A pine wood nematode disease prevention and treatment agent and a preparation method thereof

Abstract

The present application relates to nematode disease prevention and treatment agent technical field, especially to a kind of pine wood nematode disease prevention and treatment agent and preparation method thereof, by weight parts, including the following components: sophocarpidine 10-30 parts;Azadirachtin 5-20 parts;Turpentine 40-70 parts;Emulsifier 5-15 parts;Efficiency agent 1-5 parts;Stabilizer 0.5-3 parts, the synergistic effect of sophocarpidine and azadirachtin constitutes the core of the present application;Quinolone ring and pyridine ring structure in sophocarpidine molecule endow it with strong insecticidal activity, mainly by inhibiting acetylcholinesterase to interfere with the nerve conduction of nematode;And the complex triterpenoid structure of azadirachtin can interfere with the hormone system of nematode, inhibit its growth and development and reproduction.The two different mechanisms of action form complementation at molecular level, both can quickly kill nematode, and can long-term inhibit its population growth;Second, turpentine as natural terpene compound, the permeability of other active ingredients is enhanced.

Description

Technical Field

[0001] This invention relates to the field of nematode control agents, and in particular to a pine wilt disease control agent and its preparation method. Background Technology

[0002] Pine wilt disease is a catastrophic disease that seriously threatens the survival of pine trees. Caused by the pine wilt nematode (Bursaphelenchus xylophilus), it has caused enormous ecological and economic losses in many countries and regions worldwide. Traditional control methods mainly include chemical pesticide spraying, trunk injection, and biological control; however, each of these methods has its own limitations.

[0003] While chemical pesticide spraying offers rapid results, it often suffers from short-lasting effects, environmental pollution, and significant harm to non-target organisms. Furthermore, frequent use of single-component pesticides can lead to resistance in pine wilt nematodes, reducing their effectiveness. Trunk injection, though delivering pesticides directly into the tree, is largely limited by single-component formulations, hindering long-lasting control. Biological control methods, while environmentally friendly, are often inconsistent in their effectiveness and difficult to implement in large-scale forests.

[0004] In recent years, plant-derived pesticides have received widespread attention due to their low toxicity, low residue, and environmental friendliness. However, single-plant-derived pesticide components often suffer from drawbacks such as insufficient activity and easy degradation, making it difficult to meet actual control needs. Therefore, developing a highly efficient, long-lasting, and environmentally friendly pesticide for controlling pine wilt disease has become an urgent need in the field of forestry plant protection. Summary of the Invention

[0005] This invention aims to address the problems of poor efficacy, poor sustainability, and high environmental risk in existing pine wilt disease control technologies. By innovatively optimizing and combining multiple plant-derived components such as matrine, azadirachtin, and turpentine, and employing a refined preparation process, this invention has successfully developed a multi-mechanism, low-dose, highly effective, long-lasting, and environmentally friendly pine wilt disease control agent.

[0006] The purpose of this invention is to provide a pine wilt disease control agent, which, by weight, comprises the following components:

[0007] Matrine 10-30 parts;

[0008] Azadirachtin 5-20 parts;

[0009] 40-70 parts turpentine;

[0010] 5-15 parts emulsifier;

[0011] Synergist 1-5 parts;

[0012] Stabilizer 0.5-3 parts.

[0013] Preferably, the emulsifier is polyoxyethylene castor oil.

[0014] Preferably, the synergist is piperonyl butyl ether.

[0015] Preferably, the stabilizer is butylated hydroxytoluene.

[0016] Preferably, the purity of matrine is ≥98%, and the purity of azadirachtin is ≥95%.

[0017] Preferably, the turpentine oil contains 30-45% α-pinene, 15-25% β-pinene, and 5-10% Δ3-carene.

[0018] A method for preparing the aforementioned pine wilt disease control agent includes the following steps:

[0019] (1) First, add 40-70 parts of turpentine oil to the reactor and stir at 60-70°C;

[0020] (2) Next, slowly add 5-15 parts of polyoxyethylene castor oil and stir well;

[0021] (3) Then, dissolve 10-30 parts of matrine in 2-3 times the weight of matrine in ethanol, slowly add it to the reaction vessel, and stir for 30-60 minutes.

[0022] (4) Next, dissolve 5-20 parts of azadirachtin in 2-3 times the weight of azadirachtin in ethanol, slowly add it to the reaction vessel, and stir for 30-60 minutes.

[0023] (5) Next, add 1-5 parts of piperine butyl ether and stir for 15-30 minutes;

[0024] (6) Then, add 0.5-3 parts of butylated hydroxytoluene and stir until homogeneous;

[0025] (7) Further, remove ethanol by vacuum distillation at 70-80℃ until no more ethanol is distilled off;

[0026] (8) Finally, cool to room temperature, filter to remove insoluble matter, fill the filtrate into a sealed container and store it away from light.

[0027] Preferably, the reactor is equipped with a stirrer, a thermometer, and a condenser.

[0028] Preferably, the vacuum distillation in step (7) is carried out at a pressure of 50-100 mmHg.

[0029] A method of using the aforementioned pine wilt disease control agent includes the following steps:

[0030] (1) Dilute the medicine with an equal volume of water;

[0031] (2) Drill a hole with a diameter of 0.5-1 cm and a depth of 3-5 cm every 20-30 cm on the main trunk of the pine tree, 0.5-1.5 meters above the ground;

[0032] (3) Inject 20-50 ml of diluted drug solution into each well;

[0033] (4) After injection, seal the injection hole with wax;

[0034] (5) Apply during the early stage of pine wilt disease or during the prevention period, once each in spring and autumn.

[0035] From the perspective of chemical mechanism, the innovation of this invention is mainly reflected in the following aspects:

[0036] First, the synergistic effect of matrine and azadirachtin constitutes the core of this invention. The quinolone and pyridine ring structures in the matrine molecule endow it with potent insecticidal activity, mainly by interfering with the nerve conduction of nematodes through the inhibition of acetylcholinesterase. Meanwhile, the complex triterpenoid structure of azadirachtin can interfere with the nematode's hormone system, inhibiting its growth, development, and reproduction. These two different mechanisms of action complement each other at the molecular level, enabling both rapid killing of nematodes and long-term inhibition of their population growth.

[0037] Secondly, turpentine oil, as a natural terpene compound, not only acts as a solvent and carrier, but also enhances the permeability of other active ingredients through its unique molecular structure. The cyclic structures of major components such as α-pinene and β-pinene facilitate the diffusion and sustained release of the pesticide within the tree, thereby prolonging its efficacy.

[0038] Furthermore, the addition of the synergist piperityl butyl ether further enhances the overall efficacy of the formulation. Piperityl butyl ether slows the metabolism of the active ingredient by inhibiting the cytochrome P450 enzyme system in nematodes, thereby prolonging the duration of action. This molecular-level interaction demonstrates the thoughtful formulation design of this invention.

[0039] Finally, the addition of the stabilizer butylated hydroxytoluene significantly improved the storage stability of the drug. Its antioxidant effect effectively prevents the oxidative degradation of the active ingredient during storage, ensuring the long-term effectiveness of the drug from a molecular perspective.

[0040] The technical solution of this invention not only solves the problems in the prior art, but also brings a number of unexpected beneficial effects:

[0041] 1. Low dosage and high efficiency: Through the synergistic effect of multiple components, this invention achieves better prevention and control effects with lower dosage of active ingredients, which not only reduces costs but also minimizes the impact on the environment.

[0042] 2. Long-lasting effect: The carefully designed formula and preparation process enable the active ingredients to be slowly released into the tree, achieving a continuous control effect for up to 6 months and significantly reducing the frequency of application.

[0043] 3. Excellent environmental friendliness: Experimental results show that the invention has minimal impact on non-target organisms, demonstrating high selectivity, which makes it possible to apply in environmentally sensitive areas.

[0044] 4. Promotes plant growth: It was unexpectedly discovered that this invention not only did not inhibit the growth of pine trees, but also slightly promoted the growth and photosynthesis of trees. This effect provides a new approach to forest health management.

[0045] 5. Excellent stability: The formulation of this invention exhibits excellent stability under various storage conditions, which not only extends the shelf life of the product but may also reduce transportation and storage costs.

[0046] 6. Innovation in preparation process: The refined preparation process of this invention plays a key role in realizing the synergistic effect of multiple components, which provides a new paradigm for the preparation technology of agricultural and forestry pesticides.

[0047] In summary, this invention, through innovative formulation design and preparation process, has successfully developed a highly efficient, long-lasting, and environmentally friendly agent for the control of pine wilt disease. This not only solves the problems of existing technologies but also brings several unexpected beneficial effects. This achievement provides new technical support for the integrated management of pine wilt disease and also offers new ideas and possibilities for the development of agricultural and forestry pest and disease control. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0049] Example 1: A pesticide for controlling pine wilt disease and its preparation method

[0050] This embodiment provides a control agent for pine wilt disease, comprising the following components by weight: 10 parts matrine, 5 parts azadirachtin, 70 parts turpentine oil, 5 parts emulsifier, 1 part synergist, and 0.5 parts stabilizer. The emulsifier is polyoxyethylene castor oil, the synergist is piperonyl butyl ether, and the stabilizer is butylated hydroxytoluene.

[0051] Preferably, in embodiments of the present invention, the purity of matrine is 98%, and the purity of azadirachtin is 95%. The turpentine oil contains 45% α-pinene, 15% β-pinene, and 5% Δ3-carene. Matrine exerts its insecticidal effect by inhibiting acetylcholinesterase in nematodes, while azadirachtin inhibits the growth and reproduction of nematodes by interfering with their hormone system; the combined effect of both significantly reduces the risk of nematodes developing resistance.

[0052] In this embodiment, the preparation method of the pine wilt disease control agent includes the following steps:

[0053] First, 70 parts of turpentine oil were added to a reactor equipped with a stirrer, thermometer, and condenser, and stirred at 60°C. Next, 5 parts of polyoxyethylene castor oil were slowly added and stirred until homogeneous. Then, 10 parts of matrine were dissolved in 20 parts of ethanol and slowly added to the reactor, stirring for 30 minutes. Next, 5 parts of azadirachtin were dissolved in 10 parts of ethanol and slowly added to the reactor, stirring for 30 minutes. Then, 1 part of piperine butyl ether was added and stirred for 15 minutes. Subsequently, 0.5 parts of butylated hydroxytoluene were added and stirred until homogeneous. Further, the ethanol was removed by vacuum distillation at 70°C and 50 mmHg until no more ethanol distilled off. Finally, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was bottled into a sealed container and stored away from light.

[0054] Example 2: A pesticide for controlling pine wilt disease and its preparation method

[0055] This embodiment provides a control agent for pine wilt disease, comprising the following components by weight: 20 parts matrine, 12.5 parts azadirachtin, 55 parts turpentine oil, 10 parts emulsifier, 3 parts synergist, and 1.75 parts stabilizer. The emulsifier is polyoxyethylene castor oil, the synergist is piperonyl butyl ether, and the stabilizer is butylated hydroxytoluene.

[0056] Preferably, in the embodiments of the present invention, the purity of matrine is 99%, and the purity of azadirachtin is 97%. The turpentine oil contains 37.5% α-pinene, 20% β-pinene, and 7.5% Δ3-carene. Turpentine oil not only acts as a solvent but also enhances the permeability of matrine and azadirachtin, improving their distribution and utilization within the pine tree. Piperyl butyl ether, as a synergist, can inhibit the cytochrome P450 enzyme system in nematodes, further enhancing the efficacy.

[0057] In this embodiment, the preparation method of the pine wilt disease control agent includes the following steps:

[0058] First, 55 parts of turpentine oil were added to a reactor equipped with a stirrer, thermometer, and condenser, and stirred at 65°C. Next, 10 parts of polyoxyethylene castor oil were slowly added and stirred until homogeneous. Then, 20 parts of matrine were dissolved in 50 parts of ethanol and slowly added to the reactor, stirring for 45 minutes. Next, 12.5 parts of azadirachtin were dissolved in 30 parts of ethanol and slowly added to the reactor, stirring for 45 minutes. Then, 3 parts of piperine butyl ether were added and stirred for 22 minutes. Subsequently, 1.75 parts of butylated hydroxytoluene were added and stirred until homogeneous. Further, the ethanol was removed by vacuum distillation at 75°C and 75 mmHg until no more ethanol distilled off. Finally, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was bottled into a sealed container and stored away from light.

[0059] Example 3: A pesticide for controlling pine wilt disease and its preparation method

[0060] This embodiment provides a control agent for pine wilt disease, comprising the following components by weight: 30 parts matrine, 20 parts azadirachtin, 40 parts turpentine oil, 15 parts emulsifier, 5 parts synergist, and 3 parts stabilizer. The emulsifier is polyoxyethylene castor oil, the synergist is piperonyl butyl ether, and the stabilizer is butylated hydroxytoluene.

[0061] Preferably, in the embodiments of the present invention, the purity of matrine is 100%, and the purity of azadirachtin is 99%. The turpentine oil contains 30% α-pinene, 25% β-pinene, and 10% Δ3-carene. Azadirachtin has a sustained-release effect, which can prolong the shelf life of the agent. Butylated hydroxytoluene, as an antioxidant, can improve the stability and shelf life of the agent. This formulation combination is not only effective against pine wilt nematodes, but may also have a certain control effect on other pests that harm pine trees.

[0062] In this embodiment, the preparation method of the pine wilt disease control agent includes the following steps:

[0063] First, 40 parts of turpentine oil were added to a reaction vessel equipped with a stirrer, thermometer, and condenser, and stirred at 70°C. Next, 15 parts of polyoxyethylene castor oil were slowly added and stirred until homogeneous. Then, 30 parts of matrine were dissolved in 90 parts of ethanol and slowly added to the reaction vessel, stirring for 60 minutes. Next, 20 parts of azadirachtin were dissolved in 60 parts of ethanol and slowly added to the reaction vessel, stirring for 60 minutes. Then, 5 parts of piperine butyl ether were added and stirred for 30 minutes. Subsequently, 3 parts of butylated hydroxytoluene were added and stirred until homogeneous. Further, the ethanol was removed by vacuum distillation at 80°C and 100 mmHg until no more ethanol distilled off. Finally, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was bottled into a sealed container and stored away from light.

[0064] Example 4: A pesticide for controlling pine wilt disease and its preparation method

[0065] This embodiment provides a control agent for pine wilt disease, comprising the following components by weight: 15 parts matrine, 10 parts azadirachtin, 60 parts turpentine oil, 7.5 parts emulsifier, 2 parts synergist, and 1 part stabilizer. The emulsifier is polyoxyethylene castor oil, the synergist is piperonyl butyl ether, and the stabilizer is butylated hydroxytoluene.

[0066] Preferably, in the embodiments of the present invention, the purity of matrine is 98.5%, and the purity of azadirachtin is 96%. The turpentine oil contains 35% α-pinene, 22% β-pinene, and 8% Δ3-carene. This formulation, by adjusting the proportions of each component, can reduce costs and environmental risks while ensuring control efficacy. The use of plant-derived pesticide components reduces negative impacts on the environment and non-target organisms, demonstrating the environmentally friendly nature of the present invention.

[0067] In this embodiment, the preparation method of the pine wilt disease control agent includes the following steps:

[0068] First, 60 parts of turpentine oil were added to a reactor equipped with a stirrer, thermometer, and condenser, and stirred at 67°C. Next, 7.5 parts of polyoxyethylene castor oil were slowly added and stirred until homogeneous. Then, 15 parts of matrine were dissolved in 37.5 parts of ethanol and slowly added to the reactor, stirring for 40 minutes. Next, 10 parts of azadirachtin were dissolved in 25 parts of ethanol and slowly added to the reactor, stirring for 40 minutes. Then, 2 parts of piperine butyl ether were added and stirred for 20 minutes. Subsequently, 1 part of butylated hydroxytoluene was added and stirred until homogeneous. Further, the solution was distilled under reduced pressure at 77°C and 85 mmHg to remove ethanol until no more ethanol distilled off. Finally, the solution was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was bottled into a sealed container and stored away from light.

[0069] The application method of the pine wilt disease control agent described in Examples 1-4 includes the following steps:

[0070] (1) Dilute the medicine with an equal volume of water;

[0071] (2) Drill a hole with a diameter of 0.5-1 cm and a depth of 3-5 cm every 20-30 cm on the main trunk of the pine tree, 0.5-1.5 meters above the ground;

[0072] (3) Inject 20-50 ml of diluted drug solution into each well;

[0073] (4) After injection, seal the injection hole with wax;

[0074] (5) Apply during the early stage of pine wilt disease or during the prevention period, once each in spring and autumn.

[0075] Comparative Example 1: Matrine-only formulation

[0076] This comparative example aims to verify the synergistic effect of the multi-component formulation in Example 1. The formulation consists of: 10 parts matrine, 80 parts turpentine oil, and 5 parts emulsifier. The emulsifier is polyoxyethylene castor oil.

[0077] The preparation method is similar to that of Example 1, but only matrine is added, omitting the steps of adding azadirachtin, synergist, and stabilizer. The specific steps include: First, adding 80 parts of turpentine oil to a reaction vessel and stirring at 60°C. Second, slowly adding 5 parts of polyoxyethylene castor oil and stirring until homogeneous. Then, dissolving 10 parts of matrine in 20 parts of ethanol and slowly adding it to the reaction vessel, stirring for 30 minutes. Finally, removing the ethanol by vacuum distillation at 70°C and 50 mmHg, followed by cooling, filtration, and filling.

[0078] This comparative example lacks the synergistic effect of azadirachtin, and therefore cannot reduce the risk of nematode resistance through multiple mechanisms of action. Furthermore, due to the absence of synergists and stabilizers, its efficacy and durability are expected to be inferior to those of Example 1.

[0079] Comparative Example 2: Azadirachtin-based formulation

[0080] This comparative example aims to verify the synergistic effect of the multi-component formulation in Example 2. The formulation consists of: 12.5 parts azadirachtin, 77.5 parts turpentine oil, and 10 parts emulsifier. The emulsifier is polyoxyethylene castor oil.

[0081] The preparation method is similar to that of Example 2, but only azadirachtin is added, omitting the steps of adding matrine, synergist, and stabilizer. The specific steps include: First, adding 77.5 parts of turpentine oil to a reaction vessel and stirring at 65°C. Second, slowly adding 10 parts of polyoxyethylene castor oil and stirring until homogeneous. Then, dissolving 12.5 parts of azadirachtin in 30 parts of ethanol and slowly adding it to the reaction vessel, stirring for 45 minutes. Finally, removing the ethanol by vacuum distillation at 75°C and 75 mmHg, followed by cooling, filtration, and filling.

[0082] This comparative example lacks the rapid insecticidal effect of matrine, and its control efficacy is expected to be lower than that of Example 2. Furthermore, the lack of synergists and stabilizers may affect its efficacy and stability.

[0083] Comparative Example 3: Formula without synergist

[0084] This comparative example aims to verify the effect of the synergist in Example 3. The formulation consists of: 30 parts matrine, 20 parts azadirachtin, 45 parts turpentine oil, 15 parts emulsifier, and 3 parts stabilizer. The emulsifier is polyoxyethylene castor oil, and the stabilizer is butylated hydroxytoluene.

[0085] The preparation method is similar to that of Example 3, but the step of adding the synergist is omitted. The specific steps include: First, adding 45 parts of turpentine oil to a reaction vessel and stirring at 70°C. Second, slowly adding 15 parts of polyoxyethylene castor oil and stirring until homogeneous. Then, dissolving 30 parts of matrine in 90 parts of ethanol and slowly adding it to the reaction vessel, stirring for 60 minutes. Next, dissolving 20 parts of azadirachtin in 60 parts of ethanol and slowly adding it to the reaction vessel, stirring for 60 minutes. Finally, adding 3 parts of butylated hydroxytoluene, removing the ethanol by vacuum distillation at 80°C and 100 mmHg, cooling, filtering, and filling.

[0086] This comparative example lacks the role of piperitylbutyrate as a synergist, and its control effect is expected to be less than that of Example 3. The efficacy may be reduced due to the absence of a synergist that inhibits the cytochrome P450 enzyme system in nematodes.

[0087] Comparative Example 4: Stabilizer-free formulation

[0088] This comparative example aims to verify the effect of the stabilizer in Example 4. The formulation consists of: 15 parts matrine, 10 parts azadirachtin, 61 parts turpentine oil, 7.5 parts emulsifier, and 2 parts synergist. The emulsifier is polyoxyethylene castor oil, and the synergist is piperonyl butyl ether.

[0089] The preparation method is similar to that of Example 4, but the step of adding the stabilizer is omitted. The specific steps include: First, adding 61 parts of turpentine oil to a reaction vessel and stirring at 67°C. Second, slowly adding 7.5 parts of polyoxyethylene castor oil and stirring until homogeneous. Then, dissolving 15 parts of matrine in 37.5 parts of ethanol and slowly adding it to the reaction vessel, stirring for 40 minutes. Next, dissolving 10 parts of azadirachtin in 25 parts of ethanol and slowly adding it to the reaction vessel, stirring for 40 minutes. Finally, adding 2 parts of piperonyl butyl ether, removing the ethanol by vacuum distillation at 77°C and 85 mmHg, cooling, filtering, and filling.

[0090] This comparative example lacks the role of butylated hydroxytoluene as a stabilizer, and its stability and shelf life are expected to be inferior to those of Example 4. Due to the absence of an antioxidant, the agent may be more susceptible to oxidation during storage, affecting its long-term efficacy.

[0091] Comparative Example 5: High-dose single-component formulation

[0092] This comparative example aims to verify the superiority of the multi-component, low-dose formulation in Example 1. The formulation consists of: 40 parts matrine, 55 parts turpentine oil, and 5 parts emulsifier. The emulsifier is polyoxyethylene castor oil.

[0093] The preparation method is similar to that of Example 1, but only a high dose of matrine is used. The specific steps include: First, adding 55 parts of turpentine oil to a reaction vessel and stirring at 60°C. Second, slowly adding 5 parts of polyoxyethylene castor oil and stirring until homogeneous. Then, dissolving 40 parts of matrine in 80 parts of ethanol and slowly adding it to the reaction vessel, stirring for 60 minutes. Finally, removing the ethanol by vacuum distillation at 70°C and 50 mmHg, followed by cooling, filtration, and filling.

[0094] This comparative study used high doses of a single active ingredient, which may show strong insecticidal effects in the short term, but lacks multiple mechanisms of action and may more easily lead to resistance in nematodes. Furthermore, high-dose use may increase the impact on non-target organisms and environmental risks.

[0095] Comparative Example 6: Conventional Mixed Formulation

[0096] This comparative example aims to verify the effect of the unique preparation process of this invention on the efficacy. The formulation composition is the same as in Example 2, but a simple mixing preparation method is used.

[0097] The preparation method includes the following steps: First, 55 parts of turpentine oil, 10 parts of polyoxyethylene castor oil, 20 parts of matrine, 12.5 parts of azadirachtin, 3 parts of piperine butyl ether, and 1.75 parts of butylated hydroxytoluene are added sequentially to a mixing container. Then, the mixture is stirred at room temperature for 30 minutes until homogeneous. Finally, insoluble matter is removed by filtration, and the mixture is filled into a sealed container and stored away from light.

[0098] Although this comparative example uses the same components and proportions, it lacks refined preparation processes such as dissolution, stepwise addition, and vacuum distillation. Therefore, the compatibility and stability of its components are expected to be inferior to those in Example 2. Furthermore, the absence of a heating process may affect the solubility and uniformity of the active ingredients, thereby impacting efficacy.

[0099] To comprehensively evaluate the effectiveness and superiority of the pine wilt disease control agent of the present invention, we designed the following experiment:

[0100] 1. In vitro insecticidal activity test;

[0101] 2. Test on the duration of drug efficacy after trunk injection;

[0102] 3. Impact assessment on non-target organisms;

[0103] 4. Drug stability testing;

[0104] 5. Impact assessment on pine tree growth.

[0105] Experiment 1: In vitro insecticidal activity test

[0106] Experimental methods:

[0107] First, we isolated and cultured pine wilt nematodes from pine trees infected with pine wilt disease. Then, we mixed different concentrations of pesticide solutions (0.1%, 0.5%, 1%, and 5%) with culture media containing 500 nematodes. Next, after incubation at 25°C for 24, 48, and 72 hours, the number of surviving nematodes was counted. Finally, the mortality rate was calculated and statistical analysis was performed.

[0108] Experimental results:

[0109] Table 1. In vitro insecticidal activity (mortality rate %) of different formulations against pine wood nematode

[0110]

[0111]

[0112] Analysis: Examples 1-4 all exhibited excellent insecticidal activity, especially at low concentrations and over short periods. In contrast, Comparative Examples 1 and 2 (single active ingredient) showed significantly weaker insecticidal effects, confirming the synergistic effect of the multi-component formulation. Comparative Examples 3 (without synergist) and 4 (without stabilizer) were slightly less effective than the examples, indicating that synergists and stabilizers play an important role in improving efficacy. Comparative Example 5 (high-dose single component) performed well at high concentrations, but its effect was less than the examples at low concentrations, demonstrating that the formulation of the present invention is more effective at low doses. Comparative Example 6 (simple mixture) showed effects close to but slightly lower than the examples, demonstrating the importance of a refined preparation process.

[0113] Experiment 2: Duration of drug efficacy test after trunk injection

[0114] Experimental methods:

[0115] Sixty healthy 5-year-old Pinus sylvestris trees were selected and randomly divided into 10 groups (Examples 1-4, Comparative Examples 1-6), with 6 trees in each group. First, holes were drilled 1 meter above the ground and the pesticide was injected (50 ml of 1% solution per tree). Second, samples were taken from different parts of each tree (lower, middle, and upper parts) at 1 week, 1 month, 3 months, and 6 months after injection to collect and count the number of nematodes. Finally, the control rate was calculated and statistically analyzed.

[0116] Experimental results:

[0117]

[0118]

[0119] Analysis: Examples 1-4 maintained high preventive and therapeutic effects 6 months after injection, with Example 3 showing the best performance. Comparative Examples 1 and 2 showed significantly poorer persistence, further confirming the superiority of multi-component formulations. The effects of Comparative Examples 3 and 4 declined rapidly over time, indicating that synergists and stabilizers are crucial for maintaining long-term efficacy. Comparative Example 5 showed good initial effects but poor persistence, possibly due to the higher dose of a single component being more easily metabolized or degraded. The persistence of Comparative Example 6 was also inferior to the examples, further demonstrating the importance of refined preparation processes.

[0120] Experiment 3: Assessment of the impact on non-target organisms

[0121] Experimental methods:

[0122] Common soil microorganisms (bacteria and fungi), beneficial nematodes, earthworms, and herbivorous insects were selected as test subjects. These organisms were exposed to different concentrations of the pesticide (0.1%, 0.5%, and 1%), and their survival rate and behavioral changes were observed over 7 days. A blank control group was also included.

[0123] Experimental results:

[0124] Table 3. Effects of different formulations on non-target organisms (survival rate %) after 7 days

[0125]

[0126]

[0127] Analysis: Examples 1-4 had minimal impact on non-target organisms; even at a concentration of 1%, the survival rate of most organisms remained above 90%. In contrast, Comparative Example 5 (high-dose single component) had a significantly greater impact on non-target organisms, especially at higher concentrations. This result demonstrates the environmentally friendly nature of the formulation of this invention, likely due to the synergistic effect of low-dose multi-component formulations, which effectively controls pine wilt disease while minimizing impact on the ecosystem.

[0128] Experiment 4: Drug Stability Test

[0129] Experimental methods:

[0130] Each formulation of the pesticide was dispensed into sealed containers and stored at 4°C (refrigerated), 25°C (room temperature), and 40°C (high temperature), respectively. Samples were taken after 0, 1, 3, and 6 months of storage to determine changes in the content of the active ingredient and pH value, and insecticidal activity was tested (methods were the same as in Experiment 1).

[0131] Experimental results:

[0132] Table 4. Stability of different formulations under storage conditions at 25℃ (retention rate of active ingredients %)

[0133] formula 1 month 3 months 6 months Example 1 98.7 95.3 91.8 Example 2 99.1 96.2 93.1 Example 3 99.5 97.1 94.6 Example 4 99.3 96.8 93.9 Comparative Example 3 97.2 91.5 85.3 Comparative Example 4 95.8 88.7 80.9 Comparative Example 6 96.5 90.1 83.6

[0134] Note: Only some key data are listed in the table. The actual test results include all formulations under three temperature conditions.

[0135] Analysis: Examples 1-4 exhibited excellent stability under various storage conditions, especially Example 3. Comparative Examples 3 (without synergist) and 4 (without stabilizer) showed significantly poorer stability, particularly at high temperatures, confirming the important role of synergists and stabilizers in maintaining the long-term stability of the pharmaceutical preparation. The stability of Comparative Example 6 (simple mixture) was also inferior to the examples, further illustrating the impact of refined preparation processes on pharmaceutical quality.

[0136] It is worth noting that Examples 1-4 still retained more than 90% of the active ingredients after 6 months, which means they may have a long shelf life, which is very advantageous for practical application and storage.

[0137] Experiment 5: Assessment of the impact on pine tree growth

[0138] Experimental methods:

[0139] One hundred healthy 3-year-old Pinus sylvestris var. mongolica saplings were selected and randomly divided into 10 groups (Examples 1-4, Comparative Examples 1-6, and a blank control group), with 10 saplings in each group. The trunks were injected with the recommended dosage. At 3 and 6 months post-treatment, the tree height growth, diameter at breast height (DBH) growth, and leaf color, density, and other growth indicators were measured. Simultaneously, nutrient content in the tree tissues was sampled and analyzed.

[0140] Experimental results:

[0141] Table 5. Effects of different formulations on pine tree growth (percentage relative to the control group)

[0142] formula High growth Increase in chest diameter Chlorophyll content Photosynthetic rate Example 1 101.5 100.8 99.7 100.3 Example 2 102.1 101.2 100.2 100.7 Example 3 102.8 101.7 100.5 101.2 Example 4 102.3 101.4 100.3 100.9 Comparative Example 1 98.7 99.1 97.8 98.5 Comparative Example 2 97.9 98.5 96.9 97.8 Comparative Example 3 100.6 100.2 99.1 99.8 Comparative Example 4 101.1 100.5 99.4 100.1 Comparative Example 5 95.3 96.8 94.2 95.7 Comparative Example 6 100.2 99.8 98.7 99.5 control group 100 100 100 100

[0143] Analysis: Examples 1-4 not only showed no significant inhibitory effect on pine tree growth, but actually slightly promoted tree growth and photosynthesis. This may be because the agent effectively controlled potential pests and diseases, allowing the trees to devote more energy to growth. Comparative Examples 1 and 2 (single active ingredient) had a slight inhibitory effect on tree growth, possibly because the single high-dose component produced some toxicity to the trees. Comparative Example 5 (high-dose single component) showed the most significant inhibitory effect on tree growth, further demonstrating the superiority of the multi-component low-dose formulation of this invention.

[0144] Comprehensive analysis and unexpected technical effects:

[0145] 1. Synergistic Effect: Experimental results show that the multi-component formulation of this invention exhibits excellent performance in all tests. Particularly in terms of in vitro insecticidal activity and persistence after trunk injection, Examples 1-4 are significantly superior to the single-component comparative Examples 1 and 2. This synergistic effect may stem from the different mechanisms of action of matrine and azadirachtin: matrine primarily kills nematodes rapidly by inhibiting acetylcholinesterase, while azadirachtin inhibits nematode growth and reproduction by interfering with the hormone system. The combination of these two mechanisms not only improves the overall control effect but may also reduce the risk of nematodes developing resistance.

[0146] 2. High Efficiency with Low Dosage: Surprisingly, the low-dose multi-component formulations of this invention (Examples 1-4) are even more effective at killing insects than the high-dose single-component formulations (Comparative Example 5). This finding has significant practical implications because it means that better control can be achieved with fewer active ingredients, thereby reducing costs and environmental risks.

[0147] 3. Long-lasting effect: The results of Experiment 2 show that the formulation of this invention maintains a high level of control efficacy even 6 months after trunk injection. This long-lasting effect is likely due to the carefully designed formulation and preparation process, which allows the active ingredients to be slowly released within the tree. This characteristic is extremely important for practical applications because it can reduce the number of applications, lower labor costs, and reduce environmental burden.

[0148] 4. Environmental friendliness: The results of Experiment 3 are encouraging, showing that the formulation of this invention has minimal impact on non-target organisms. This high selectivity is likely due to the synergistic effect of low doses of multiple components, which effectively controls pine wilt disease while minimizing disturbance to the ecosystem. This characteristic makes this invention promising for application in environmentally sensitive areas.

[0149] 5. Excellent stability: Experiment 4 reveals the excellent storage stability of the formulation of this invention. In particular, Example 3 retained 94.6% of the active ingredients after being stored at 25°C for 6 months. This high stability not only extends the product's shelf life but may also reduce transportation and storage costs.

[0150] 6. Promoting Plant Growth: An unexpected finding in Experiment 5 was that the formulation of this invention not only did not inhibit the growth of pine trees, but actually slightly promoted tree growth and photosynthesis. This positive effect may be due to the effective control of potential pests and diseases by the agent, allowing the trees to devote more energy to growth. This finding suggests that this invention may have additional application potential, such as promoting forest growth or increasing yield.

[0151] 7. The crucial role of the preparation process: Comparative Example 6 (simple mixing) performed worse than the Examples in all tests, highlighting the crucial role of a refined preparation process in achieving synergistic effects among multiple components. Specific dissolution sequences, temperature control, and vacuum distillation may have promoted interactions between the components, resulting in effects beyond simple physical mixing.

[0152] The pine wilt disease control agent of this invention achieves highly efficient, long-lasting, and environmentally friendly control effects through multi-component synergistic action, refined preparation process, and trunk injection administration. Its unexpected technical effects, such as low-dose high efficiency, excellent stability, and potential growth-promoting effects on plants, not only solve the problem of pine wilt disease control but also provide new ideas and possibilities for the field of agricultural and forestry pest and disease control. These innovations and advantages make this invention have great potential for practical application and commercialization.

[0153] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A pesticide for controlling pine wilt disease, characterized in that, By weight parts, including the following components: Matrine 10-30 parts; Azadirachtin 5-20 parts; Turpentine 40-70 parts; Emulsifier 5-15 parts; Synergist 1-5 parts; Stabilizer 0.5-3 parts; The emulsifier is polyoxyethylene castor oil; The synergist is piperonyl butoxide; The stabilizer is dibutyl hydroxytoluene; The turpentine contains 30-45% of α-pinene, 15-25% of β-pinene, and 5-10% of Δ3-carene.

2. The agent for preventing and / or treating pine wilt disease according to claim 1, wherein The purity of the matrine is ≥98%, and the purity of the azadirachtin is ≥95%.

3. A method for preparing the Bursaphelenchus xylophilus disease control agent according to any one of claims 1 to 2, characterized by, Including the following steps: (1) First, add 40-70 parts of turpentine to the reaction kettle and stir at 60-70°C; (2) Second, slowly add 5-15 parts of polyoxyethylene castor oil and stir evenly; (3) Then, dissolve 10-30 parts of matrine in ethanol with a weight of 2-3 times that of matrine, slowly add to the reaction kettle, and stir for 30-60 minutes; (4) Again, dissolve 5-20 parts of azadirachtin in ethanol with a weight of 2-3 times that of azadirachtin, slowly add to the reaction kettle, and stir for 30-60 minutes; (5) Next, add 1-5 parts of piperonyl butoxide and stir for 15-30 minutes; (6) Then, add 0.5-3 parts of dibutyl hydroxytoluene and stir evenly; (7) Further, distill under reduced pressure at 70-80°C to remove ethanol until no ethanol is distilled out; (8) Finally, cool to room temperature, filter out the insoluble matter, and fill the filtrate into a sealed container for light-proof storage.

4. The production method according to claim 3, characterized by, The reaction kettle is equipped with a stirrer, a thermometer, and a condenser.

5. The preparation method according to claim 3, characterized in that, The reduced pressure distillation in step (7) is carried out at a pressure of 50-100 mmHg.

6. A method for using the Bursaphelenchus xylophilus disease control agent according to any one of claims 1 to 2, characterized by, Including the following steps: (1) Dilute the medicament with an equal volume of water; (2) Drill a hole with a diameter of 0.5-1 cm and a depth of 3-5 cm every 20-30 cm at a distance of 0.5-1.5 meters from the ground on the main trunk of the pine tree; (3) Inject 20-50 ml of the diluted liquid into each hole; (4) After injection, seal the injection hole with wax; (5) Use at the initial stage of pine wood nematode disease or during the prevention period, and apply once in spring and autumn each year.

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