Broad-spectrum nematicide compounded with fungal metabolite and nano-carrier and preparation method of broad-spectrum nematicide

Abstract

The invention discloses a broad-spectrum nematicide compounding fungal metabolites and a nano-carrier and a preparation method, and relates to the technical field of insecticides. The broad-spectrum nematicide compounding the fungal metabolite and the nano-carrier is composed of an active component and the nano-carrier, the active component is formed by compounding fermentation broth of paecilomyces lilacinus and pochonia chlamydosporia after extraction, and the nano-carrier is core-shell structure nano-particles encapsulating the nano-carrier. According to the broad-spectrum nematicide containing the composite fungus metabolite and the nano-carrier and the preparation method, the metabolite of paecilomyces lilacinus and the metabolite of pochonia chlamydosporia are scientifically compounded, so that the synergistic effect of active ingredients with different action mechanisms is exerted, the comprehensive poisoning effect on nematodes is remarkably enhanced, the insecticidal spectrum is widened, and the broad-spectrum nematicide has a good application prospect. The chitosan-TPP core and sodium alginate shell double-layer structure provides double protection for active components, and the core ensures high encapsulation efficiency and effective load.

Description

Technical Field

[0001] This invention relates to the field of insecticide technology, and in particular to a broad-spectrum nematicide composed of a compound fungal metabolite and a nanocarrier, and its preparation method. Background Technology

[0002] Currently, nematode control still heavily relies on chemically synthesized nematicides, such as thiazophos and abamectin. However, the long-term use of chemical pesticides has brought about many problems, including pesticide residues, environmental pollution, destruction of soil microbiota, the development of nematode resistance, and potential toxicity to humans and animals.

[0003] Biological control has become a research hotspot due to its environmental friendliness and sustainability. Among them, the use of fungi and their metabolites to control nematodes has shown great potential. Paecilomyces lilacinus and Proconia chlamydosporium are two recognized highly effective biocontrol fungi. However, the direct application of their natural metabolites has obvious drawbacks: (1) poor stability, easily degraded by light, heat and microorganisms; (2) short duration of effectiveness, easily leached or decomposed in the soil, requiring frequent application; (3) difficult formulation processing, poor water solubility or unstable physicochemical properties, affecting their commercial application. Summary of the Invention

[0004] The purpose of this invention is to provide a broad-spectrum nematicide composed of a compound fungal metabolite and a nanocarrier, and a method for its preparation, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a broad-spectrum nematicide composed of a compound fungal metabolite and a nanocarrier, comprising an active ingredient and a nanocarrier. The active ingredient is a compound formed by extracting fermentation broth from Paecilomyces lilacinus and Proconia chlamydosporium. The nanocarrier is a core-shell structured nanoparticle encapsulating a nanocarrier, wherein the core structure is formed by cross-linking chitosan with sodium tripolyphosphate, and the shell structure is a sodium alginate layer.

[0006] Preferably, the total content of lysimachia lilacin A+B in the extract of the Paecilomyces lilacin fermentation broth is not less than 3.0 mg / g, and the Paecilomyces lilacin metabolite extract has a 48-hour lethality of not less than 70% against second-instar larvae of Southern root-knot nematodes at a concentration of 100 μg / mL.

[0007] Preferably, the extract of the *Prconia thunbergii* fermentation broth contains a rotamycin content of not less than 1.0 mg / g.

[0008] Preferably, the mass ratio of the Paecilomyces lilacinus metabolite extract to the Proconia chrysophagus metabolite extract is 1:(1-3).

[0009] Preferably, the mass ratio of chitosan to sodium alginate is (2-4):1.

[0010] Another technical problem to be solved by the present invention is to provide a method for preparing a broad-spectrum nematicide consisting of a composite fungal metabolite and a nanocarrier, comprising the following steps: Step 1: Prepare fermentation broths of Paecilomyces lilacinus and Proconia chrysophagus separately. After centrifugation, extraction and concentration, obtain crude extracts of their respective metabolites. Mix the two crude extracts in a certain proportion and redissolve them in water to obtain a composite fungal metabolite aqueous solution. Step 2: Dissolve chitosan in acetic acid solution to form chitosan solution, dissolve sodium tripolyphosphate in deionized water to form TPP solution, and dissolve sodium alginate in water to form sodium alginate solution; under stirring, mix the aqueous solution of the compound fungal metabolites from Step 1 with the chitosan solution, and slowly add TPP solution dropwise using a syringe pump to form drug-loaded chitosan-TPP nanonuclei, then add sodium alginate solution dropwise, stir for 20-30 minutes, and the reaction forms a nanoparticle suspension, which is then post-treated to obtain the nematicide.

[0011] Preferably, the solvent used for extraction in step one is ethyl acetate.

[0012] Preferably, in step two, the concentration of the chitosan solution is 1.0-2.0 mg / mL, and the concentration of the sodium alginate solution is 0.5-1.0 mg / mL.

[0013] Preferably, the concentration of the TPP solution is 0.2-0.5 mg / mL, and the dropping rate of the TPP solution is 0.3-0.8 mL / min.

[0014] Preferably, the post-processing in step two includes the following steps: centrifugation at 10,000 rpm for 15 min, washing with deionized water 2-3 times, and redispersing in water containing 0.5% trehalose to obtain a stable nano suspension.

[0015] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides a broad-spectrum nematicide based on a composite fungal metabolite and a nanocarrier, along with its preparation method. By scientifically combining metabolites of Paecilomyces lilacinus and Proconia chlamydosporium, the synergistic effect of active ingredients with different mechanisms of action is achieved, enhancing the comprehensive toxicity against nematodes and broadening the insecticidal spectrum. The chitosan-TPP core and sodium alginate shell double-layer structure provide dual protection for the active ingredients. The core ensures high encapsulation efficiency and effective loading, while the shell acts as a controlled-release barrier and stabilizes the core, jointly achieving stable and continuous release of the active ingredients. This effectively overcomes the shortcomings of natural metabolites, such as short duration of action and easy degradation. The active ingredients are derived from natural fungal metabolites, and the carrier material is a biodegradable polysaccharide. The entire system has good environmental compatibility, avoiding chemical pesticide residues and pollution problems. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying tables. 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. Example

[0017] Fermentation of the strains: *Paecilomyces lilacinus* and *Prconia chlamydosporium* were inoculated onto PDA plates and activated at 28°C for 5 days. The mycelial cakes were then picked and inoculated into Erlenmeyer flasks containing 100 mL of PDB liquid medium. The flasks were cultured at 28°C and 180 rpm for 3 days to prepare the seed culture. The seed culture was transferred at a 5% inoculum to a fermenter containing 1 L of optimized fermentation medium. The optimized fermentation medium contained 0.5% colloidal chitin as an inducer. Fermentation was carried out at 28°C, with an aeration rate of 1:1 vvm and a stirring speed of 200 rpm for 6 days.

[0018] Extraction and standardization: The fermentation broth was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected. The supernatant was then extracted three times with an equal volume of ethyl acetate. The organic phases were combined and concentrated under reduced pressure using a rotary evaporator in a 40℃ water bath to obtain crude extracts of Paecilomyces lilacinus metabolites and Procognacia chlamydosporidis metabolites. HPLC analysis showed that the total content of oxytetracycline A+B in the Paecilomyces lilacinus crude extract was 3.5 mg / g, and the content of aureomycin in the Procognacia chlamydosporidis crude extract was 1.3 mg / g.

[0019] Bioassays showed that the extract of Paecilomyces lilacinus had a 48-hour lethality of 78% against second-instar larvae of southern root-knot nematodes at a concentration of 100 μg / mL. Example

[0020] The crude extracts of Paecilomyces lilacinus metabolites and Proconia chrysophagus metabolites from Example 1 were mixed at a mass ratio of 1:2, and an appropriate amount of deionized water was added. The mixture was then sonicated to prepare an aqueous solution of the compound fungal metabolites with a total concentration of 10 mg / mL.

[0021] To prepare the carrier solution: Weigh 20 mg of chitosan (degree of deacetylation ≥90%), dissolve it in 20 mL of 1% (v / v) acetic acid solution, and stir magnetically until completely dissolved to prepare a 1.0 mg / mL solution. Filter the solution through a 0.45 μm filter membrane. Weigh 4 mg of sodium tripolyphosphate (TPP), dissolve it in 20 mL of deionized water to prepare a 0.2 mg / mL TPP solution. Weigh 5 mg of sodium alginate, dissolve it in 10 mL of deionized water to prepare a 0.5 mg / mL sodium alginate solution.

[0022] Formation of drug-loaded nanonuclei: Take 10 mL of the aqueous solution of the composite fungal metabolites prepared in Example 1, mix it with 10 mL of chitosan solution under magnetic stirring at 400 rpm, and slowly add 5 mL of TPP solution to the above mixture at a rate of 0.5 mL / min using a syringe pump. After the addition is completed, the solution will show an opalescent appearance, indicating that the drug-loaded chitosan-TPP nanonuclei have been formed.

[0023] Constructing the sodium alginate shell: Under stirring conditions, 5 mL of sodium alginate solution was added dropwise to the above nanocore suspension at a rate of 1 mL / min using a syringe pump. After the addition was complete, stirring was continued for 25 minutes to allow the sodium alginate to fully coat the surface of the nanocore through electrostatic interaction, forming a complete core-shell structure.

[0024] Post-processing and storage: The suspension after reaction was centrifuged at 10,000 rpm for 15 minutes, the supernatant was carefully discarded, the precipitate was resuspended with deionized water and washed twice to remove unencapsulated free metabolites, the obtained nanoparticle precipitate was redispersed in 10 mL of an aqueous solution containing 0.5% (w / v) trehalose, and vortexed to disperse it evenly, thus obtaining the core-shell structured nano-nematicide of the present invention, which was stored at 4°C in the dark. Example

[0025] In this embodiment, the crude extract of Paecilomyces lilacinus metabolites and the crude extract of Proconia chrysophagus metabolites are in a 1:1 ratio, and the mass ratio of chitosan to sodium alginate is maintained at 3:1. Except for the ratio of active ingredients, the rest is the same as in Example 2. Example

[0026] In this embodiment, the crude extract of Paecilomyces lilacinus metabolites and the crude extract of Proconia chrysophagus metabolites are in a ratio of 1:3, and the mass ratio of chitosan to sodium alginate is maintained at 3:1. Except for the ratio of active ingredients, the rest is the same as in Example 2. Example

[0027] In this embodiment, the crude extract of Paecilomyces lilacinus metabolites and the crude extract of Proconia chrysophagus metabolites are in a ratio of 1:2. The mass ratio of chitosan to sodium alginate is maintained at 1.5:1. The mass ratio is changed by adjusting the concentration and volume of the chitosan solution and the sodium alginate solution. The rest of the contents are the same as in Example 2.

[0028] Comparative Example 1: The mixture of crude extracts of complex fungal metabolites obtained in Example 1 was directly dissolved in water to prepare a solution with the same concentration of active ingredients as the nano-preparation.

[0029] Comparative Example 2: Except for not adding sodium alginate solution, the other steps are exactly the same as in Example 2.

[0030] Comparative Example 3: This comparative example does not use sodium tripolyphosphate solution. Instead, the aqueous solution of the complex fungal metabolites is directly mixed with the chitosan solution, and then sodium alginate solution is added dropwise. Conventional polyelectrolyte complex nanoparticles are formed through the direct electrostatic interaction between chitosan and sodium alginate. The active ingredients, carrier ratio, and other contents are the same as in Example 2.

[0031] The insecticides prepared in Examples 2-5 and Comparative Examples 1-3 were subjected to performance and toxicity tests, and the test methods are as follows: The average particle size was determined using the dynamic light scattering method. The nano suspension was appropriately diluted with ultrapure water to a semi-transparent state, placed in a cuvette, and measured at 25°C using a particle size analyzer such as the Malvern Zetasizer Nano ZS. The result was the average of three measurements. PDI represents the uniformity of particle size distribution. The smaller the PDI, the narrower the distribution and the more uniform the system. Zeta potential represents the net charge on the surface of nanoparticles. It is determined by laser Doppler electrophoresis using the same instrument as DLS, equipped with a folded capillary potential cell. The diluted sample is injected into the cell, and the migration velocity of the particles under the action of the electric field is measured. The Zeta potential is calculated using the Smoluchowski model. Encapsulation efficiency refers to the percentage of encapsulated active ingredient relative to the total dosage. A combination of ultracentrifugation and high-performance liquid chromatography (HPLC) is used. An appropriate amount of nanoparticle suspension is taken and ultracentrifuged (14,000 rpm, 30 min) to precipitate the nanoparticles. The supernatant is carefully collected, and the concentration of unencapsulated free drug is determined using a validated HPLC method. The encapsulation efficiency is calculated as: (Total dosage - Free drug) / Total dosage × 100%.

[0032] In the indoor toxicity assay, the test nematodes were second-instar larvae of *Sinopteryx chinensis*. A 24-well plate immersion method was used, with incubation at 25°C. Nematode mortality was examined after 48 and 72 hours. The 48-hour LC50 (μg / mL) represents the concentration of the agent that caused 50% mortality of the test nematodes after 48 hours of exposure, and the 72-hour LC50 (μg / mL) represents the concentration that caused 50% mortality of the test nematodes after 72 hours of exposure. The lethality rate @100 μg / mL indicates the direct killing effect of the test sample on second-instar larvae of *Sinopteryx chinensis* at a uniform, fixed concentration of 100 μg / mL.

[0033] Soil pot experiment: Tomato seedlings were planted, and each pot was artificially inoculated with 1500 second-instar larvae of the southern root-knot nematode. One week after inoculation, the nano-nematicide of this invention was applied to Examples 2-5 and Comparative Examples 1-3, respectively. The root-knot index and the number of nematodes in the soil were investigated after 60 days.

[0034]

[0035] The table above shows that Example 4 exhibits the best overall performance and can be considered the optimal choice. The overall performance decline in Example 5 demonstrates that an excessively low chitosan ratio can compromise the stability and performance of the particles. In the 48-hour toxicity test, Comparative Example 2 showed a lower LC50 concentration. 50 The difference compared to Example 2 was relatively small, indicating that its initial release and toxicity were acceptable. This is because the exposed chitosan-TPP core had a higher positive charge (+35.8 mV), which may have made it easier to bind to the negatively charged nematode surface. However, in the 72-hour toxicity test, the difference widened. The sodium alginate shell played a crucial role in controlling the release barrier, preventing the sudden release of the drug within the core and achieving a stable and continuous release, thus ensuring long-term efficacy. Simultaneously, the shell also protected the core from rapid destruction in complex soil environments. The efficacy of Example 2 was higher than that of Comparative Example 2. Comparative Example 3 lacked a densely cross-linked core, resulting in a loose carrier structure, low encapsulation rate, and poor efficacy. This indicates that the chitosan-TPP core and the sodium alginate shell form a synergistic organic whole, neither of which can be omitted.

[0036] 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. A broad-spectrum nematicide combining a complex of fungal metabolites and a nanocarrier, characterized in that, Composed of active ingredients and nanocarriers, the active ingredients are extracted from the fermentation broth of Paecilomyces lilacinus and Proconia chlamydosporium and then compounded. The nanocarriers are core-shell structured nanoparticles encapsulating nanocarriers, wherein the core structure is formed by cross-linking chitosan with sodium tripolyphosphate, and the shell structure is a sodium alginate layer.

2. The broad-spectrum nematicide based on a composite fungal metabolite and nanocarrier according to claim 1, characterized in that, The total content of lysimachia lilacin A+B in the extract of the fermentation broth of Paecilomyces lilacinus is not less than 3.0 mg / g, and the 48-hour lethality of Paecilomyces lilacinus metabolite extract against second-instar larvae of southern root-knot nematodes is not less than 70% at a concentration of 100 μg / mL.

3. The broad-spectrum nematicide based on a composite fungal metabolite and nanocarrier according to claim 1, characterized in that, The extract of the fermentation broth of *Prconia thunbergii* contains no less than 1.0 mg / g of aureomycin.

4. The broad-spectrum nematicide based on a composite fungal metabolite and nanocarrier according to claim 1, characterized in that, The mass ratio of the Paecilomyces lilacinus metabolite extract to the Proconia thunbergii metabolite extract is 1:(1-3).

5. The broad-spectrum nematicide based on a composite fungal metabolite and nanocarrier according to claim 1, characterized in that, The mass ratio of chitosan to sodium alginate is (2-4):

1.

6. A method for preparing a broad-spectrum nematicide comprising a compound fungal metabolite and a nanocarrier as described in any one of claims 1-5, characterized in that, Includes the following steps, Step 1: Prepare fermentation broths of Paecilomyces lilacinus and Proconia chrysophagus separately. After centrifugation, extraction and concentration, obtain crude extracts of their respective metabolites. Mix the two crude extracts in a certain proportion and redissolve them in water to obtain a composite fungal metabolite aqueous solution. Step 2: Dissolve chitosan in acetic acid solution to form chitosan solution, dissolve sodium tripolyphosphate in deionized water to form TPP solution, and dissolve sodium alginate in water to form sodium alginate solution; under stirring, mix the aqueous solution of the compound fungal metabolites from Step 1 with the chitosan solution, and slowly add TPP solution dropwise using a syringe pump to form drug-loaded chitosan-TPP nanonuclei, then add sodium alginate solution dropwise, stir for 20-30 minutes, and the reaction forms a nanoparticle suspension, which is then post-treated to obtain the nematicide.

7. The method for preparing a broad-spectrum nematicide based on a composite fungal metabolite and nanocarrier according to claim 6, characterized in that, The solvent used for extraction in step one is ethyl acetate.

8. The method for preparing a broad-spectrum nematicide based on a composite fungal metabolite and a nanocarrier according to claim 6, characterized in that, In step two, the concentration of the chitosan solution is 1.0-2.0 mg / mL, and the concentration of the sodium alginate solution is 0.5-1.0 mg / mL.

9. The method for preparing a broad-spectrum nematicide based on a composite fungal metabolite and a nanocarrier according to claim 6, characterized in that, The concentration of the TPP solution is 0.2-0.5 mg / mL, and the dropping rate of the TPP solution is 0.3-0.8 mL / min.

10. The method for preparing a broad-spectrum nematicide based on a composite fungal metabolite and a nanocarrier according to claim 6, characterized in that, The post-processing in step two The process includes the following steps: centrifugation at 10,000 rpm for 15 minutes, washing with deionized water 2-3 times, and redispersing in water containing 0.5% trehalose to obtain a stable nano-suspension.

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