Environment-friendly composite pesticide based on marine microorganisms and preparation method of environment-friendly composite pesticide

Abstract

The invention relates to the technical field of pesticides, in particular to an environment-friendly composite pesticide based on marine microorganisms and a preparation method thereof.The environment-friendly composite pesticide is prepared from, by weight, 40%-52% of functional microspheres, 5%-6% of matrine, 2%-3% of rotenone, 3%-4% of rhamnolipid, 1%-1.5% of sodium alginate, 30%-35% of kieselguhr, 1%-2% of zinc sulfate, 2%-4% of hydroxyapatite and the balance water. Natural marine materials such as algin, sodium alginate and kieselguhr are used as carriers, plant source active ingredients and a biological surfactant are matched, matrine serves as a cholinesterase inhibitor, rotenone serves as a mitochondrial compound I inhibitor, a dual-action mechanism is formed, and meanwhile pollution caused by chemical pesticide residues is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticides, and in particular to an environment-friendly composite pesticide based on marine microorganisms and a preparation method thereof. Background Art

[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 pest resistance. Traditional pesticides are mainly synthesized chemically, and their high residue and difficult degradation characteristics pose a long-term threat to the environment and human health. In recent years, biological pesticides have attracted much attention due to their natural sources, strong targeting, and good environmental compatibility. The marine environment has extreme characteristics such as high salinity, high pressure, and low oxygen, which have promoted microorganisms to evolve metabolic pathways different from those of terrestrial strains and can produce novel antibacterial, insecticidal, or antiviral active substances (such as polyketides, peptides, and terpenoids). Research shows that the secondary metabolites of some marine actinomycetes, bacilli, 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] The invention patent with the publication number CN106577774A discloses a production method of a marine animal-derived pesticide. Using starfish as raw material, an environment-friendly animal-derived pesticide is prepared through gradient alcohol extraction, salting-out precipitation combined with immobilized protease hydrolysis, chromatography purification, and compounding processes. The control effect of this pesticide on pests such as Bradysia odoriphaga and Pieris rapae is equivalent to that of natural pyrethrins. However, the secondary metabolites of marine microorganisms (such as saponins and polyketides) are easily degraded by ultraviolet rays, high temperatures, or pH fluctuations in the field environment, resulting in poor persistence of drug efficacy and the need for frequent application. In addition, the compatibility problem between microbial-derived active ingredients and chemical auxiliaries (such as emulsifiers) is prone to stratification and precipitation, resulting in uneven spraying or clogging of the application equipment. Summary of the Invention

[0004] In order to solve the problems mentioned in the above background art, the present invention provides an environment-friendly composite pesticide based on marine microorganisms and a preparation method thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: An environment-friendly composite pesticide based on marine microorganisms, comprising the following raw materials by weight percentage: 40 - 52% of functionalized microspheres, 5 - 6% of matrine, 2 - 3% of rotenone, 3 - 4% of rhamnolipid, 1 - 1.5% of sodium alginate, 30 - 35% of diatomite, 1 - 2% of zinc sulfate, and 2 - 4% of hydroxyapatite, with the balance being water.

[0006] Further, the functionalized microspheres are prepared through the following steps: S1. Dissolve alginate in 0.85% sterile physiological saline, stir until completely dissolved, cool to 30°C, add chitosan oligosaccharide, fucoxanthin and concentrated bacterial solution, magnetically stir at a controlled temperature for 30 - 60 min, then add 0.05 M calcium chloride solution dropwise at 4°C for 15 - 20 min, centrifuge, discard the supernatant, wash twice with PBS solution, and resuspend in a cryoprotectant solution containing 5% trehalose and 1% glycerol to obtain an alginate microsphere solution; S2. Add polyaspartic acid and octyl glycidyl ether to dimethyl sulfoxide, add triethylamine, under nitrogen protection, stir and react at a controlled temperature for 24 - 48 h, pour the reaction solution into absolute ethanol, filter and collect the precipitate, dry to constant weight to obtain modified polyaspartic acid; S3. Add the alginate microsphere solution and modified polyaspartic acid to 0.1 M phosphate buffer solution with a pH of 6.5, ultrasonically treat for 5 - 10 min, add genipin, stir and react at a controlled temperature for 4 h, then centrifuge to collect the precipitate, and freeze-dry at 0°C for 24 h to obtain functionalized microspheres.

[0007] Further, in step S1, the controlled temperature is 30 - 35°C, and the magnetic stirring speed is 100 - 200 rpm.

[0008] 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).

[0009] Further, the concentrated bacterial solution in step S1 includes Bacillus amyloliquefaciens and Pseudomonas pseudoalteromonas. The concentration of Bacillus amyloliquefaciens is 1×10 7 CFU / mL, and the concentration of Pseudomonas pseudoalteromonas is 1×10 6 CFU / mL.

[0010] The concentrated bacterial solution is prepared by the following steps: Streak the glycerol-preserved strain of Bacillus amyloliquefaciens on an LB solid medium (containing 1.5% agar), culture at 30°C for 24 h, pick a single colony and inoculate it into an LB liquid medium, culture in a shaker at 30°C and 200 rpm until OD 600 ≈0.8, centrifuge and discard the supernatant, wash the bacterial cells twice with sterile physiological saline to obtain a Bacillus amyloliquefaciens bacterial suspension. Streak the glycerol-preserved strain of Pseudomonas pseudoalteromonas on a 2216E marine solid medium (containing 1.5% agar), culture at 25°C for 24 h, pick a single colony and inoculate it into a 2216E liquid medium, culture in a shaker at 25°C and 150 rpm until OD 600≈0.8, centrifuge to discard the supernatant, wash the thallus twice with sterile normal saline to obtain a Pseudoalteromonas suspension. Mix the Bacillus amyloliquefaciens suspension and the Pseudoalteromonas suspension at a volume ratio of 10:1, add sterile normal saline to adjust the final concentration, and centrifuge and concentrate until the concentration of Bacillus amyloliquefaciens is 1×10 7 CFU / mL and the concentration of Pseudoalteromonas is 1×10 6 CFU / mL, and store at 4°C to obtain a concentrated bacterial solution. Among them, the composition of the LB liquid medium is 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl, the pH value is 7.0, and the composition of the 2216E liquid medium is 5 g / L of peptone, 1 g / L of yeast extract, and the filtered and sterilized aged seawater, and the pH value is 7.5. 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).

[0011] Further, in step S2, the temperature is controlled at 60 - 65°C, and the stirring speed is 200 - 300 rpm.

[0012] Further, in step S3, the mass ratio of the alginate microsphere solution, modified polyaspartic acid, phosphate buffer solution and genipin is (1 - 1.1):(0.5 - 0.7):(100 - 110):(0.15 - 0.18).

[0013] Further, in step S3, the temperature of the ultrasonic treatment is 4 - 7°C, the power of the ultrasonic treatment is 40 - 50 W, the temperature is controlled at 30 - 35°C, and the stirring speed is 200 - 300 rpm.

[0014] According to another aspect of the present invention, a preparation method of the above-mentioned environment-friendly composite pesticide is provided, including the following steps: Dry-mix the functionalized microspheres and diatomite, ball-mill to a particle size ≤50 μm, sequentially add matrine, rotenone, and rhamnolipid, and stir at 35 - 37°C for 2 - 3 h to obtain material A. Dissolve sodium alginate in hot water at 50 - 55°C, add zinc sulfate and hydroxyapatite, and homogenize and emulsify at a speed of 5000 rpm for 5 min to obtain material B. Mix material A and material B, dry to a constant weight, and pass through a 100-mesh sieve to obtain the environment-friendly composite pesticide.

[0015] The beneficial effects of the present invention: 1. In the technical solution of the present invention, the functionalized microspheres are formed by crosslinking alginate - Ca²⁺ to form the microsphere core, and the modified polyaspartic acid introducing octyl hydrophobic chains and genipin are crosslinked secondarily to form the shell. Through the ionic crosslinking network of alginate and the positive charge modification of chitosan oligosaccharides, a dynamic loading system is formed. Its internal multi - level pore structure regulates the diffusion path of pesticide molecules through gradient pore sizes, and combines with the synergistic effect of the hydrophobic - hydrophilic interface to achieve the directional adsorption and long - term slow release of active ingredients, significantly improving the utilization efficiency of pesticides and reducing environmental loss.

[0016] 2. In the technical solution of the present invention, the microsphere surface can effectively block the damage of external light, heat, and oxidation factors to the microorganisms and pesticides in the core, create a stable micro - environment for the microorganisms, significantly extend the preservation period of the microbial activity, and at the same time delay the oxidative degradation of the active ingredients, ensuring the long - term stability of pesticides.

[0017] 3. In the technical solution of the present invention, natural marine materials such as alginate, sodium alginate, and diatomite are used as carriers, combined with plant - derived active ingredients and biosurfactants. Among them, matrine is used as a cholinesterase inhibitor, and rotenone is used as a mitochondrial complex I inhibitor, forming a dual - action mechanism while avoiding chemical pesticide residue pollution. The functionalized microspheres loaded with Bacillus amyloliquefaciens and Pseudomonas pseudoalteromonas can target and inhibit soil - borne pathogens, and at the same time promote the balance of soil micro - ecology. The microsphere skeleton materials and excipients all have biodegradable characteristics and are naturally decomposed into small molecules in the environment, avoiding secondary pollution. Detailed implementation mode

[0018] Next, the technical solution of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0019] Unless otherwise specified, the raw materials used in the present invention are all conventional products purchased from the market.

[0020] Preparation Example 1 The functionalized microspheres are prepared through the following steps: S1. Dissolve 30 g of alginate in 1000 g of 0.85% sterile physiological saline, stir until completely dissolved, cool to 30 °C, and add 20 g of chitosan oligosaccharides, 20 g of fucoxanthin, and 20 g of concentrated bacterial solution. Among them, the concentration of Bacillus amyloliquefaciens is 1×10 7 CFU / mL, and the concentration of Pseudomonas pseudoalteromonas is 1×10 6CFU / mL. After magnetic stirring 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, followed by centrifugation. The supernatant was discarded, and the precipitate was washed twice with PBS solution and resuspended in a cryoprotectant solution containing 5% trehalose and 1% glycerol to obtain an alginate microsphere solution; S2. 100 g of polyaspartic acid and 1.6 g of octyl glycidyl ether were added to 1100 g of dimethyl sulfoxide, and 0.1 g of triethylamine was added. Under nitrogen protection, the mixture was stirred and reacted at 60°C at 200 rpm for 24 h. The reaction solution was poured into absolute ethanol, and the precipitate was collected by filtration and dried to constant weight to obtain modified polyaspartic acid; S3. 10 g of the alginate microsphere solution and 5 g of modified polyaspartic acid were added to 1000 g of 0.1 M phosphate buffer solution with a pH value of 6.5, and ultrasonic treatment was carried out at 4°C for 5 min with a power of 40 W. 1.5 g of genipin was added, and the mixture was stirred and reacted at 30°C at 200 rpm for 4 h. Then, the precipitate was collected by centrifugation and freeze-dried at 0°C for 24 h to obtain functionalized microspheres.

[0021] Preparation Example 2 The functionalized microspheres were prepared by the following steps: S1. 35 g of alginate was dissolved in 1000 g of 0.85% sterile physiological saline, stirred until completely dissolved, cooled to 30°C, and 25 g of chitosan oligosaccharide, 23 g of fucoxanthin, and 22 g of concentrated bacterial solution were added. Among them, the concentration of Bacillus amyloliquefaciens was 1×10 7 CFU / mL, and the concentration of Pseudomonas pseudoalteromonas was 1×10 6 CFU / mL. After magnetic stirring 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, followed by centrifugation. The supernatant was discarded, and the precipitate was washed twice with PBS solution and resuspended in a cryoprotectant solution containing 5% trehalose and 1% glycerol to obtain an alginate microsphere solution; S2. 110 g of polyaspartic acid and 1.8 g of octyl glycidyl ether were added to 1120 g of dimethyl sulfoxide, and 0.15 g of triethylamine was added. Under nitrogen protection, the mixture was stirred and reacted at 62°C at 250 rpm for 36 h. The reaction solution was poured into absolute ethanol, and the precipitate was collected by filtration and dried to constant weight to obtain modified polyaspartic acid; S3. 10.8 g of the alginate microsphere solution and 6 g of modified polyaspartic acid were added to 1050 g of 0.1 M phosphate buffer solution with a pH value of 6.5, and ultrasonic treatment was carried out at 5°C for 8 min with a power of 45 W. 1.7 g of genipin was added, and the mixture was stirred and reacted at 32°C at 250 rpm for 4 h. Then, the precipitate was collected by centrifugation and freeze-dried at 0°C for 24 h to obtain functionalized microspheres.

[0022] Preparation Example 3 The functionalized microspheres were prepared through the following steps: S1. Dissolve 40 g of alginate in 1000 g of 0.85% sterile physiological saline, stir until completely dissolved, cool to 30 °C, add 30 g of chitosan oligosaccharide, 25 g of fucoxanthin, and 25 g of concentrated bacterial solution. Among them, the concentration of Bacillus amyloliquefaciens is 1×10 7 CFU / mL, and the concentration of Pseudomonas pseudoalteromonas is 1×10 6 CFU / mL. After magnetic stirring at 35 °C at a speed of 200 rpm for 60 min, add 1100 g of 0.05 M calcium chloride solution dropwise at 4 °C for 20 min, then centrifuge, discard the supernatant, wash twice with PBS solution, and resuspend in a cryoprotectant containing 5% trehalose and 1% glycerol to obtain an alginate microsphere solution; S2. Add 120 g of polyaspartic acid and 2 g of octyl glycidyl ether to 1150 g of dimethyl sulfoxide, add 0.2 g of triethylamine, under nitrogen protection, stir and react at 65 °C at a speed of 300 rpm for 48 h, pour the reaction solution into absolute ethanol, filter and collect the precipitate, and dry to constant weight to obtain modified polyaspartic acid; S3. Add 11 g of alginate microsphere solution and 7 g of modified polyaspartic acid to 1100 g of 0.1 M phosphate buffer solution with a pH value of 6.5, ultrasonically treat at 7 °C for 10 min, the power of ultrasonication is 50 W, add 1.8 g of genipin, stir and react at 35 °C at a speed of 300 rpm for 4 h, then centrifuge to collect the precipitate, and freeze-dry at 0 °C for 24 h to obtain functionalized microspheres.

[0023] Example 1 A preparation method of an environment-friendly composite pesticide includes the following steps: Dry-mix 4.5 g of the functionalized microspheres prepared in Preparation Example 1 and 3.5 g of diatomite, ball-mill to a particle size of 50 μm, successively add 0.55 g of matrine, 0.25 g of rotenone, and 0.35 g of rhamnolipid, stir at 35 °C for 2 h to obtain Material A. Dissolve 0.12 g of sodium alginate in 0.18 g of hot water at 50 °C, add 0.15 g of zinc sulfate and 0.3 g of hydroxyapatite, and homogenize and emulsify at a speed of 5000 rpm for 5 min to obtain Material B. Mix Material A and Material B, dry to constant weight, and pass through a 100-mesh sieve to obtain the environment-friendly composite pesticide.

[0024] Example 2 A preparation method of an environment-friendly composite pesticide includes the following steps: The functionalized microspheres prepared in Preparation Example 2 (5 g) and 3.2 g of diatomite were dry-mixed and ball-milled to a particle size of 50 μm. Then, 0.6 g of matrine, 0.2 g of rotenone, and 0.4 g of rhamnolipid were added in sequence, and the mixture was stirred at 36 °C for 2.5 h to obtain Material A. 0.1 g of sodium alginate was dissolved in 0.1 g of hot water at 52 °C, and 0.18 g of zinc sulfate and 0.22 g of hydroxyapatite were added. The mixture was homogenized and emulsified at a speed of 5000 rpm for 5 min to obtain Material B. Material A and Material B were mixed, dried to a constant weight, and passed through a 100-mesh sieve to obtain the environment-friendly composite pesticide.

[0025] Example 3 A preparation method of an environment-friendly composite pesticide, comprising the following steps: The functionalized microspheres prepared in Preparation Example 3 (5.2 g) and 3.3 g of diatomite were dry-mixed and ball-milled to a particle size of 40 μm. Then, 0.52 g of matrine, 0.28 g of rotenone, and 0.32 g of rhamnolipid were added in sequence, and the mixture was stirred at 37 °C for 3 h to obtain Material A. 0.14 g of sodium alginate was dissolved in 0.18 g of hot water at 55 °C, and 0.13 g of zinc sulfate and 0.31 g of hydroxyapatite were added. The mixture was homogenized and emulsified at a speed of 5000 rpm for 5 min to obtain Material B. Material A and Material B were mixed, dried to a constant weight, and passed through a 100-mesh sieve to obtain the environment-friendly composite pesticide.

[0026] Comparative Example 1 The difference between this comparative example and Example 1 is that alginate was used instead of the functionalized microspheres, and the remaining steps were the same as those in Example 1.

[0027] Comparative Example 2 The difference between this comparative example and Example 2 is that polyaspartic acid was used instead of the functionalized microspheres, and the remaining steps were the same as those in Example 2.

[0028] Comparative Example 3 The difference between this comparative example and Example 3 is that chitosan oligosaccharide was used instead of the functionalized microspheres, and the remaining steps were the same as those in Example 3.

[0029] Third-instar Plutella xylostella larvae were selected, with 30 larvae in each group and 3 groups in total. The 10 mg pesticide samples of Examples 1-3 and Comparative Examples 1-3 were respectively diluted with deionized water to 0.1% (w / v), and the diluted solutions were evenly sprayed with a sprayer into a pest rearing container with a diameter of 10 cm, and the spraying amount per dish was 1 mL. Another control group was set up, and the control group was sprayed with an equal amount of clear water. The treated pests were placed in an artificial climate chamber, where the temperature of the artificial climate chamber was 25 ± 1 °C, the humidity was 70%, and the light cycle was 16L:8D. The number of dead pests was recorded at 24 h and 48 h after treatment. Taking the complete immobility of the insect body as the death standard, the corrected mortality rate (%) was calculated. Corrected mortality rate (%) = (mortality rate of the treatment group - mortality rate of the control group) / (1 - mortality rate of the control group) × 100. The results are shown in Table 1: Table 1. Corrected Mortality Rates of Examples 1-3 and Comparative Examples 1-3

[0030] Prepare matrine standard methanol solutions with concentration gradients of 0.1, 0.5, 1.0, 5.0, and 10.0 μg / mL. Take 10 mg of the pesticide samples of Examples 1-3 and Comparative Examples 1-3 and add them to 50 mL of phosphate buffer with a pH of 6.5. Place them in a constant temperature oscillator at 37°C and 100 rpm. Sample and measure the matrine concentration at 0.5 h, 2 h, 6 h, 12 h, 24 h, and 48 h respectively. Take 1 mL of the solution each time, filter it through a 0.22 μm filter membrane, and then supplement an equal amount of fresh buffer. Calculate the 48 h cumulative release rate. The 48 h cumulative release rate (%) = . Wherein, Ct is the matrine concentration (μg / mL) measured at each time point (0.5 h, 2 h, 6 h, 12 h, 24 h, 48 h); V t is the volume of each sampling (1 mL); M 总 is the total drug loading of matrine in the pesticide sample (μg). The results are shown in Table 2: Table 2. 48 h Cumulative Release Rates of Examples 1-3 and Comparative Examples 1-3

[0031] As can be seen from Table 1, the 48-hour corrected mortality rates of Examples 1-3 (92.9 - 93.5%) are much higher than those of the comparative examples (47.2 - 60.3%). The functionalized microspheres may form a porous network through the cross-linked structure of alginate and modified polyaspartic acid, effectively loading and protecting active ingredients such as matrine and rotenone and the bacterial solution. The synergistic effect between the bacterial community and the plant-derived pesticide enhances the insecticidal activity. The genipin-crosslinked microspheres may release drugs through pH or enzyme response, enabling the active ingredients to be slowly released in the pests and prolonging the action time. However, the single materials such as alginate and polyaspartic acid in Comparative Examples 1-3 may lack controlled release ability, resulting in burst release or premature degradation of the drugs.

[0032] As can be seen from Table 2, the 48-hour cumulative release rates of the examples reach over 95%, while those of the comparative examples are only 86 - 90%. The cross-linked structure of the functionalized microspheres balances the drug encapsulation and release rates, which can not only avoid excessive release in the initial stage but also ensure 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 stage.

[0033] In summary, the optimization of the processes of the functionalized microspheres prepared in Preparation Examples 1-3 and Examples 1-3 realizes the efficient loading, targeted controlled release, and synergistic enhancement of the active ingredients and the bacterial solution, thereby improving the insecticidal effect and release stability of the pesticide.

[0034] In the description of the specification, the descriptions referring to terms such as "preparation example", "embodiment", "each embodiment", etc. mean that the specific features, structures, materials or characteristics described in connection with that embodiment or preparation example are included in at least one embodiment or preparation example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or preparation example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or preparation examples.

[0035] As described above, only the preferred specific embodiments of the present invention are provided, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. An environment-friendly composite pesticide based on marine microorganisms, characterized in that, It comprises the following raw materials by weight percentage: 40-55% of functionalized microspheres, 5-6% of matrine, 2-3% of rotenone, 3-4% of rhamnolipid, 1-1.5% of sodium alginate, 30-40% of diatomite, 1-2% of zinc sulfate, and 2-4% of hydroxyapatite, with the balance being water.

2. The environmentally friendly composite pesticide based on marine microorganisms according to claim 1, wherein The functionalized microspheres are prepared through the following steps: S1. Dissolve sodium alginate in physiological saline, stir until completely dissolved, cool to 30 °C, add chitosan oligosaccharide, fucoxanthin, and concentrated bacterial solution. After magnetically stirring for 30-60 min while controlling the temperature, add calcium chloride solution dropwise at 4 °C for 15-20 min, then centrifuge, wash with PBS solution, and resuspend in cryoprotectant to obtain sodium alginate microsphere solution; S2. Add polyaspartic acid and octyl glycidyl ether to dimethyl sulfoxide, add triethylamine, and under nitrogen protection, stir and react at a controlled temperature for 24-48 h, filter, and dry to obtain modified polyaspartic acid; S3. Add the sodium alginate microsphere solution and modified polyaspartic acid to phosphate buffer, ultrasonically treat for 5-10 min, add genipin, stir and react at a controlled temperature for 4 h, then centrifuge to collect the precipitate and freeze-dry to obtain functionalized microspheres.

3. An environment-friendly composite pesticide based on marine microorganisms according to claim 2, characterized in that, In step S1, the temperature is controlled at 30-35 °C, and the magnetic stirring speed is 100-200 rpm.

4. An environment-friendly composite pesticide based on marine microorganisms according to claim 2, characterized in that, In step S1, the mass ratio of sodium 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).

5. The environmentally friendly composite pesticide based on marine microorganisms according to claim 2, characterized in that, In step S1, the concentrated bacterial liquid includes Bacillus amyloliquefaciens and Pseudoalteromonas, and the concentration of Bacillus amyloliquefaciens is 1×10 7 CFU / mL, and the concentration of Pseudoalteromonas is 1×10 6 CFU / mL.

6. An environment-friendly composite pesticide based on marine microorganisms according to claim 2, 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).

7. An environment-friendly composite pesticide based on marine microorganisms according to claim 2, characterized in that, In step S2, the temperature is controlled at 60-65 °C, and the stirring speed is 200-300 rpm.

8. An environment-friendly composite pesticide based on marine microorganisms according to claim 2, characterized in that, In step S3, the mass ratio of sodium alginate microsphere solution, modified polyaspartic acid, phosphate buffer, and genipin is (1-1.1):(0.5-0.7):(100-110):(0.15-0.18).

9. An environment-friendly composite pesticide based on marine microorganisms according to claim 2, characterized in that, In step S3, the temperature of ultrasonic treatment is 4-7 °C, the power of ultrasonic treatment is 40-50 W, the temperature is controlled at 30-35 °C, and the stirring speed is 200-300 rpm.

10. A preparation method of an environment-friendly composite pesticide based on marine microorganisms as described in any one of claims 1-9, characterized in that, It comprises the following steps: Dry-mix the functionalized microspheres and diatomite, ball-mill until the particle size ≤ 50 μm, sequentially add matrine, rotenone, and rhamnolipid, stir at 35-37 °C for 2-3 h to obtain material A. Dissolve sodium alginate in hot water at 50-55 °C, add zinc sulfate and hydroxyapatite, and homogenize and emulsify to obtain material B. Mix material A and material B, dry, and pass through a 100-mesh sieve to obtain an environment-friendly composite pesticide.

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