A nematicidal pharmaceutical composition and use thereof

Abstract

The present application relates to the technical field of pesticide preparation, and discloses a nematicidal pharmaceutical composition and application thereof.The methyl isothiocyanate and the abamectin are compounded in the present application, and the killing activity on the second instar larvae and eggs of southern root-knot nematode can be significantly improved.The indoor biological determination results show that the two kinds of agents after compounding exhibit different degrees of synergistic effect on different development stages of southern root-knot nematode.The synergistic effect on the second instar larvae of southern root-knot nematode is the most significant when the mass ratio of methyl isothiocyanate to abamectin is 50:1, and the synergistic coefficient reaches 8.31;the inhibitory effect on the egg hatching of southern root-knot nematode is the most significant when the mass ratio is 15:1, and the synergistic coefficient reaches 2.14.The composition has the advantages of significant synergistic effect, low dosage, stable control effect and the like, and can be used for the green and efficient prevention and control of root-knot nematode diseases of protected vegetables, fruit trees and economic crops.

Description

Technical Field

[0001] This invention relates to the field of pesticide formulation technology, and to a nematicidal drug composition and its application, specifically to a nematicidal composition containing ivermectin and methyl isothiocyanate and its application in controlling plant parasitic nematodes, particularly its application in controlling southern root-knot nematodes. Background Technology

[0002] Root-knot nematode disease is an important soil-borne disease caused by root-knot nematodes (Meloidogyne spp.) infecting plant roots. It widely affects tomatoes, cucumbers, peppers, watermelons, and many other economic crops. After invading the plant root system, the nematodes form root knots, damaging the normal structure and function of the root system, leading to stunted plant growth, reduced yield, and lower quality. In severe cases, it can cause significant yield reduction or even crop failure. Currently, the control of root-knot nematodes mainly relies on chemical agents, biological control, and agricultural control measures. Ivermectin, a macrolide biopesticide, has good contact and stomach poison effects against plant-parasitic nematodes; methyl isothiocyanate (MITC) is a broad-spectrum soil fumigant that effectively inhibits pathogens, weed seeds, and nematodes in the soil. However, using ivermectin alone has drawbacks such as a short residual effect and its efficacy being greatly affected by environmental factors; using MITC alone involves large dosages and high costs. Therefore, developing a compound composition that can fully leverage the advantages of ivermectin and MITC and achieve synergistic effects is of great significance for improving the control efficacy of root-knot nematodes, reducing the amount of pesticides used, and decreasing control costs. Summary of the Invention

[0003] The purpose of this invention is to provide a nematicidal composition containing ivermectin and methyl isothiocyanate and its application. By combining ivermectin with methyl isothiocyanate, the toxic activity against southern root-knot nematodes can be significantly improved, the dosage of the nematicide can be reduced, and the control effect can be enhanced.

[0004] The present invention provides a nematicide composition, wherein the nematicide composition is obtained by mixing methyl isothiocyanate and ivermectin at an active ingredient mass ratio of 10:1 to 100:1, and the total effective concentration of methyl isothiocyanate and ivermectin is 0.2-4.0 ppm.

[0005] The methyl isothiocyanate and ivermectin are mixed in the form of mother liquors. The methyl isothiocyanate is dissolved in acetone and then diluted with distilled water to obtain the methyl isothiocyanate mother liquor; the ivermectin emulsifiable concentrate is directly diluted with distilled water to obtain the ivermectin mother liquor. The mass concentration of methyl isothiocyanate in the methyl isothiocyanate mother liquor is 800-1200 mg / L; the mass concentration of ivermectin in the ivermectin mother liquor is 800-1200 mg / L. Specifically, the mass concentration of methyl isothiocyanate in the methyl isothiocyanate mother liquor is 1000 mg / L; and the mass concentration of ivermectin in the ivermectin mother liquor is 1000 mg / L.

[0006] Preferably, the preparation of the methyl isothiocyanate mother liquor includes the following steps: dissolving methyl isothiocyanate in acetone and diluting it with distilled water. The preparation of the ivermectin mother liquor includes the following steps: directly diluting ivermectin emulsifiable concentrate with distilled water.

[0007] In a preferred embodiment, when controlling second-instar larvae of the southern root-knot nematode, the mass ratio of the active ingredients of methyl isothiocyanate to ivermectin is 30:1-90:1 (preferably 40-60:1); when controlling the hatching of southern root-knot nematode eggs, the mass ratio of the active ingredients of methyl isothiocyanate to ivermectin is 11:1-50:1 (preferably 12-18:1).

[0008] In another embodiment, when controlling second-instar larvae of the southern root-knot nematode, the mass ratio of the active ingredient methyl isothiocyanate to ivermectin is 48-52:1 (specifically, 50:1); when controlling the hatching of southern root-knot nematode eggs, the mass ratio of the active ingredient methyl isothiocyanate to ivermectin is 14-16:1 (e.g., 15:1).

[0009] This invention also provides the application of the above-described nematicidal drug composition in the control of plant parasitic nematodes. Preferably, the plant parasitic nematode is the southern root-knot nematode.

[0010] Specifically, it is used to kill second-instar larvae of the southern root-knot nematode, or to inhibit the hatching of southern root-knot nematode eggs.

[0011] The present invention also provides a nematicide composition, which is obtained by mixing 42% thiamethoxam and 0.5% ivermectin emulsifiable concentrate in a mass ratio of 100-130:1. Preferably, the nematicide composition is obtained by mixing 42% thiamethoxam and 0.5% ivermectin emulsifiable concentrate in a mass ratio of 110-120:1.

[0012] The present invention also provides the application of the nematicidal drug composition in the control of root-knot nematodes.

[0013] The nematicidal composition of this invention exhibits a synergistic effect between its two active ingredients. The combined use of methyl isothiocyanate and ivermectin effectively enhances the control of target organisms while reducing pesticide usage. Experimental results show that the nematicidal composition of this invention can improve the toxic activity against southern root-knot nematodes and reduce pesticide dosage. Detailed Implementation

[0014] In this invention, the nematicidal drug composition is obtained by mixing methyl isothiocyanate stock solution and ivermectin stock solution according to the mass ratio of active ingredients; the mass-volume concentration of methyl isothiocyanate in the methyl isothiocyanate stock solution is 1000 mg / L; the mass-volume concentration of ivermectin in the ivermectin stock solution is 1000 mg / L. This invention, by combining methyl isothiocyanate stock solution with a concentration of 1000 mg / L and ivermectin stock solution with a concentration of 1000 mg / L, can maximize the efficacy of each of methyl isothiocyanate and ivermectin, significantly improving the drug activity, enhancing the control effect against southern root-knot nematodes, and greatly reducing the dosage and cost of the drug. This method is more effective in controlling crop root-knot nematodes than using these two stock solutions alone.

[0015] In this invention, the preparation of the methyl isothiocyanate mother liquor includes the following steps: dissolving methyl isothiocyanate in acetone and diluting with distilled water. This invention does not specifically limit the source of the methyl isothiocyanate; commercially available methyl isothiocyanate known to those skilled in the art can be used.

[0016] In this invention, the preparation of the ivermectin mother liquor includes the following steps: directly diluting the ivermectin emulsifiable concentrate with distilled water. In this invention, the ivermectin emulsifiable concentrate is preferably a 0.5% by mass emulsifiable concentrate. This invention does not specifically limit the source of the ivermectin; commercially available ivermectin products are acceptable. The ivermectin emulsifiable concentrate used in this invention was purchased from Shun Yi Co., Ltd.

[0017] The present invention will be further described in detail below with reference to specific embodiments. The technical solutions of the present invention include, but are not limited to, the following embodiments.

[0018] Example 1: Test on the toxicity of the nematicide composition against second-instar (J2) larvae of the southern root-knot nematode.

[0019] Step 1: Preparation of the medicinal solution

[0020] Preparation of stock solution: Dissolve and dilute methyl isothiocyanate (MITC) in acetone to prepare a 1000 ppm MITC stock solution; dilute ivermectin with distilled water to prepare a 1000 ppm ivermectin stock solution for later use.

[0021] Preparation of compound drug solutions: Based on the mass ratio of the active ingredients of methyl isothiocyanate and ivermectin, the MITC mother liquor and ivermectin mother liquor were compounded in ratios of 10:1, 25:1, 30:1, 50:1, 70:1 and 100:1, respectively, to obtain compound drug solutions with different mass ratios for later use.

[0022] Concentration Setting: Based on the preliminary test results, a series of final effective concentrations were set for both single-agent and compound formulations. Specifically, the final effective concentrations for methyl isothiocyanate single agent were set to 0.5, 1.0, 1.5, 2.0, 4.5, 6.0, and 7.5 ppm; the final effective concentrations for ivermectin single agent were set to 0.01, 0.02, 0.05, 0.1, 0.2, 0.4, and 1.0 ppm; and the final effective concentrations for each compound formulation of methyl isothiocyanate and ivermectin at active ingredient mass ratios of 10:1, 25:1, 30:1, 50:1, 70:1, and 100:1 were all set to 0.05, 0.1, 0.3, 0.5, 0.8, and 1.0 ppm. The drug solution added during the experiment was pre-prepared at twice the above final effective concentrations and mixed with an equal volume of nematode suspension to achieve the corresponding set concentrations. Each treatment was set up with 4 replicates, and a distilled water control was also included.

[0023] Step 2: Test nematodes

[0024] The test nematode was a second-instar larva of the southern root-knot nematode (Meloidogyne incognita), provided by Zhejiang Jidun Biotechnology Co., Ltd. The larvae were cultured at 25℃ before the experiment.

[0025] Step 3: Method for determining toxicity

[0026] The toxicity of the pesticide against second-instar larvae of the southern root-knot nematode was determined using the immersion method. In 0.2 mL centrifuge tubes, 100 μL of a nematode suspension containing approximately 150–200 viable second-instar larvae was added to each tube, followed by 100 μL of the pre-prepared pesticide solution from step 1. The mixture was thoroughly combined with the nematode suspension, resulting in a total volume of 200 μL per tube. After equal volumes of pesticide and nematode suspension were mixed, the final effective concentration of the pesticide in each treatment system reached the concentration set in step 1. Four replicates were set for each treatment. The treated centrifuge tubes were incubated at 25°C for 24 h, and the number of dead nematodes in each treatment was observed and recorded under a stereomicroscope.

[0027] Step 4: Result Calculation and Analysis

[0028] The lifespan of nematodes is determined by needle prick. Nematodes that are stiff and do not react after being pricked are considered dead; nematodes that are bent or react after being pricked are considered alive.

[0029] Calculate the mortality rate and adjusted mortality rate using the following formulas:

[0030] Mortality rate (%) = Number of dead nematodes / Total number of tested nematodes × 100

[0031] Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (100 - control mortality rate) × 100

[0032] Regression analysis was used to analyze the logarithmic values ​​of drug concentrations and the corrected mortality probability values. The LC50 values ​​of each single agent and compound agent were calculated, and the Wadley method was used to calculate the synergistic coefficient. The combined effect of methyl isothiocyanate and ivermectin on the second instar larvae of Southern Root-knot Nematode was evaluated. The results of the indoor toxicity test on the second instar larvae of Southern Root-knot Nematode are shown in Table 1.

[0033] Table 1. Results of indoor toxicity assays of the combination of methyl isothiocyanate and ivermectin on second-instar larvae of *Strombus heterophylla*.

[0034]

[0035] Table 1 shows that both methyl isothiocyanate and ivermectin, as single agents, exhibited toxic activity against second-instar larvae of the southern root-knot nematode. The LC50 of methyl isothiocyanate was 1.730 ppm, and that of ivermectin was 0.076 ppm. When the two agents were combined, all ratios showed a synergistic effect, with synergistic coefficients ranging from 2.02 to 8.31, all greater than 1.5. This indicates that the combination of methyl isothiocyanate and ivermectin has a significant synergistic effect against second-instar larvae of the southern root-knot nematode.

[0036] Among different compound ratios, the compound with the highest LC50 (0.146 ppm) and synergistic effect (8.31) was achieved when the mass ratio of methyl isothiocyanate to ivermectin was 50:1, indicating the most significant synergistic effect between the two agents against second-instar larvae of the southern root-knot nematode. When the mass ratios of methyl isothiocyanate to ivermectin were 30:1, 70:1, and 100:1, the synergistic effects were 3.67, 3.84, and 3.65, respectively, also showing a significant synergistic effect. Based on the combined LC50 and synergistic effect results, 50:1 is the optimal compound ratio for controlling second-instar larvae of the southern root-knot nematode under the conditions of this experiment.

[0037] Example 2: Test on the inhibitory effect of the pharmaceutical composition of the present invention on the hatching of southern root-knot nematode eggs.

[0038] Step 1: Preparation of the medicinal solution

[0039] Preparation of stock solution: Dissolve and dilute methyl isothiocyanate (MITC) in acetone to prepare a 1000 ppm MITC stock solution; dilute ivermectin with distilled water to prepare a 1000 ppm ivermectin stock solution for later use.

[0040] Preparation of compound drug solutions: Based on the mass ratio of the active ingredients of methyl isothiocyanate and ivermectin, the MITC mother liquor and ivermectin mother liquor were compounded in proportions of 10:1, 15:1, 25:1, 50:1, 70:1 and 100:1, respectively, to obtain compound drug solutions with different mass ratios for later use.

[0041] Concentration Setting: Based on the preliminary test results, a series of final effective concentrations were set for both single-agent and compound formulations. Specifically, the final effective concentrations for methyl isothiocyanate single agent were set to 0.5, 1.5, 2.0, 3.0, 4, 0, and 4.5 ppm; the final effective concentrations for ivermectin single agent were set to 0.05, 0.1, 0.2, 0.5, 0.6, and 0.8 ppm; and the final effective concentrations for each compound formulation prepared by mixing methyl isothiocyanate and ivermectin at active ingredient mass ratios of 10:1, 15:1, 25:1, 50:1, 70:1, and 100:1 were all set to 0.2, 0.5, 0.8, 1.0, 2.0, and 4.0 ppm. The test solutions were all pre-prepared at twice the above final effective concentrations and mixed with an equal volume of insect egg suspension to achieve the corresponding set final effective concentrations. Four replicates were set up for each concentration treatment, and a distilled water control was set up to determine the inhibitory activity of each single agent and compound agent on the hatching of Southern root-knot nematode eggs.

[0042] Step 2: Test insect eggs

[0043] The test eggs were those of the southern root-knot nematode (Meloidogyne incognita), provided by Zhejiang Jidun Biotechnology Co., Ltd. Before the experiment, the eggs were cultured at 25℃, and eggs with consistent developmental stages were selected for the hatching inhibition test.

[0044] Step 3: Egg Hatching Inhibition Test

[0045] The inhibitory effects of methyl isothiocyanate, ivermectin, and compound formulations with different active ingredient ratios on the hatching of southern root-knot nematodes were determined using the egg hatching method. In a 96-well cell culture plate, 100 μL of egg suspension (approximately 200–250 eggs) was added to each well, followed by 100 μL of the corresponding concentration of the drug solution prepared in step 1. The drug solution and egg suspension were thoroughly mixed, and each treatment was performed in quadruplicate.

[0046] All test solutions were prepared at twice the final test concentration and mixed with an equal volume of insect egg suspension to bring each treatment system to the final test concentration set in step 1.

[0047] The treated culture plates were placed in a constant temperature incubator at 25℃ for 7–9 days. After the culture was completed, the hatching of the eggs in each treatment was observed and recorded under a stereomicroscope, and the number of second-instar larvae that hatched and the number of unhatched eggs were counted.

[0048] Step 4: Result Calculation and Analysis

[0049] Based on the number of second-instar larvae hatched and the number of unhatched eggs in each treatment, the egg hatching rate and hatching inhibition rate were calculated.

[0050] Egg hatching rate (%) = Number of second-instar larvae hatched / Total number of eggs tested × 100

[0051] Hatching inhibition rate (%) = (Control hatching rate - Treatment hatching rate) / Control hatching rate × 100

[0052] Regression analysis was used to analyze the logarithmic values ​​of pesticide concentrations and the probability values ​​of hatching inhibition rates, and the EC50 of each single agent and compound agent on the hatching of southern root-knot nematode eggs was calculated. 50 The combined effect of methyl isothiocyanate and ivermectin on the hatching of southern root-knot nematode eggs was evaluated by calculating the synergistic coefficient using the Wadley method. The results of the inhibitory effect on the hatching of southern root-knot nematode eggs are shown in Table 2.

[0053] Table 2. Results of indoor toxicity assays of methyl isothiocyanate combined with ivermectin on southern root-knot nematode eggs.

[0054]

[0055] Table 2 shows that both methyl isothiocyanate and ivermectin alone can inhibit the hatching of southern root-knot nematode eggs. Methyl isothiocyanate, however, has a lower EC50. 50 The EC50 of ivermectin was 1.865 ppm. 50 The concentration was 0.116 ppm. The two agents, when combined, exhibited varying degrees of synergistic effects on the hatching of southern root-knot nematodes eggs. The combinations at ratios of 15:1, 25:1, and 50:1 showed a synergistic effect, with synergistic coefficients ranging from 1.90 to 2.14, indicating that the combination of methyl isothiocyanate and ivermectin can enhance the inhibitory effect on the hatching of southern root-knot nematodes eggs.

[0056] In different compounding ratios, when the mass ratio of methyl isothiocyanate to ivermectin is 15:1, the compound agent EC 50The lowest concentration was 0.448 ppm, with the highest synergistic effect coefficient of 2.14, indicating that the two agents had the most significant synergistic inhibitory effect on the hatching of southern root-knot nematodes eggs at this ratio. As the proportion of methyl isothiocyanate further increased, the inhibitory effect of the combination on egg hatching gradually decreased. The 25:1 and 50:1 ratios still showed a synergistic effect, with synergistic coefficients of 1.98 and 1.90, respectively. When the ratio was increased to 70:1 and 100:1, the synergistic coefficients decreased to 1.29 and 1.07, respectively, showing an additive effect. The results indicate that excessively high proportions of methyl isothiocyanate do not further enhance the synergistic inhibitory effect of ivermectin on the hatching of southern root-knot nematodes eggs.

[0057] This invention has discovered that methyl isothiocyanate and ivermectin have different optimal synergistic ratios for different developmental stages of southern root-knot nematodes. A mass ratio of 50:1 is more suitable for killing second-instar larvae, while a mass ratio of 15:1 is more suitable for inhibiting egg hatching. This indicates that the composition of this invention has synergistic control advantages targeting different developmental stages.

[0058] Example 3: Field control efficacy of ivermectin combined with ivermectin against sweet potato root-knot nematodes.

[0059] Step 1: Test site and test crop

[0060] The field trial was conducted in Chengmai County, Hainan Province, with sweet potato as the test crop. Fields with relatively uniform root-knot nematode infestation were selected as experimental sites. Soil samples were collected from each plot before pesticide application using a five-point sampling method to determine the initial root-knot nematode population density in the soil.

[0061] Step 2: Test reagents and treatment setup

[0062] The test agents were 42% valerate (valerate decomposes to produce methyl isothiocyanate) and 0.5% ivermectin emulsifiable concentrate. Based on the results of combined indoor toxicity assays, treatments were set up including valerate single-agent, ivermectin single-agent, and a combination of both, with a blank control included. Each treatment had four replicates, arranged in a randomized block design, with each plot area being 25 m². 2 Specific treatment and dosage instructions are shown in Table 3.

[0063] Step 3: Pesticide application and field management

[0064] The pesticides were applied according to the prescribed dosages for each treatment. The 42% ivermectin single-agent treatment was diluted with water and applied to the soil via soil injection; the 0.5% ivermectin emulsifiable concentrate single-agent treatment was diluted with water and applied to the soil in the same manner; the compound treatment was prepared by mixing 42% ivermectin aqueous solution and 0.5% ivermectin emulsifiable concentrate at the prescribed dosages, diluting with water, and then applying the compound solution to the soil via soil injection in a single application. After application, the soil was covered with plastic film for treatment. After the set treatment time, the film was removed for ventilation, and sweet potatoes were planted after the pesticides had fully dispersed. Except for the type and dosage of pesticides, all other field management practices remained consistent across treatments.

[0065] Step 4: Investigation and Evaluation of Prevention and Control Effectiveness

[0066] Soil samples were collected from each treatment before and 60 days after application to determine the number of root-knot nematodes in the soil. At sweet potato harvest, 20 plants were randomly selected from each plot, their roots were completely dug up and cleaned, and the roots were graded according to the degree of root knot formation. The number of plants with root knots at each grade was recorded, the root knot index for each treatment was calculated, and the root knot control efficacy was calculated. The root knot grading standard used a scale of 0 to 5, where grade 0 indicates no root knots and grade 5 indicates the most severe root knot formation. The root knot index was calculated using the following formula: Root knot index (%) = Σ(Number of plants with root knots at each grade × Corresponding grade) / (Total number of plants surveyed × Highest grade) × 100. Root knot control efficacy (%) = (Root knot index of blank control - Root knot index of treatment) / Root knot index of blank control × 100.

[0067] Step 5: Test Results

[0068] The field control efficacy of methyl isothiocyanate (produced after decomposition) combined with ivermectin against sweet potato root-knot nematodes is shown in Table 3. All treatments reduced the number of root-knot nematodes in the rhizosphere soil of sweet potatoes and lessened the degree of root knot formation at harvest. At 60 days post-application, the nematode densities of methyl isothiocyanate (155.00), ivermectin (147.50), and the combined treatment were 42.50 nematodes / 100g soil, respectively, all lower than the control group. The combined treatment showed the lowest nematode density and a control efficacy of 92.06%, significantly higher than the control efficacy of the two single-agent treatments.

[0069] The root knot survey results at harvest showed that the root knot index of the compound treatment was 5.0, and the root knot control efficacy reached 93.06%, which was higher than that of methyl isothiocyanate alone (72.22%) and ivermectin alone (75.00%). The results indicate that the compound treatment of methyl isothiocyanate (which decomposes to produce methyl isothiocyanate) and ivermectin can effectively reduce the population of root-knot nematodes in the rhizosphere soil of sweet potato, reduce root knot formation, and has a good field control effect on sweet potato root-knot nematodes.

[0070] Table 3. Field control efficacy of basil and ivermectin combined with sweet potato root-knot nematode.

[0071] 。

Claims

1. A nematicide composition, characterized in that, The nematicide composition is obtained by mixing methyl isothiocyanate and ivermectin at an active ingredient mass ratio of 10:1 to 100:1, and the total effective concentration of methyl isothiocyanate and ivermectin is 0.2-4.0 ppm.

2. The nematicidal composition according to claim 1, characterized in that, The methyl isothiocyanate and ivermectin are mixed in the form of a mother liquor, wherein the methyl isothiocyanate is dissolved in acetone and then diluted with distilled water to obtain the methyl isothiocyanate mother liquor; and the ivermectin emulsifiable concentrate is directly diluted with distilled water to obtain the ivermectin mother liquor.

3. The nematicide composition according to claim 2, characterized in that, The mass concentration of the mother liquor for methyl isothiocyanate is 800-1200 mg / L, and the mass concentration of the mother liquor for ivermectin is 800-1200 mg / L.

4. The nematicide composition according to claim 2, characterized in that, When controlling second-instar larvae of the southern root-knot nematode, the mass ratio of the active ingredients of methyl isothiocyanate to ivermectin is 30:1-90:1; when controlling the hatching of southern root-knot nematode eggs, the mass ratio of the active ingredients of methyl isothiocyanate to ivermectin is 11:1-50:

1.

5. The nematicidal composition according to claim 4, characterized in that, When controlling second-instar larvae of the southern root-knot nematode, the mass ratio of the active ingredients of methyl isothiocyanate to ivermectin is 48-52:1; when controlling the hatching of southern root-knot nematode eggs, the mass ratio of the active ingredients of methyl isothiocyanate to ivermectin is 14-16:

1.

6. The use of the nematicidal drug composition according to any one of claims 1 to 5 in the control of southern root-knot nematodes.

7. The application as described in claim 6, characterized in that, It is used to kill second-instar larvae of the southern root-knot nematode or to inhibit the hatching of southern root-knot nematode eggs.

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