A choline-based tobacco root-knot nematode inhibitor and its preparation method

Abstract

The present invention relates to the technical field of pest repellents, and discloses a choline-based tobacco root-knot nematode inhibitor and its preparation method. The choline-based tobacco root-knot nematode inhibitor provided by the present invention comprises the following raw materials, in parts by weight: 10-20 parts of a choline derivative, 5-10 parts of a mixed plant essential oil, 3-5 parts of a composite microbial agent, 1-3 parts of sodium alginate, 3-5 parts of an emulsifier, and 40-60 parts of water. The choline-based tobacco root-knot nematode inhibitor provided by the present invention has a strong inhibitory effect on tobacco root-knot nematodes, effectively blocking the parasitic pathways of the root-knot nematodes and restoring normal plant growth. The choline-based tobacco root-knot nematode inhibitor provided by the present invention also has a high biosafety rating.

Description

Technical Field

[0001] The present invention relates to the technical field of pest repellents, and in particular to a choline-based tobacco root-knot nematode inhibitor and a preparation method thereof. Background Art

[0002] Tobacco root-knot nematode disease is a major threat to tobacco production. Once the root-knot nematodes invade tobacco roots, they stimulate root cell proliferation, forming root knots of varying sizes. This severely impacts the tobacco root system's ability to absorb water and nutrients, leading to weak plant growth, yellowing leaves, reduced yield, and decreased quality. Therefore, the development of tobacco root-knot nematode inhibitors is of great significance to tobacco cultivation. Summary of the Invention

[0003] The present invention aims to provide a choline-based tobacco root-knot nematode inhibitor and a preparation method thereof. The choline-based tobacco root-knot nematode inhibitor has a good inhibitory effect on tobacco root-knot nematodes and can effectively block the parasitic channels of the root-knot nematodes and restore the normal growth of the plants.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] One of the technical solutions of the present invention:

[0006] A choline-based tobacco root-knot nematode inhibitor, comprising the following raw materials in parts by weight:

[0007] 10-20 parts of choline derivatives, 5-10 parts of mixed plant essential oils, 3-5 parts of composite microbial agents, 1-3 parts of sodium alginate, 3-5 parts of emulsifiers and 40-60 parts of water.

[0008] Furthermore, the structural formula of the choline derivative is:

[0009] .

[0010] Furthermore, the preparation method of the choline derivative comprises the following steps:

[0011] Choline chloride is first dissolved in dichloromethane to form a choline chloride solution, triethylamine is then added to the choline chloride solution to form a mixed solution, and 4-fluorobenzoyl chloride is finally added dropwise to the mixed solution. After the addition is completed, the mixture is reacted and purified to obtain the choline derivative.

[0012] Furthermore, the molar ratio of choline chloride, triethylamine and 4-fluorobenzoyl chloride is 1:1.2:1.

[0013] Furthermore, the dripping rate is 30d / min.

[0014] Furthermore, the reaction is specifically carried out at room temperature for 6 hours.

[0015] Furthermore, the purification is specifically as follows: filtration, washing with 5% dilute hydrochloric acid, saturated sodium bicarbonate, and water in sequence, and rotary evaporation.

[0016] In the present invention, the synthesis formula of the choline derivative is:

[0017] .

[0018] Furthermore, the preparation method of the mixed plant essential oil comprises the following steps:

[0019] 1) mint leaves and rosemary branches and leaves are mixed and crushed to obtain a mixed plant essential oil raw material;

[0020] 2) placing the mixed plant essential oil raw material obtained in step 1) in water, performing steam distillation, and separating the oil and water to obtain the mixed plant essential oil.

[0021] Furthermore, in step 1), the mass ratio of the mint leaves to the rosemary branches and leaves is 1:(0.4-0.6).

[0022] Furthermore, in step 2), the mass volume ratio of the mixed plant essential oil raw material and water is 1:(4-6).

[0023] Furthermore, in step 2), the steam distillation is specifically: steam distillation at 95-105° C. for 2-4 hours.

[0024] Furthermore, the composite microbial agent is formed by mixing Bacillus subtilis and Bacillus amyloliquefaciens in a mass ratio of 1:1.

[0025] The second technical solution of the present invention:

[0026] The preparation method of the above-mentioned choline-based tobacco root-knot nematode inhibitor comprises the following steps:

[0027] 1) Adding the choline derivative, the composite microbial agent and sodium alginate to water and stirring to obtain a premixed solution;

[0028] 2) Adding the mixed plant essential oil and the emulsifier to the premixed solution obtained in step 1), stirring and emulsifying, thereby obtaining the choline-based tobacco root-knot nematode inhibitor.

[0029] Furthermore, in step 1), the water needs to be preheated to 50-60°C.

[0030] Furthermore, in step 1), the stirring is specifically: maintaining the temperature at 50-60° C. and stirring for 40-60 minutes.

[0031] Furthermore, in step 2), the stirring and emulsification is specifically: stirring and emulsifying at 50-60° C. for 1-2 hours.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The choline-based tobacco root-knot nematode inhibitor provided by the present invention, when directly acting on the second-instar larvae of tobacco root-knot nematodes, can achieve a 24-hour mortality rate of 76% and a 48-hour mortality rate of 88%. Compared with the commonly used insecticide avermectin, the choline-based tobacco root-knot nematode inhibitor provided by the present invention has a better direct killing effect on tobacco root-knot nematodes;

[0034] After root irrigation, the choline-based tobacco root-knot nematode inhibitor provided by the present invention can achieve a root knot index of 1, a nematode reduction rate of 82%, a plant height of 117 cm, and a SPAD value of 42. This indicates that the choline-based tobacco root-knot nematode inhibitor provided by the present invention has a good inhibitory effect on tobacco root-knot nematodes and can effectively block the parasitic pathways of root-knot nematodes, restoring normal plant growth.

[0035] When the choline-based tobacco root-knot nematode inhibitor provided by the present invention is directly sprayed on the surface of K326 tobacco leaves, the leaves do not suffer from symptoms such as burns and deformities, and have low contact toxicity to bees, which indicates that the choline-based tobacco root-knot nematode inhibitor provided by the present invention has high biosafety.

[0036] The present invention provides a choline-based tobacco root-knot nematode inhibitor, wherein the choline derivative has targeted neurotoxicity to tobacco root-knot nematodes. Specifically, the choline derivative provided by the present invention can interfere with the synthesis, release, and conduction of neurotransmitters such as acetylcholine, and the 4-fluorobenzoic acid released after hydrolysis can interfere with the energy metabolism of tobacco root-knot nematodes, thereby achieving an inhibitory effect on tobacco root-knot nematodes.

[0037] In the choline-based tobacco root-knot nematode inhibitor provided by the present invention, the volatile odor of the mixed plant essential oil can repel root-knot nematodes, keeping them away from tobacco roots and reducing the risk of tobacco being infected by root-knot nematodes. At the same time, the active ingredients contained in the plant essential oil can directly act on the root-knot nematodes, interfering with their physiological and biochemical processes and inhibiting their growth and reproduction, thereby reducing the damage caused by root-knot nematodes to tobacco.

[0038] The present invention provides a choline-based tobacco root-knot nematode inhibitor. The composite microbial agent can produce a variety of antimicrobial substances during metabolism, such as lipopeptides and protein antibiotics, which have a direct inhibitory effect on root-knot nematodes. In addition, the composite microbial agent can also colonize on the surface of tobacco roots, forming a protective film to prevent root-knot nematodes from invading and reduce the risk of tobacco being infected by root-knot nematodes.

[0039] In the choline-based tobacco root-knot nematode inhibitor provided by the present invention, sodium alginate can form a protective film on the surface of the tobacco root system, preventing the invasion of root-knot nematodes and reducing the risk of tobacco being infected by root-knot nematodes;

[0040] The present invention provides a choline-based tobacco root-knot nematode inhibitor. The choline derivative can interfere with the nervous system of the root-knot nematode, inhibiting its growth and reproduction. The plant essential oil is volatile and bioactive, and can repel and inhibit the root-knot nematode. The composite microbial agent can produce a variety of antibacterial substances, which have an inhibitory effect on the root-knot nematode. The sodium alginate can form a protective film on the tobacco roots, preventing the invasion of the root-knot nematode. The four ingredients work synergistically to greatly improve the inhibitory effect on tobacco root-knot nematodes. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0042] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] In the following examples, the Bacillus subtilis was purchased from the China General Microorganism Culture Collection Center with a deposit number of CGMCC.No. 1.9086; the Bacillus amyloliquefaciens was purchased from the China General Microorganism Culture Collection Center with a deposit number of CGMCC.No. 1.3450.

[0047] In the following examples, a method for preparing a choline-based tobacco root-knot nematode inhibitor comprises the following steps:

[0048] 1. Preparation of choline derivatives:

[0049] According to the molar ratio of choline chloride, triethylamine and 4-fluorobenzoyl chloride being 1:1.2:1, choline chloride is first dissolved in dichloromethane to form a choline chloride solution, and then triethylamine is added to the choline chloride solution to form a mixed solution. Finally, 4-fluorobenzoyl chloride is added dropwise to the mixed solution at a dripping rate of 30 d / min. After the dripping is completed, the mixture is reacted at room temperature for 6 h, filtered, washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate and water in sequence, and rotary evaporated to obtain the choline derivative;

[0050] 2. Preparation of mixed plant essential oils:

[0051] 1) mixing the mint leaves and rosemary branches and leaves in a mass ratio of 1:(0.4-0.6), crushing the mint leaves and rosemary branches and leaves into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0052] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:(4-6), and steam distilling at 95-105° C. for 2-4 hours to separate the oil and water to obtain the mixed plant essential oil;

[0053] 3. Preparation of composite microbial agents:

[0054] Bacillus subtilis and Bacillus amyloliquefaciens are mixed in a mass ratio of 1:1 to obtain the composite microbial agent;

[0055] 4. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0056] 1) Weigh the raw materials according to the following parts by mass:

[0057] 10-20 parts of choline derivatives, 5-10 parts of mixed plant essential oils, 3-5 parts of composite microbial agents, 1-3 parts of sodium alginate, 3-5 parts of emulsifiers, and 40-60 parts of water;

[0058] 2) heating the water weighed in step 1) to 50-60° C., then adding the choline derivative, composite microbial agent, and sodium alginate weighed in step 1) to the heated water, maintaining the temperature at 50-60° C. and stirring for 40-60 minutes to obtain a premixed solution;

[0059] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 50-60° C. for 1-2 hours to obtain the choline-based tobacco root-knot nematode inhibitor.

[0060] Example 1

[0061] A choline-based tobacco root-knot nematode inhibitor

[0062] 1. Preparation of choline derivatives:

[0063] Choline chloride (1.39 g, 10 mmol) was dissolved in dichloromethane to form a choline chloride solution. Triethylamine (1.66 mL, 12 mmol) was then added to the choline chloride solution to obtain a mixed solution. 4-Fluorobenzoyl chloride (1.19 mL, 10 mmol) was added dropwise to the mixed solution at a drip rate of 30 d / min. After the dripping was completed, the mixture was reacted at room temperature for 6 h, filtered, washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate, and water, and rotary evaporated to obtain a choline derivative.

[0064] 2. Preparation of mixed plant essential oils:

[0065] 1) mixing the mint leaves and rosemary branches and leaves in a mass ratio of 1:0.4, and crushing the mixture into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0066] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:4, and subjecting the mixed plant essential oil raw material to steam distillation at 95° C. for 2 hours to separate the oil and water, thereby obtaining the mixed plant essential oil;

[0067] 3. Preparation of composite microbial agents:

[0068] Bacillus subtilis and Bacillus amyloliquefaciens are mixed in a mass ratio of 1:1 to obtain the composite microbial agent;

[0069] 4. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0070] 1) Weigh the raw materials according to the following parts by mass:

[0071] 10 parts of choline derivatives, 5 parts of mixed plant essential oils, 3 parts of composite microbial agents, 1 part of sodium alginate, 3 parts of emulsifiers, and 40 parts of water;

[0072] 2) heating the water weighed in step 1) to 50° C., then adding the choline derivative, composite microbial agent, and sodium alginate weighed in step 1) to the heated water, maintaining the temperature at 50° C. and stirring for 40 minutes to obtain a premixed solution;

[0073] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 50° C. for 1 hour to obtain the choline-based tobacco root-knot nematode inhibitor.

[0074] Example 2

[0075] A choline-based tobacco root-knot nematode inhibitor

[0076] 1. Preparation of choline derivatives:

[0077] Choline chloride (1.39 g, 10 mmol) was dissolved in dichloromethane to form a choline chloride solution. Triethylamine (1.66 mL, 12 mmol) was then added to the choline chloride solution to obtain a mixed solution. 4-Fluorobenzoyl chloride (1.19 mL, 10 mmol) was added dropwise to the mixed solution at a drip rate of 30 d / min. After the dripping was completed, the mixture was reacted at room temperature for 6 h, filtered, washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate, and water, and rotary evaporated to obtain a choline derivative.

[0078] 2. Preparation of mixed plant essential oils:

[0079] 1) mixing the mint leaves and rosemary branches and leaves in a mass ratio of 1:0.5, and crushing the mixture into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0080] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:5, and subjecting the mixed plant essential oil raw material to steam distillation at 100° C. for 3 hours to separate the oil and water, thereby obtaining the mixed plant essential oil;

[0081] 3. Preparation of composite microbial agents:

[0082] Bacillus subtilis and Bacillus amyloliquefaciens are mixed in a mass ratio of 1:1 to obtain the composite microbial agent;

[0083] 4. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0084] 1) Weigh the raw materials according to the following parts by mass:

[0085] 15 parts of choline derivative, 8 parts of mixed plant essential oil, 4 parts of composite microbial agent, 2 parts of sodium alginate, 4 parts of emulsifier, 50 parts of water;

[0086] 2) heating the water weighed in step 1) to 55° C., then adding the choline derivative, composite microbial agent, and sodium alginate weighed in step 1) to the heated water, maintaining the temperature at 55° C. and stirring for 50 minutes to obtain a premixed solution;

[0087] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 55° C. for 1.5 hours to obtain the choline-based tobacco root-knot nematode inhibitor.

[0088] Example 3

[0089] A choline-based tobacco root-knot nematode inhibitor

[0090] 1. Preparation of choline derivatives:

[0091] Choline chloride (1.39 g, 10 mmol) was dissolved in dichloromethane to form a choline chloride solution. Triethylamine (1.66 mL, 12 mmol) was then added to the choline chloride solution to obtain a mixed solution. 4-Fluorobenzoyl chloride (1.19 mL, 10 mmol) was added dropwise to the mixed solution at a drip rate of 30 d / min. After the dripping was completed, the mixture was reacted at room temperature for 6 h, filtered, washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate, and water, and rotary evaporated to obtain a choline derivative.

[0092] 2. Preparation of mixed plant essential oils:

[0093] 1) mixing the mint leaves and rosemary branches and leaves in a mass ratio of 1:0.6, and crushing the mixture into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0094] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:6, and subjecting the mixed plant essential oil raw material to steam distillation at 105° C. for 4 hours to separate the oil and water, thereby obtaining the mixed plant essential oil;

[0095] 3. Preparation of composite microbial agents:

[0096] Bacillus subtilis and Bacillus amyloliquefaciens are mixed in a mass ratio of 1:1 to obtain the composite microbial agent;

[0097] 4. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0098] 1) Weigh the raw materials according to the following parts by mass:

[0099] 20 parts of choline derivative, 10 parts of mixed plant essential oil, 5 parts of composite microbial agent, 3 parts of sodium alginate, 5 parts of emulsifier, 60 parts of water;

[0100] 2) heating the water weighed in step 1) to 60° C., then adding the choline derivative, composite microbial agent, and sodium alginate weighed in step 1) to the heated water, maintaining the temperature at 60° C. and stirring for 60 minutes to obtain a premixed solution;

[0101] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 60° C. for 2 hours to obtain the choline-based tobacco root-knot nematode inhibitor.

[0102] Comparative Example 1

[0103] A choline-based tobacco root-knot nematode inhibitor

[0104] 1. Preparation of mixed plant essential oils:

[0105] 1) mixing the mint leaves and rosemary branches and leaves in a mass ratio of 1:0.5, and crushing the mixture into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0106] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:5, and subjecting the mixed plant essential oil raw material to steam distillation at 100° C. for 3 hours to separate the oil and water, thereby obtaining the mixed plant essential oil;

[0107] 2. Preparation of composite microbial agents:

[0108] Bacillus subtilis and Bacillus amyloliquefaciens are mixed in a mass ratio of 1:1 to obtain the composite microbial agent;

[0109] 3. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0110] 1) Weigh the raw materials according to the following parts by mass:

[0111] 15 parts of choline chloride, 8 parts of mixed plant essential oils, 4 parts of composite microbial agent, 2 parts of sodium alginate, 4 parts of emulsifier, and 50 parts of water;

[0112] 2) heating the water weighed in step 1) to 55° C., then adding the choline chloride, composite microbial agent, and sodium alginate weighed in step 1) to the heated water, maintaining the temperature at 55° C. and stirring for 50 minutes to obtain a premixed solution;

[0113] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 55° C. for 1.5 hours to obtain the choline-based tobacco root-knot nematode inhibitor.

[0114] Comparative Example 2

[0115] A choline-based tobacco root-knot nematode inhibitor

[0116] 1. Preparation of choline derivatives:

[0117] Choline chloride (1.39 g, 10 mmol) was dissolved in dichloromethane to form a choline chloride solution. Triethylamine (1.66 mL, 12 mmol) was then added to the choline chloride solution to obtain a mixed solution. 4-Fluorobenzoyl chloride (1.19 mL, 10 mmol) was added dropwise to the mixed solution at a drip rate of 30 d / min. After the dripping was completed, the mixture was reacted at room temperature for 6 h, filtered, washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate, and water, and rotary evaporated to obtain a choline derivative.

[0118] 2. Preparation of mixed plant essential oils:

[0119] 1) mixing mint leaves and lavender in a mass ratio of 1:0.5 and crushing the mixture into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0120] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:5, and subjecting the mixed plant essential oil raw material to steam distillation at 100° C. for 3 hours to separate the oil and water, thereby obtaining the mixed plant essential oil;

[0121] 3. Preparation of composite microbial agents:

[0122] Bacillus subtilis and Bacillus amyloliquefaciens are mixed in a mass ratio of 1:1 to obtain the composite microbial agent;

[0123] 4. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0124] 1) Weigh the raw materials according to the following parts by mass:

[0125] 15 parts of choline derivative, 8 parts of mixed plant essential oil, 4 parts of composite microbial agent, 2 parts of sodium alginate, 4 parts of emulsifier, 50 parts of water;

[0126] 2) heating the water weighed in step 1) to 55° C., then adding the choline derivative, composite microbial agent, and sodium alginate weighed in step 1) to the heated water, maintaining the temperature at 55° C. and stirring for 50 minutes to obtain a premixed solution;

[0127] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 55° C. for 1.5 hours to obtain the choline-based tobacco root-knot nematode inhibitor.

[0128] Comparative Example 3

[0129] A choline-based tobacco root-knot nematode inhibitor

[0130] 1. Preparation of choline derivatives:

[0131] Choline chloride (1.39 g, 10 mmol) was dissolved in dichloromethane to form a choline chloride solution. Triethylamine (1.66 mL, 12 mmol) was then added to the choline chloride solution to obtain a mixed solution. 4-Fluorobenzoyl chloride (1.19 mL, 10 mmol) was added dropwise to the mixed solution at a drip rate of 30 d / min. After the dripping was completed, the mixture was reacted at room temperature for 6 h, filtered, washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate, and water, and rotary evaporated to obtain a choline derivative.

[0132] 2. Preparation of mixed plant essential oils:

[0133] 1) mixing lavender and rosemary branches and leaves in a mass ratio of 1:0.5, and crushing the mixture into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0134] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:5, and subjecting the mixed plant essential oil raw material to steam distillation at 100° C. for 3 hours to separate the oil and water, thereby obtaining the mixed plant essential oil;

[0135] 3. Preparation of composite microbial agents:

[0136] Bacillus subtilis and Bacillus amyloliquefaciens are mixed in a mass ratio of 1:1 to obtain the composite microbial agent;

[0137] 4. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0138] 1) Weigh the raw materials according to the following parts by mass:

[0139] 15 parts of choline derivative, 8 parts of mixed plant essential oil, 4 parts of composite microbial agent, 2 parts of sodium alginate, 4 parts of emulsifier, 50 parts of water;

[0140] 2) heating the water weighed in step 1) to 55° C., then adding the choline derivative, composite microbial agent, and sodium alginate weighed in step 1) to the heated water, maintaining the temperature at 55° C. and stirring for 50 minutes to obtain a premixed solution;

[0141] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 55° C. for 1.5 hours to obtain the choline-based tobacco root-knot nematode inhibitor.

[0142] Comparative Example 4

[0143] A choline-based tobacco root-knot nematode inhibitor

[0144] 1. Preparation of choline derivatives:

[0145] Choline chloride (1.39 g, 10 mmol) was dissolved in dichloromethane to form a choline chloride solution. Triethylamine (1.66 mL, 12 mmol) was then added to the choline chloride solution to obtain a mixed solution. 4-Fluorobenzoyl chloride (1.19 mL, 10 mmol) was added dropwise to the mixed solution at a drip rate of 30 d / min. After the dripping was completed, the mixture was reacted at room temperature for 6 h, filtered, washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate, and water, and rotary evaporated to obtain a choline derivative.

[0146] 2. Preparation of mixed plant essential oils:

[0147] 1) mixing the mint leaves and rosemary branches and leaves in a mass ratio of 1:0.5, and crushing the mixture into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0148] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:5, and subjecting the mixed plant essential oil raw material to steam distillation at 100° C. for 3 hours to separate the oil and water, thereby obtaining the mixed plant essential oil;

[0149] 3. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0150] 1) Weigh the raw materials according to the following parts by mass:

[0151] 15 parts of choline derivative, 8 parts of mixed plant essential oil, 4 parts of Bacillus subtilis, 2 parts of sodium alginate, 4 parts of emulsifier, 50 parts of water;

[0152] 2) heating the water weighed in step 1) to 55° C., then adding the choline derivative, Bacillus subtilis, and sodium alginate weighed in step 1) to the heated water, maintaining the temperature at 55° C. and stirring for 50 minutes to obtain a premixed solution;

[0153] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 55° C. for 1.5 hours to obtain the choline-based tobacco root-knot nematode inhibitor.

[0154] Comparative Example 5

[0155] A choline-based tobacco root-knot nematode inhibitor

[0156] 1. Preparation of choline derivatives:

[0157] Choline chloride (1.39 g, 10 mmol) was dissolved in dichloromethane to form a choline chloride solution. Triethylamine (1.66 mL, 12 mmol) was then added to the choline chloride solution to obtain a mixed solution. 4-Fluorobenzoyl chloride (1.19 mL, 10 mmol) was added dropwise to the mixed solution at a drip rate of 30 d / min. After the dripping was completed, the mixture was reacted at room temperature for 6 h, filtered, washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate, and water, and rotary evaporated to obtain a choline derivative.

[0158] 2. Preparation of mixed plant essential oils:

[0159] 1) mixing the mint leaves and rosemary branches and leaves in a mass ratio of 1:0.5, and crushing the mixture into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0160] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:5, and subjecting the mixed plant essential oil raw material to steam distillation at 100° C. for 3 hours to separate the oil and water, thereby obtaining the mixed plant essential oil;

[0161] 3. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0162] 1) Weigh the raw materials according to the following parts by mass:

[0163] 15 parts of choline derivative, 8 parts of mixed plant essential oil, 4 parts of Bacillus amyloliquefaciens, 2 parts of sodium alginate, 4 parts of emulsifier, 50 parts of water;

[0164] 2) heating the water weighed in step 1) to 55° C., then adding the choline derivative, Bacillus amyloliquefaciens, and sodium alginate weighed in step 1) to the heated water, maintaining the temperature at 55° C. and stirring for 50 minutes to obtain a premixed solution;

[0165] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 55° C. for 1.5 hours to obtain the choline-based tobacco root-knot nematode inhibitor.

[0166] Comparative Example 6

[0167] A choline-based tobacco root-knot nematode inhibitor

[0168] 1. Preparation of choline derivatives:

[0169] Choline chloride (1.39 g, 10 mmol) was dissolved in dichloromethane to form a choline chloride solution. Triethylamine (1.66 mL, 12 mmol) was then added to the choline chloride solution to obtain a mixed solution. 4-Fluorobenzoyl chloride (1.19 mL, 10 mmol) was added dropwise to the mixed solution at a drip rate of 30 d / min. After the dripping was completed, the mixture was reacted at room temperature for 6 h, filtered, washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate, and water, and rotary evaporated to obtain a choline derivative.

[0170] 2. Preparation of mixed plant essential oils:

[0171] 1) mixing the mint leaves and rosemary branches and leaves in a mass ratio of 1:0.5, and crushing the mixture into 0.2-0.4 cm fragments to obtain a mixed plant essential oil raw material;

[0172] 2) placing the mixed plant essential oil raw material obtained in step 1) in water at a mass volume ratio of the mixed plant essential oil raw material to water of 1:5, and subjecting the mixed plant essential oil raw material to steam distillation at 100° C. for 3 hours to separate the oil and water, thereby obtaining the mixed plant essential oil;

[0173] 3. Preparation of composite microbial agents:

[0174] Bacillus subtilis and Bacillus amyloliquefaciens are mixed in a mass ratio of 1:1 to obtain the composite microbial agent;

[0175] 4. Preparation of choline-based tobacco root-knot nematode inhibitors:

[0176] 1) Weigh the raw materials according to the following parts by mass:

[0177] 15 parts of choline derivative, 8 parts of mixed plant essential oil, 4 parts of composite microbial agent, 4 parts of emulsifier, 50 parts of water;

[0178] 2) heating the water weighed in step 1) to 55° C., then adding the choline derivative and composite microbial agent weighed in step 1) to the heated water, maintaining the temperature at 55° C. and stirring for 50 minutes to obtain a premixed solution;

[0179] 3) Adding the mixed plant essential oil and emulsifier weighed in step 1) to the premixed solution obtained in step 2), stirring and emulsifying at 55° C. for 1.5 hours to obtain the choline-based tobacco root-knot nematode inhibitor.

[0180] Effect verification

[0181] 1. Laboratory in vitro activity test

[0182] Second-instar larvae (J2) of tobacco root-knot nematodes were isolated from infected tobacco root knots and suspended in sterile water;

[0183] 1 mL of the test reagent (the choline-based tobacco root-knot nematode inhibitor prepared in Examples 1 to 3 and Comparative Examples 1 to 6, at a concentration of 50 ppm) and 100 J2 larvae were added to each well of a multi-well plate. The plates were incubated in the dark at 25°C. After 24 and 48 hours, microscopic examinations were performed to calculate the mortality rate (stiffness was considered death). The mortality rate calculation results are shown in Table 1.

[0184] Table 1 Mortality rate (%)

[0185]

[0186] As can be seen from the data in Table 1, when the choline-based tobacco root-knot nematode inhibitor provided by the present invention directly acts on the second-instar larvae of tobacco root-knot nematodes, the 24-hour mortality rate of the second-instar larvae of tobacco root-knot nematodes can reach 76%, and the 48-hour mortality rate can reach 88%. Compared with the commonly used insecticide avermectin, the choline-based tobacco root-knot nematode inhibitor provided by the present invention has a better direct killing effect on tobacco root-knot nematodes.

[0187] 2. Potted Plant Experiment

[0188] K326 tobacco plants were sown in pots (5 plants per pot). Two weeks after sowing, 1000 J2 larvae were inoculated in each pot. 24 hours after inoculation, the roots were irrigated with 50 mL / plant of pesticide (choline-based tobacco root-knot nematode inhibitor prepared in Examples 1 to 3 and Comparative Examples 1 to 6, concentration of 50 ppm; avermectin, concentration of 50 ppm; water). Three weeks after the application, the number of nematodes and plant growth indicators were tested. The results of the potted plant experiment are shown in Table 2.

[0189] Table 2 Potted plant test results

[0190]

[0191] As can be seen from the data in Table 2, after root irrigation and application of the choline-based tobacco root-knot nematode inhibitor provided by the present invention, the root knot index can reach 1, the nematode reduction rate can reach 82%, the plant height can reach 117 cm, and the SPAD value can reach 42. This shows that the choline-based tobacco root-knot nematode inhibitor provided by the present invention has a good inhibitory effect on tobacco root-knot nematodes, and can effectively block the parasitic channels of root-knot nematodes and restore normal growth of plants.

[0192] 3. Biosafety Testing

[0193] The biosafety test of the choline-based tobacco root-knot nematode inhibitor prepared in Example 2 was conducted. The test showed that when the choline-based tobacco root-knot nematode inhibitor at a concentration of 1000 ppm was sprayed on the surface of K326 tobacco leaves, the leaves showed no symptoms such as burns or deformities.

[0194] After testing, it was found that after bees were exposed to a choline-based tobacco root-knot nematode inhibitor at a concentration of 1000 ppm, the 48-hour mortality rate was only 23%, indicating low contact toxicity to bees.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered by the scope of protection of the claims of the present invention.

Claims

1. A choline-based tobacco root-knot nematode inhibitor, characterized in that: Including the following raw materials by weight: 10-20 parts of choline derivatives, 5-10 parts of mixed plant essential oils, 3-5 parts of composite microbial agents, 1-3 parts of sodium alginate, 3-5 parts of emulsifiers and 40-60 parts of water; The structural formula of the choline derivative is: ; The mixed plant essential oil is obtained by distilling a mixture of mint leaves and rosemary branches and leaves, wherein the mass ratio of the mint leaves to the rosemary branches and leaves is 1: (0.4-0.6); The composite microbial agent is prepared by mixing Bacillus subtilis and Bacillus amyloliquefaciens in a mass ratio of 1:1; The Bacillus subtilis was purchased from the China General Microorganism Culture Collection Center with a deposit number of CGMCC.No.1.9086; the Bacillus amyloliquefaciens was purchased from the China General Microorganism Culture Collection Center with a deposit number of CGMCC.No.1.3450.

2. The choline-based tobacco root-knot nematode inhibitor according to claim 1, characterized in that: The preparation method of the choline derivative comprises the following steps: Choline chloride is first dissolved in dichloromethane to form a choline chloride solution, triethylamine is then added to the choline chloride solution to form a mixed solution, and 4-fluorobenzoyl chloride is finally added dropwise to the mixed solution. After the addition is completed, the mixture is reacted and purified to obtain the choline derivative.

3. The choline-based tobacco root-knot nematode inhibitor according to claim 2, characterized in that: The molar ratio of choline chloride, triethylamine and 4-fluorobenzoyl chloride is 1:1.2:1; the dripping rate is 30 d / min; the reaction is specifically carried out at room temperature for 6 hours; the purification is specifically carried out by filtration, washing with 5% dilute hydrochloric acid, saturated sodium bicarbonate and water in sequence, and rotary evaporation.

4. The choline-based tobacco root-knot nematode inhibitor according to claim 1, characterized in that: The preparation method of the mixed plant essential oil comprises the following steps: 1) mint leaves and rosemary branches and leaves are mixed and crushed to obtain a mixed plant essential oil raw material; 2) placing the mixed plant essential oil raw material obtained in step 1) in water, performing steam distillation, and separating the oil and water to obtain the mixed plant essential oil.

5. The choline-based tobacco root-knot nematode inhibitor according to claim 4, characterized in that: In step 2), the mass volume ratio of the mixed plant essential oil raw material and water is 1: (4-6); in step 2), the steam distillation is specifically: steam distillation at 95-105° C. for 2-4 hours.

6. A method for preparing the choline-based tobacco root-knot nematode inhibitor according to any one of claims 1 to 5, characterized in that: The following steps are involved: 1) Adding the choline derivative, the composite microbial agent and sodium alginate to water and stirring to obtain a premixed solution; 2) Adding the mixed plant essential oil and the emulsifier to the premixed solution obtained in step 1), stirring and emulsifying, thereby obtaining the choline-based tobacco root-knot nematode inhibitor.

7. The preparation method according to claim 6, characterized in that In step 1), the water needs to be preheated to 50-60°C; the stirring is specifically: maintaining the temperature at 50-60°C and stirring for 40-60 minutes; In step 2), the stirring and emulsification is specifically: stirring and emulsifying at 50-60° C. for 1-2 hours.

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