Composition and preparation for preventing and treating pine wood nematodes and application of composition and preparation

Abstract

The invention belongs to the technical field of pesticide compounding, and particularly relates to a composition for preventing and treating pine wood nematodes, a preparation and application of the composition and the preparation. Effective active components in the composition comprise an active component A and an active component B, the concentration interaction ratio of the active component A to the active component B is 1: (0.1-10), the active component A is abamectin or emamectin benzoate, and the active component B is rotenone. Plant source active components and chemical pesticide components are compounded to improve the prevention and control effect, the pesticide dosage is reduced, the environmental risk is reduced, a binary compound system of rotenone and abamectin chemical agents is innovatively constructed, and the pesticide composition has a good application prospect in prevention and control of rotenone and abamectin chemical agents. Through cooperation of different action mechanisms of inhibition of mitochondrial respiratory chains of the two and blocking of glutamic acid gated chloride ion channels, multi-target, low-risk and high-efficiency prevention and control targets are achieved, and a green and efficient compound pesticide is provided for green and sustainable prevention and control of pine wood nematode diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide compounding, and in particular relates to a composition and a preparation for preventing and controlling pine wood nematodes and applications thereof. Background Art

[0002] Pine wilt disease is caused by the pine nematode ( Bursaphelenchus xylophilus Pine wilt disease is a global quarantine disease caused by pinewood, which infects 47 pine species and 14 non-pine species, posing a serious threat to the forestry economy and ecological security. As a key tree species with both economic and ecological value, pine trees are widely used in papermaking, construction, furniture, and other fields. They also play an important role in ecological functions such as windbreak and sand fixation and air purification. Currently, the main technologies used to control pine wilt disease include plant quarantine, chemical control, physical control, and biological control. Chemical control is one of the main means of controlling pine wilt disease. Main measures include fumigating pine wood products, injecting pesticides into tree trunks, and drone-based chemical spraying to control pine wilt nematodes and their host insects. Commonly used nematicides for pine wilt are primarily composed of avermectin and emamectin benzoate (emamectin benzoate). However, the long-term and large-scale use of single chemical agents may lead to the development of pesticide resistance in nematodes, resulting in problems such as pesticide residues and environmental pollution.

[0003] In order to delay drug resistance and reduce the risk of drug use, pesticide compounding technology has become a research hotspot. Existing compounding schemes mostly focus on chemical pesticide combinations, such as fluopyram + emamectin benzoate (mass ratio 3:5, LC 50 =3.23 mg / L, co-toxicity coefficient 334.67) and avermectin + sulfoxaflor (LC 50 =2.4 mg / L); For another example, the combination system of 2% avermectin and 6% fluopyram showed a significant synergistic effect in the control of pine wood nematodes. The LC 50 The value was 2.0208 mg / L, which was lower than that of single dose 5% fluopyram (LC 50 =21.6071 mg / L) and 5% avermectin (LC 50 =51.1536 mg / L) were reduced by 10.7 times and 25.3 times respectively; sublethal concentration (LC 20 ) after treatment with the combination agent, the nematode population was 1,425 (CK > 20,000), and the egg hatching rate at 3 mg / L was only 20.32%. Further testing confirmed that the nematicidal activity of this system increased with increasing temperature within the 15°C to 35°C temperature range. However, most research results indicate that fluopyram is more effective than avermectin in killing nematicides, but the proportion of fluopyram is three times that of avermectin. Although the combination strategy reduces the single dosage of avermectin, the cumulative use of fluopyram is unavoidable.

[0004] Plant-based pesticides are gradually gaining popularity due to their high efficiency, low toxicity, diverse targets, and environmental friendliness. There are also many cases of using plant-based active ingredients in the prevention and control of pine wood nematode disease, such as Astragalus-Zanthoxylum crude extract (LC 50 =67.67 μg / mL), marigold-willow leaf compound (LC 50 =245.64 μg / mL), etc. Although environmentally friendly, their toxicity levels are significantly lower than those of chemical compound preparations, making it difficult to meet the needs of rapid prevention and control during the outbreak of pine wilt disease. In addition, the complex interactions between multiple components in crude extracts may reduce targeting. For example, although α-terthiophene in marigold extracts is phototoxic, its effect depends on ultraviolet radiation conditions, and its biological activity is greatly attenuated in pine forests with high density. Although plant-based preparations have advantages in ecological safety, their low efficiency and short-term effects limit their value for large-scale application, especially during the critical period of epidemic outbreak prevention and control, when it is difficult to replace chemical agents. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a composition, preparation and application of pine wood nematode prevention and control. The present invention adopts a compound of plant-derived active ingredients and chemical pesticide ingredients to improve the prevention and control effect, reduce the amount of pesticide used and reduce environmental risks, and innovatively constructs a binary compound system of rotenone and avermectin chemical agents. Through the synergy of two different mechanisms of action of mitochondrial respiratory chain inhibition and glutamate-gated chloride ion channel blocking, the multi-target, low-risk and high-efficiency prevention and control goals are achieved, which not only alleviates the defect of insufficient efficacy of plant-derived preparations, but also reduces the vulnerability of high residual amount of chemical agents, providing a green and efficient compound pesticide for the green and sustainable prevention and control of pine wood nematode disease.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The first object of the present invention is to provide a composition for controlling pine wood nematodes, wherein the effective active ingredients in the composition are composed of active ingredient A and active ingredient B, the concentration interaction ratio of the active ingredient A and the active ingredient B is 1:0.1-10, the active ingredient A is avermectin or avermectin benzoate, and the active ingredient B is rotenone.

[0008] Furthermore, the active ingredient A is avermectin, and the concentration ratio of the active ingredient A to the active ingredient B is 1:0.2-9.

[0009] Furthermore, when the active ingredient A is emamectin benzoate, the weight ratio of the active ingredient A to the active ingredient B is 1:0.2-1 or 1:8.8-9.

[0010] Furthermore, the final concentration of the active ingredient A and the active ingredient B in the composition is 1 mg / L to 10 mg / L.

[0011] Furthermore, the final concentration of the active ingredient A and the active ingredient B in the composition is 5.5 mg / L to 8 mg / L.

[0012] The second object of the present invention is to provide a preparation for controlling pine wood nematodes, which comprises the above-mentioned composition and auxiliary ingredients permitted in forestry.

[0013] Furthermore, the preparation is a suspension concentrate, emulsifiable concentrate or wettable powder.

[0014] The third object of the present invention is to provide the use of the above-mentioned preparation for controlling pine wood nematodes in controlling pine wood nematode disease.

[0015] Furthermore, the final concentration of the preparation is 400 mg / L to 4000 mg / L.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The composition for preventing and controlling pine wood nematodes provided by the present invention adopts a combination of plant-derived active ingredients and chemical pesticide ingredients to improve the prevention and control effect, reduce the amount of pesticides used, and reduce environmental risks. It innovatively constructs a binary compound system of rotenone and avermectin-type chemical agents. Through the synergistic action of the two agents' different mechanisms of action, namely, mitochondrial respiratory chain inhibition and glutamate-gated chloride ion channel blocking, it achieves a multi-target, low-risk, and high-efficiency prevention and control goal. It not only alleviates the defect of insufficient efficacy of plant-derived preparations, but also reduces the amount of high-residue chemical agents used, providing a green and efficient compound pesticide for the green and sustainable prevention and control of pine wood nematode disease.

[0017] (2) When the active ingredient A of the present invention is emamectin benzoate, in the interactive assay test, when the interactive ratio of emamectin benzoate to rotenone is 7:3, 6:4, 5:5 or 8:2, the toxicity ratios are 1.52, 1.41, 1.36 and 1.34 respectively; in the co-toxicity coefficient test, the optimal interactive ratio of emamectin benzoate to rotenone is 7:3, LC 50 It is 4.00 mg / L, and the co-toxicity coefficient reaches 149.1986.

[0018] (3) When the active ingredient A of the present invention is avermectin, in the interactive determination method, the interactive ratios of avermectin and rotenone are 7:3, 6:4, 5:5 and 4:6, and the toxicity ratios are 1.69, 1.68, 1.62 and 1.62 respectively. In the co-toxicity coefficient test, as the most preferred solution of the present invention, the interactive ratio of avermectin and rotenone is 7:3, LC 50The concentration of avermectin in the pine wood nematode is 3.18 mg / L, and the co-toxicity coefficient is 231.0893. It has a significant inhibitory effect on the egg production, egg hatching rate, and population size of female pine wood nematodes, can reduce the dosage of the chemical pesticide avermectin, and increase its nematicidal effect, showing broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the effect of different treatments of the present invention on the egg production of pine wood nematode.

[0020] Figure 2 This is a graph showing the hatching rate of pine wood nematode eggs over time after treatment with different pesticides in the present invention. DETAILED DESCRIPTION

[0021] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0022] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0023] On the one hand, the present invention provides a composition for controlling pine wood nematodes, wherein the effective active ingredients in the composition are composed of active ingredient A and active ingredient B, the concentration interaction ratio of active ingredient A and active ingredient B is 1:0.1-10, the active ingredient A is avermectin or avermectin benzoate, and the active ingredient B is rotenone.

[0024] It should be noted that the composition for controlling pine wood nematodes provided by the present invention is based on rotenone as a specific inhibitor of mitochondrial respiratory chain complex I (NADH dehydrogenase), which can block electron transfer and inhibit ATP synthesis, leading to the collapse of nematode energy metabolism, excellent nematicidal activity, a half-life of 1-3 days, and is extremely easily degraded in the environment. Therefore, it has great potential in the control of pine wood nematodes. The plant-based rotenone active ingredient is compounded with chemical pesticide ingredients to improve the control effect, reduce the amount of pesticide used, and reduce environmental risks. An innovative binary compound system of rotenone and avermectin-type chemical agents is constructed. Through the synergy of the different mechanisms of action of the two, namely mitochondrial respiratory chain inhibition and glutamate-gated chloride ion channel blocking, a multi-target, low-risk, and high-efficiency control goal is achieved, which not only alleviates the defect of insufficient efficacy of plant-based preparations, but also reduces the amount of high-residue chemical agents, providing a green and efficient compound pesticide for the green and sustainable control of pine wood nematode disease.

[0025] In some embodiments, the active ingredient A is avermectin, and the concentration interaction ratio of the active ingredient A and the active ingredient B is 1:0.2 to 9. As a preferred embodiment of the present invention, when the active ingredient A is avermectin, the concentration interaction ratio of the active ingredient A and the active ingredient B is 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 or 1:9. Abamectin and rotenone have a synergistic effect after compounding, and the toxicity ratio is greater than 1.25. As a more preferred embodiment of the present invention, the weight ratio of avermectin and rotenone is 7:3, 6:4 or 5:5, and the toxicity ratios are 1.69, 1.68 and 1.62, respectively. As the most preferred embodiment of the present invention, the weight ratio of avermectin and rotenone is 7:3, LC 50 It is 3.18 mg / L, and the co-toxicity coefficient is 231.0893. It has a significant inhibitory effect on the egg laying amount, egg hatching rate, and population size of female pine wood nematodes. It can reduce the dosage of the chemical pesticide avermectin and increase its nematicidal effect. It has broad application prospects.

[0026] In some embodiments, the active ingredient A is emamectin benzoate, and the concentration interaction ratio of the active ingredient A and the active ingredient B is 1:0.2-1 or 1:8.8-9. As a preferred embodiment of the present invention, when the active ingredient A is emamectin benzoate, the concentration interaction ratio of emamectin benzoate and rotenone is 8:2, 7:3, 6:4, 5:5 or 1:9. After emamectin benzoate and rotenone are compounded, they have a synergistic effect, and the toxicity ratio is greater than 1.25. As a more preferred embodiment of the present invention, the weight ratio of avermectin and rotenone is 7:3, and the toxicity ratio is 1.52, LC 50 The value is 4.00 mg / L, and the co-toxicity coefficient reaches 149.1986.

[0027] In some embodiments, the final concentration of the active ingredient A and the active ingredient B in the composition is 1 mg / L to 10 mg / L.

[0028] In some embodiments, the final concentration of the active ingredient A and the active ingredient B in the composition is 5.5 mg / L to 8 mg / L.

[0029] On the other hand, the present invention also provides a preparation for controlling pine wood nematodes, which comprises the above-mentioned composition and auxiliary ingredients permitted in forestry.

[0030] It should be noted that the auxiliary components can be selected from one or more of wetting agents, dispersants, emulsifiers, thickeners, disintegrants, antifreeze agents, defoaming agents, solvents, preservatives, stabilizers, synergists, and carriers.

[0031] The wetting agent can be at least one of sodium dodecylbenzene sulfate, sodium dodecylbenzene sulfonate, soap powder, alkyl sulfate, alkyl polyoxyethylene ether succinate sulfonate, etc.; the dispersant is selected from at least one of polycarboxylates, lignin sulfonates, naphthalene or alkylnaphthalene formaldehyde condensate sulfonates, alkylbenzene calcium sulfonate, alkylphenol polyoxyethylene phosphate, polymerized alkylaryl sulfonate, etc.; the emulsifier is selected from at least one of fatty alcohol polyoxyethylene ether, fatty alcohol ethylene oxide-propylene oxide copolymer, phenylethylphenol polyoxyethylene polyoxypropylene ether, alkylphenol polyoxyethylene ether, etc.; the thickener is selected from at least one of xanthan gum, polyvinyl alcohol, organic bentonite, magnesium aluminum silicate, carboxymethyl cellulose, etc.; the disintegrant is selected from at least one of aluminum chloride, sucrose, modified starch, cellulose, urea, sodium carbonate, sodium bicarbonate, citric acid and tartaric acid, etc.; the antifreeze is selected from at least one of alcohols, alcohol ethers, inorganic salts, etc.; the defoaming agent is selected from silicone oil, C 10 ~C 20 At least one of saturated fatty acid compounds or silicone compounds; the solvent is selected from at least one of benzene, toluene, xylene, methanol, ethanol, isopropanol, n-butanol, etc.; the preservative is selected from at least one of sorbic acid, potassium sorbate, benzoic acid, sodium benzoate, sodium p-hydroxybenzoate, etc.; the stabilizer is selected from at least one of succinic acid, adipic acid, epoxidized vegetable oil, etc.; the carrier is selected from at least one of kaolin, bentonite, light calcium carbonate, diatomaceous earth, etc.; In some embodiments, the formulation is a suspension concentrate, emulsifiable concentrate, or wettable powder.

[0032] The present invention also provides use of the above-mentioned preparation for preventing and controlling pine wood nematodes in preventing and controlling pine wood nematode disease.

[0033] In some embodiments, the formulation is used at a final concentration of 400 mg / L to 4000 mg / L.

[0034] It should be noted that the preparation provided by the present invention can also be added with a commercial tree penetrant so that the preparation is applied in a spray form. The amount of the commercial tree penetrant is 1% of the total mass of the preparation, which can control the incidence of the disease and the number of nematodes.

[0035] The following is further described through specific examples.

[0036] Example 1. Toxicity of a single agent to pine wood nematodes Rotenone was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. with a purity of ≥99%.

[0037] Abamectin was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a purity of ≥97%.

[0038] Emamectin benzoate (emamectin benzoate) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. with a purity of ≥95%.

[0039] The present invention adopts the insect immersion method to determine the nematicidal activities of rotenone, avermectin and emamectin benzoate.

[0040] Nematicidal activity indoor toxicity assay: Plates cultured with Pseudomonas spp. that had been fed by pine nematodes for approximately 3–4 days were washed with sterile water to obtain a suspension of pine nematodes, which was then diluted to approximately 100 nematodes / 100 μL. 10 mg of rotenone, avermectin, and emamectin benzoate stock solutions were weighed and added to 1 mL of dimethyl sulfoxide (DMSO) to prepare stock solutions. The stock solutions were then diluted sequentially to 1000, 500, 200, 100, and 50 mg / L. 1 μL of each pesticide dilution and 99 μL of the nematode suspension were added to 48-well cell culture plates, resulting in final concentrations of 10 mg / L, 5 mg / L, 2 mg / L, 1 mg / L, and 0.5 mg / L, respectively. Treatments containing DMSO and sterile water served as controls. Each treatment was repeated three times, with at least two biological replicates. After 24 hours of treatment, the number of pine nematode deaths was recorded. Corrected mortality rates, toxicity regression equations, and median lethal concentrations (LC) were calculated using Excel. 50 , correlation coefficient R and LC 50 95% confidence interval of .

[0041] Control mortality (%) = number of deaths in the control / number of test insects in the control × 100%.

[0042] Adjusted mortality rate (%) = (average mortality rate of treatment group - mortality rate of control group) / (1-mortality rate of control group) × 100%.

[0043] Method for determining whether nematodes are dead or alive: any nematode that moves or has an "S" shape, curled shape, wavy shape, or spiral shape is judged as alive; any nematode that does not move and has a "J" shape or "C" shape, or has a rigid body and a non-refractive body wall, and does not move when poked with an insect needle, is judged as dead.

[0044] The nematicidal activities of the three active ingredients are shown in Table 1. The LC values ​​of rotenone are 50 The LC value of avermectin is 5.94 mg / L. 50 The LC value of emamectin benzoate is 8.18 mg / L. 50 It is 5.98 mg / L.

[0045] Table 1 Nematicidal activity of rotenone, avermectin and emamectin benzoate 2. Interactive determination method to screen the optimal ratio The LC of the two single doses of rotenone combined with avermectin and emamectin benzoate was 50 The values ​​were divided into 10 equal parts, and 12 treatment combinations were set for measurement (as shown in Table 2). In Table 2, A is active ingredient A (avermectin or emamectin benzoate), and B is active ingredient B (rotenone). Each experiment was repeated three times, with at least two biological replicates.

[0046] Table 2 Interaction ratio of single agent concentration in each treatment The calculation formula of the measurement results is as follows: Actual mortality rate = observed mortality rate - CK mortality rate.

[0047] Expected mortality rate = Pesticide A LC 50 Actual mortality rate × proportion of pesticide A + LC of pesticide B 50 The actual mortality rate is calculated by multiplying the proportion of pesticide B.

[0048] Toxicity ratio = actual mortality rate / expected mortality rate.

[0049] A toxicity-effect ratio greater than 1.25 indicates a synergistic effect, a toxicity-effect ratio less than 0.75 indicates an antagonistic effect, and a toxicity-effect ratio of around 1 indicates an additive effect.

[0050] A reciprocal assay was used to preliminarily screen the nematicidal activity of rotenone with avermectin and emamectin benzoate against pine wood nematodes (see Table 3). Among the rotenone / avermectin combinations, additive effects were observed at the 10:0, 9:1, and 0:10 concentration ratios, while synergistic effects were observed at all other ratios. The highest toxicity-to-effect ratios (1.69, 1.68, and 1.62) were observed at avermectin / rotenone ratios of 7:3, 6:4, and 5:5, respectively. Synergistic effects were observed at the 8:2, 7:3, 6:4, 5:5, and 1:9 concentration ratios of emamectin / rotenone. The most significant synergistic effect was observed at a 7:3 ratio, with a toxicity-to-effect ratio of 1.52.

[0051] Table 3 Nematicidal activity of different compound ratios of avermectin, emamectin benzoate and rotenone against pine wood nematodes 3. Confirm the optimal ratio by using the co-toxicity coefficient method The three optimal ratios of rotenone, avermectin and emamectin benzoate were selected for testing, and the LC values ​​were calculated based on the indoor nematicidal activity test. 50 The evaluation method was based on the co-toxicity coefficient method. Each treatment was repeated three times, with at least two biological replicates.

[0052] Single-dose toxicity index = (LC of standard agent) 50 / LC of the test single dose 50 )×100.

[0053] The theoretical toxicity index of the mixture (A+B) = the toxicity index of agent A × the mass proportion of agent A in the mixture + the toxicity index of agent B × the mass proportion of agent B in the mixture.

[0054] The measured toxicity index of the mixture (A+B) = [LC of the standard agent] 50 / LC of mixture (A+B) 50 ]×100.

[0055] Co-toxicity coefficient = [measured toxicity index of the mixture (A+B) / theoretical toxicity index of the mixture (A+B)] × 100.

[0056] Evaluation criteria: a co-toxicity coefficient greater than 120 indicates a synergistic effect; a co-toxicity coefficient less than 80 indicates an antagonistic effect; and a co-toxicity coefficient of 80 ≤ ≤ 120 indicates an additive effect.

[0057] The co-toxicity coefficient method was used to test the nematicidal activity of rotenone with abamectin and emamectin benzoate against pine wood nematodes in the three optimal combination ratios (as shown in Table 4). Both abamectin and emamectin benzoate showed the highest activity with rotenone at a ratio of 7:3, especially abamectin and rotenone, LC 50The optimal value was 3.18 mg / L, and the co-toxicity coefficient reached 231.0893. Therefore, the optimal compound combination and ratio was a 7:3 concentration interaction ratio of avermectin and rotenone.

[0058] Table 4 Combined toxicity test of the three optimal ratios of avermectin and rotenone against pine wood nematodes 4. Effects of different treatments on pine wood nematode egg production The two single agents and a combined combination (avermectin:rotenone weight ratio = 7:3) of the optimal combination were selected for subsequent testing. Four groups were established: the DMSO control (CK), rotenone (SCY) (1.782 mg / L), avermectin (SCA) (5.726 mg / L), and the combined combination (SCF) (rotenone 1.782 mg / L + avermectin 5.726 mg / L). Ten male and female adults were placed in a 48-well plate. 99 μL of sterile water was added, and 1 μL of the drug solution was added according to the final concentration. After 12, 24, 36, 48, and 60 hours of darkness, egg production was counted at the corresponding time points. Each treatment was replicated three times, with at least two biological replicates.

[0059] The average number of eggs laid by a female = total number of eggs laid / 10.

[0060] The results of the analysis of the effects of different pesticide treatments on the egg production of female pine wood nematodes are as follows: Figure 1 shown. Figure 1 This is the effect of different treatments of the present invention on the egg production of pine wood nematode. Figure 1 Different lowercase letters indicate that the differences between the groups reached a significant level (p < 0.05). Figure 1 As shown, all treatments exhibited significant egg-laying inhibition compared to CK, but no significant differences were found among treatment groups. The SCF treatment exhibited the most significant sustained inhibitory effect, maintaining egg production at 2.6 ± 0.25 eggs. This remained unchanged with prolonged treatment, demonstrating a sustained inhibitory effect on pine wood nematode egg production. SCA treatment slightly increased egg production, with average egg production at 12, 24, 36, 48, and 60 h being 2.94 ± 0.40, 3.28 ± 0.49, 3.58 ± 0.45, 4.16 ± 0.51, and 4.18 ± 0.53 eggs, respectively, indicating a gradual weakening of its inhibitory effect with prolonged treatment. The SCY treatment exhibited an average egg production of 4.06 ± 0.39 eggs at 60 h, demonstrating a similar inhibitory effect to SCA. The egg production of CK showed a significant positive correlation with time, and its egg production continued to increase from (5.28 ± 0.49) eggs to (9.26 ± 0.37) eggs.

[0061] 5. Effects of different treatments on the hatching rate of pine wood nematode eggs Equal amounts of mixed-age insect liquid were placed in a 48-well plate and laid eggs in the dark at 25°C for 12 hours. The supernatant was discarded and the eggs were washed two to three times with sterile water. 99 μL of sterile water was added, followed by 1 μL of the drug solution according to the final concentration. Each treatment was replicated three times, with at least two biological replicates. After 12, 24, 36, 48, and 60 hours, the number of hatched larvae and eggs from each treatment was observed at the corresponding time points.

[0062] The hatching rate of insect eggs = [number of larvae / (number of eggs + number of larvae)] × 100%.

[0063] Figure 2 This is a graph showing the hatching rate of pine wood nematode eggs over time after treatment with different pesticides in the present invention. Figure 2 Different lowercase letters indicate that the differences between the groups reached a significant level (p < 0.05). Figure 2 As shown, the hatching rate in the CK group increased significantly with treatment time, reaching a peak of 98.68% ± 0.01% at 60 h. The SCF group exhibited the strongest hatching inhibition, with a significantly lower hatching rate (13.09% ± 0.02%) at 60 h compared to the other treatment groups. No statistical differences were observed between time points, confirming the agent's ability to continuously inhibit embryonic development. The SCA and SCY groups exhibited some time-dependent recovery, with hatching rates at 60 h (49.59% ± 0.03%) and (44.89% ± 0.07%), respectively, but both were significantly lower than those in the CK group.

[0064] 6. Effects of different treatments on pine wood nematode populations Wash to obtain a mixed-age suspension of 3000 nematodes / 0.99 mL. 10 μL of the drug solution was added based on the final concentration. After 24 h of treatment, the suspension was centrifuged, the supernatant removed, and the suspension washed three times with sterile water. Mixed-age pine nematodes were aspirated and placed on plates completely covered with Pseudomonas spp. in the dark at 25°C. Nematodes were then separated using the Baermann funnel method. The total number of nematodes, adults, larvae, female adults, and male adults in 20 μL of the suspension was counted under a light microscope three times. The total number of the corresponding indicators was calculated based on the total volume of the washing solution. Ten female adults and ten male adults were randomly selected and heated to death using an alcohol lamp. Body length was then measured under an inverted microscope. Each treatment was replicated five times, with at least two biological replicates.

[0065] Table 5 shows the effects of different pesticide treatments on the physiological parameters of PWN populations. The CK treatment significantly increased the population size (3751.00 ± 632.03), the number of adults (1938.45 ± 403.90), and the number of larvae (1812.56 ± 244.24) compared to all other treatments. The SCY treatment exhibited an inhibitory effect, with the population size, adult size, and larval size reduced by 70.83%, 70.64%, and 71.03%, respectively, compared to the CK treatment. The SCA treatment exhibited a stronger inhibitory effect, with the adult size reduced by 84.55% compared to the CK treatment, while the adult and larval sizes remained similar. The SCF treatment exhibited the strongest inhibitory effect, with the total population size (143.60 ± 14.63) being only 3.83% of that of the CK treatment, and the number of larvae and adults being 59.93 ± 9.20 and 83.67 ± 10.66, respectively.

[0066] Table 5 Effects of different treatments on pine wood nematode populations Analysis of variance revealed that, as shown in Table 5 (where a, b, and ab indicate significant differences between groups, p < 0.05), the adult / larvae ratio did not differ statistically among the treatment groups. In sex ratio analysis, the female-to-male ratio in the CK group was 3.55 ± 0.91, while the proportion of males increased significantly in the SCY (0.84 ± 0.38), SCA (0.28 ± 0.09), and SCF (0.95 ± 0.36) treatments. Morphological measurements revealed that male body length in the SCF group (762.07 ± 22.58 μm) was significantly greater than that in the CK (709.77 ± 25.36 μm) and SCY (677.13 ± 21.09 μm) treatments. No statistical differences were observed in female body length parameters among the treatments.

[0067] In summary, the present invention determined that the optimal compound combination of abamectin and rotenone in a ratio of 7:3 exhibited the highest activity through systematic nematicidal activity screening and compound optimization testing. 50 The optimal combination significantly inhibited female pine wood nematode egg production, egg hatching rate, and population size, reducing the use of the chemical pesticide avermectin and increasing its nematicidal effect, suggesting broad application prospects.

[0068] It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0069] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A composition for controlling pine wood nematodes, characterized in that: The effective active ingredients in the composition consist of active ingredient A and active ingredient B, the concentration mutual ratio of the active ingredient A to the active ingredient B is 1:0.1-10, the active ingredient A is avermectin or avermectin benzoate, and the active ingredient B is rotenone.

2. The composition for controlling pine wood nematodes according to claim 1, characterized in that: The active ingredient A is avermectin, and the concentration interaction ratio of the active ingredient A to the active ingredient B is 1:0.2-9.

3. The composition for controlling pine wood nematodes according to claim 1, characterized in that: The active ingredient A is emamectin benzoate, and the concentration interaction ratio of the active ingredient A to the active ingredient B is 1:0.2-1 or 1:8.8-9.

4. The composition for controlling pine wood nematodes according to claim 1, characterized in that: The final concentration of the active ingredient A and the active ingredient B in the composition is 1 mg / L to 10 mg / L.

5. The composition for controlling pine wood nematodes according to claim 1, characterized in that: The final concentration of the active ingredient A and the active ingredient B in the composition is 5.5 mg / L to 8 mg / L.

6. A preparation for controlling pine wood nematodes, characterized in that: The preparation comprises the composition according to any one of claims 1 to 5 and auxiliary ingredients permitted in forestry.

7. The preparation for controlling pine wood nematodes according to claim 6, characterized in that: The preparation is a suspension concentrate, emulsifiable concentrate or wettable powder.

8. Use of the preparation for controlling pine wood nematodes according to claim 6 or claim 7 in controlling pine wood nematode disease.

9. The use according to claim 8, characterized in that The final concentration of the preparation is 400 mg / L to 4000 mg / L.

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