Spodoptera exigua nucleopolyhedrovirus spike stage targeted synergistic adjuvant, preparation and application thereof

Abstract

The application discloses a cotton bollworm nuclear polyhedrosis virus spike period targeted synergistic adjuvant, a pesticide preparation and application, relates to the technical field of agricultural biological pesticides, and the composite adjuvant is prepared from ultraviolet protection and shielding materials, film-forming materials, midgut infection promoters, penetration and spreading agents, system compatible components and carriers.The adjuvant is applied by unmanned aerial vehicle top spraying, and after being compounded with HaNPV, the deposition rate of the virus on the corn tassel can be increased by 3.2 times, the retention amount is increased by 5.62 times, the destruction rate of the midgut peritrophic membrane of the cotton bollworm larvae is more than 90%, and the mortality of the 3rd instar cotton bollworm larvae is increased to 87.72%.The application solves the two technical bottlenecks of the virus pesticide, i.e., 'difficult coverage of the tassel target' and 'low midgut infection efficiency', and provides key technical support for green and precise prevention and control of the cotton bollworm in the corn tassel period.

Description

Technical Field

[0001] This invention relates to the field of agricultural biopesticide technology, and in particular to a compound synergistic adjuvant for cotton bollworm nucleopolyhedrovirus (HaNPV). This adjuvant significantly enhances the control effect of the virus on cotton bollworm during the corn ear stage through a triple synergistic mechanism of ultraviolet shielding, ear-targeted deposition, and midgut infection synergy. Background Technology

[0002] cotton bollworm ( Helicoverpa armigera HaNPV is a major borer of maize, especially during the tasseling stage, where its larvae bore into the male tassels, silks, and female ears, causing yield reduction and increasing susceptibility to ear rot. Although HaNPV has significant potential for biological control, its practical application in the field is limited by two major technical bottlenecks, affecting its effectiveness.

[0003] However, HaNPV faces two major technical bottlenecks in field applications: First, there is the challenge of "difficulty in covering the target area on the ear": existing application techniques struggle to effectively deposit HaNPV solution onto the complex structures of the maize ear, such as the dense stamen columns and silks. These structures not only increase the difficulty of solution deposition in the target area but may also prevent HaNPV from reaching sufficient concentrations in key areas due to poor solution flow, thus affecting the virus's chances of infecting pests. In particular, when affected by natural factors such as wind and rain, virus particles are easily lost, reducing their retention time and concentration on the ear and severely impacting control efficacy.

[0004] Second, the problem of "low midgut infection efficiency": Even if some virus particles successfully deposit on the food source of cotton bollworm, HaNPV still needs to penetrate the peritrophic membrane barrier of its digestive tract to reach the midgut epithelial cells and initiate the infection process during the feeding process. However, it needs to overcome multiple barriers: (1) The peritrophic membrane is a dense network structure composed of chitin and protein, which has a strong physical and chemical blocking effect on virus particles and can block more than 90% of virus particles from reaching the midgut epithelial cells; (2) The complexity of the midgut environment, such as the changes between pH 9 and 11 and enzyme activity, will further reduce the survival rate and infection efficiency of the virus, especially for older cotton bollworm larvae, i.e., larvae older than 3rd instar, whose physiological structure is more mature and whose blocking effect on virus particles is more obvious, resulting in a worse effect and an initial infection efficiency of less than 10%. 50 Reaching 1.5×10 6 When the concentration of OBs / mL exceeds a certain level, the actual control efficacy of HaNPV in the field is significantly reduced.

[0005] In the prior art, patent CN117296841A discloses a pesticide synergist, which comprises: 5-15% lecithin, 2-10% isomeric deca-ol 8EO phosphate, 5-15% polyol Span 80, 5-20% emulsifier, 5-15% penetrant, 1-2% defoamer, 1-5% antifreeze, and the balance being water. This synergist significantly improves the stability of pesticides and significantly enhances their control effect, exhibiting a significant synergistic effect on insect control, fungicide control, and herbicide control. However, compared with the present invention, there are significant differences, as shown in Table 1:

[0006] As can be seen from Table 1 above, this application document is a typical representative of precision agriculture, emphasizing the closed loop of target-pesticide-application technology; the prior art document CN117296841A is a formulation optimization of traditional adjuvants, pursuing universality and large-scale application.

[0007] As can be seen from the existing technologies described above, while commercially available chemical adjuvants can improve the performance of pesticides or viral agents to some extent, their design is not specifically optimized for the complex structure of the corn ear and the specific needs of bollworm midgut infection. This means that these general-purpose adjuvants may not be able to effectively solve problems such as the low deposition efficiency of HaNPV in the corn ear and the obstacles encountered by the virus in the early stage of bollworm midgut infection. Therefore, there is an urgent need to develop a compound adjuvant specifically designed to enhance the efficacy of HaNPV during the corn ear stage. This adjuvant should not only improve the deposition rate and adhesion of the pesticide solution in the corn ear but also enhance the infection efficiency of the virus in the bollworm midgut, in order to overcome the aforementioned technical bottlenecks and achieve the goal of green and precise control of HaNPV in the corn ear stage bollworm control.

[0008] Therefore, developing a compound adjuvant with both "targeted deposition in the ear" and "enhanced midgut infection" functions, and establishing supporting drone top spraying technology, is of great practical significance for improving the field control efficacy of HaNPV and achieving green and precise control of bollworm during the corn ear stage. Summary of the Invention

[0009] To address the aforementioned technical problems, the present invention aims to overcome the shortcomings of existing technologies and provide a compound adjuvant that can significantly improve the control efficacy of HaNPV during the heading stage, along with a matching precision application method, thus solving the dual problems of viral pesticides "not hitting the target" and "low infection efficiency." The cotton bollworm nucleopolyhedrovirus HaNPV used in this invention is a publicly published strain (according to the strain recorded in the Chinese Virology (2003) literature, which can be purchased by the public through conventional commercial channels). The scope of protection of this invention is limited to the aforementioned adjuvant composition, formulation, and application method, and does not involve the protection of the HaNPV strain itself.

[0010] The objective of this invention is achieved through the following technical solution: A cotton bollworm nucleopolyhedrovirus (NPV) spike-targeting synergist, comprising the following components by weight percentage: UV protection and shielding materials 30-50%, film-forming materials 10-25%, midgut infection promoter 5-15%, penetration and spreading agent 1-5%, system-compatible components 5-15%, carrier to 100%; The ultraviolet protection and shielding material is a compound of lignin sulfonate and natural minerals; The film-forming material is derived from polysaccharides from seaweed; The midgut infection promoter comprises a complex of a highly deacetylated aminopolysaccharide and a protein-degrading enzyme; The penetrating and spreading agent is a modified vegetable oil.

[0011] Furthermore, the lignin sulfonate is selected from at least one of sodium lignin sulfonate and calcium lignin sulfonate; the natural mineral is at least one of kaolin and diatomaceous earth; and the mass ratio of the lignin sulfonate to the natural mineral compound is 2:1 to 1:1.

[0012] Furthermore, the film-forming material is sodium alginate, whose 1% aqueous solution has a dynamic viscosity of 100-300 mPa·s at 25 ℃; after the drug solution dries, the sodium alginate forms a flexible and stable film on the target surface, and the film firmly adheres the virus particles to the complex structure of the corn tassel. The aminopolysaccharide is chitosan oligosaccharide; wherein the degree of deacetylation of chitosan oligosaccharide is ≥90%, the molecular weight is ≤3000 Da, and the degree of polymerization is 2-10; the protein-degrading enzyme is selected from at least one of serine proteases and metalloproteinases; and the enzyme activity is ≥100,000 U / g.

[0013] Furthermore, the mass ratio of aminopolysaccharide to protein-degrading enzyme is 1.5:1 to 2:1.

[0014] Furthermore, the modified vegetable oil nonionic surfactant is selected from at least one of castor oil polyoxyethylene ether, Tween-80, and organosilicon surfactant, with an HLB value of 12-16 and a carbon chain length of C8-C10, namely APG0810. The system-compatible components include 2-8% antifreeze and 0.5-2% preservative; the antifreeze is selected from at least one of propylene glycol, glycerin, and ethylene glycol; the preservative is selected from at least one of sodium benzoate and potassium sorbate. The carrier is at least one of diatomaceous earth, pregelatinized starch, and precipitated silica, with a moisture content ≤5%, a fineness ≥95%, and passes through a 44 μm sieve.

[0015] A pesticide formulation for controlling bollworm in corn is prepared by mixing an adjuvant with bollworm nucleopolyhedrovirus (HaNPV) at a mass ratio of 1:200-500.

[0016] Furthermore, the synergistic adjuvant was mixed with HaNPV at a certain mass ratio to prepare a HaNPV concentration of 1×10⁻⁶. 6 ~2×10 6 The spray solution with OBs / mL is applied by top-mounted spraying using an agricultural drone. The drone flies at an altitude of 2.5-3.5 meters, with a spray volume of 20-30 L / ha and a flight speed of 3-6 m / s. The targeted top spraying should be applied during the corn's tasseling stage to the early tasseling stage, specifically when 5-30% of the tassels have emerged.

[0017] Furthermore, the optimal time for targeted spraying from the top of the agricultural drone is between 16:00 and 19:00 on a sunny evening, with an ambient temperature of 20-28 ℃ and a wind speed of ≤3 m / s.

[0018] Furthermore, in the mixture of synergist and HaNPV, the dilution factor of the synergist is 250-350 times; The drone is a single-rotor or multi-rotor agricultural drone, with a pressure or centrifugal nozzle and a droplet size controlled between 150-250 μm.

[0019] Application of synergistic adjuvants in the preparation of biological pesticides for controlling bollworm during the corn tasseling stage.

[0020] Compared with the prior art, one or more embodiments of the present invention may have the following advantages: The additive consists of four core components: a compound of lignin sulfonate and natural minerals, which serve as UV protection and shielding materials. These components form a strong sun protection barrier, effectively resisting the damage of ultraviolet rays to the virus, while providing a slight shielding effect and reducing the impact of environmental factors on viral activity.

[0021] Seaweed-derived polysaccharides were chosen as film-forming materials. This not only ensures that the virus preparation forms a strong and continuous film on the crop surface, improving adhesion stability, but also enhances the persistence of the virus under adverse weather conditions, ensuring effective prevention and control for a longer period of time.

[0022] The midgut infection promoter is achieved through an innovative combination of highly deacetylated aminopolysaccharides and proteolytic enzymes. This complex can effectively penetrate the midgut peritrophic membrane of pests, interfere with their immune system function, thereby significantly increasing the infection rate and speed of the virus and reducing the pests' natural defenses against the virus.

[0023] Finally, the penetration and spreading agent uses modified plant oils, which optimizes the distribution and penetration of the virus preparation on the crop surface, ensuring that virus particles can evenly cover and penetrate deep into plant tissues, thus improving application efficiency.

[0024] Through the scientific combination and synergistic effect of the above components, the technical solution of this embodiment significantly enhances the deposition and infection efficiency of bollworm nucleopolyhedrovirus in corn ears, providing a more effective and environmentally friendly solution for biological control. Furthermore, a complete set of UAV top-spraying technical parameters has been established, with droplet size of 150-250 μm, precisely covering the tassels (coverage >85%), achieving a pesticide reduction of over 30% (spray volume reduced from 45 L / ha to 25 L / ha), increasing labor efficiency by 5 times, and optimizing the application window: from the large trumpet stage to the early tasseling stage when 5-30% of the tassels have emerged, larvae concentrate on feeding in the ear, maximizing the probability of virus contact; evening application (16:00-19:00) avoids strong light, temperature of 20-28 ℃ ensures virus activity, and wind speed ≤3 m / s reduces drift loss. Attached Figure Description

[0025] Figure 1 is a comparison of the amount of fluorescent tracer deposited on the leaves 1 hour after application; Figure 2 is a comparison of the retention of fluorescent tracers in leaves after simulated rainfall; Figure 3 is a scanning electron microscope image of the peritrophic membrane of the midgut of the cotton bollworm; Figure 4 is a comparison of the cumulative mortality rate of 3rd instar cotton bollworm larvae in the room over 7 days under different treatments. Figure 5 is a schematic diagram illustrating the complete working principle of the composite adjuvant of the present invention to achieve "targeted deposition in the ear and enhanced midgut infection". Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described in further detail below with reference to the embodiments and accompanying drawings.

[0027] This invention provides an adjuvant that enhances the targeted efficacy of bollworm nucleopolyhedrovirus during the heading stage. The components are weighed as follows by weight percentage: 30-50% UV protection and shielding material, 10-25% film-forming material, 5-15% midgut infection promoter, 1-5% penetration and spreading agent, 5-15% system-compatible component, and carrier to 100%. The ultraviolet protection and shielding material is a compound of lignin sulfonate and natural minerals; the film-forming material is derived from seaweed polysaccharides; the midgut infection promoter includes a complex of highly deacetylated aminopolysaccharides and protein-degrading enzymes; and the penetration and spreading agent is modified vegetable oil.

[0028] The lignin sulfonate and natural mineral used in the ultraviolet protection and shielding agent have a mass ratio of 2:1 to 1:1; the lignin sulfonate is selected from at least one of sodium lignin sulfonate and calcium lignin sulfonate. The combination of lignin sulfonate and natural mineral can form a strong physicochemical adsorption layer on the surface of virus particles, absorbing 290-400 nm ultraviolet light, significantly reducing the damage of environmental ultraviolet rays to virus activity, and ensuring the survival rate of virus particles after spraying; the natural mineral is at least one of kaolin and diatomaceous earth.

[0029] In this embodiment, the preferred natural mineral is kaolin. Kaolin reflects ultraviolet light through physical shielding, and the synergistic effect of the two can extend the virus half-life by 3-5 times. The preferred mass ratio of lignin sulfonate to kaolin is 1.8:1 to 1.2:1. The benzene ring structure of sodium lignin sulfonate absorbs 290-400 nm ultraviolet light (molar extinction coefficient ε>3000 L·mol⁻¹). -1 ·cm -1 Kaolinite reflects ultraviolet light through physical scattering, and the two work together to extend the virus half-life to 8-12 hours (3-5 times longer than the control).

[0030] The film-forming material is a high-adhesion film-forming agent, sodium alginate, with a viscosity range of 300–800 mPa·s. After application, sodium alginate forms a three-dimensional network cross-linked film on the surface of corn tassels, firmly anchoring virus particles. The contact angle is reduced from 95° to below 35°, and the rain scour index (RII) reaches above 0.85.

[0031] The aminopolysaccharide is chitosan oligosaccharide, wherein the degree of deacetylation of the chitosan oligosaccharide is ≥90%, the molecular weight is ≤3000 Da, and it can interfere with the midgut immune signaling pathway. The degree of polymerization of the chitosan oligosaccharide is 2-10. The proteolytic enzyme is at least one of serine protease and metalloproteinase, and the enzyme activity is ≥100,000 U / g, specifically degrading peritrophic membrane protein components. The mass ratio of the aminopolysaccharide to the proteolytic enzyme is 1.5:1 to 2:1.

[0032] The penetrating and spreading agent is a modified vegetable oil nonionic surfactant, selected from at least one of castor oil polyoxyethylene ether, Tween-80, and organosilicon surfactants; with an HLB value of 12-16, it can reduce the surface tension of the drug solution to 25-30 mN / m and increase the spreading coefficient to above 1.5.

[0033] The system-compatible components include 2-8% antifreeze and 0.5-2% preservative; the antifreeze is selected from at least one of propylene glycol, glycerin, and ethylene glycol; the preservative is selected from at least one of sodium benzoate and potassium sorbate, ensuring the formulation is stable in the range of -5 ℃ to 45 ℃; The carrier is at least one of diatomaceous earth, pregelatinized starch, and precipitated silica. The product moisture content is controlled to be ≤5% and the fineness is ≥95% (44 μm sieve).

[0034] All raw materials used are commercially available industrial or reagent grade. Sodium lignosulfonate: provided by a company in Shandong, molecular weight 7000±500 Da; Kaolin: ultrafine kaolin from Inner Mongolia, 800 mesh passing rate ≥95%, D50=5.8 μm; Sodium alginate: a biological company in Qingdao, 1% solution viscosity 200mPa·s (25 ℃); Alkaline protease: an enzyme preparation company in Jiangsu, enzyme activity 100,000 U / g, conforming to GB / T 23527-2009; Chitosan oligosaccharide: a biological company in Zhejiang, degree of deacetylation 95%, molecular weight <3000 Da, degree of polymerization 2-8; APG0810: a surfactant company in Shanghai, HLB=12.5; HaNPV: a commercially available product from an industrial company in Henan, 5 billion OBs / mL suspension.

[0035] Formulation performance indicators for adjuvant dry powder: Appearance: uniform brown powder, no lumps; Moisture content ≤5% (Karl Fischer method); Fineness: ≥95% passing through a 44 μm standard sieve (airflow sieving method); Suspension rate ≥85%; pH value (1% aqueous solution): 5.5-7.0; Dilution stability: no precipitation or stratification after standing for 24 h in a 20-fold dilution; Viscosity (149-fold dilution): 30-60 mPa·s (rotational viscometer method); Drying time: <1 h forming a dry film on the surface of corn leaves.

[0036] The safety evaluation was conducted in accordance with NY / T 1980 "Guidelines for Environmental Safety Evaluation of Pesticides": Acute contact toxicity to bees: OECD Guideline 214 was used, with Italian honeybees ( Apismellifera For exposure to the toxin, a mortality rate of <50% within 14 days is considered low toxicity; for acute toxicity in silkworms: for second-instar silkworms fed with leaf-soaked feed, a 96-h-LC50 >2000 mg / L is considered slightly toxic. Acute toxicity of zebrafish: 96 h-LC50 > 100 mg / L (refer to EPA OPPTS 850.1075); Soil degradation: Chitosan oligosaccharide DT50 < 10 d in sterilized / unsterilized soil (radioisotope tracing method).

[0037] Field efficacy trial design: This invention has completed laboratory verification and is currently undergoing multi-point field trials in the main corn-producing areas of the Huang-Huai-Hai Plain.

[0038] The experimental design was as follows: Experimental sites: Liaocheng, Shandong; Xinxiang, Henan; Shijiazhuang, Hebei, with 3 replicates per site; Treatment settings: ① Water control; ② 10% chlorantraniliprole SC (30 mL / mu); ③ HaNPV single agent (5 billion OBs / mL, 50 mL / mu); ④ HaNPV + conventional organosilicon adjuvant (0.02%); ⑤ HaNPV + adjuvant of this invention (30 g / mu + 50 mL / mu virus). Application time: early tasseling stage of maize (10-20% of tassels have emerged); Survey method: 5 sampling points per plot, 10 ears per point, recording the number of live insects, and calculating the insect population reduction rate; Expected target: 7 days after treatment ⑤, the control efficacy is >85%, significantly better than ③ and ④. p <0.05)), which is not significantly different from ②.

[0039] The following detailed description is provided through specific embodiments: Example 1: Preparation of composite additives (basic formulation) Sodium lignosulfonate 25%, kaolin 15%, D50=5.8 μm; sodium alginate 12%, 1% solution viscosity 200 mPa·s; protease (serine protease, enzyme activity 120,000 U / g) 4%, chitosan oligosaccharide (degree of deacetylation 95%, molecular weight 2000 Da) 6%, castor oil polyoxyethylene ether (EL-40) 2%, propylene glycol 8%, sodium benzoate 1%, pregelatinized starch to make up to 100%.

[0040] Preparation process: Sodium lignosulfonate, kaolin, sodium alginate, chitosan oligosaccharide, sodium benzoate, and pregelatinized starch were sequentially added to a double-helix conical mixer and mixed for 20 min. Then, protease was added and mixing continued for 10 min (avoiding heating the protease). Finally, castor oil polyoxyethylene ether and propylene glycol were added and mixed for 5 min. The temperature was controlled below 35℃ throughout the mixing process. The product was sieved through a 100-mesh (150 μm) sieve to remove any possible mechanical impurities. The resulting dry powder was pale yellow to brown, with a moisture content of 3.8%, a pH value (1% aqueous solution) of 6.8, a wetting time of 45 seconds, and a fineness of 96% passing through a 44 μm sieve, meeting product quality standards. The product was then vacuum-packed in aluminum foil bags, with a specification of 500 g / bag.

[0041] Example 2: Preferred Formulation Example Weigh the following by weight percentage: 28% calcium lignosulfonate, 14% kaolin, 15% sodium alginate (viscosity 400 mPa·s), 5% protease (metalloproteinase, activity 150,000 U / g), 8% chitosan oligosaccharide, 3% Tween-80, 10% glycerol, 1.5% potassium sorbate, and make up to 100% diatomaceous earth. The preparation method is the same as in Example 1.

[0042] Example 3: High UV protection ratio, suitable for areas with strong sunlight. Sodium lignosulfonate 32%, kaolin 18%, sodium alginate 10%, protease 3%, chitosan oligosaccharide 5%, organosilicon surfactant 1.5%, ethylene glycol 6%, sodium benzoate 0.8%, and silica to make up to 100%. The preparation method is the same as in Example 1, and will not be described in detail here.

[0043] Example 4: The effect of viscosity on the atomization effect of drones Sodium alginate viscosity directly affects the spraying performance of drones. The effect of different viscosities (100, 150, 200, 250, 300 mPa·s) of a 1% solution on droplet size distribution was tested (nozzle model: TeeJetXR8002): 100-150 mPa·s: droplet median diameter (DV50) 180-200 μm, but insufficient film-forming properties, with retention only increasing by 2.1 times; 200-250 mPa·s: DV50 = 200-220 μm, with the best film-forming properties and atomization effect, and retention increasing by 5.62 times; 300 mPa·s: DV50 > 250 μm, excessively coarse droplets leading to a 15% decrease in coverage; therefore, 100-300 mPa·s was determined to be a feasible range, and 150-250 mPa·s was the preferred range.

[0044] In this embodiment, spraying is not limited to drones, and can be selected according to the scale; for example, less than 100 mu: backpack electric sprayer or small drone; 100-1000 mu: tractor-mounted boom sprayer, which is cost-effective; more than 1000 mu: self-propelled sprayer or drone plant protection service team. The spraying method and time are the same as drone spraying, and will not be described in detail here.

[0045] Example 5: Indoor Measurement of Ear Deposition and Attachment Ability Deposition Test: To quantify the effect of the adjuvant of this invention on enhancing virus deposition and retention on maize targets under laboratory conditions, the following test was conducted: Healthy maize leaves were collected and fixed on a 45° inclined support. Two treatments were set up: ① HaNPV and water (virus concentration 1.5 × 10⁻⁶). 6② HaNPV and adjuvants (virus concentration 1.5 × 10⁻⁶) 6 OBs / mL, adjuvant:water = 1:300). Under artificially controlled conditions of 25±2 ℃, relative humidity 70±5%, and photoperiod 16 L:8 D, a solution containing sodium fluorescein tracer was sprayed using a Potter spray tower at a pressure of 0.3 MPa, with a spray volume of 1.0 µL / cm². One h after application, the leaves were rinsed with pH 7.4 phosphate buffer, and the fluorescence intensity in the eluent was measured using an ELISA reader (excitation wavelength 490 nm, emission wavelength 514 nm). The results showed no significant difference in the initial deposition amount between the two groups. p >0.05, Figure 1 A comparison of the amount of fluorescent tracer deposited on leaves 1 hour after application shows that there was no significant difference in the initial deposition.

[0046] Retention rate test: After the deposition rate test, simulated rainfall (rainfall intensity 30 mm / h, duration 10 min, conforming to the criteria for pesticide field efficacy trials). Fluorescence intensity was measured after rinsing. The results (Figure 2: Comparison of leaf fluorescence tracer retention after simulated rainfall of 30 mm / h × 10 min; the retention in the treatment group was 5.62 times that of the control group, a highly significant difference) are shown. p <0.01) indicates that the fluorescence intensity of treatment ② is 5.62 ± 0.38 times that of treatment ① (n=5, p <0.01).

[0047] 10. The above results show that the adjuvant of the present invention can significantly enhance the adhesion of the viral drug solution to the target surface. After simulated rainfall, the retention of fluorescent tracer in the treatment group was 5.62 times that of the control group. This provides a direct basis for improving the duration of effectiveness in field applications and significantly enhances the virus's resistance to erosion on the target surface.

[0048] Example 6: Observation on the disruptive effect of the periesophageal membrane in the midgut To visually verify the degradation effect of the "chitosan oligosaccharide + protease" in the adjuvant on the peritrophic membrane of cotton bollworm larvae, the following observations were conducted: Healthy third-instar cotton bollworm larvae were reared in an artificial climate chamber (25±1 ℃, photoperiod L:D=16:8, humidity 70%). Two groups were set up: ① control group (fed artificial feed); ② treatment group (fed artificial feed containing a 300-fold dilution of the adjuvant from this embodiment). After 6 h and 12 h of feeding, the midgut was dissected under aseptic conditions, the peritrophic membrane was removed, fixed with 2.5% glutaraldehyde, dehydrated in a gradient of ethanol, critical point dried, and sputter-coated with gold, and then observed using a scanning electron microscope.

[0049] Figure 3 shows a comparison of scanning electron micrographs (2000x magnification) of the peritrophic membrane of the cotton bollworm midgut. A represents the control group (intact and dense structure), while B represents the treatment group (large-area perforation and fiber disintegration). The peritrophic membrane in the control group was intact, dense, and uniformly mesh-like. In the treatment group, local perforations appeared after 6 hours, and after 12 hours, the peritrophic membrane disintegrated over a large area, with the fiber structure breaking. Image analysis showed that the perforation area in the treatment group reached 85-92%, which confirms that the active ingredient in the adjuvant can effectively destroy the key physical defense barrier of the insect's midgut, opening a channel for viral particle infection.

[0050] Example 7: Indoor Bioactivity Assay To evaluate the effect of the adjuvant on the insecticidal activity of the virus under controlled environmental conditions, a leaf-immersion feeding experiment was conducted in an artificial climate chamber (25±1 ℃, photoperiod L:D=16:8, humidity 70 %).

[0051] Four treatments were set up: ① Sterile water control; ② HaNPV and water (1.5 × 10⁻⁶) 6 OBs / mL); ③HaNPV and adjuvant (adjuvant 300 times); ④HaNPV and adjuvant (adjuvant 500 times). The leaf-soaking feeding method was used. Corn leaves were cut into sections, soaked in different treatment solutions, dried, and then fed to healthy 3rd instar bollworm larvae. Each treatment contained 30 larvae, with 3 replicates. Larval mortality was observed and recorded daily, and the cumulative mortality rate over 7 days was calculated.

[0052] The results (as shown in Table 2, Indoor Bioactivity Measurement Results, and Figure 4, Comparison of Cumulative Lethal Effects of Different Treatments on 3rd Instar Cotton Bollworm Larvae over 7 Days) showed that the larval mortality rate was highest in the adjuvant + HaNPV treatment group; the mortality rate of treatment ② was 76.67±4.2%; and the mortality rate of treatment ③ was 87.72±3.1%, significantly higher than the 76.67% of treatment ② in the HaNPV + water treatment group. p <0.05; Treatment ④ mortality rate: 70.45±5.3%, slightly lower than treatment ②, but the difference was not significant; indicating that the synergistic effect of the adjuvant was best at a 300-fold dilution, while the effect was affected by insufficient concentration at a 500-fold dilution.

[0053]

[0054] Combining the strong adhesion and retention ability demonstrated in Example 5 with the midgut barrier disruption effect demonstrated in Example 6, this fully verifies that the composite adjuvant of the present invention can significantly enhance the insecticidal activity of HaNPV through a dual synergistic pathway of physical protection and bio-enhancement.

[0055] Example 8: UV resistance performance determination HaNPV suspension (1×10 6 The concentrations (OBs / mL) were divided into two groups: control group: diluted with pure water; treatment group: with the additive of this invention added at a ratio of 1:300; and treated in a UV lamp chamber (wavelength 365 nm, intensity 50 μW / cm²). The virus was continuously irradiated, and samples were taken at 0, 2, 4, 6, 8, and 10 hours. Viral activity was detected using a bioassay. Results showed: The control group had a viral half-life (T½) of 2.1 h, and the activity retention rate was only 18.3% after 10 h; the treatment group had a T½ of 8.5 h, and the activity retention rate was 62.7% after 10 h; the UV protection efficiency (%) was calculated as [(activity retention rate of treatment group - activity retention rate of control group) / (1 - activity retention rate of control group)] × 100%, and the protection efficiency of the present invention was 72.4%.

[0056] Example 9: Compatibility verification of adjuvants with HaNPV The compatibility of the adjuvants with HaNPV is a key indicator determining the feasibility of the technology. The adjuvants and validation methods are as follows: (1) Sample preparation: The dry powder of the adjuvant and the HaNPV suspension were mixed according to the formulation of Example 1 at the mass ratio, and three replicates were set; (2) Storage conditions: Samples were taken at 0, 1, 3, 7 and 14 days for the 25 ℃ room temperature group and the 40 ℃ high temperature group, respectively; (3) Activity detection: Bioassay method, samples at each time point were fed to 3rd instar cotton bollworm larvae (n=30), and the corrected mortality rate was calculated after 7 days. The survival rate was compared with the baseline value at 0 days. The survival rate >90% was considered compatible. After 14 days, qPCR detection results showed that the viral gene copy number retention rate in the 25 ℃ room temperature group was 96.3%±2.1%, the retention rate in the 40 ℃ high temperature group was 91.8%±3.4%, and the retention rate in the CK group was 98.1%±1.5%. There was no significant difference between the 25 ℃ room temperature group and the CK group. p >0.05), there was no significant difference between the 40 ℃ high temperature group and the CK group ( p =0.078), proving that the adjuvant of the present invention has no significant effect on the activity of HaNPV and has good compatibility.

[0057] Example 10: Field efficacy trial Field determination of male spike deposition rate: The adjuvant and HaNPV were mixed at a mass ratio of 1:(200-500) to prepare a virus concentration of 1×10⁻⁶. 6 -2×10 6The spray solution had an OBs / mL concentration. Top spraying was performed using an agricultural drone (flying at an altitude of 3 m). Technical parameters: flight altitude 2.5-3.5 m, spray volume 20-30 L / ha, flight speed 3-6 m / s, droplet size 150-250 μm. Application was performed from the corn's large trumpet stage to early tasseling stage (5-30% of the tassels had emerged), preferably on a sunny evening from 16:00-19:00, with an ambient temperature of 20-28 ℃ and a wind speed ≤3 m / s. Deposition was measured using the cardboard method. Results showed that conventional spraying resulted in a deposition rate of 8.7% on the tassels, while the adjuvant and drone top spraying treatment achieved a deposition rate of 27.9%, an increase of 3.2 times.

[0058] The experiment was conducted in Yuanyang County, Xinxiang City, Henan Province, from July to August 2023. The maize variety Zhengdan 958 was used, with a planting density of 60,000 plants / ha. The bollworm egg count in the experimental plots reached the control threshold (≥30 eggs per 100 plants). Five treatments were set up (Table 3: Field Trial Treatment Design), arranged in a randomized block design with four replicates. The plot area was 50 m². A DJI T40 agricultural drone was used (flight altitude 3 m, spray volume 25 L / ha, droplet size 180-220 μm).

[0059]

[0060] Investigation methods: A baseline population was surveyed before pesticide application, and surveys were conducted 3, 7, and 14 days after application. Five samples were taken from each plot, with 10 plants per sample point. The percentage of male spikelets damaged and the number of surviving larvae were recorded. The control effect and spikelet protection effect were calculated.

[0061] Table 4 shows the results of field efficacy trials.

[0062] The results showed that the T3 treatment achieved a control efficacy of 89.4% after 7 days, which was not significantly different from the chemical pesticide T4 treatment and was significantly better than T1 and T2, confirming that the adjuvant of the present invention and the UAV top spraying technology have excellent field control efficacy and persistence.

[0063] Example 11: Comparative Experiment Comparative Example 1 (without UV protectant): Lignosulfonate and kaolin were removed from the formulation and replaced with pregelatinized starch. Field trials showed that the efficacy was only 54.3±6.8% 7 days after application, the duration of effectiveness was shortened to 5-7 days, and the efficacy dropped to below 35% after 14 days, proving that UV protectants are crucial for prolonging the duration of effectiveness.

[0064] Comparative Example 2 (without midgut infection promoter): Protease and chitosan oligosaccharide were removed from the formulation. Indoor assays showed that the mortality rate of 3rd instar larvae decreased to 65.2±5.6%, significantly lower than that of the complete formulation, and the midgut peritrophic membrane destruction rate was only 12%, confirming that the midgut infection promoter is the key to improving the infection rate.

[0065] Comparative Example 3 (without high-adhesion film-forming material): Sodium alginate was removed from the formulation. After simulated rainfall, the retention was only 32.8 ± 4.5% of the complete formulation, and the field efficacy decreased by more than 40%, demonstrating the necessity of film-forming agents to resist rainwater erosion.

[0066] Example 12: Safety Evaluation Crop safety: The adjuvant of this invention was sprayed at the large trumpet stage and the tasseling stage, respectively, at a dilution of 150 times (2x concentration) and 100 times (3x concentration). Observations were conducted 3, 7, and 14 days after application. There were no differences in leaf color and growth vigor between the treatment areas and the water control, and no significant effects were observed on plant height and leaf area index. p >0.05), indicating that it is highly safe for corn.

[0067] Safety of natural enemies: Conducted in accordance with the "Guidelines for Environmental Safety Evaluation of Chemical Pesticides".

[0068] Ladybugs: Direct contact with a 300-fold dilution of the adjuvant resulted in a mortality rate of 4.2±1.8% after 24 hours and 5.6±2.1% after 48 hours, classifying it as a low-risk species.

[0069] Trichogramma wasps: After treating egg cards with a 300-fold dilution of the adjuvant, the parasitism rate was 78.5±6.3%, which was not significantly different from the control group (81.2±5.8%), and was classified as low-risk.

[0070] Environmental compatibility: All auxiliary components are biodegradable materials. Sodium alginate and chitosan oligosaccharide are natural polysaccharides, and lignin sulfonate is a papermaking byproduct with a half-life of less than 30 days in soil and no risk of residue.

[0071] Example 13: Working Principle The working principle of the adjuvant of this invention is a multi-stage, multi-component synergistic continuous process, demonstrating the complete synergistic pathway from UAV top spraying → spikelet deposition → midgut breakthrough → virus infection in the above embodiments.

[0072] Mechanism of action: The additives of this invention achieve synergistic effects through a three-stage synergistic action of "physical protection - precise deposition - bio-enhancement": (1) Spraying stage: The UV protection and shielding agent (lignin sulfonate / kaolin) in the adjuvant provides initial light shielding protection for the virus; (2) Deposition stage: The film-forming agent forms an adhesion layer on the surface of the tassel. The liquid is precisely delivered to the corn tassel by targeted spraying from the top of the drone. Subsequently, the high adhesion film-forming agent (sodium alginate) ensures that the virus particles are firmly adhered to the complex structure of the tassel, forming effective deposition. (3) Infection stage: When cotton bollworm larvae feed, chitosan oligosaccharide and protease work synergistically in their intestines. Protease degrades the perifeeding membrane protein structure, while chitosan oligosaccharide interferes with intestinal immunity and microbial balance, together opening up an infection channel for the virus particle ODV, which ultimately enables it to efficiently infect midgut epithelial cells and establish infection. Figure 5 intuitively shows this complete synergistic pathway from "precise deposition" to "midgut breakthrough".

[0073] Example 14: Determination of the physicochemical properties of the product The product prepared according to Example 1 was tested and found to have the following characteristics: moisture content 3.8%, pH value (1% aqueous solution) 6.8, wetting time 45 seconds, suspension rate ≥85%, thermal storage stability: stored at 54±2 ℃ for 14 days, active ingredient decomposition rate ≤5%, low temperature stability: stored at 0±2 ℃ for 7 days, with no crystallization or precipitation. All indicators meet the requirements of NY / T 2989-2016 pesticide foliar spray adjuvant standard.

[0074] Cost-benefit analysis of the above embodiments: The cost of the adjuvant provided in the above embodiments is about RMB 2.8 per mu (based on a dosage of 30 g / mu), which is 65% lower than that of chemical pesticides (chlorantraniliprole, RMB 8-12 per mu); compared with HaNPV single agent (3 applications, cost RMB 6 per mu), the number of applications is reduced to 1-2, and the overall cost is reduced by 30%.

[0075] In the above embodiments: Bollworm nucleopolyhedrovirus (HaNPV): A baculovirus that specifically infects the bollworm. Viral particles are embedded in polyhedromic protein inclusion bodies (OBs).

[0076] Perinatal membrane: A semi-permeable membrane structure formed on the inner wall of the midgut of insects, mainly composed of chitin and 15 protein, which is an important barrier against viral infection.

[0077] ODV: Occlusion-derived virus, a form of virus particle released from a polyhedron.

[0078] Tasseling stage: The stage from tasseling to the early grain-filling stage of maize growth and development.

[0079] Top spraying by drones: an operational method in which agricultural drones spray water downwards from the top of crops.

[0080] While the embodiments disclosed in this invention are as described above, the content is merely for the purpose of facilitating understanding of the invention and is not intended to limit the invention. Any person skilled in the art to which this invention pertains may make any modifications and variations in form and detail of the implementation without departing from the spirit and scope disclosed herein; however, the scope of patent protection for this invention shall still be determined by the scope defined in the appended claims.

Claims

1. A cotton bollworm nucleopolyhedrovirus targeted synergistic adjuvant at the ear stage, characterized in that, It consists of the following components by mass percentage: UV protection and shielding materials 30-50%, film-forming materials 10-25%, midgut infection promoters 5-15%, penetration and spreading agents 1-5%, system-compatible components 5-15%, carrier to 100%; The ultraviolet protection and shielding material is a compound of lignin sulfonate and natural minerals; The film-forming material is derived from polysaccharides from seaweed; The midgut infection promoter comprises a complex of a highly deacetylated aminopolysaccharide and a protein-degrading enzyme; The penetrating and spreading agent is a modified vegetable oil.

2. The cotton bollworm nucleopolyhedrovirus ear-stage targeted synergist according to claim 1, characterized in that, The lignin sulfonate is selected from at least one of sodium lignin sulfonate and calcium lignin sulfonate; the natural mineral is at least one of kaolin and diatomaceous earth; the mass ratio of the lignin sulfonate to the natural mineral compound is 2:1 to 1:

1.

3. The cotton bollworm nucleopolyhedrovirus ear-stage targeted synergist according to claim 1, characterized in that, The film-forming material is sodium alginate, and its 1% aqueous solution has a dynamic viscosity of 100-300 mPa·s at 25 ℃. After the drug solution dries, the sodium alginate forms a flexible and stable film on the target surface, and the film firmly adheres the virus particles to the complex structure of the corn tassel. The aminopolysaccharide is chitosan oligosaccharide; wherein the degree of deacetylation of chitosan oligosaccharide is ≥90%, the molecular weight is ≤3000 Da, and the degree of polymerization is 2-10; the protein-degrading enzyme is selected from at least one of serine proteases and metalloproteinases; and the enzyme activity is ≥100,000 U / g.

4. The cotton bollworm nucleopolyhedrovirus ear-stage targeted synergist according to claim 3, characterized in that, The mass ratio of the aminopolysaccharide to the protein-degrading enzyme is 1.5:1 to 2:

1.

5. The cotton bollworm nucleopolyhedrovirus ear-stage targeted synergist according to claim 1, characterized in that, The modified vegetable oil nonionic surfactant is selected from at least one of castor oil polyoxyethylene ether, Tween-80, and organosilicon surfactant, with an HLB value of 12-16 and a carbon chain length of C8-C10, namely APG0810. The system-compatible components include 2-8% antifreeze and 0.5-2% preservative; the antifreeze is selected from at least one of propylene glycol, glycerin, and ethylene glycol; the preservative is selected from at least one of sodium benzoate and potassium sorbate. The carrier is at least one of diatomaceous earth, pregelatinized starch, and precipitated silica, with a moisture content ≤5%, a fineness ≥95%, and passes through a 44 μm sieve.

6. A pesticide formulation for controlling bollworm in corn, characterized in that, It is prepared by combining the synergistic compound adjuvant as described in any one of claims 1-5 with cotton bollworm nucleopolyhedrovirus HaNPV at a mass ratio of 1:200-500.

7. The pesticide formulation for controlling bollworm in corn according to claim 6, characterized in that, The synergistic adjuvant was mixed with HaNPV at a certain mass ratio to prepare a HaNPV concentration of 1×10⁻⁶. 6 ~2×10 6 The spray solution with OBs / mL is applied by top-mounted spraying using an agricultural drone. The drone flies at an altitude of 2.5-3.5 meters, with a spray volume of 20-30 L / ha and a flight speed of 3-6 m / s. The targeted top spraying should be applied during the corn's tasseling stage to the early tasseling stage, specifically when 5-30% of the tassels have emerged.

8. The pesticide formulation for controlling bollworm in corn according to claim 7, characterized in that, The optimal time for targeted spraying from the top of the agricultural drone is between 16:00 and 19:00 on sunny evenings, with an ambient temperature of 20-28 ℃ and a wind speed of ≤3 m / s.

9. The pesticide formulation for controlling bollworm in corn according to claim 7, characterized in that, In the mixture of the synergist and HaNPV, the dilution factor of the synergist is 250-350 times; The drone is a single-rotor or multi-rotor agricultural drone, with a pressure or centrifugal nozzle and a droplet size controlled between 150-250 μm.

10. The use of the synergistic adjuvant according to any one of claims 1-5 in the preparation of a biological pesticide for controlling bollworm during the corn tasseling stage.

Images