartic acid, octyl glycidyl ether, dimethyl sulfoxide and triethylamine is (10-12):(0.16-0.2):(110-115):(0.01-0.02).

6. The environmentally friendly compound pesticide based on marine microorganisms according to claim 1, characterized in that, In step S2, the temperature is controlled at 60-65℃ and the stirring speed is 200-300 rpm.

7. The environmentally friendly compound pesticide based on marine microorganisms according to claim 1, characterized in that, In step S3, the mass ratio of alginate microsphere solution, modified polyaspartic acid, phosphate buffer and genipin is (1-1.1):(0.5-0.7):(100-110):(0.15-0.18).

8. The environmentally friendly compound pesticide based on marine microorganisms according to claim 1, characterized in that, In step S3, the ultrasonic treatment temperature is 4-7℃, the ultrasonic treatment power is 40-50W, the temperature is controlled at 30-35℃, and the stirring speed is 200-300rpm.

9. A method for preparing an environmentally friendly compound pesticide based on marine microorganisms as described in any one of claims 1-8, characterized in that, Includes the following steps: Functionalized microspheres and diatomaceous earth were dry-mixed and ball-milled until the particle size was ≤50μm. Matrine, rotenone, and rhamnolipid were added sequentially, and the mixture was stirred at 35-37℃ for 2-3 hours to obtain material A. Sodium alginate was dissolved in hot water at 50-55℃, and zinc sulfate and hydroxyapatite were added. The mixture was homogenized and emulsified to obtain material B. Material A and material B were mixed, dried, and passed through a 100-mesh sieve to obtain an environmentally friendly compound pesticide.

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