Application of 2-methylindoline in prevention and control of nematodes

Abstract

The invention discloses an application of 2-methyl indoline in prevention and control of nematodes. The invention relates to an application of 2-methylindoline in prevention and control of nematodes. The 2-methylindoline is diluted to 12.5-50 [mu] g / ml for use. The nematodes are root-knot nematodes. The nematodes are meloidogyne incognita. The preparation method of the 2-methylindoline comprises the following steps: after cigar fermented tobacco leaves are extracted with methanol, extracting with ethyl acetate to obtain an extract containing the 2-methylindoline. A nematode pesticide comprises the 2-methyl indoline in any one of the applications mentioned above. The nematode insecticide is a botanical insecticide. The small molecule compound 2-methylindoline is applied to prevention and control of nematodes, can significantly kill root-knot nematodes, has a good prevention and control effect, and has high application potential.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, and in particular to the application of 2-methyldihydroindole in the control of nematodes. Background Technology

[0002] The southern root-knot nematode (Meloidogyne incognita), one of the world's most harmful soil-borne plant parasitic nematodes, poses a serious threat to agricultural production. According to statistics from the International Plant Protection Convention (IPPC), this nematode has infested 3,147 plant species, including those in the Solanaceae, Cucurbitaceae, and Fabaceae families, causing direct economic losses exceeding tens of billions of US dollars annually. Its unique biological characteristics and pathogenic mechanisms make it a major challenge to sustainable agricultural development. Faced with the problems of pesticide resistance and ecological risks associated with traditional chemical pesticides, the development of green control technologies is urgently needed. In recent years, plant-derived pesticides have received considerable attention due to their multiple biological activities and environmental safety; therefore, developing plant-derived pesticides is an important way to build an environmentally friendly integrated pest management system. Summary of the Invention

[0003] The purpose of this invention is to provide the application of 2-methyldihydroindole in the control of nematodes. This compound can significantly kill southern root-knot nematodes, thereby improving the biological control effect against southern root-knot nematodes.

[0004] This invention provides the application of 2-methyldihydroindole in the control of nematodes, wherein 2-methyldihydroindole is diluted to 12.5-50 μg / ml before use.

[0005] In the above application, the nematode is the root-knot nematode.

[0006] In the above application, the nematode is the southern root-knot nematode.

[0007] In the above applications, the method for preparing 2-methyldihydroindole includes extracting fermented tobacco leaves of cigars with methanol and then extracting them with ethyl acetate to obtain an extract containing 2-methyldihydroindole.

[0008] The present invention also provides a nematode insecticide comprising the 2-methyldihydroindole used in any of the above-described applications.

[0009] The nematode insecticides mentioned above are plant-derived insecticides.

[0010] The present invention also provides a method for preparing the above-mentioned nematode insecticide, comprising extracting the fermented tobacco leaves of cigars with methanol, extracting with ethyl acetate, concentrating, and obtaining the agent.

[0011] The present invention has the following beneficial effects:

[0012] The small molecule compound 2-methyldihydroindole of this invention can be used to control nematodes. It can significantly kill root-knot nematodes, with good control effect and high application potential.

[0013] In this invention, 2-methyldihydroindole, a small molecule compound that can effectively kill root-knot nematodes, was screened from compounds detected in cigar tobacco extracts, providing a green and safe biopesticide raw material for the control of southern root-knot nematodes. Traditionally, the synthesis cost of dihydroindole compounds is relatively high. This invention explores a new method for preparing 2-methyldihydroindole, reducing application costs and practical difficulties.

[0014] The nematode insecticide of the present invention has a significant killing effect on root-knot nematodes, especially on southern root-knot nematodes, and is environmentally friendly, biodegradable and pollution-free in the process of control. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention.

[0016] Application of 2-methyldihydroindole in the control of nematodes. The molecular structure of 2-methyldihydroindole is as follows:

[0017]

[0018] 2-Methyldihydroindole is diluted to 12.5-50 μg / ml before use, with 50 μg / ml being the preferred concentration. It is effective against nematodes including the southern root-knot nematode and the northern root-knot nematode, and is particularly effective against the southern root-knot nematode.

[0019] Example 1

[0020] The fermented tobacco leaves of the "Yunxue 39" cigar used in the examples were collected from the World Tobacco Plantation in Yuxi, Yunnan.

[0021] The methanol extract of Yunxue 39 was obtained by extraction with ethyl acetate, and the metabolites in the extract were detected by LC-MS. The specific substances producing nematicidal activity in the Yunxue 39 extract were analyzed.

[0022] 1. The preparation method of Yunxue 39 extract includes:

[0023] A. Yunxue 39 fermented tobacco leaves are dried and then thoroughly pulverized to 1-5mm using a pulverizer to obtain coarsely pulverized material;

[0024] B. Extract the coarsely ground material obtained in step A with methanol by cold soaking for 24-48 hours (the mass ratio of coarsely ground material to methanol is 10 kg: 1 kg), repeat 3-5 times to obtain cigar tobacco fermentation extract.

[0025] C. The extract obtained in step B is repeatedly extracted with an organic solvent to obtain an extract, which is then concentrated to 5 kg under pressure using a rotary evaporator.

[0026] 2. The LC-MS detection method for metabolites in Yunxue 39 extract includes:

[0027] A. Remove the sample from the -80℃ freezer and thaw it. Vortex for 1 min. Add 70% methanol-water internal standard extraction solution pre-cooled at -20℃ (600 μL of extraction solution per 50 mg sample). Vortex for 15 min. The internal standard extraction solution is prepared by dissolving 1 mg of standard in 1 mL of 70% methanol-water to prepare a 1000 μg / mL standard stock solution. The 1000 μg / mL stock solution is further diluted with 70% methanol to prepare a 250 μg / mL internal standard solution. Centrifuge at 12000 r / min and 4℃ for 3 min. Take an appropriate amount of supernatant and filter it through a microporous membrane (0.22 μm pore size). Store it in a sample vial for LC-MS analysis.

[0028] B.LC conditions: The chromatographic column was Waters ACQUITY Premier HSS T3 (2.1 mm × 100 mm, 1.8 μm); the mobile phase A was 0.1% formic acid aqueous solution; the mobile phase was acetonitrile solution (containing 0.1% formic acid); the instrument column temperature was 40℃; the flow rate was 0.4 mL / min; and the injection volume was 4 μL.

[0029] 3. The test results are shown in Table 1.

[0030] The results in Table 1 show that the main nematicidal component in cigar extract is 2-methyldihydroindole.

[0031] Table 1

[0032]

[0033]

[0034] Example 2

[0035] Purified compounds were purchased and analyzed. The toxic activity of the compound solution was determined using the immersion method. Healthy, uniformly sized nematodes were selected as test nematodes. The experiment was conducted in 24-well cell culture plates. Nematode suspension was injected into the sample wells. The compound was accurately weighed and dissolved in sterile water to prepare the agent. This agent was then mixed thoroughly with the nematode suspension to achieve concentrations of 50 μg / ml, 25 μg / ml, and 12.5 μg / ml. The plates were incubated at 25°C with three replicates per treatment, using 100 nematodes per replicate. A 5% avermectin solution was used as a positive control, and sterile water treatment was used as a blank control. Nematode mortality and survival were checked at 24, 48, and 72 hours after treatment. Nematode mortality and corrected mortality were calculated using formulas (1) and (2), respectively.

[0036] Nematode mortality rate (%) = number of dead nematodes / number of tested nematodes × 100 (1),

[0037] Corrected mortality rate (%) = (nematode mortality rate in treatment group - nematode mortality rate in control group) / (1 - nematode mortality rate in control group) × 100 (2).

[0038] Methods for determining whether nematodes are alive or dead: Under a microscope, nematodes that are swimming or whose bodies are in an "S" shape are considered alive. Nematodes that show a significant change in shape after being gently touched with an inoculation needle while still are considered alive; otherwise, they are considered dead.

[0039] The toxic activity of 2-methyldihydroindole against nematodes was determined by the drug immersion method, and the toxicity regression equation was calculated. The results are shown in Tables 2 and 3.

[0040] Table 2.2 The toxic effects of 2-methyldihydroindole on southern root-knot nematodes.

[0041]

[0042] Table 3. Toxicity and toxicity curves of 2-methyldihydroindole against Southern root-knot nematode.

[0043]

[0044] Tables 2 and 3 show that 2-methyldihydroindole exhibits strong toxic activity against *Strombus haemolyticus* (southern root-knot nematode). The toxicity of this compound against *Strombus haemolyticus* is concentration-dependent, and the effect increases significantly with treatment time. Treatment with 50 μg / ml resulted in a corrected mortality rate of 34.36% after 72 hours. The LC50 values ​​for *Strombus haemolyticus* at 24 hours, 48 ​​hours, and 72 hours were 88.070 μg / ml, 80.535 μg / ml, and 62.625 μg / ml, respectively. At the same treatment time, its toxic activity against *Strombus haemolyticus* was significantly higher than that of 5% abamectin.

[0045] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. The above descriptions are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art.

Claims

The application of 1,2-methyldihydroindole in the control of nematodes is characterized by, 2-Methyldihydroindole should be diluted to 12.5-50 μg / ml before use.

2. The application according to claim 1, characterized in that, The nematode is a root-knot nematode.

3. The application according to claim 2, characterized in that, The nematode in question is the southern root-knot nematode.

4. The application according to claim 1, characterized in that, The method for preparing the 2-methyldihydroindole includes extracting fermented tobacco leaves of cigars with methanol and then extracting them with ethyl acetate to obtain an extract containing the 2-methyldihydroindole.

5. A nematode insecticide, characterized in that, Includes the 2-methyldihydroindole in any of the applications described in claims 1-4.

6. The nematode insecticide according to claim 5, characterized in that, It is a plant-derived insecticide.

7. The method for preparing the nematode insecticide according to claim 6, characterized in that, The process involves extracting the fermented tobacco leaves of the cigar with methanol, then extracting with ethyl acetate, and concentrating the extract to obtain a pharmaceutical agent.

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