A plant source and microbial source pesticide composition for killing apple snails and a preparation method and application thereof

Abstract

This invention provides a plant-derived and microbial-derived pesticide composition for attracting and killing golden apple snails, its preparation method, and its application, belonging to the field of pesticide technology. The pesticide composition of this invention includes: duckweed extract, betaine, plant-derived components, microbial-derived components, and excipients; the plant-derived components consist of catnip extract, pokeweed extract, and areca nut extract, and the microbial-derived component is microencapsulated Bacillus thuringiensis. This invention utilizes the combination of duckweed extract and betaine to actively attract golden apple snails for feeding; the ternary combination of plant-derived components synergistically destroys the snail's mucous membrane and interferes with its physiological metabolism, rapidly killing the golden apple snail; the microbial-derived component colonizes in the snail's intestines, releasing molluscicides, forming an internal and external snail-killing mechanism. Tests show that the pesticide composition of this invention has high safety for zebrafish and significant killing effects on juvenile and adult golden apple snails, exhibiting strong attraction, thorough snail killing, and environmental friendliness, making it suitable for green control of golden apple snails in aquaculture waters.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, and in particular relates to a plant-derived and microbial pesticide composition for attracting and killing golden apple snails, its preparation method and application. Background Technology

[0002] Golden apple snails are a malignant invasive species that pose multiple threats to agriculture, aquaculture, the ecological environment, and public health. This species has a high reproductive capacity and a diverse diet, feeding heavily on aquatic crops such as rice, easily causing crop loss, reduced yields, or even total crop failure. It also competes with native aquatic organisms for resources and disrupts the aquatic ecosystem. Therefore, the control of this species has become a pressing pest problem in agriculture and aquaculture.

[0003] Currently, the control of golden apple snails mainly relies on chemical molluscicides, such as metaldehyde and molluscicides. While these agents have some molluscicidal effect, they have revealed many problems in practical applications. First, long-term use of chemical molluscicides easily leads to drug resistance in golden apple snails, reducing control effectiveness and increasing dosage and control costs. Second, chemical agents are difficult to degrade in the environment, easily remaining in water bodies and soil, causing continuous pollution to farmland ecosystems, and spreading to surrounding waters through runoff, toxic to farmed aquatic organisms such as fish, shrimp, and crabs, and even affecting the quality and safety of aquatic products. In addition, some chemical molluscicides are also toxic to humans and animals, posing safety hazards. To avoid the shortcomings of chemical agents, single-plant or microbial molluscicides have been gradually developed and applied, but these preparations have significant technical shortcomings: poor attraction, making it difficult to actively attract golden apple snails; slow onset of action and incomplete molluscicidal action; no effect on snail eggs; and difficulty in effectively controlling golden apple snail populations when used alone.

[0004] In summary, existing control technologies suffer from the dual problems of chemical residues and resistance, as well as insufficient efficacy of biological agents. Developing an environmentally friendly, highly efficient, rapid, and thorough control product that is also safe for non-target aquatic organisms has become an urgent need for green control of golden apple snails in agricultural planting and breeding industries. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a plant-derived and microbial-derived pesticide composition for attracting and killing golden apple snails, its preparation method and application. The pesticide composition provided by this invention can not only effectively attract and kill golden apple snails and is environmentally friendly, but also has high safety for non-target aquatic organisms.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a plant-derived and microbial pesticide composition for attracting and killing golden apple snails, comprising the following raw materials in parts by weight: 3-8 parts of duckweed extract, 1-3 parts of betaine, 5-15 parts of plant-derived components, 3-5 parts of microbial components, and 50-60 parts of excipients; the plant-derived components are composed of catnip extract, pokeweed extract, and areca nut extract in a mass ratio of (2-5):(1-4):(2-6).

[0007] Preferably, the duckweed extract is prepared by ultrasonic-assisted water extraction and anhydrous ethanol precipitation of duckweed.

[0008] Preferably, the catnip extract is prepared by extracting catnip leaves with a eutectic solvent and purifying them with a macroporous adsorption resin; the eutectic solvent is prepared by mixing choline chloride, citric acid, 1,4-butanediol and water.

[0009] Preferably, the pokeweed extract is obtained by anaerobic fermentation of a compound microorganism; the compound microorganism consists of Bifidobacterium adolescentis and Lactobacillus plantarum.

[0010] Preferably, the areca nut extract is obtained by ultrasonic extraction of areca nut leaves with ethanol solution.

[0011] Preferably, the microbial source component is prepared by microencapsulation of Bacillus thuringiensis.

[0012] More preferably, the microencapsulated wall material includes at least one of konjac glucomannan, chitosan, and sodium alginate.

[0013] Preferably, the excipients include at least one of diatomaceous earth, soluble starch, sodium lignosulfonate, and xanthan gum.

[0014] The present invention also provides a method for preparing the above-mentioned plant-derived and microbial pesticide composition, comprising: mixing duckweed extract, betaine, plant-derived components, microbial components and excipients, followed by wet granulation, drying and granulation to obtain the plant-derived and microbial pesticide composition.

[0015] The present invention also provides an application of the above-mentioned plant-derived and microbial-derived pesticide composition in the preparation of products for controlling golden apple snails.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a plant- and microbial pesticide composition for attracting and killing golden apple snails, comprising: an attractant, a plant-derived component, a microbial component, and excipients. The attractant includes duckweed extract and betaine; the plant-derived component includes catnip extract, pokeweed extract, and areca nut extract; and the microbial component is Bacillus thuringiensis. The attractant is a natural attractant that actively attracts golden apple snails; the plant-derived component can damage the mucous membrane on the surface of the golden apple snail, inhibiting mucus secretion and rapidly killing both juvenile and adult snails; Bacillus thuringiensis can colonize and multiply in the intestines, producing molluscicides. This pesticide composition can be mixed with water to form a suspension, which can be sprayed by drones. It has a specific killing effect on golden apple snails and is safe, non-toxic, and harmless to non-target aquatic animals in planting and aquaculture. Detailed Implementation

[0017] This invention provides a plant-derived and microbial pesticide composition for attracting and killing golden apple snails, comprising the following raw materials in parts by weight: 3-8 parts of duckweed extract, 1-3 parts of betaine, 5-15 parts of plant-derived components, 3-5 parts of microbial components, and 50-60 parts of excipients; the plant-derived components are composed of catnip extract, pokeweed extract, and areca nut extract in a mass ratio of (2-5):(1-4):(2-6).

[0018] The duckweed extract of this invention is preferably obtained by ultrasonic-assisted water extraction and anhydrous ethanol precipitation of duckweed. The preferred preparation method of the duckweed extract includes: mixing duckweed powder with deionized water at a material-to-liquid ratio of 1:10-15 g / mL, ultrasonically extracting 1-3 times at a temperature of 50-55℃, a power of 250-350W, and a frequency of 25-30kHz, each time for 30-90 min, centrifuging at 3500-4500 rpm for 15-20 min, combining the supernatants, concentrating the supernatant to 1 / 3-1 / 5 of its original volume, adding anhydrous ethanol until the ethanol volume fraction in the system reaches 90%-95%, stirring for 10 min, allowing it to stand at 4℃ for 12 h, centrifuging at 3000-4000 rpm for 20-30 min, collecting the precipitate, and drying it to a water content of 2wt%-5wt% to obtain the duckweed extract.

[0019] Duckweed is an aquatic plant that the golden apple snail naturally feeds on. The duckweed extract obtained by this invention retains the active ingredients that have a strong attraction to the golden apple snail, and can actively attract the snail.

[0020] The pesticide composition of this invention combines duckweed extract and betaine to form a natural attraction system, exhibiting a significant synergistic effect in attracting insects. Duckweed extract leverages the natural feeding habits of the golden apple snail to provide a specific chemotactic attraction, retaining natural active ingredients that align with its feeding habits, achieving targeted attraction. Betaine, as a natural olfactory and gustatory chemotactic agent, has strong diffusion and rapid onset of action, enhancing the attraction signal from a sensory perspective. The combination of these two substances creates a dual attraction effect, resulting in a complementary effect of rapid attraction and sustained absorption.

[0021] The catnip extract of this invention is preferably obtained by extracting catnip leaves with a eutectic solvent and purifying them with a macroporous adsorption resin. The preferred preparation method of the catnip extract includes: pulverizing catnip leaves and mixing them with a eutectic solvent at a material-to-liquid ratio of 1:12-18 g / mL; ultrasonically extracting the mixture for 30-40 min at a temperature of 55-60℃, a power of 200-300W, and a frequency of 30-40kHz; centrifuging the mixture; adsorbing the supernatant onto an AB-8 type macroporous adsorption resin column; eluting the supernatant with deionized water; eluting the supernatant with a 60%-65% (v / v) ethanol solution; collecting the eluent of the 60%-65% (v / v) ethanol solution; concentrating the eluent; and drying the eluent to a water content of 2wt%-5wt% to obtain the catnip extract. The eutectic solvent is preferably prepared by mixing choline chloride, citric acid, 1,4-butanediol, and water. More preferably, the preparation method of the eutectic solvent includes: mixing choline chloride, citric acid, and 1,4-butanediol in a molar ratio of 1:(0.8-1.2):(0.3-0.5), adding 25%-35% of the total mass of the eutectic solvent to deionized water, and stirring at 60-80°C for 40-60 min to obtain the eutectic solvent. The macroporous adsorption resin is preferably an AB-8 type macroporous adsorption resin column.

[0022] The pokeweed extract of this invention is preferably obtained by anaerobic fermentation using a compound microorganism; the compound microorganism is preferably composed of *Bifidobacterium adolescentis* and *Lactobacillus plantarum*. The *Bifidobacterium adolescentis* is preferably *Bifidobacterium adolescentis* XJ8, sourced from the China Agricultural Microbiological Culture Collection Center, accession number ACCC 60429, and the *Lactobacillus plantarum* is preferably *Lactobacillus plantarum* IFFI 6015, sourced from the China Agricultural Microbiological Culture Collection Center, accession number ACCC 11095. The preferred method for preparing the pokeweed extract includes: mixing pulverized pokeweed root, glucose, soluble starch, and water, sterilizing the mixture, inoculating with the compound microorganism, anaerobic fermenting at 25-30℃ for 3-5 days (replenishing glucose and soluble starch every 15-20 hours), sterilizing, filtering, centrifuging, collecting the supernatant, concentrating, and drying to a water content of 2wt%-5wt% to obtain the pokeweed extract.

[0023] The areca nut extract of this invention is preferably obtained by ultrasonic extraction of areca nut leaves with ethanol solution. The preferred method for preparing the areca nut extract includes: pulverizing areca nut leaves, mixing them with a 50%-60% ethanol solution at a material-to-liquid ratio of 1:10-15 g / mL, ultrasonically extracting 1-3 times at a temperature of 50-60℃, a power of 250-300W, and a frequency of 25-30kHz, each time for 40-60 minutes, centrifuging, concentrating the supernatant, and drying to a water content of 2wt%-5wt% to obtain the areca nut extract.

[0024] The core plant-derived molluscicidal component of this composition is a combination of extracts from Nepeta cataria, Phytolacca acinosa, and Areca catechu. These three ingredients work synergistically, destroying the surface structure and interfering with internal physiology to achieve highly efficient killing of both juvenile and adult Pomacea canaliculata. Furthermore, they form an internal-external linkage molluscicidal mechanism with the microbial components, making them a key carrier for green molluscicidal treatment. Nepeta cataria extract, after extraction and purification using a low-melting-point solvent, has a highly concentrated active ingredient, which can effectively destroy the surface mucosa of Pomacea canaliculata and inhibit mucus secretion, causing the snail to lose its body wall protection and osmotic pressure regulation capabilities. Phytolacca acinosa extract, through anaerobic fermentation with compound microorganisms, combines the natural active ingredients of Phytolacca acinosa with microbial metabolites, which can synergistically destroy the snail's mucosa and penetrate the snail's body to interfere with its normal physiological metabolism, accelerating snail death. Areca catechu extract, after ultrasonic extraction with ethanol, enriches its active ingredients, which, in addition to enhancing the effect of destroying the surface mucosa, can also interfere with the feeding and digestive physiology of Pomacea canaliculata, further improving the killing efficiency. The combination of these three ingredients achieves complementary targets and enhanced molluscicidal effects, solving the problems of incomplete molluscicidal action and slow onset of action of single plant-derived components. The plant-derived ingredients of this invention are natural extracts, which are in line with the concept of green research and development. They are non-toxic to non-target aquatic organisms, can be naturally degraded in the environment, and leave no residual pollution. Furthermore, when combined with microencapsulated Bacillus thuringiensis, they form a dual-action system of "surface killing and intestinal poisoning", which greatly improves the snail-killing efficiency and thoroughness of the overall snail-killing composition and meets the green control needs of golden apple snails.

[0025] The microbial source component of this invention is preferably prepared by microencapsulation of Bacillus thuringiensis. The preferred preparation method of the microbial source component includes: centrifuging the Bacillus thuringiensis fermentation broth at 2000-3000 rpm for 8-12 min, collecting the mixture of bacterial cells and spores, resuspending it in a 3%-5% (w / w) trehalose solution, and adjusting the concentration to (3-6) × 10⁻⁶. 10CFU / mL was used to obtain a bacterial suspension. The bacterial suspension was mixed with the wall material mixture at a volume ratio of 1:(3-5), emulsified at 1300-1700 rpm for 10-15 min, and then added dropwise to a 2%-3% calcium chloride solution. The mixture was allowed to stand and solidify at 25°C for 2-3 h, filtered, and the microcapsule particles were collected and dried to a water content of 4wt%-8wt% to obtain the microbial source component. The microencapsulated wall material preferably includes at least one of konjac glucomannan, chitosan, and sodium alginate. The preparation method of the wall material mixture preferably includes: mixing chitosan with a 1%-1.5% acetic acid solution, adding sodium alginate and konjac glucomannan, stirring, and obtaining the wall material mixture. The wall material mixture contains 0.8%-1.2% chitosan, 3%-3.5% sodium alginate, and 1.7%-2.3% konjac glucomannan.

[0026] Microencapsulated microbial components can maintain the activity of Bacillus thuringiensis spores, preventing them from being inactivated by contact with other components in the composition. At the same time, it improves storage stability and environmental adaptability, ensuring efficacy in field applications. The microcapsule wall material can slowly degrade in the intestine of Pomacea canaliculata, achieving targeted release and colonization of the bacteria. The specific molluscicidal toxins produced during its reproduction can destroy the intestinal tissue and physiological metabolism of Pomacea canaliculata. This, combined with the mucosal destructive effect of plant-derived components, forms an internal and external synergy, improving the thoroughness of molluscicidal action. It can kill both juvenile and adult snails, while avoiding the development of drug resistance in Pomacea canaliculata, making it suitable for long-term control.

[0027] The excipients of this invention include at least one of diatomaceous earth, soluble starch, sodium lignosulfonate, and xanthan gum.

[0028] The excipients of this invention improve the processability, stability, and efficacy of pesticide compositions. Diatomaceous earth serves as a carrier, increasing the dispersibility and adsorption of the formulation; soluble starch acts as a filler, adjusting the formulation ratio and improving its formability; sodium lignosulfonate acts as a dispersant, helping the formulation to disperse uniformly in water, preventing aggregation, and improving efficacy; xanthan gum thickens and stabilizes, maintaining the homogeneity of the suspension system and extending the duration of action. The excipients are compatible with wet granulation processes, ensuring efficient formulation forming, drying, and granulation. All excipients are environmentally friendly, compatible with the natural main components, do not affect molluscicidal activity, are safe for non-target organisms, and pose no residue risk.

[0029] The present invention also provides a method for preparing the above-mentioned plant-derived and microbial pesticide composition, comprising: mixing duckweed extract, betaine, plant-derived components, microbial components and excipients, followed by wet granulation, drying and granulation to obtain the plant-derived and microbial pesticide composition.

[0030] The present invention also provides an application of the above-mentioned plant-derived and microbial-derived pesticide composition in the preparation of products for controlling golden apple snails.

[0031] The application method of the pesticide composition described in this invention for controlling golden apple snails in rice paddies is as follows: Apply the pesticide 3-7 days after rice seedling transplanting, or at the 2-3 leaf stage of seedlings in direct-seeded fields, or during the peak period of golden apple snail infestation. Choose a windless / light wind (wind speed ≤3) and rainless early morning or evening for operation. Dilute the pesticide composition with water 17,000-22,000 times, stir evenly, filter through an 80-mesh filter, and add it to the drone's storage tank. Set the drone's flight altitude to 50-80cm above the rice canopy, flight speed to 4-6m / s, spray width to 3-4m, and application volume to 15-20L per mu (approximately 0.067 hectares). Spray evenly along the rice rows, focusing on areas where golden apple snails congregate, such as around the paddy dikes and water inlets, ensuring that the pesticide solution evenly covers the paddy field surface and the base of the seedlings, avoiding over-spraying and missed areas. Reapplication principle: If heavy rain occurs within 24 hours after application, reapplication is necessary, with the reapplication amount being 50%-60% of the original application weight.

[0032] The method for applying the pesticide composition described in this invention to control golden apple snails in aquaculture water is as follows: Apply the pesticide 7-10 days after pond cleaning and before stocking, or apply a low dose when golden apple snails occur sporadically during the aquaculture period. The best effect is achieved when the water temperature is 18-30℃, avoiding the peak molting period of fish, shrimp, and crabs. Dilute the pesticide composition with water 17,000-22,000 times, stir evenly, filter through a 100-mesh filter, and add it to the drone's pesticide storage tank. Set the drone's flight altitude to 30-50cm above the water surface, flight speed to 3-5m / s, spray width to 2-3m, and application volume to 20-25L per mu (approximately 0.067 hectares). Focus spraying along the edges of the aquaculture pond, in shallow water areas, and in areas with aquatic plants where golden apple snails are concentrated, while evenly distributing the pesticide in deeper water areas.

[0033] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the following embodiments are all conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Bifidobacterium adolescentis XJ8 was obtained from the China Agricultural Microbial Culture Collection Center, with accession number ACCC60429; Lactobacillus plantarum IFFI 6015 was obtained from the China Agricultural Microbial Culture Collection Center, with accession number ACCC11095; Bifidobacterium longum BB536 was obtained from the China Industrial Microbial Culture Collection Center, with strain number CICC24632; and Lactobacillus delbrueckii MSC7 was obtained from the China Industrial Microbial Culture Collection Center, with strain number CICC23485.

[0037] The preparation method of duckweed powder is as follows: after washing fresh duckweed, dry it at 55℃ to a water content of 7wt%, and then crush it through a 100-mesh sieve to obtain duckweed powder. Example 1

[0038] (1) Preparation of duckweed extract Duckweed powder and deionized water were mixed at a material-to-liquid ratio of 1:12 g / mL. The mixture was ultrasonically extracted twice at 52℃, 300W, and 28kHz for 40 min each time. After extraction, the mixture was centrifuged at 4000 rpm for 18 min. The supernatants were combined and concentrated to 1 / 4 of their original volume. Anhydrous ethanol was added until the ethanol volume fraction in the system reached 92%. The mixture was stirred for 10 min and then allowed to stand at 4℃ for 12 h. After centrifugation at 3500 rpm for 25 min, the precipitate was collected and dried to a water content of 2.64 wt% to obtain the duckweed extract.

[0039] (2) Preparation of plant-derived components Catnip extract: Dried catnip leaves were pulverized and mixed with a eutectic solvent at a material-to-liquid ratio of 1:15 g / mL. The mixture was ultrasonically extracted for 35 min at a temperature of 58℃, a power of 250W, and a frequency of 35kHz. After centrifugation, the supernatant was adsorbed onto an AB-8 macroporous adsorption resin column, eluted with deionized water, and then eluted with a 63% ethanol solution. The eluent of the 63% ethanol solution was collected, concentrated, and dried to a water content of 3.04 wt% to obtain catnip extract. The method for preparing the eutectic solvent is as follows: choline chloride, citric acid, and 1,4-butanediol are mixed in a molar ratio of 1:1:0.4, and then 30% of the total mass of the eutectic solvent is added to deionized water. The mixture is stirred at 70°C for 50 minutes to obtain the eutectic solvent. Phytolacca extract: Powdered phytolacca root, glucose, soluble starch, and water were mixed in a weight ratio of 100:4:3:1000 and sterilized. The mixture was then inoculated with a compound microbial system (composed of *Bifidobacterium adolescentis* XJ8 and *Lactobacillus plantarum* IFFI 6015; the concentration of *Bifidobacterium adolescentis* XJ8 in the fermentation system was 2 × 10⁻⁶). 7 CFU / mL, the concentration of Lactobacillus plantarum IFFI 6015 is 2×10⁻⁶.7 The mixture was anaerobic fermented at 28°C for 4 days (with glucose and soluble starch added every 18 hours, each time 4g of glucose and 3g of soluble starch were added per kilogram of fermentation system), sterilized, filtered, centrifuged, the supernatant was collected, concentrated, and dried to a water content of 3.27wt% to obtain the pokeweed extract. Areca nut extract: The areca nut leaves were crushed and mixed with 55% ethanol solution at a ratio of 1:12 g / mL. The mixture was ultrasonically extracted twice at a temperature of 55℃, a power of 280W, and a frequency of 28kHz, for 50 min each time. After centrifugation, the supernatant was concentrated and dried to a water content of 3.52 wt% to obtain the areca nut extract. The plant-derived components were obtained by mixing catnip extract, pokeweed extract and areca nut extract in a mass ratio of 3:2:4.

[0040] (3) Preparation of microbial-derived components The Bacillus thuringiensis fermentation broth was centrifuged at 2500 rpm for 10 min, the precipitate was collected, resuspended in a 4% (w / w) trehalose solution, and the bacterial concentration was adjusted to 5 × 10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension. The bacterial suspension was mixed with the wall material mixture at a volume ratio of 1:4. After emulsification at 1500 rpm for 12 min, it was added dropwise to a 2.5% calcium chloride solution. The mixture was allowed to stand and solidify at 25℃ for 2.5 h. After filtration, the microcapsule particles were collected and dried to a water content of 4.29 wt% to obtain the microbial source component. The preparation method of the wall material mixture is as follows: chitosan is mixed with acetic acid solution with a weight content of 1.2%, sodium alginate and konjac glucomannan are added, and the mixture is stirred to obtain the wall material mixture; the mass fraction of chitosan in the wall material mixture is 1%, the mass fraction of sodium alginate is 3.2%, and the mass fraction of konjac glucomannan is 2%.

[0041] (4) Weighing Accurately weigh the following components according to their weight percentages: 5 parts duckweed extract, 2 parts betaine, 10 parts plant-derived components, 4 parts microbial-derived components, 42 parts diatomaceous earth, 6 parts soluble starch, 4 parts sodium lignosulfonate, and 3 parts xanthan gum.

[0042] (5) Preparation of pesticide compositions Duckweed extract, betaine, plant-derived components, and microbial-derived components were mixed at 120 rpm for 18 min to obtain an active mixed powder. The mixed powder, diatomaceous earth, soluble starch, sodium lignosulfonate, and xanthan gum were mixed at 100 rpm for 22 min to obtain a total mixed powder. 28% of the total mixed powder mass of deionized water was added to the total mixed powder, and the mixture was stirred at 80 rpm for 12 min. The mixture was then extruded and granulated, and hot-air dried at a particle thickness of 2 ± 0.2 cm, a temperature of 50 °C, and an air velocity of 1 m / s until the particle moisture content was 2.73 wt%. The granules were then sized to obtain a plant-derived and microbial-derived pesticide composition. Example 2

[0043] (1) Preparation of duckweed extract Duckweed powder and deionized water were mixed at a material-to-liquid ratio of 1:10 g / mL. The mixture was ultrasonically extracted three times at 50℃, 250W, and 25kHz for 30 min each time. After centrifugation at 3500 rpm for 20 min, the supernatants were combined and concentrated to 1 / 3 of their original volume. Anhydrous ethanol was added until the ethanol volume fraction in the system reached 90%. The mixture was stirred for 10 min and then allowed to stand at 4℃ for 12 h. After centrifugation at 3000 rpm for 30 min, the precipitate was collected and dried to a water content of 3.28 wt% to obtain the duckweed extract.

[0044] (2) Preparation of plant-derived components Catnip extract: Dried catnip leaves were pulverized and mixed with a eutectic solvent at a material-to-liquid ratio of 1:12 g / mL. The mixture was ultrasonically extracted at 55℃, 200W, and 30kHz for 40 min. After centrifugation, the supernatant was adsorbed onto an AB-8 macroporous adsorption resin column, eluted with deionized water, and then eluted with a 60% ethanol solution. The eluent of the 60% ethanol solution was collected, concentrated, and dried to a water content of 2.73 wt% to obtain catnip extract. The method for preparing the eutectic solvent is as follows: choline chloride, citric acid, and 1,4-butanediol are mixed in a molar ratio of 1:0.8:0.5, and then 25% of the total mass of the eutectic solvent is added to deionized water. The mixture is stirred at 60°C for 60 min to obtain the eutectic solvent. Phytolacca extract: Powdered phytolacca root, glucose, soluble starch, and water were mixed in a weight ratio of 100:3:2:1000 and sterilized. The mixture was then inoculated with a compound microbial system (composed of *Bifidobacterium adolescentis* XJ8 and *Lactobacillus plantarum* IFFI 6015; the concentration of *Bifidobacterium adolescentis* XJ8 in the fermentation system was 1.5 × 10⁻⁶). 7 The concentration of Lactobacillus plantarum IFFI 6015 was 1.5 × 10 CFU / mL. 7The mixture was anaerobic fermented at 25°C for 5 days (with glucose and soluble starch added every 20 hours, each time 3g of glucose and 2g of soluble starch were added per kilogram of fermentation system), sterilized, filtered, centrifuged, the supernatant was collected, concentrated, and dried to a water content of 2.93wt% to obtain the pokeweed extract. Areca nut extract: The areca nut leaves were crushed and mixed with 50% ethanol solution at a ratio of 1:10 g / mL. The mixture was ultrasonically extracted three times at a temperature of 50℃, a power of 250W, and a frequency of 25kHz, for 40 minutes each time. After centrifugation, the supernatant was concentrated and dried to a water content of 3.71 wt% to obtain the areca nut extract. The plant-derived components were obtained by mixing catnip extract, pokeweed extract and areca nut extract in a mass ratio of 2:1:2.

[0045] (3) Preparation of microbial-derived components The Bacillus thuringiensis fermentation broth was centrifuged at 2000 rpm for 12 min, the precipitate was collected, resuspended in a 3% (w / w) trehalose solution, and the bacterial concentration was adjusted to 3 × 10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension. The bacterial suspension was mixed with the wall material mixture at a volume ratio of 1:3. After emulsification at 1300 rpm for 15 min, it was added dropwise to a 2% (w / w) calcium chloride solution. The mixture was allowed to stand and solidify at 25°C for 2 h. After filtration, the microcapsule particles were collected and dried to a water content of 5.63 wt% to obtain the microbial source component. The preparation method of the wall material mixture is as follows: chitosan is mixed with acetic acid solution with a percentage content of 1%, sodium alginate and konjac glucomannan are added, and the mixture is stirred to obtain the wall material mixture; the mass fraction of chitosan in the wall material mixture is 0.8%, the mass fraction of sodium alginate is 3%, and the mass fraction of konjac glucomannan is 1.7%.

[0046] (4) Weighing Accurately weigh the following components according to their weight proportions: 3 parts duckweed extract, 1 part betaine, 5 parts plant-derived components, 3 parts microbial-derived components, 40 parts diatomaceous earth, 5 parts soluble starch, 3 parts sodium lignosulfonate, and 2 parts xanthan gum.

[0047] (5) Preparation of pesticide compositions Duckweed extract, betaine, plant-derived components, and microbial-derived components were mixed at 100 rpm for 20 min to obtain an active mixed powder. The mixed powder, diatomaceous earth, soluble starch, sodium lignosulfonate, and xanthan gum were mixed at 80 rpm for 25 min to obtain a total mixed powder. 25% of the total mixed powder mass of deionized water was added to the total mixed powder, and the mixture was stirred at 100 rpm for 10 min. The mixture was then extruded and granulated, and hot-air dried at a particle thickness of 1.7 ± 0.2 cm, a temperature of 45 °C, and an air velocity of 0.8 m / s until the particle moisture content was 3.46 wt%. The granules were then sized to obtain a plant-derived and microbial-derived pesticide composition. Example 3

[0048] (1) Preparation of duckweed extract Duckweed powder and deionized water were mixed at a material-to-liquid ratio of 1:15 g / mL and ultrasonically extracted for 90 min at 55℃, 350 W, and 30 kHz. The mixture was then centrifuged at 4500 rpm for 15 min. The supernatants were combined and concentrated to 1 / 5 of their original volume. Anhydrous ethanol was added until the ethanol volume fraction in the system reached 95%. The mixture was stirred for 10 min and then allowed to stand at 4℃ for 12 h. The mixture was then centrifuged at 4000 r / min for 20 min, the precipitate was collected, and dried to a water content of 2.53 wt% to obtain the duckweed extract.

[0049] (2) Preparation of plant-derived components Catnip extract: Dried catnip leaves were pulverized and mixed with a eutectic solvent at a material-to-liquid ratio of 1:18 g / mL. The mixture was ultrasonically extracted for 30 min at 60℃, 300W, and 40kHz. After centrifugation, the supernatant was adsorbed onto an AB-8 macroporous adsorption resin column, eluted with deionized water, and then eluted with a 65% ethanol solution. The eluent of the 65% ethanol solution was collected, concentrated, and dried to a water content of 2.50 wt% to obtain catnip extract. The method for preparing the eutectic solvent is as follows: choline chloride, citric acid, and 1,4-butanediol are mixed in a molar ratio of 1:1.2:0.3, and then 35% of the total mass of the eutectic solvent is added to deionized water. The mixture is stirred at 80°C for 40 minutes to obtain the eutectic solvent. Phytolacca extract: Powdered phytolacca root, glucose, soluble starch, and water were mixed in a weight ratio of 100:5:4:1000 and sterilized. The mixture was then inoculated with a compound microbial system (composed of *Bifidobacterium adolescentis* XJ8 and *Lactobacillus plantarum* IFFI 6015; the concentration of *Bifidobacterium adolescentis* XJ8 in the fermentation system was 3 × 10⁻⁶). 7 CFU / mL, the concentration of Lactobacillus plantarum IFFI 6015 is 3×10⁻⁶. 7The mixture was anaerobic fermented at 30°C for 3 days (with glucose and soluble starch added every 15 hours, each time 5g of glucose and 4g of soluble starch were added per kilogram of fermentation system), sterilized, filtered, centrifuged, the supernatant was collected, concentrated, and dried to a water content of 2.77wt% to obtain the pokeweed extract. Areca nut extract: Areca nut leaves were crushed and mixed with 60% ethanol solution at a material-liquid ratio of 1:15 g / mL. The mixture was ultrasonically extracted at 60℃, 300W, and 30kHz for 60 min. After centrifugation, the supernatant was concentrated and dried to a water content of 3.25 wt% to obtain areca nut extract. The plant-derived components were obtained by mixing catnip extract, pokeweed extract and areca nut extract in a mass ratio of 5:4:6.

[0050] (3) Preparation of microbial-derived components The Bacillus thuringiensis fermentation broth was centrifuged at 3000 rpm for 8 min, the precipitate was collected, resuspended in 5% (w / w) trehalose solution, and the bacterial concentration was adjusted to 6 × 10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension. The bacterial suspension was mixed with the wall material mixture at a volume ratio of 1:5. After emulsification at 1700 rpm for 10 min, it was added dropwise to a 3% calcium chloride solution. The mixture was allowed to stand and solidify at 25℃ for 3 h. After filtration, the microcapsule particles were collected and dried to a water content of 4.74 wt% to obtain the microbial source component. The preparation method of the wall material mixture is as follows: chitosan is mixed with acetic acid solution with a percentage content of 1.5%, sodium alginate and konjac glucomannan are added, and the mixture is stirred to obtain the wall material mixture; the mass fraction of chitosan in the wall material mixture is 1.2%, the mass fraction of sodium alginate is 3.5%, and the mass fraction of konjac glucomannan is 2.3%.

[0051] (4) Weighing Accurately weigh the following components according to their weight percentages: 8 parts duckweed extract, 3 parts betaine, 15 parts plant-derived components, 5 parts microbial-derived components, 43 parts diatomaceous earth, 8 parts soluble starch, 5 parts sodium lignosulfonate, and 4 parts xanthan gum.

[0052] (5) Preparation of pesticide compositions Duckweed extract, betaine, plant-derived components, and microbial-derived components were mixed at 150 rpm for 15 min to obtain an active mixed powder. The mixed powder, diatomaceous earth, soluble starch, sodium lignosulfonate, and xanthan gum were mixed at 120 rpm for 20 min to obtain a total mixed powder. 30% of the total mixed powder mass of deionized water was added to the total mixed powder, and the mixture was stirred at 70 rpm for 15 min. The mixture was then extruded and granulated, and hot-air dried at a particle thickness of 2.3 ± 0.2 cm, a temperature of 52 °C, and an air velocity of 1.2 m / s until the particle moisture content was 2.38 wt%. The granules were then sized to obtain a plant-derived and microbial-derived pesticide composition.

[0053] Comparative Example 1 The difference between this comparative example and Example 1 is that the preparation of duckweed extract in step (1) is as follows: duckweed powder and deionized water are mixed at a ratio of 1:12 g / mL, and ultrasonically extracted twice at a temperature of 52℃, a power of 300W, and a frequency of 28kHz, for 40 min each time. After centrifugation at 4000 rpm for 18 min, the supernatants are combined, concentrated, and dried to a water content of 2.28 wt% to obtain duckweed extract.

[0054] Comparative Example 2 The difference between this comparative example and Example 1 is that the plant-derived component in step (2) is only catnip extract.

[0055] Comparative Example 3 The difference between this comparative example and Example 1 is that the plant-derived component in step (2) is only the extract of Phytolacca acinosa.

[0056] Comparative Example 4 The difference between this comparative example and Example 1 is that the plant-derived component in step (2) is only areca nut extract.

[0057] Comparative Example 5 The difference between this comparative example and Example 1 is that the plant-derived components in step (2) consist of pokeweed extract and areca nut extract in a mass ratio of 2:4.

[0058] Comparative Example 6 The difference between this comparative example and Example 1 is that the plant-derived components in step (2) consist of catnip extract and areca nut extract in a mass ratio of 3:4.

[0059] Comparative Example 7 The difference between this comparative example and Example 1 is that the plant-derived components in step (2) consist of Nepeta cataria extract and Phytolacca extract in a mass ratio of 3:2.

[0060] Comparative Example 8 The difference between this comparative example and Example 1 is that the preparation method of the catnip extract in step (2) is as follows: the dried catnip leaves are crushed and mixed with a 63% ethanol solution at a material-to-liquid ratio of 1:15 g / mL. The mixture is then ultrasonically extracted for 35 min at a temperature of 58℃, a power of 250W, and a frequency of 35kHz. After centrifugation, the supernatant is concentrated and dried to a water content of 2.95 wt% to obtain the catnip extract. Comparative Example 9 The difference between this comparative example and Example 1 is that the complex microorganisms in the pokeweed extract in step (2) consist of Bifidobacterium longum BB536 and Lactobacillus delbrueckii MSC7, and the concentration of Bifidobacterium longum BB536 in the fermentation system is 2×10⁻⁶. 7 The concentration of Lactobacillus delbrueckii MSC7 was 2 × 10 CFU / mL. 7 (CFU / mL), other conditions remained unchanged.

[0061] Comparative Example 10 The difference between this comparative example and Example 1 is that the preparation method of the microbial source component in step (3) is as follows: the Bacillus thuringiensis fermentation broth is centrifuged at 2500 rpm for 10 min, the precipitate is collected, and it is resuspended in a 4% (w / w) trehalose solution. The bacterial concentration is then adjusted to 5 × 10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension, which was dried to a water content of 4.29 wt% to obtain the microbial-derived components.

[0062] Experimental Example 1 Safety testing 1. Experimental Materials 1.1 Test organisms Zebrafish (AB strain): 2 months after hatching, body length 2.5±0.3cm, healthy and disease-free, domesticated in the laboratory for 7 days, survival rate ≥95% during domestication.

[0063] Test samples: pesticide compositions prepared in Examples 1 and Comparative Examples 1-10, Positive 1 (30% tea saponin aqueous solution, Wuhan Guokege Green Ecological Technology Co., Ltd.), Positive 2 (siloxane ethanolamine salt wettable powder, Shanghai Hulian Biopharmaceutical (Xiayi) Co., Ltd.).

[0064] 1.2 Water used in experiments Deionized water aerated for more than 24 hours, with the following water quality indicators: pH 7.0±0.5, dissolved oxygen ≥6mg / L, hardness 100±20mg / L CaCO3, and water temperature controlled at 25±1℃.

[0065] 2. Pesticide solution preparation The samples were diluted with water by 0.5,000 times, 10,000 times, 20,000 times and 40,000 times respectively, stirred evenly and then allowed to stand for 10 minutes.

[0066] The dilution water was used as a blank control group.

[0067] All medicines are prepared and used immediately.

[0068] 3. Experimental Procedure 3.1 Experimental Environment Temperature 25±1℃, light cycle 12L:12D (light intensity 1000-1500lx), no feeding throughout, 4 replicates per group, 10 zebrafish per replicate.

[0069] 3.2 Zebrafish Exposure Treatment Experimental container preparation: Add 800 mL of the corresponding concentration of drug solution / water to each glass, and label the group and concentration; Zebrafish release: The domesticated zebrafish were randomly divided into groups of 10 each and slowly released into beakers (to avoid stress). The release time was recorded as 0h. Exposure condition control: slight aeration throughout the process (fine bubbles, without impacting the zebrafish) to ensure dissolved oxygen ≥6mg / L; replace 50% of the same concentration of fresh medicine solution every 24 hours, avoiding water temperature fluctuations >1℃ during replacement; record water temperature, pH, and dissolved oxygen regularly every day to ensure water quality stability.

[0070] 4. Observation and Recording The mortality rate of zebrafish was observed and recorded 96 hours after release.

[0071] Mortality criteria: The zebrafish stops swimming, the gill covers stop opening and closing, and there is no response when the tail is lightly touched with a straw. The dead individuals are immediately removed and the number is recorded. The sum of the number of zebrafish deaths in 4 replicates is counted.

[0072] 5 Results The results of the effects of the drug solutions prepared for each sample on the total number of zebrafish deaths are shown in Table 1.

[0073] Table 1. Total number of zebrafish deaths for each sample. Table 1 uses the number of zebrafish deaths as an indicator to investigate the safety of various pesticide compositions to non-target aquatic organisms at different dilution ratios. There were no deaths in the blank control group, while positive controls 1 and 2 showed significant toxicity. In particular, positive control 2 caused all or a large number of zebrafish to die at all dilution ratios, highlighting the environmental risks of traditional molluscicides.

[0074] In Example 1 and the comparative examples of this invention, no zebrafish mortality was observed at dilution ratios of 10,000 times or higher. Only a small number of deaths occurred at a high concentration of 5,000 times, and the number of deaths was far lower than that of the positive control. This indicates that the plant-derived and microbial pesticide composition has extremely low toxicity to non-target aquatic organisms and excellent environmental compatibility. The small difference in safety between the comparative examples and Example 1 indicates that even with adjustments to the preparation process of each component and changes in the proportion of plant-derived components, the composition still exhibits low toxicity characteristics, meets the safety requirements of green pesticides, and is suitable for the control of golden apple snails in aquatic environments such as rice paddies and aquaculture water bodies.

[0075] Experimental Example 2 Indoor Killing of Golden Apple Snails 1. Experimental Materials 1.1 Test Target Organism Golden apple snails: Select healthy individuals that are of uniform size and have strong activity (3-5cm shell height and 0.5-1.0cm shell height) and have been temporarily housed indoors for more than 7 days. During the temporary housing period, feed them duckweed and green vegetable leaves, change 1 / 3 of the water daily, and maintain the water temperature at 25±2℃. Stop feeding 24 hours before the experiment and select undamaged individuals that are actively crawling as test snails.

[0076] 1.2 Test Sample Example 1: Pesticide compositions prepared in Comparative Examples 1-10.

[0077] 1.3 Water used in experiments Deionized water aerated for more than 24 hours, with the following water quality indicators: pH 7.0±0.5, dissolved oxygen ≥6mg / L, hardness 100±20mg / L CaCO3, and water temperature controlled at 25±1℃.

[0078] 2. Sample drug solution preparation The pesticide compositions prepared in Example 1 and Comparative Examples 1-10 were diluted 20,000 times with water, stirred evenly, and allowed to stand for 10 minutes to obtain sample solutions.

[0079] The dilution water was used as a blank control group.

[0080] All medicines are prepared and used immediately.

[0081] 3. Experimental Grouping and Setup Grouping principle: Divide into adult snail group and juvenile snail group, with 4 parallel repetitions in each group, and 10 golden apple snails in each repetition; Add liquid to the container: Add 4L of water and 0.2mL of sample solution diluted 20,000 times to each 5L glass square tank (size 20cm×15cm×18cm), put in an aeration stone for slight aeration (to ensure dissolved oxygen ≥6mg / L, and the bubbles are fine and do not impact the snail body), and let stand for 15min to stabilize the solution. Snail release: Using sterile tweezers, slowly place the selected golden apple snails into the experimental container, allowing the snails to sink naturally to the bottom of the water. Record the release time (marked as 0h). After release, seal the container with sterile gauze to prevent the golden apple snails from escaping.

[0082] Ambient temperature: maintained at 25±1℃ throughout the process; Lighting conditions: A 12L:12D light cycle (light intensity 1000-1500 lx) was used to simulate the natural aquatic environment; Water quality maintenance: Do not change the water during the experiment. Monitor the water temperature, pH and dissolved oxygen daily. If the dissolved oxygen is <5mg / L, increase the aeration rate appropriately. If the water becomes turbid, remove the parallel group and repeat the experiment. 4. Observation and Recording The number of dead golden apple snails was observed and recorded 48 hours after release.

[0083] Mortality determination: When the golden apple snail's shell is turned outwards and its soft body is exposed, and the snail's flesh and foot are gently touched with sterile forceps without any contraction or movement, and the snail remains motionless for 48 hours without any signs of life, it is considered dead. The dead individuals are immediately removed with sterile forceps, the number of deaths is recorded, and the mortality rate is calculated. The total number of deaths is the sum of the deaths in four parallel trials. Mortality rate (%) = (Dead golden apple snails / Total number of snails released) × 100%.

[0084] 5 Results The results of the total number of deaths and mortality rates of juvenile and adult snails after the preparation of the drug solution for each sample are shown in Table 2.

[0085] Table 2. Mortality rate of golden apple snails in each sample (%) Table 2 shows that no deaths occurred in the blank control group, verifying the effectiveness of the experimental system and excluding the influence of environmental factors on the survival of golden apple snails. Example 1 achieved a 100% kill rate for juvenile golden apple snails and a 95% kill rate for adult snails, demonstrating the highly efficient molluscicidal effect of the pesticide composition and preparation process of this invention.

[0086] The kill rates of single plant-derived components in Comparative Examples 2-4 were significantly low, and the effects of binary compound components in Comparative Examples 5-7 were still not optimal. This demonstrates that the ternary compound of extracts from Nepeta cataria, Phytolacca acinosa, and Areca catechu is key to achieving complementary target sites and enhanced molluscicidal efficacy, solving the problem of incomplete molluscicidal action by single plant-derived components. In Comparative Example 10, the kill rate decreased significantly due to the lack of microencapsulation of Bacillus thuringiensis. This is because the unencapsulated Bacillus thuringiensis interacted directly with the plant-derived components during preparation, leading to a reduction in the activity of both the microbial and plant-derived components. In Comparative Examples 1, 8, and 9, the molluscicidal effect slightly decreased due to changes in the purification process of the duckweed extract, the solvent for Nepeta cataria extraction, and the fermentation microbial strain of Phytolacca acinosa. This indicates that the extraction process and strain type selected in this invention maximize the preservation of active ingredient efficacy, which is an important condition for ensuring molluscicidal effect. The above data show that the pesticide composition of this invention, through the scientific compounding and process optimization of each component, achieves highly efficient killing of juvenile and adult Pomacea canaliculata, completely solving the technical shortcomings of insufficient efficacy of traditional single biological agents.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A plant-derived and microbially-derived pesticide composition for luring and killing Pomacea canaliculata, characterized by comprising: (a) a plant-derived pesticide; and (b) a microbially-derived pesticide. The ingredients include the following parts by weight: 3-8 parts duckweed extract, 1-3 parts betaine, 5-15 parts plant-derived ingredients, 3-5 parts microbial-derived ingredients, and 50-60 parts excipients. The plant-derived components consist of catnip extract, pokeweed extract and areca nut extract in a mass ratio of (2-5):(1-4):(2-6).

2. The pesticidal composition of plant and microbial origin according to claim 1, characterized in that, The duckweed extract was prepared by ultrasonic-assisted water extraction and anhydrous ethanol precipitation of duckweed.

3. The pesticidal composition of plant and microbial origin according to claim 1, characterized in that, The extract of catnip was prepared by extracting catnip leaves with a eutectic solvent and purifying them with a macroporous adsorption resin; the eutectic solvent was prepared by mixing choline chloride, citric acid, 1,4-butanediol and water.

4. The pesticidal composition of plant and microbial origin according to claim 3, characterized in that, The pokeweed extract was obtained by anaerobic fermentation of a complex of microorganisms; the complex of microorganisms consisted of Bifidobacterium adolescentis and Lactobacillus plantarum.

5. The pesticidal composition of plant and microbial origin according to claim 1, characterized in that, Areca nut extract is prepared by ultrasonic extraction of areca nut leaves with ethanol solution.

6. The pesticidal composition of plant and microbial origin according to claim 1, characterized in that, The microbial source component was prepared by microencapsulation of Bacillus thuringiensis.

7. The plant-derived and microbial-derived pesticide composition according to claim 6, characterized in that, The microencapsulated wall material includes at least one of konjac glucomannan, chitosan, and sodium alginate.

8. The plant-derived and microbial-derived pesticide composition according to claim 1, characterized in that, The excipients include at least one of diatomaceous earth, soluble starch, sodium lignosulfonate, and xanthan gum.

9. A method for preparing a plant-derived and microbial-derived pesticide composition according to any one of claims 1-8, characterized in that, include: Duckweed extract, betaine, plant-derived ingredients, microbial-derived ingredients, and excipients are mixed, and then wet-granulated, dried, and sized to obtain a combination of plant-derived and microbial-derived pesticides.

10. The use of a plant-derived and microbial pesticide composition according to any one of claims 1-8 in the preparation of a product for controlling golden apple snails.

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