A microbial insecticide, its preparation method, and its application in controlling pine wilt disease.

Abstract

This invention discloses a microbial insecticide, its preparation method, and its application in controlling pine wilt disease. The insecticide is a compound of *Arthrum sinense* (CGMCC No. 3.6755) and *Bacillus sicca* (CGMCC No. 1.8513), using dendritic mesoporous silica modified with propyl gallate as a carrier, achieving stepwise release through a double-layer coating. This invention achieves targeted delivery to the xylem and sustained stable release, combining rapid nematode killing with long-lasting control. Field efficacy reaches over 75% within 24 months, reducing chemical pesticide usage, with a residual effect exceeding 2 years, and good environmental compatibility.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a microbial insecticide, its preparation method, and its application in the control of pine wilt disease. Background Technology

[0002] Pine wilt disease, known as "pine cancer," is one of the most devastating quarantine diseases in global forest ecosystems, caused by the pine wilt nematode (Pinus wiltus). Bursaphelenchus xylophilus It is caused by the pine sawyer beetle and spreads through its vector insect.

[0003] Currently, the control of pine wilt disease mainly relies on chemical nematicides, such as abamectin, avermectin, and fluopyram. However, chemical agents have the following problems: (1) Long-term and large-scale use leads to drug resistance in pine wilt nematodes, and the control effect declines year by year; (2) The effective period is short, generally only 3-6 months, requiring multiple applications, which increases the control cost and environmental pressure; (3) After trunk injection, the agent is unevenly distributed in the tree and is difficult to reach all nematode parasitic sites.

[0004] Biological control, as an environmentally friendly method of control, has received widespread attention in recent years. Reported biocontrol microorganisms include Bacillus thuringiensis, Paecilomyces lilacinus, Metarhizium anisopliae, and E. elanis nematode fungi. However, existing biological control agents have the following shortcomings: (1) slow nematode killing speed, making it difficult to quickly control the epidemic; (2) poor colonization ability of microorganisms in trees, easily affected by environmental factors, resulting in unstable control effects; (3) lack of an effective delivery system, making it difficult for microbial spores to reach the xylem where nematodes reside.

[0005] To improve control efficacy, existing technologies attempt to combine microorganisms with chemical agents, but simple combinations fail to solve the problems of difficult microbial colonization and short-lasting effects of the agents. Furthermore, existing carriers are mostly spherical microspheres or nanoparticles, which have poor targeting and are difficult to specifically adsorb onto the xylem, leading to significant agent loss.

[0006] Therefore, there is an urgent need to develop a novel microbial insecticide that combines rapid nematode killing with long-term control, synergistic effects of microorganisms and chemical agents, and stable release, in order to meet the pressing needs of pine wilt disease control. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a microbial insecticide, its preparation method, and its application in the control of pine wilt disease. This insecticide utilizes a synergistic microbial combination of *Arthrobacter sinensis* and *Bacillus sicca*, combined with a dendritic mesoporous silica nanocarrier specifically modified with tannic acid-propyl gallate composite lignin. This achieves targeted delivery, intelligent release, and synergistic effect of multiple components, significantly improving the control effect of pine wilt disease, extending the duration of effectiveness, and reducing the amount of chemical agents used and production costs.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A microbial insecticide comprising a core functional microbial composition, a chemical nematicide, and a dendritic mesoporous silica nanocarrier, wherein the core functional microbial composition is derived from Arachnium chinense (Syngonium sinense). Arthrobotrys sinensis ) and Bacillus sicca ( Bacillus siamensis )composition.

[0009] Furthermore, the strain number of *Arthrozoa sinensis* is CGMCC No. 3.6755; and the strain number of *Bacillus sicca* is CGMCC No. 1.8513.

[0010] Furthermore, the original preservation date of the Chinese arthrozoon is November 15, 2004, and the original preservation date of the Siamese Bacillus is September 1, 2008. Both strains can be publicly obtained through the China General Microbiological Culture Collection Center.

[0011] Furthermore, the preparation method of the core functional microbial composition is as follows: inoculate *Arthrum chinense* strain into PDA medium, culture in the dark at 25°C for 7-10 days, collect conidia, and prepare spore powder; then inoculate *Bacillus sicca* strain into LB liquid medium, culture at 37°C and 180 rpm for 24-36 hours, collect spores by centrifugation, and prepare spore powder; the spore powder and spore powder are then freeze-dried under vacuum and stored separately.

[0012] The PDA formula is: 200 g potato, 20 g glucose, 15-20 g agar, 1000 mL water, natural pH value; The LB formula is: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 1000 mL water, natural pH; all culture media should be sterilized before use.

[0013] Furthermore, the spore concentration of the *Arthropoda sinensis* is ≥1.0 × 10⁻⁶. 9 CFU / g, the spore concentration of the *Bacillus sicca* is ≥1.0 × 10⁻⁶. 10 CFU / g.

[0014] Furthermore, the chemical nematicide is emamectin benzoate.

[0015] Furthermore, the preparation method of the dendritic mesoporous silica nanocarrier is as follows: A. Template method for preparing nanoparticles: 1.0 g of hexadecyltrimethylammonium bromide (CTAB) was dissolved in 480 mL of deionized water, 3.5 mL of 25% ammonia solution was added, the pH was adjusted to 11-12, and after stirring evenly, 5 mL of tetraethyl orthosilicate (TEOS) was slowly added dropwise. The mixture was stirred at 30 °C for 24 h. After the reaction was completed, the product was collected by centrifugation, washed three times each with ethanol and deionized water, and calcined at 550 °C for 6 h at a rate of 5 °C / min to remove the template, thus obtaining dendritic mesoporous silica nanoparticles. B. Aminoation modification: 1.0 g of nanoparticles were dispersed in 100 mL of anhydrous ethanol, and 0.5 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 60 °C for 6 h. After centrifugation and washing, the nanoparticles were dried under vacuum to obtain aminoated nanoparticles. C. Surface Modification: 0.5 g of aminated nanoparticles were dispersed in 50 mL of ethanol-water solution (1:1 v / v). 0.1 g of tannic acid, 0.05 g of propyl gallate, and 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added. EDC served as a reaction promoter to improve the covalent grafting efficiency of polyphenols and amino groups. The reaction was carried out at room temperature in the dark with shaking for 8 h. After centrifugation, washing, and vacuum drying, a dendritic mesoporous silica nanocarrier with a polyphenol-modified surface was obtained.

[0016] This invention uses the CTAB soft template method to prepare dendritic mesoporous silica, and the formation process of its unique structure is as follows: 1. Self-assembly of dendritic templates: In an alkaline aqueous solution with pH 11-12, cationic surfactant CTAB molecules self-assemble into rod-shaped primary micelles through hydrophobic interactions; as the system concentration increases, the rod-shaped micelles further aggregate in a radial manner from the center outward to form dendritic precursor templates.

[0017] 2. Silica framework deposition: Tetraethyl orthosilicate (TEOS) is slowly added and hydrolyzes under alkaline conditions to generate negatively charged silicate ions. These ions are adsorbed onto the surface of positively charged CTAB micelles through electrostatic interaction and gradually undergo condensation reaction, depositing along the periphery of the dendritic template to form a silica framework.

[0018] 3. Template removal and pore formation: After calcination at 550℃, the CTAB organic template is completely decomposed and removed, ultimately retaining three-dimensional dendritic mesoporous silica nanoparticles that complement the template structure.

[0019] Following surface modification, the surface polyphenol layer is composed of tannic acid and propyl gallate. The ortho-dihydroxyl groups of tannic acid and propyl gallate are oxidized to ortho-quinone intermediates under weakly alkaline aerobic conditions. The ortho-quinone then undergoes a Michael addition reaction with the primary amino groups on the surface of the aminated nanoparticles to form stable covalent bonds. Stable binding of the polyphenol layer to the carrier is achieved through the ortho-quinone-amino covalent bond. It is further speculated that EDC may interact with the ortho-quinone intermediate or promote its reaction efficiency with the amino group, thereby contributing to an increase in the grafting rate of the polyphenol layer. The polyphenol layer can specifically bind to pine wood phenol through hydrogen bonding, π-π stacking, and hydrophobic interactions, achieving targeted xylem delivery of the carrier. It also exhibits pH-responsive drug release characteristics, accelerating drug release in the slightly acidic environment caused by pine wilt nematode infection.

[0020] The overall morphology of the dendritic mesoporous silica nanocarrier is shown in Figure 2: Scanning electron microscopy (SEM) image (a) shows that the nanoparticles are spherical with a diameter of 100-200 nm and have a large number of radial dendritic protrusions on the surface; transmission electron microscopy (TEM) image (b) clearly shows that there are open, interconnected mesoporous channels extending from the center to the periphery inside the particles. This unique structure not only has a larger specific surface area and higher drug loading capacity, but also provides a smooth diffusion channel for drug molecules, ensuring the slow and sustained release of chemical nematicides.

[0021] A method for preparing a microbial insecticide includes the following steps: (1) Preparation of core functional microbial compositions; (2) Preparation of dendritic mesoporous silica nanocarriers: (3) Drug loading and coating: a. Disperse 20-30g of dendritic mesoporous silica nanocarrier in 200-300mL of deionized water, add 30mL of ethanol solution containing 0.5-1.5g of emamectin benzoate, stir and adsorb at room temperature in the dark for 12h; collect the product by centrifugation at 8000rpm for 10min, wash twice with deionized water, and vacuum dry at 40℃ to obtain drug-loaded nanoparticles; b. Core mixing: Mix 20g of drug-loaded nanoparticles with 10g of Bacillus sicca spore powder evenly to obtain a core mixture; c. Coating: Dissolve 12g of sodium alginate in 1000mL of deionized water, add 8g of Aralia elata spore powder, and stir well; then add the kernel mixture and continue stirring for 30min; drip the mixture into 2000mL of an aqueous solution containing 1% chitosan and 2% calcium chloride through a syringe, and solidify for 30min; collect the microspheres by centrifugation at 8000rpm for 10min, wash twice with deionized water, and freeze-dry to obtain the microbial insecticide.

[0022] The dendritic mesoporous silica nanocarrier has a particle size of 100-200 nm; the loading of emamectin benzoate is 2-6 wt%.

[0023] The application of a microbial insecticide in the control of pine wilt disease was carried out by trunk injection. The dosage was 5-8g per 10cm diameter pine tree, and the application period was during the spring budding period of pine trees or one month before the emergence of the pine sawyer beetle.

[0024] Beneficial effects: 1. This invention achieves synergistic nematode control through the combination of *Syngonium chinense* and *Bacillus sicca*. *Syngonium chinense* can prey on pine wood nematodes by capturing organs and can colonize the wood of pine trees to continuously control nematodes. *Bacillus sicca* can secrete nematode-killing active substances to rapidly reduce the activity of pine wood nematodes, while also promoting the growth and colonization of *Syngonium chinense*, thus overcoming the shortcomings of unstable nematode-killing effects and slow nematode-killing speed of single microorganisms.

[0025] 2. This invention uses dendritic mesoporous silica nanoparticles modified with tannic acid-propyl gallate composite as a carrier. This carrier can specifically adsorb pine wood, achieving targeted delivery of the agent and reducing agent loss. At the same time, the porous structure of the nanoparticles can realize the slow and intelligent release of chemical nematicides, prolonging the agent's effective period and reducing the amount of chemical agents used.

[0026] 3. This invention achieves the stepwise release of functional components through a double-layer coating structure: the outer coating of Ardisia crenata can first colonize the xylem, the inner chemical nematicide can quickly kill nematodes, and Bacillus sicca continues to play a role. The synergistic effect of multiple components achieves rapid control and long-term prevention and control. The control effect is significantly better than that of single chemical agents and simple compound microbial preparations. The effective period against pine wilt disease can reach more than 2 years. It has good environmental compatibility and is suitable for widespread application. Attached Figure Description

[0027] Figure 1 A cross-culture diagram showing the antagonistic interaction between *Arthrobacter chinensis* strain CGMCC No. 3.6755 and *Bacillus sicca* strain CGMCC No. 1.8513; Figure 2 The images shown are scanning electron microscope (SEM) images (a) and transmission electron microscope (TEM) images (b) of the dendritic mesoporous silica nanocarrier of the present invention. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0029] Example 1 1. Preparation of core functional microbial compositions The strain *C. chinensis* (CGMCC No. 3.6755) was inoculated onto PDA solid medium (potato 200 g / L, glucose 20 g / L, agar 20 g / L, pH natural) and cultured in the dark at 25°C for 8 days. Conidia on the plate surface were washed with sterile physiological saline, and mycelia were removed by filtration through four layers of sterile gauze. The spore pellet was collected by centrifugation at 4000 rpm for 10 min and then freeze-dried at -50°C for 24 h to obtain a spore concentration of 1.2 × 10⁻⁶. 9 CFU / g of Arthropoda spore powder from China.

[0030] Bacillus sicca (CGMCC No. 1.8513) strain was inoculated into LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0), and cultured at 37°C with shaking at 180 rpm for 30 h. The vegetative cells were then killed by heating in an 80°C water bath for 15 min. The spore pellet was collected by centrifugation at 6000 rpm for 15 min and then freeze-dried at -50°C for 24 h to obtain a spore concentration of 1.5 × 10⁻⁶. 10 CFU / g Bacillus sicca spore powder.

[0031] 2. Preparation of dendritic mesoporous silica nanocarriers A. Template-based preparation of nanoparticles: 1.0 g of hexadecyltrimethylammonium bromide (CTAB) was dissolved in 480 mL of deionized water. 3.5 mL of 25% ammonia solution was added to adjust the pH to 11.5. After stirring at 300 rpm until homogeneous, 5 mL of tetraethyl orthosilicate (TEOS) was slowly added dropwise at a rate of 1 mL / min. The reaction was carried out at 30 °C with stirring for 24 h. After the reaction was complete, the product was collected by centrifugation at 8000 rpm for 10 min. The product was washed three times each with anhydrous ethanol and deionized water. The template was removed by calcination at 550 °C for 6 h at a rate of 5 °C / min, yielding dendritic mesoporous silica nanoparticles.

[0032] B. Amination Modification: 1.0 g of the above nanoparticles were dispersed in 100 mL of anhydrous ethanol and ultrasonically dispersed for 30 min (200 W). 0.5 mL of 3-aminopropyltriethoxysilane (APTES) was added, and the mixture was refluxed at 60 °C for 6 h. After the reaction was complete, the mixture was centrifuged at 8000 rpm for 10 min, washed three times with anhydrous ethanol, and vacuum dried at 40 °C for 12 h to obtain amination-modified nanoparticles.

[0033] C. Surface-targeted modification: 0.5 g of aminated nanoparticles were dispersed in 50 mL of ethanol-water solution (1:1 v / v), ultrasonically dispersed for 20 min, and then 0.1 g of tannic acid, 0.05 g of propyl gallate, and 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) crosslinking agent were added. The mixture was reacted at 150 rpm for 8 h at room temperature in the dark. After the reaction, the mixture was centrifuged at 8000 rpm for 10 min, washed three times with deionized water, and vacuum dried at 40 °C for 12 h to obtain dendritic mesoporous silica nanocarriers. The particle size of the dendritic mesoporous silica nanocarriers was 100-200 nm.

[0034] The carrier exhibits a clear dendritic porous structure as observed by transmission electron microscopy (TEM). The surface morphology is shown in scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images, as follows: Figure 2 As shown.

[0035] 3. Drug loading and coating a. Drug loading: 25g of the above carrier was dispersed in 250mL of deionized water and ultrasonically dispersed for 30min. 30mL of anhydrous ethanol solution containing 1.0g of emamectin benzoate was added, and the mixture was stirred at 200rpm for 12h at room temperature in the dark. The product was collected by centrifugation at 8000rpm for 10min, washed twice with deionized water, and vacuum dried at 40℃ for 12h to obtain drug-loaded nanoparticles with a drug loading of 3.9wt%.

[0036] The drug loading was determined using a differential method combined with high-performance liquid chromatography (HPLC): Since directly measuring the mass of drug on the carrier is difficult, the adsorption amount was inferred by measuring the mass of unadsorbed free drug in actual experiments. Drug loading (wt%) = (Drug dosage - Free drug content in supernatant) / Dry mass of drug-loaded nanoparticles × 100%.

[0037] The drug loading was determined using this method in subsequent examples and comparative examples.

[0038] b. Core mixing: Place 20g of drug-loaded nanoparticles and 10g of Bacillus sicca spore powder in a three-dimensional mixer and mix at 30 rpm for 15 min to obtain a homogeneous core mixture.

[0039] c. Double-layer coating: Dissolve 12g of sodium alginate in 1000mL of deionized water and stir until completely dissolved. Add 8g of *Ardisia crenata* spore powder and stir evenly. Then add the above core mixture and continue stirring for 30 minutes to form a uniform suspension. Drop the suspension through a 0.8mm needle syringe into 2000mL of an aqueous solution containing 1% chitosan and 2% calcium chloride, and allow it to solidify at room temperature for 30 minutes. Centrifuge at 8000rpm for 10 minutes to collect the microspheres, wash twice with deionized water, and freeze-dry under vacuum at -50℃ for 24 hours to obtain the microbial insecticide of this embodiment.

[0040] Example 2 1. Preparation of core functional microbial compositions Same as in Example 1, 1.2 × 10⁻⁶ spore powder of *Arthropoda sinensis* was prepared. 9 CFU / g) and Bacillus sicca spore powder (1.5×10 10 CFU / g).

[0041] 2. Preparation of dendritic mesoporous silica nanocarriers Following the same steps AC as in Example 1, a dendritic mesoporous silica nanocarrier modified with surface polyphenols was obtained.

[0042] 3. Drug loading and coating a. Drug loading: 25g of the above carrier was dispersed in 250mL of deionized water and ultrasonically dispersed for 30min. 30mL of anhydrous ethanol solution containing 0.5g of emamectin benzoate was added, and the mixture was stirred at 200rpm for 12h at room temperature in the dark. The product was collected by centrifugation at 8000rpm for 10min, washed twice with deionized water, and vacuum dried at 40℃ for 12h to obtain drug-loaded nanoparticles with a drug loading of 2.0wt%.

[0043] b. Core mixing: Same as in Example 1, mix 20g of drug-loaded nanoparticles with 10g of Bacillus sicca spore powder evenly.

[0044] c. Double coating: Same as step c in Example 1, freeze-dry to obtain the microbial insecticide of this example.

[0045] Example 3 1. Preparation of core functional microbial compositions Same as in Example 1, 1.2 × 10⁻⁶ spore powder of *Arthropoda sinensis* was prepared. 9 CFU / g) and Bacillus sicca spore powder (1.5×10 10 CFU / g).

[0046] 2. Preparation of dendritic mesoporous silica nanocarriers Following the same steps AC as in Example 1, a dendritic mesoporous silica nanocarrier modified with surface polyphenols was obtained.

[0047] 3. Drug loading and coating a. Drug loading: 25g of the above carrier was dispersed in 250mL of deionized water and ultrasonically dispersed for 30min. 30mL of anhydrous ethanol solution containing 0.8g of emamectin benzoate was added, and the mixture was stirred and adsorbed at 200rpm for 12h at room temperature in the dark. The product was collected by centrifugation at 8000rpm for 10min, washed twice with deionized water, and vacuum dried at 40℃ for 12h to obtain drug-loaded nanoparticles with a drug loading of 3.2wt%.

[0048] b. Core mixing: Same as in Example 1, mix 20g of drug-loaded nanoparticles with 10g of Bacillus sicca spore powder evenly.

[0049] c. Double coating: Same as step c in Example 1, freeze-dry to obtain the microbial insecticide of this example.

[0050] Example 4 1. Preparation of core functional microbial compositions Same as in Example 1, 1.2 × 10⁻⁶ spore powder of *Arthropoda sinensis* was prepared. 9 CFU / g) and Bacillus sicca spore powder (1.5×10 10 CFU / g).

[0051] 2. Preparation of dendritic mesoporous silica nanocarriers Following the same steps AC as in Example 1, a dendritic mesoporous silica nanocarrier modified with surface polyphenols was obtained.

[0052] 3. Drug loading and coating a. Drug loading: 25g of the above carrier was dispersed in 250mL of deionized water and ultrasonically dispersed for 30min. 30mL of anhydrous ethanol solution containing 1.2g of emamectin benzoate was added, and the mixture was stirred at 200rpm for 12h at room temperature in the dark. The product was collected by centrifugation at 8000rpm for 10min, washed twice with deionized water, and vacuum dried at 40℃ for 12h to obtain drug-loaded nanoparticles with a drug loading of 4.5wt%.

[0053] b. Core mixing: Same as in Example 1, mix 20g of drug-loaded nanoparticles with 10g of Bacillus sicca spore powder evenly.

[0054] c. Double coating: Same as step c in Example 1, freeze-dry to obtain the microbial insecticide of this example.

[0055] Example 5 1. Preparation of core functional microbial compositions Same as Example 1.

[0056] 2. Preparation of dendritic mesoporous silica nanocarriers Same as Example 1.

[0057] 3. Drug loading and coating a. Drug loading: 25g of the above carrier was dispersed in 250mL of deionized water and ultrasonically dispersed for 30min. 30mL of anhydrous ethanol solution containing 1.5g of emamectin benzoate was added, and the mixture was stirred at 200rpm for 12h at room temperature in the dark. The product was collected by centrifugation at 8000rpm for 10min, washed twice with deionized water, and vacuum dried at 40℃ for 12h to obtain drug-loaded nanoparticles with a drug loading of 5.7wt%.

[0058] b. Kernel hybrid: Same as Example 1.

[0059] c. Double coating: Same as step c in Example 1, freeze-dry to obtain the microbial insecticide of this example.

[0060] Comparative Example 1 In this comparative example, only *Arthropoda sinensis* was used in the preparation of the core functional microbial composition; all other steps and processes were the same as in Example 1. That is: 1. Preparation of core functional microorganisms: Same as in Example 1, except that *Arthropoda sinensis* spore powder (1.2 × 10⁻⁶) was prepared. 9 CFU / g).

[0061] 2. Preparation of dendritic mesoporous silica nanocarriers: Same as in Example 1.

[0062] 3. Drug loading and coating a. Drug loading: Same as step a in Example 1, to obtain drug-loaded nanoparticles with a drug loading of 3.9 wt%.

[0063] b. Core mixing: Mix 20g of drug-loaded nanoparticles with 10g of sterile starch (instead of Bacillus sicca spore powder) until homogeneous.

[0064] c. Double coating: Dissolve 12g of sodium alginate in 1000mL of deionized water, add 8g of Aralia elata spore powder, and stir well. Subsequent curing and drying steps are the same as in Example 1 to obtain this comparative formulation.

[0065] Comparative Example 2 In this comparative example, only *Bacillus sicca* was used in the preparation of the core functional microbial composition; all other steps and processes were the same as in Example 1. That is: 1. Preparation of core functional microorganisms: Same as in Example 1, except that Bacillus sicca spore powder (1.5 × 10⁻⁶) was prepared. 10 CFU / g).

[0066] 2. Preparation of dendritic mesoporous silica nanocarriers: Same as in Example 1.

[0067] 3. Drug loading and coating a. Drug loading: Same as step a in Example 1, to obtain drug-loaded nanoparticles with a drug loading of 3.9 wt%.

[0068] b. Core mixing: Mix 20g of drug-loaded nanoparticles with 10g of Bacillus sicca spore powder until homogeneous.

[0069] c. Double coating: Dissolve 12g of sodium alginate in 1000mL of deionized water, add 8g of sterile starch (instead of Chinese knotweed spore powder), and stir well. Subsequent steps are the same as in Example 1 to obtain this comparative formulation.

[0070] Comparative Example 3 Except for the use of other species of *Arthropoda sinensis* in the preparation of the core functional microbial composition, the remaining steps and processes in this comparative example are the same as in Example 1. That is: 1. Preparation of core functional microbial compositions The strain *Arthrum chinense* (CGMCC No. 3.6627) was inoculated onto PDA solid medium (potato 200 g / L, glucose 20 g / L, agar 20 g / L, pH natural) and incubated in the dark at 25°C for 8-10 days. Conidia on the plate surface were washed with sterile physiological saline, and mycelia were removed by filtration through four layers of sterile gauze. The spore pellet was collected by centrifugation at 4000 rpm for 10 min and then freeze-dried at -50°C for 24 h. After adjusting the incubation time as needed, a spore concentration of 1.2 × 10⁻⁶ was obtained. 9 CFU / g of Arthropoda spore powder from China.

[0071] The strain of *Arthrum chinense* described is CGMCC No. 3.6627, purchased from the China General Microbiological Culture Collection Center, with an original deposit date of May 13, 2003.

[0072] Comparative Example 4 Except for the use of other species of Bacillus sicca in the preparation of the core functional microbial composition, the remaining steps and processes in this comparative example are the same as in Example 1. That is: 1. Preparation of core functional microbial compositions Bacillus sicca (GDMCC 822593) strain was inoculated into LB broth (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0) and cultured at 28°C with shaking at 180 rpm for 30–40 h. The vegetative cells were then killed by heating in an 80°C water bath for 15 min. The spore pellet was collected by centrifugation at 6000 rpm for 15 min and then freeze-dried at -50°C for 24 h. With appropriate adjustments to the culture time, a spore concentration of 1.5 × 10⁻⁶ was obtained. 10 CFU / g Bacillus sicca spore powder.

[0073] The strain of Bacillus sicca mentioned is numbered GDMCC 822593, purchased from Guangdong Provincial Microbial Culture Collection Center, with an original deposit date of June 3, 2025.

[0074] Comparative Example 5 In this comparative example, except that dendritic mesoporous silica nanocarriers are not used, and nano-silica of the same particle size is substituted, the remaining steps are completely the same as in Example 1, namely: 1. Preparation of core functional microbial composition: Same as in Example 1.

[0075] 2. Drug loading and coating a. Drug loading: 25g of nano-silica (particle size 100-200nm) was dispersed in 250mL of deionized water and ultrasonically dispersed for 30min. Then, 30mL of anhydrous ethanol solution containing 1.0g of emamectin benzoate was added, and the mixture was stirred at 200rpm for 12h at room temperature in the dark. The product was collected by centrifugation at 8000rpm for 10min, washed twice with deionized water, and vacuum dried at 40℃ for 12h to obtain drug-loaded nanoparticles.

[0076] b. Core mixing: Place 20g of drug-loaded nanoparticles and 10g of Bacillus sicca spore powder in a three-dimensional mixer and mix at 30 rpm for 15 min to obtain a homogeneous core mixture.

[0077] c. Double-layer coating: Dissolve 12g of sodium alginate in 1000mL of deionized water and stir until completely dissolved. Add 8g of *Ardisia crenata* spore powder and stir evenly. Then add the above core mixture and continue stirring for 30 minutes to form a uniform suspension. Drop the suspension through a 0.8mm needle syringe into 2000mL of an aqueous solution containing 1% chitosan and 2% calcium chloride, and allow it to solidify at room temperature for 30 minutes. Centrifuge at 8000rpm for 10 minutes to collect the microspheres, wash twice with deionized water, and freeze-dry under vacuum at -50℃ for 24 hours to obtain a microbial insecticide.

[0078] Comparative Example 6 The chemical nematicide used is 5% commercially available emamectin benzoate emulsifiable concentrate, applied according to the recommended dosage in the instructions: 10g of formulation per 10cm of breast diameter.

[0079] Performance testing Test for antagonistic effects of strains: The antagonistic effect of the strains was tested using the streak-over method: PDA solid medium (200g potato, 20g glucose, 20g agar, 1000mL water, sterilized at 121℃ for 20min) and LB liquid medium (10g tryptone, 5g yeast extract, 10g sodium chloride, 1000mL water, sterilized at 121℃ for 20min) were prepared. Activated Bacillus sicca (CGMCC No. 1.8513) was inoculated into LB liquid medium and cultured at 37℃ with shaking at 180rpm for 24h. After inactivating the vegetative cells by water bath at 80℃ for 15min, the spore concentration was adjusted to 1×10⁻⁶. 8 CFU / mL, and simultaneously take a plate of Arthropoda sinensis (CGMCC No. 3.6755) cultured at 25℃ in the dark for 7-10 days, wash off the conidia with sterile physiological saline and adjust the concentration to 1×10⁻⁶ CFU / mL. 7 CFU / mL, using an inoculating loop, streak a straight line with *Bacillus sicca* spore suspension in the center of a sterile PDA plate. Then, using an inoculating loop perpendicular to this line, streak a cross line with *Arthrasma sinense* spore suspension. Separate controls were plates streaked only with *Bacillus sicca* and only with *Arthrasma sinense*. All plates were incubated in the dark at 25°C. Colony growth in the cross-section area was observed daily. If no inhibition zone formed at the cross-section, and colonies grew normally to the cross-section area, and the streak length of both strains was not significantly different from the corresponding control group on day 7, then the two strains were considered to have no antagonistic effect. Conversely, if a clear inhibition zone appeared at the cross-section or the growth of either strain was inhibited, then antagonistic effect was considered. The cross-culture diagram is shown below. Figure 1 The results show that no inhibition zone was observed at the intersection of the two strains, indicating that the two strains can be used in combination.

[0080] Indoor pine wilt nematode activity test: 1. Materials and Methods Test nematode: Pine wood nematode (Bursaphelenchus xylophilus), collected from the xylem of diseased black pine in Linyi City, Shandong Province. After isolation and purification in the laboratory, it was cultured in the dark at 25°C for 7 days on Botrytis cinerea plates. The nematodes were collected using the Bellman funnel method, washed 3 times with sterile water, and the nematode concentration was adjusted to 1000 nematodes / mL for later use.

[0081] Test formulations: microbial insecticides prepared in Examples 1-5, control formulations prepared in Comparative Examples 1-6, and commercially available 5% emamectin benzoate emulsifiable concentrate in Comparative Example 6.

[0082] Test method: The nematicidal activity was determined by the immersion method. 2 mL of nematode suspension was placed in a sterile centrifuge tube, and 2 mL of the test formulation suspension (each experimental group's formulation was prepared as a 10 g / L suspension) was added. The mixture was thoroughly mixed and incubated in the dark at 25°C. Nematode mortality was observed under a microscope at 24 h, 48 h, and 72 h. Nematodes were considered dead when their bodies were stiff and showed no response to needle prick. Each treatment was repeated three times, with sterile water treatment serving as a blank control. All results were taken as the arithmetic mean.

[0083] Calculation formula: Mortality rate (%) = (Number of dead nematodes / Number of dead nematodes) × 100% Corrected mortality rate (%) = (Treatment group mortality rate - Control group mortality rate) / (1 - Control group mortality rate) × 100% Table 1 Corrected mortality rate (%) of different formulations against pine wood nematode The results showed that the microbial insecticide prepared in this invention had excellent killing effect on pine wilt nematodes. The 72-hour corrected mortality rate of Examples 1-5 all reached 100%, slightly higher than that of the commercially available chemical agent Comparative Example 6, and the long-lasting nematicidal effect was more stable. The nematicidal effect of Comparative Example 1 (containing only Arthropoda sinensis) and Comparative Example 2 (containing only Bacillus sicca) was significantly lower than that of Example 1, proving that Arthropoda sinensis and Bacillus sicca have a significant synergistic nematicidal effect. The nematicidal effects of Comparative Examples 3 and 4, which used other preserved strains, were also significantly weaker than the specific strain selected in this invention, indicating that strain specificity has a key influence on the final nematicidal activity. The drug loading, adsorption and release effect of Comparative Example 5, which used ordinary nano-silica instead of dendritic mesoporous silica nanocarrier, was worse, and the overall nematicidal activity was also significantly reduced, proving that the carrier structure selected in this invention is beneficial to improving the effect of the agent. Comparative Example 6 (commercially available abamectin emulsifiable concentrate) showed relatively rapid initial nematicidal activity at 24 h. However, the insecticides prepared in Examples 1-5 of this invention exhibited significantly higher corrected mortality rates at 48 h and 72 h than Comparative Example 6, demonstrating superior long-lasting nematicidal effects. This reflects that the microbial preparations of this invention can enhance the efficacy of chemical agents to a certain extent. Field test for control of pine wilt disease: Materials and Methods Experimental site: A forest area in Lanshan District, Linyi City, Shandong Province where pine wilt disease occurs. The forest stands are mainly composed of 20-year-old black pines with a diameter at breast height of 10-15 cm and a pine wilt disease incidence rate of about 15%.

[0084] Test formulations: formulations prepared in Examples 1-3 and Comparative Examples 1-6.

[0085] Experimental design: A randomized block design was adopted, with 3 replicate plots for each treatment, 20 pine trees in each plot, and a water treatment as a blank control.

[0086] Application method: Use trunk injection method, apply 5g of the preparation per 10cm of diameter at breast height, and apply in April 2024 (spring pine budding period).

[0087] Methods: Disease incidence in pine trees in each plot was investigated at 6, 12, and 24 months after application of the pesticide. The number of diseased trees was recorded, and the disease index and control effect were calculated. All results were expressed as the arithmetic mean.

[0088] Disease severity grading criteria: Level 0: No symptoms; Grade 1: The needles below 1 / 4 of the tree crown are yellow and wilted; Level 2: 1 / 4-1 / 2 of the tree crown has yellowing and wilting needles; Level 3: 1 / 2-3 / 4 of the tree crown has dead needles; Level 4: The entire plant has died.

[0089] Calculation formula: Disease index = Σ(Number of diseased plants at each level × Corresponding level) / (Total number of plants × Highest level) × 100 Prevention and control efficacy (%) = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100 Table 2. Field control efficacy (%) of different formulations against pine wilt disease The results showed that Examples 1-5 of this invention exhibited excellent field control efficacy against pine wilt disease, maintaining a control efficacy of over 75% for 24 months after application, significantly superior to all comparative examples. Comparative example 6 (commercially available abamectin emulsifiable concentrate) showed good control efficacy for 6 months after application, but the efficacy dropped sharply to 52.7% after 12 months and only 19.8% after 24 months, with a much shorter duration of effectiveness than the formulation of this invention. The control efficacy of Comparative examples 1-5 was significantly lower than that of Example 1, further verifying the synergistic effect of the microbial combination, targeting carrier, and double-layer coating structure of this invention. The formulation of this invention has a duration of effectiveness of over 2 years, which can significantly reduce the number of applications, lower control costs, and reduce environmental pressure.

[0090] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A microbial insecticide, characterized in that, It includes a core functional microbial composition, a chemical nematicide, and a dendritic mesoporous silica nanocarrier. The core functional microbial composition is derived from Arachnium chinense (Syngonium sinense). Arthrobotrys sinensis ) and Bacillus sicca ( Bacillus siamensis )composition.

2. The microbial insecticide according to claim 1, characterized in that, The strain number of *Arthrozoa sinensis* is CGMCC No. 3.6755; the strain number of *Bacillus sicca* is CGMCC No. 1.8513.

3. The microbial insecticide according to claim 1, characterized in that, The preparation method of the core functional microbial composition is as follows: Inoculate *Arthrum chinense* strain into PDA medium, culture in the dark at 25°C for 7-10 days, collect conidia, and prepare spore powder; then inoculate *Bacillus sicca* strain into LB liquid medium, culture at 37°C and 180 rpm for 24-36 hours, collect spores by centrifugation, and prepare spore powder; after vacuum freeze-drying, the spore powder and spore powder can be stored separately.

4. The microbial insecticide according to claim 3, characterized in that, The spore concentration of *Arthropoda sinensis* is ≥1.0 × 10⁻⁶. 9 CFU / g, the spore concentration of the *Bacillus sicca* is ≥1.0 × 10⁻⁶. 10 CFU / g.

5. The microbial insecticide according to claim 1, characterized in that, The chemical nematicide is emamectin benzoate.

6. The microbial insecticide according to claim 1, characterized in that, The method for preparing the dendritic mesoporous silica nanocarrier is as follows: A. Template method for preparing nanoparticles: 1.0 g of cetyltrimethylammonium bromide (CTAB) was dissolved in 480 mL of deionized water, 3.5 mL of 25% ammonia solution was added, the pH was adjusted to 11-12, and after stirring evenly, 5 mL of tetraethyl orthosilicate (TEOS) was slowly added dropwise. The reaction was stirred at 30 °C for 24 h. After the reaction was completed, the product was collected by centrifugation, washed three times each with ethanol and deionized water, and calcined at 550 °C for 6 h to remove the template, yielding dendritic mesoporous silica nanoparticles. B. Aminoation modification: 1.0 g of nanoparticles were dispersed in 100 mL of anhydrous ethanol, and 0.5 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 60 °C for 6 h. After centrifugation and washing, the nanoparticles were dried under vacuum to obtain aminoated nanoparticles. C. Surface modification: 0.5 g of aminated nanoparticles were dispersed in 50 mL of ethanol-water solution with a volume ratio of 1:

1. 0.1 g of tannic acid, 0.05 g of propyl gallate and 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added. The mixture was shaken at room temperature in the dark for 8 h. After centrifugation, washing and vacuum drying were performed to obtain dendritic mesoporous silica nanocarriers.

7. A method for preparing a microbial insecticide according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of core functional microbial compositions; (2) Preparation of dendritic mesoporous silica nanocarriers: (3) Drug loading and coating: a. Disperse 20-30g of dendritic mesoporous silica nanocarrier in 200-300mL of deionized water, add 30mL of ethanol solution containing 0.5-1.5g of emamectin benzoate, stir and adsorb at room temperature in the dark for 12h; collect the product by centrifugation at 8000rpm for 10min, wash twice with deionized water, and vacuum dry at 40℃ to obtain drug-loaded nanoparticles; b. Core mixing: Mix 20g of drug-loaded nanoparticles with 10g of Bacillus sicca spore powder evenly to obtain a core mixture; c. Coating: Dissolve 12g of sodium alginate in 1000mL of deionized water, add 8g of Aralia elata spore powder, and stir well; then add the kernel mixture and continue stirring for 30min; drip the mixture into 2000mL of an aqueous solution containing 1% chitosan and 2% calcium chloride through a syringe, and solidify for 30min; collect the microspheres by centrifugation at 8000rpm for 10min, wash twice with deionized water, and freeze-dry to obtain the microbial insecticide.

8. The application of a microbial insecticide as described in any one of claims 1 to 6 in the control of pine wilt disease, characterized in that, The application method is trunk injection, with a dosage of 5-8g per 10cm diameter pine tree.

9. The application of the microbial insecticide according to claim 8 in the control of pine wilt disease, characterized in that, The application period is during the spring pine budding period or one month before the emergence of the pine sawyer beetle.

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