Insect pathogenic nematode gel formulation for controlling cerambycid and carpenter moth and method for preparing the same

Abstract

The application discloses an insect pathogenic nematode gel preparation for preventing and treating cerambycids and woodborers and a preparation method thereof. Two types of high-affinity nematodes are obtained through in-vivo propagation of Galleria mellonella larvae, and the infection activity is improved through low-temperature activation of trehalose; a target attractant is prepared by compounding pine oil with cerambycid and woodborer larva excrement extract, which can guide the nematodes to migrate into deep tunnel of the trunk; the gel matrix is compounded by sodium alginate, sodium carboxymethyl cellulose and xanthan gum, and moisture-retention and anti-reversal agents and potassium sorbate preservatives are used in combination, so that the adhesion, stability and nematode cell protection effect of the gel are considered. Field tests show that the corrected control rate of the preparation on cerambycid and woodborer larvae can reach more than 85% in 15 days, the nematodes can invade the insect bodies in 24 hours and kill the pests in 48 hours; the preparation can be stored for 6 months in a cold storage at 2-8 DEG C, the survival rate of the nematodes is higher than 70% in 15 days at normal temperature, the gel is resistant to rainwater washing, and the nematodes can maintain the effect for more than 7 days in the forest.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for forest pests, and more specifically to a gel preparation of entomopathogenic nematodes for controlling longhorn beetles and wood-boring moths, and its preparation method. Background Technology

[0002] Longhorn beetles and wood-boring moths are serious trunk-boring pests that cause significant damage in forestry and landscaping. Their larvae hide inside tree trunks, boring and damaging the tree's vascular tissue, leading to tree decline, branch dieback, and in severe cases, the death of the entire tree. This results in substantial economic and ecological losses to forestry ecology and landscaping. Currently, the main control methods for these trunk-boring pests include chemical control, physical control, and biological control. However, all these methods have significant technical shortcomings, making it difficult to achieve efficient, green, and large-scale pest control.

[0003] Chemical control methods, such as trunk injection, fumigation of borer tunnels, and trunk application, offer advantages like rapid effectiveness and short-term pest control. However, they also present several problems: long-term application can lead to pesticide residues, environmental pollution, and harm to natural enemies, disrupting the ecological balance; it can also induce pesticide resistance in pests, resulting in a continuous decline in control efficacy. Furthermore, pesticides struggle to penetrate deep into the borer tunnels, offering limited protection against older larvae and leaving significant blind spots in pest control.

[0004] Physical control methods mainly include manual capture, light trapping, and felling and clearing of affected trees. They are ecologically safe, but are cumbersome to operate, have high labor costs, and low efficiency. They are only suitable for small-scale, low-density pest scenarios and cannot meet the needs of large-scale, contiguous trunk-boring pest control in forest areas, thus greatly limiting their large-scale application.

[0005] Biological control, with its advantages of being environmentally friendly, lacking drug resistance, and providing strong and sustained control, has become the mainstream research direction for the green control of stem-boring pests. Among them, entomopathogenic nematodes (EPNs), with *Nematoda stearothermiae* and *Heterobalacillus* as core dominant strains, can synergize with symbiotic bacteria, inducing septicemia and death in pests within 24–48 hours after infection. They possess strong active target-finding ability, a wide host range, and are safe for humans and animals, making them suitable for controlling the concealed damage caused by stem-boring pests. Nematodes can precisely infect insects through tunnels, frass holes, and spiracles, and can multiply and reproduce within the insect carcasses, continuously infecting new pests, thus possessing long-term pest control potential. Existing research has confirmed that superior nematode strains such as *Nematoda stearothermiae* A24 have excellent control efficacy against stem-boring pests such as the litchi wood-boring moth and the longhorn beetle.

[0006] To address the challenges of storing and transporting entomopathogenic nematodes and their low survival rates in the field, the industry has largely adopted formulation-based processes to prepare nematode preparations. Among these, Chinese patent document "CN102550606A" discloses a colloidal silica-based entomopathogenic nematode gel preparation and its preparation method. This method uses a colloidal silica solution with specific parameters as a carrier, combined with entomopathogenic nematodes and alcohol solvents to form a uniform and stable suspension gel system. By strictly controlling the solution pH, particle size, and ion concentration, and employing a low-temperature static curing process, the gel preparation can be prepared. The finished product can be stored at room temperature and applied directly or after dilution in the field.

[0007] However, this solution and traditional calcium alginate gel-based nematode formulations are both general-purpose formulations, not specifically optimized for the concealed tunneling characteristics of longhorn beetles and wood-boring moths, or for the complex forest environment. Therefore, they have significant technical shortcomings for controlling stem-boring pests, as detailed below: 1) Existing gel formulations do not contain targeted attractant components, and the compatible nematode strains have poor affinity with the larvae of borers and insufficient infectivity. The formulation development only focuses on the storage and transportation activity of nematodes and does not adapt to the special environment of the closed and hidden tunnels. Nematodes cannot accurately locate the target larvae in the deep tunnels, and the field control rate is generally less than 60%, which is difficult to meet the requirements of forestry control.

[0008] 2) Existing colloidal silica gels lack sufficient moisture retention and flexibility, while calcium alginate gels have weak mechanical strength. Furthermore, no moisturizing, adhesive, or stress-resistance additives have been added, and the gel pore structure is not optimized. The formulation is prone to dehydration and cracking at room temperature, leading to nematode inactivation. After application in the field, it exhibits poor adhesion to tree trunks and is easily washed away by rainwater. The nematode survival rate is less than 50% after 15 days of storage at room temperature, and less than 30% after 3 days of application in the forest, indicating a lack of long-term control capabilities.

[0009] 3) This scheme requires strict control of precise parameters such as matrix particle size, ion concentration, and pH, and requires long-term low-temperature static curing, which is complicated and energy-intensive.

[0010] In summary, the preparation of existing entomopathogenic nematode gel formulations is inadequate. Therefore, this invention is proposed. Summary of the Invention

[0011] The purpose of this invention is to address the problems mentioned in the background section by providing a gel formulation for controlling longhorn beetles and wood-boring moths, and its preparation method. This formulation is suitable for pest control on various plants affected by longhorn beetles and wood-boring moths, including London plane trees, ash trees, Chinese scholar trees, poplar trees, willow trees, pine trees, and fruit trees. It achieves efficient, green, and sustainable control of concealed borer pests, avoiding environmental pollution and pesticide resistance issues associated with chemical pesticides.

[0012] To achieve the above objectives, the present invention specifically adopts the following technical solution: A method for preparing an insect pathogenic nematode gel formulation for controlling longhorn beetles and wood-boring moths, comprising steps S1 to S4.

[0013] S1. Select the A11 strain of *Strombus stearothermiae* and the H06 strain of *Strombus heterotrophus*, and artificially culture them using *Hemibarbus thunbergii* larvae as live hosts. Collect L3 stage nematodes of the two types of nematodes. The artificial culture process involves a temperature of 22–25℃, a relative humidity of 85–90%, and a cycle of 7–10 days.

[0014] To ensure affinity and infectivity to longhorn beetle and wood-boring moth larvae, this invention uses the A11 strain of *Strombus stearothermiae* and the H06 strain of *Heterobacter spp.*

[0015] S2. After rinsing and purifying the L3 infective stage nematodes of the two types of nematodes, they are mixed evenly at a mass ratio of 1.5 to 2.5:1 to obtain mixed nematodes. Then, the mixed nematodes are activated to enhance their activity and ensure their infectivity. This hybrid nematode exhibits high affinity and strong infectivity for longhorn beetle and wood-boring moth larvae, rapidly invading their bodies and causing their death. It can also reproduce within the insect carcasses, creating a sustained control effect.

[0016] Further, the method for activating the mixed nematodes in S2 includes: Add trehalose solution to the mixed nematodes at an amount of 0.45–0.55% of the mass of the mixed nematodes, and activate at 20–22°C for 2–3 hours.

[0017] S3. Add the activated mixed nematodes to the gel matrix solution containing the targeting attractant, and stir at a stirring rate of 100-150 r / min for 5-8 min to obtain the entomopathogenic nematode gel.

[0018] Furthermore, the method for preparing the gel matrix solution containing the targeting attractant in S3 includes: The temperature of deionized water in a constant temperature water bath is controlled at 50-60℃. Sodium alginate, sodium carboxymethyl cellulose and xanthan gum are added in sequence with stirring to obtain a mixed solution. Sodium alginate crosslinks with calcium ions to form a gel framework, sodium carboxymethyl cellulose enhances the gel's adhesion and flexibility, and xanthan gum improves the gel's stability and moisturizing properties. The three work synergistically to solve the problems of poor stability and insufficient adhesion of existing gels. Add a moisturizing and stress-resistant agent and a preservative to the mixed solution by stirring, then cool to room temperature and let stand for 10-15 minutes to obtain a gel matrix solution; The targeting attractant was stirred and added to the gel matrix solution to obtain a gel matrix solution containing the targeting attractant.

[0019] Furthermore, the moisturizing and stress-resistant agent is a mixture of glycerin and trehalose. Glycerin enhances the moisturizing properties of the gel and reduces moisture loss, while trehalose protects the cell membrane of the nematodes and enhances their tolerance to high temperatures and dryness, thus solving the problem of low nematode survival rate. The preservative is potassium sorbate, which inhibits the growth of microorganisms in the gel, prevents gel deterioration, and extends the shelf life of the preparation.

[0020] Furthermore, the method for preparing the targeted attractant includes: Take the feces of longhorn beetle larvae and wood-boring moth larvae, add deionized water to the resulting feces mixture, soak it, filter to remove the residue, and obtain feces extract; Fecal extract and turpentine oil were mixed at a mass ratio of 3:1 and stirred evenly to obtain a targeted attractant.

[0021] It is made from extracts of longhorn beetle larvae excrement and wood-boring moth larvae excrement, mixed with turpentine oil. It can specifically attract longhorn beetle and wood-boring moth larvae, while guiding nematodes in the gel to move directionally into the tunnels, thereby improving the infection efficiency of nematodes.

[0022] Furthermore, the added deionized water is 5 times the mass of the fecal mixture, and the soaking time is 24 hours.

[0023] Further, sodium alginate, sodium carboxymethyl cellulose, and xanthan gum are added sequentially by stirring at a rate of 300–400 r / min.

[0024] Furthermore, the stirring rate for adding the moisturizing and stress-resistant agent and the preservative to the mixed solution is 300-400 r / min, and the stirring time is 15-20 min.

[0025] Furthermore, the stirring rate of the targeted attractant added to the gel matrix solution is 200-300 r / min, and the stirring time is 10-15 min.

[0026] Furthermore, in S3, the stirring rate for obtaining the entomopathogenic nematode gel is 100–150 r / min, and the stirring time is 5–8 min.

[0027] S4. Place the entomopathogenic nematode gel into a sealed bag and store it under cold storage to obtain the entomopathogenic nematode gel preparation.

[0028] As an inventive concept similar to the above-described technical solutions, this invention also claims protection for a gel formulation for controlling insect pathogenic nematodes such as longhorn beetles and wood-boring moths, prepared using any of the aforementioned preparation methods, and comprising the following components by mass percentage: 1.0–3.0% entomopathogenic nematodes, 10.0–18.0% gel matrix, 0.5–1.5% targeted attractant, 3.0–5.0% moisturizing and stress-resistant agent, 0.1–0.3% preservative and the balance being deionized water.

[0029] Preferably, the entomopathogenic nematode gel formulation comprises, by weight percentage, the following components: 1.5–2.0% entomopathogenic nematodes, 12.0–15.0% gel matrix, 0.8–1.2% targeted attractant, 3.5–4.5% moisturizing and stress-resistant agent, 0.15–0.25% preservative and the balance being deionized water.

[0030] The gel matrix is ​​composed of sodium alginate, sodium carboxymethyl cellulose and xanthan gum in a mass ratio of 5:3:2. The moisturizing and stress-relieving agent is a mixture of glycerin and trehalose in a mass ratio of 2:1.

[0031] The application method for entomopathogenic nematode gel preparations is as follows: When using the gel formulation of this invention, the gel formulation can be drawn into a syringe and injected into the entrance of the borer tunnel or the frass hole of the longhorn beetle or wood-boring moth. About 5 mL is inserted into each hole (adjusted according to the size of the borer tunnel). Under the guidance of the targeted attractant, the nematodes in the gel invade the borer tunnel and infect the larvae of the longhorn beetle and wood-boring moth, thereby achieving the purpose of prevention and control.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention selects the A11 strain of *Strombus steggii* and the H06 strain of *Heterobacter spp.* as active insect sources. Both types of nematodes have good affinity and infectivity for longhorn beetle and wood-boring moth larvae. At the same time, a targeted attractant is prepared by combining extracts of longhorn beetle and wood-boring moth larvae excrement with turpentine oil. This attractant can guide L3-stage nematodes to migrate into the trunk tunnels to locate target pests, effectively solving the shortcomings of existing formulations such as weak nematode targeting and low field control efficacy against trunk-boring pests, and improving long-term and continuous pest control capabilities. In addition, after infecting and killing pests, the nematodes can multiply and proliferate in the insect carcasses, continuously infecting new larvae, thus achieving long-term control in the forest. 2. This invention uses a ternary compound gel matrix of sodium alginate, sodium carboxymethyl cellulose, and xanthan gum to synergistically enhance the strength of the gel skeleton, the adhesion to tree trunks, and the moisture retention and flexibility; it is compounded with glycerin and trehalose compound moisturizing and stress-resistant agents, combined with potassium sorbate preservative components, which not only prevents the gel from dehydrating and cracking during room temperature storage and from being washed away by rainwater in the forest, but also protects the nematode cell membrane, enhances the nematode's tolerance to high temperature and dry environment, and improves the survival rate of nematodes during storage and in the forest. 3. This invention is prepared at room temperature throughout the entire process, without the need for strict ion concentration and pH control or long-term low-temperature static curing. The process parameters are mild and controllable, and the operation steps are simplified. The raw materials used, such as alginate, plant extracts, and polysaccharide adjuvants, are readily available and inexpensive, and the production energy consumption is low. It is suitable for industrial-scale mass production and can meet the needs of large-scale application of this invention to combat borers in forest areas. 4. All components of this invention are green and non-toxic. The nematodes specifically parasitize borers, posing no harm to humans, livestock, or natural enemies in the forest. There are no pesticide residues, and it will not induce pesticide resistance in pests. It replaces traditional chemical agents to achieve green control of longhorn beetles and wood-boring moths, reducing the risk of ecological and environmental pollution caused by chemical pesticides. Attached Figure Description

[0033] Figure 1 The present invention provides a flowchart of the preparation method; Figure 2 This is a bar chart showing the test data of the forest control effect of this invention; Figure 3 This is a bar chart showing the test data of the nematode storage survival performance of the present invention. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The materials and instruments used in the following embodiments are all commercially available.

[0035] In a first aspect, embodiments of the present invention provide a method for preparing a gel formulation of insect pathogenic nematodes for controlling longhorn beetles and wood-boring moths, such as... Figure 1 As shown, it includes steps S1 to S4.

[0036] S1. Select the A11 strain of *Strombus stearothermiae* and the H06 strain of *Strombus heterotrophus*, and artificially culture them using *Hemibarbus thunbergii* larvae as live hosts. Collect L3 stage nematodes of the two types of nematodes. The artificial culture process involves a temperature of 22–25℃, a relative humidity of 85–90%, and a cycle of 7–10 days.

[0037] S2. After rinsing and purifying the L3 stage infective nematodes of the two types of nematodes, they are mixed evenly at a mass ratio of 1.5 to 2.5:1 to obtain mixed nematodes, and then the mixed nematodes are activated.

[0038] Further, the method for activating the mixed nematodes in S2 includes: Add trehalose solution to the mixed nematodes at an amount of 0.45–0.55% of the mass of the mixed nematodes, and activate at 20–22°C for 2–3 hours.

[0039] S3. Add the activated mixed nematodes to the gel matrix solution containing the targeting attractant, and stir at a stirring rate of 100-150 r / min for 5-8 min to obtain the entomopathogenic nematode gel.

[0040] Furthermore, the method for preparing the gel matrix solution containing the targeting attractant in S3 includes: The temperature of deionized water in a constant temperature water bath is controlled at 50-60℃. Sodium alginate, sodium carboxymethyl cellulose and xanthan gum are added in sequence with stirring to obtain a mixed solution. Add a moisturizing and stress-resistant agent and a preservative to the mixed solution by stirring, then cool to room temperature and let stand for 10-15 minutes to obtain a gel matrix solution; The targeting attractant was stirred and added to the gel matrix solution to obtain a gel matrix solution containing the targeting attractant.

[0041] Furthermore, the moisturizing and stress-relieving agent is a mixture of glycerin and trehalose, and the preservative is potassium sorbate.

[0042] Furthermore, the method for preparing the targeted attractant includes: Take the feces of longhorn beetle larvae and wood-boring moth larvae, add deionized water to the resulting feces mixture, soak it, filter to remove the residue, and obtain feces extract; Fecal extract and turpentine oil were mixed at a mass ratio of 3:1 and stirred evenly to obtain a targeted attractant.

[0043] Furthermore, the added deionized water is 5 times the mass of the fecal mixture, and the soaking time is 24 hours.

[0044] Further, sodium alginate, sodium carboxymethyl cellulose, and xanthan gum are added sequentially by stirring at a rate of 300–400 r / min.

[0045] Furthermore, the stirring rate for adding the moisturizing and stress-resistant agent and the preservative to the mixed solution is 300-400 r / min, and the stirring time is 15-20 min.

[0046] Furthermore, the stirring rate of the targeted attractant added to the gel matrix solution is 200-300 r / min, and the stirring time is 10-15 min.

[0047] Furthermore, in S3, the stirring rate for obtaining the entomopathogenic nematode gel is 100–150 r / min, and the stirring time is 5–8 min.

[0048] S4. Place the entomopathogenic nematode gel into a sealed bag and store it under cold storage to obtain the entomopathogenic nematode gel preparation.

[0049] Secondly, embodiments of the present invention provide a gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths, prepared by the method described above, and comprising the following components by mass percentage: 1.0–3.0% entomopathogenic nematodes, 10.0–18.0% gel matrix, 0.5–1.5% targeted attractant, 3.0–5.0% moisturizing and stress-resistant agent, 0.1–0.3% preservative and the balance being deionized water.

[0050] The present invention will be further described below with reference to specific embodiments and comparative examples.

[0051] Example 1 This embodiment provides a method for preparing an insect-pathogenic nematode gel formulation for controlling longhorn beetles and wood-boring moths, comprising the following steps: S1 nematode propagation: The A11 strain of *Streptococcus stearothermiae* and the H06 strain of *Streptococcus heterotrophus* were selected and cultured artificially with *Hemibarbus thunbergii* larvae as live hosts. The culture temperature was 23.5℃, the relative humidity was 88%, and the culture period was 8.5 days. L3 stage nematodes of the two types of nematodes were collected. Purification and activation of S2 nematodes: Two types of L3 stage invasive nematodes were rinsed three times with sterile physiological saline to remove impurities and dead nematodes, and then mixed at a mass ratio of 2:1 to obtain mixed nematodes; 0.5% of the mass of trehalose solution was added to the mixed nematodes and activated at 21℃ for 2.5h. S3 prepared a gel matrix containing a targeted attractant and then prepared a nematode gel. (1) Gel matrix preparation: Deionized water was heated to 55°C in a water bath and sodium alginate, sodium carboxymethyl cellulose, and xanthan gum (mass ratio of 5:3:2) were added sequentially under stirring at 350 r / min to prepare a mixed solution; after stirring at 350 r / min for 18 min, a moisturizing and stress-resistant agent composed of glycerol and trehalose (mass ratio of 2:1) and a potassium sorbate preservative were added, and the solution was cooled to room temperature and allowed to stand for 12 min to obtain the gel matrix solution; (2) Preparation of targeted attractant: Take equal mass of longhorn beetle larvae feces and wood-boring moth larvae feces, add 5 times the total mass of feces deionized water and soak for 24h, filter to remove residue to obtain fecal extract; mix fecal extract with turpentine oil at a mass ratio of 3:1, stir at 250r / min for 12min to obtain targeted attractant; (3) Mixing and molding: Add the targeting attractant to the gel matrix solution by stirring at 250 r / min for 12 min; then add the activated mixed nematodes and stir at 120 r / min for 6 min to obtain the entomopathogenic nematode gel; S4 Dispensing and Storage: The obtained gel is sealed in bags and refrigerated at 2-8°C to obtain the entomopathogenic nematode gel preparation.

[0052] The formulation in this embodiment is composed of the following components by weight percentage: 1.8% entomopathogenic nematodes, 13.5% gel matrix, 1.0% targeting attractant, 4.0% moisturizing and stress-resistant agent, 0.2% potassium sorbate, and the remainder being deionized water.

[0053] Example 2 This embodiment provides a method for preparing an insect-pathogenic nematode gel formulation for controlling longhorn beetles and wood-boring moths, comprising the following steps: S1 nematode propagation: The A11 strain of *Streptococcus stearothermiae* and the H06 strain of *Streptococcus heterotrophus* were selected and cultured artificially with *Hemibarbus thunbergii* larvae as live hosts. The culture temperature was 22℃, the relative humidity was 85%, and the culture period was 7 days. L3 stage nematodes of the two types of nematodes were collected. Purification and activation of S2 nematodes: Two types of L3 stage infection nematodes were rinsed three times with sterile physiological saline to remove impurities and dead nematodes, and then mixed at a mass ratio of 1.5:1 to obtain mixed nematodes; 0.45% of the mass of trehalose solution was added to the mixed nematodes and activated at 20°C for 2 hours. S3 prepared a gel matrix containing a targeted attractant and then prepared a nematode gel. (1) Gel matrix preparation: Deionized water was heated to 50°C in a water bath and sodium alginate, sodium carboxymethyl cellulose, and xanthan gum (mass ratio of 5:3:2) were added sequentially under stirring at 300r / min to prepare a mixed solution; after stirring at 300r / min for 15min, a moisturizing and stress-resistant agent composed of glycerol and trehalose (mass ratio of 2:1) and a potassium sorbate preservative were added, and the solution was cooled to room temperature and allowed to stand for 10min to obtain the gel matrix solution; (2) Preparation of targeted attractant: Take equal mass of longhorn beetle larvae feces and wood-boring moth larvae feces, add 5 times the total mass of feces deionized water and soak for 24h, filter to remove residue to obtain fecal extract; mix fecal extract with turpentine oil at a mass ratio of 3:1, stir at 200r / min for 10min to obtain targeted attractant; (3) Mixing and molding: Stir at 200 r / min for 10 min and add the targeting attractant to the gel matrix solution; then add the activated mixed nematodes and stir at 100 r / min for 5 min to obtain the entomopathogenic nematode gel; S4 Dispensing and Storage: The obtained gel is sealed in bags and refrigerated at 2-8°C to obtain the entomopathogenic nematode gel preparation.

[0054] The formulation in this embodiment is composed of the following components by weight percentage: 1.0% entomopathogenic nematodes, 10.0% gel matrix, 0.5% targeting attractant, 3.0% moisturizing and stress-resistant agent, 0.1% potassium sorbate, and the remainder being deionized water.

[0055] Example 3 This embodiment provides a method for preparing an insect-pathogenic nematode gel formulation for controlling longhorn beetles and wood-boring moths, comprising the following steps: S1 nematode propagation: The A11 strain of *Streptococcus stearothermiae* and the H06 strain of *Heterobacter spp.* were selected and artificially cultured using *Heterobacter spp.* larvae as live hosts. The culture temperature was 25℃, the relative humidity was 90%, and the culture period was 10 days. L3 stage nematodes of the two types of nematodes were collected. Purification and activation of S2 nematodes: Two types of L3 stage infection nematodes were rinsed three times with sterile physiological saline to remove impurities and dead nematodes, and then mixed at a mass ratio of 2.5:1 to obtain mixed nematodes; 0.55% of the mass of trehalose solution was added to the mixed nematodes and activated at 22℃ for 3 hours. S3 prepared a gel matrix containing a targeted attractant and then prepared a nematode gel. (1) Gel matrix preparation: Deionized water was heated to 60°C in a water bath and sodium alginate, sodium carboxymethyl cellulose, and xanthan gum (mass ratio of the three is 5:3:2) were added sequentially under stirring at 400r / min to prepare a mixed solution; after stirring at 400r / min for 20min, a moisturizing and stress-resistant agent composed of glycerol and trehalose (mass ratio of 2:1) and a potassium sorbate preservative were added, and the solution was cooled to room temperature and allowed to stand for 15min to obtain the gel matrix solution; (2) Preparation of targeted attractant: Take equal mass of longhorn beetle larvae feces and wood-boring moth larvae feces, add 5 times the total mass of feces deionized water and soak for 24h, filter to remove residue to obtain fecal extract; mix fecal extract with turpentine oil at a mass ratio of 3:1, stir at 300r / min for 15min to obtain targeted attractant; (3) Mixing and molding: Stir at 300 r / min for 15 min and add the targeting attractant to the gel matrix solution; then add the activated mixed nematodes and stir at 150 r / min for 8 min to obtain the entomopathogenic nematode gel; S4 Dispensing and Storage: The obtained gel is sealed in bags and refrigerated at 2-8°C to obtain the entomopathogenic nematode gel preparation.

[0056] The formulation in this embodiment is composed of the following components by weight percentage: 3.0% entomopathogenic nematodes, 18.0% gel matrix, 1.5% targeting attractant, 5.0% moisturizing and stress-resistant agent, 0.3% potassium sorbate, and the remainder being deionized water.

[0057] Comparative Example 1 (Primary Technology Comparison) The gel-based silica-based worm pathogen nematode gel preparation is prepared according to the process disclosed in the existing technology "CN102550606A". It uses a single common nematode, without adding targeted attractant components, ternary composite gel matrix, and glycerol-trehalose stress resistance system. The preparation process includes a low-temperature static curing step of 5-15℃.

[0058] Comparative Example 2 (Blank Control without Targeted Attractant) The difference between this comparative example and Example 1 is that no targeting inducer was added when preparing the gel matrix.

[0059] Comparative Example 3 (Single nematode control) The difference between this comparative example and Example 1 is that only the A11 strain of *Streptococcus stearothermiae* was used, without the addition of the H06 strain of *Streptococcus heterotrophus*.

[0060] The following tests were conducted on the products prepared in each embodiment and comparative example, and all test conditions were uniform.

[0061] 1. Forest control efficacy test Experimental methods: Injection into borer tunnels, forest plot experiment, and corrected mortality rates of longhorn beetle and wood-boring moth larvae were calculated 15 days after application; The test results are shown in Table 1: Table 1. Test data on forest control effectiveness From Table 1 and Figure 2 It can be seen that the control rate of Examples 1 to 3 is ≥85%, and the larvae can invade the insect body within 24 hours and kill the larvae within 48 hours; the control efficacy of Comparative Example 1 is less than 60%, and the onset of action and mortality speed are significantly delayed. Comparative Example 2 lacks the targeted attractant component, and Comparative Example 3 contains only a single nematode; the infection rate and final control efficacy of both are significantly lower than those of all Examples.

[0062] 2. Nematode storage survival performance test The test results for three groups of indicators—low-temperature refrigeration, short-term storage at room temperature, and adhesion retention on forest trunks—are shown in Table 2. Table 2. Test data on the storage survival performance of nematodes. From Table 2 and Figure 3 It can be seen that the survival rate of Examples 1-3 after 6 months of refrigeration is ≥80%, and the survival rate after 15 days at room temperature is ≥70%; the storage performance of Comparative Example 1 is significantly deteriorated. The gel matrix and moisturizing and stress-resistant system of Comparative Examples 2 and 3 are the same as those of the Examples, and the storage survival rate is close to that of the Examples; the adhesion rate of Examples 1-3 and Comparative Examples 2 and 3 are all over 90%, which can resist the washing away of heavy rain, and the nematodes survive in the forest for 7 days; the gel of Comparative Example 1 is easy to fall off, and a large number of nematodes are inactive after 3 days.

[0063] 3. Comparison test of raw material cost and production cycle Using the raw material cost of Comparative Example 1 as a baseline of 1.00, the raw material costs and preparation cycles for each group are shown in Table 3: Table 3. Statistics on raw material costs and preparation cycles for each group As shown in Table 3, Comparative Example 1 uses high-cost nano-colloidal silica and a special alcohol solvent, and requires a low-temperature curing process, resulting in high cost and long cycle. Examples 1-3 of this invention all use conventional polysaccharides and alginate raw materials, reducing raw material costs by more than 30%, and are processed at room temperature throughout, increasing production efficiency by more than 50%. Comparative Examples 2 and 3 only reduced the attractant or the number of nematode species; the raw materials and processes were basically the same as the examples, with no significant difference in cost and cycle.

Claims

1. A method for preparing a gel formulation of entomopathogenic nematodes for controlling longhorn beetles and wood-boring moths, characterized in that, Includes the following steps: S1. Select the A11 strain of *Strombus stearothermiae* and the H06 strain of *Strombus heterotrophus*, and artificially culture them using *Hemibarbus thunbergii* larvae as live hosts. Collect L3 stage nematodes of the two types of nematodes. The artificial culture process involves a temperature of 22–25℃, a relative humidity of 85–90%, and a cycle of 7–10 days. S2. After rinsing and purifying the L3 stage infection stage nematodes of the two types of nematodes, they are mixed evenly at a mass ratio of 1.5 to 2.5:1 to obtain mixed nematodes, and then the mixed nematodes are activated. S3. Add the activated mixed nematodes to the gel matrix solution containing the targeting attractant, and stir at a stirring rate of 100-150 r / min for 5-8 min to obtain the entomopathogenic nematode gel. S4. Place the entomopathogenic nematode gel into a sealed bag and store it under cold storage to obtain the entomopathogenic nematode gel preparation.

2. The method for preparing the gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths as described in claim 1, characterized in that, The method for activating the mixed nematodes in S2 includes: Add trehalose solution to the mixed nematodes at an amount of 0.45–0.55% of the mass of the mixed nematodes, and activate at 20–22°C for 2–3 hours.

3. The method for preparing the gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths as described in claim 1, characterized in that, The preparation method of the gel matrix solution containing the targeting attractant in S3 includes: The temperature of deionized water in a constant temperature water bath is controlled at 50-60℃. Sodium alginate, sodium carboxymethyl cellulose and xanthan gum are added in sequence with stirring to obtain a mixed solution. Add a moisturizing and stress-resistant agent and a preservative to the mixed solution by stirring, then cool to room temperature and let stand for 10-15 minutes to obtain a gel matrix solution; The targeting attractant was stirred and added to the gel matrix solution to obtain a gel matrix solution containing the targeting attractant.

4. The method for preparing the gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths as described in claim 3, characterized in that, The moisturizing and stress-relieving agent is a mixture of glycerin and trehalose, and the preservative is potassium sorbate.

5. The method for preparing the gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths as described in claim 3, characterized in that, The preparation method of the targeted attractant includes: Take the feces of longhorn beetle larvae and wood-boring moth larvae, add deionized water to the resulting feces mixture, soak it, filter to remove the residue, and obtain feces extract; Fecal extract and turpentine oil were mixed at a mass ratio of 3:1 and stirred evenly to obtain a targeted attractant.

6. The method for preparing the gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths as described in claim 5, characterized in that, The amount of deionized water added is 5 times the mass of the fecal mixture, and the soaking time is 24 hours.

7. The method for preparing the gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths as described in claim 3, characterized in that, Sodium alginate, sodium carboxymethyl cellulose, and xanthan gum were added sequentially while stirring at a rate of 300–400 r / min.

8. The method for preparing the gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths as described in claim 3, characterized in that, Add the moisturizing and stress-resistant agent and preservative to the mixed solution by stirring at a rate of 300-400 r / min for 15-20 min.

9. The method for preparing the gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths as described in claim 3, characterized in that, The targeted attractant was added to the gel matrix solution at a stirring rate of 200–300 r / min for 10–15 min.

10. A gel formulation for controlling entomopathogenic nematodes of longhorn beetles and wood-boring moths, characterized in that, Prepared by the preparation method according to any one of claims 1 to 9, by mass percentage, Includes the following components: 1.0–3.0% entomopathogenic nematodes, 10.0–18.0% gel matrix, 0.5–1.5% targeted attractant, 3.0–5.0% moisturizing and stress-resistant agent, 0.1–0.3% preservative and the balance being deionized water.

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