Application of cigar tobacco leaf extract separated component in preparation of nematicide

Abstract

The invention provides application of cigar tobacco leaf extract separated components to preparation of nematicides, and relates to the technical field of nematicides. According to the application of the cigar tobacco leaf extract separation component to preparation of the nematicide, cigar tobacco leaf extract, the separation component and nematodes are included, the cigar tobacco leaf extract is obtained by extracting cigar tobacco leaves through an extraction agent after the cigar tobacco leaves are extracted through an organic solvent, the separation component is the same part obtained by eluting and combining the extract with an eluent, and the nematode is tobacco meloidogyne incognita. The cigar 'Yunxue 39' tobacco leaf extract separated component is used for preparing the nematicide and has a plurality of advantages. The nematode killing activity is remarkable, and the poisoning effect on meloidogyne incognita is good; the feed additive is high in safety and harmless to human, livestock and beneficial organisms; the environment compatibility is good, and no residual poison exists; the preparation process is simple, low in cost and easy to popularize and apply, and a high-quality choice is provided for preventing and treating nematode diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nematicides, in particular to the use of separated components of cigar tobacco leaf extract in preparing nematicides. Background Art

[0002] The southern root-knot nematode (Meloidogyne incognita) belongs to the genus Meloidogyne in the family Heteroderae, order Meloidogyne. Its host range is extremely broad, encompassing vegetables, fruits, cash crops, and some food crops. Globally, it has been documented to infect over 3,000 plant species. This nematode primarily attacks plant roots, stimulating root knot formation, severely damaging the root structure, and hindering the plant's absorption of water and nutrients. This can lead to stunted plant growth, yellowing and wilting of leaves, a significant decrease in fruit yield and quality, and in severe cases, even the death of the entire plant.

[0003] The wounds caused by southern root-knot nematodes infecting plant roots serve as entry points for pathogenic fungi and bacteria, such as wilt and root rot, exacerbating the occurrence of complex diseases. Infestations typically result in crop yield reductions of 20%-50%, and in severe cases, even total crop failure in the Solanaceae and Cucurbitaceae families. Protected vegetable and cash crops are particularly affected, with losses of 30%-60% for tomatoes, cucumbers, and tobacco in some areas. Current control methods for southern root-knot nematodes face numerous challenges. While chemical pesticides are effective, they can cause environmental pollution, pesticide residues, harm human and livestock health, and kill natural enemies of pests and other beneficial organisms. Biological control methods are relatively slow to take effect and are significantly affected by environmental factors. Agricultural control measures, such as crop rotation and deep tillage, are difficult to implement effectively on a large scale due to limitations in land resources and planting practices. Therefore, the development of an effective, safe, and environmentally friendly nematicide is urgent. Summary of the Invention

[0004] Technical problems solved

[0005] In response to the deficiencies of the prior art, the present invention provides an application of separated components of cigar tobacco leaf extract for preparing a nematicide, which solves the problems of environmental pollution and poor effectiveness of existing southern root-knot nematode control methods.

[0006] Technical Solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical scheme: the use of separated components of cigar tobacco leaf extract for preparing nematicides, including cigar tobacco leaf extract, separated components and nematodes, the cigar tobacco leaf extract is an extract obtained by extracting cigar tobacco leaves with an organic solvent and then extracting them with an extractant, the separated components are the same parts of the extract eluted with an eluent and combined, and the nematode is the southern root-knot nematode of tobacco.

[0008] Preferably, the cigar tobacco leaf extract is a methanol extract of cigar tobacco leaves, the extract of the cigar tobacco leaf extract is an ethyl acetate extract of the cigar tobacco leaf extract, and the separation component is the cigar tobacco leaf extract using 100-200 mesh silica gel as the stationary phase, and the Yunxue 39 ethyl acetate extract is eluted for 10 column volumes with a mobile phase of dichloromethane: ethyl acetate (7:3, V / V), the eluent is discarded, and the same parts are eluted with a mobile phase of dichloromethane: ethyl acetate (6:4, V / V) and combined.

[0009] Preferably, the method for preparing the separated components of the cigar tobacco leaf extract specifically comprises the following steps:

[0010] S1. Yunxue 39 tobacco leaves were dried and crushed to 1-5 mm using a grinder to obtain a coarsely ground product;

[0011] S2. The coarsely ground product obtained in step A is cold-soaked in an organic solvent for 24 to 48 hours, repeated 3 to 5 times to obtain a cigar tobacco extract;

[0012] S3. The extract obtained in step B was repeatedly extracted with an organic solvent and concentrated under pressure by a rotary evaporator to obtain a cigar tobacco leaf extract;

[0013] S4. The extract obtained in step C was washed with 100-200 mesh silica gel as the stationary phase, and the Yunxue 39 ethyl acetate extract was eluted for 10 column volumes with a mobile phase of dichloromethane:ethyl acetate (7:3, V / V). This eluate was discarded, and then eluted with a mobile phase of dichloromethane:ethyl acetate (6:4, V / V) and the same fractions were combined to obtain separated components.

[0014] A cigar tobacco leaf nematode-killing agent, wherein the nematode is southern root-knot nematode of tobacco, the active component of the nematode-killing agent is a component separated from an extract of cigar tobacco leaf extract, and the preparation form of the nematode-killing agent is a soluble liquid.

[0015] Preferably, the nematicide contains 40% to 70% separated components of the Yunxue 39 tobacco leaf extract; the separated components of the Yunxue 39 tobacco leaf extract are obtained by extracting 50 kg of dried Yunxue 39 tobacco leaves with 5 to 10 kg of organic solvent, extracting the extract with an extracting liquid, concentrating the extract to 5 kg, eluting the Yunxue 39 extract part with an eluent and combining the same parts to obtain 1 kg of separated components.

[0016] A Yunxue 39 tobacco leaf nematode agent, wherein the nematode is the southern root-knot nematode, and the raw materials for preparing the nematode agent include the following components in percentage by mass: 40% to 70% of a Yunxue 39 tobacco leaf extract fraction, 5% to 20% of an organic solvent, and 5% to 10% of an antifreeze agent, the sum of the percentages by mass of the above components being 100%;

[0017] The Yunxue 39 tobacco leaf extract separation component is prepared by extracting 50 kg of dried Yunxue 39 tobacco leaves with 5 to 10 kg of organic solvent to obtain an extract, extracting the extract with an extracting solution, concentrating the extract to 5 kg, and then eluting with an eluent and combining the same parts to obtain 1 kg of separation components;

[0018] The organic solvent is methanol;

[0019] The extract is ethyl acetate;

[0020] The eluent was dichloromethane:ethyl acetate (7:3, V / V), dichloromethane:ethyl acetate (6:4, V / V).

[0021] The antifreeze agent is ethylene glycol, propylene glycol or glycerol.

[0022] Beneficial effects

[0023] The present invention provides the use of separated components of cigar tobacco leaf extract for preparing a nematicide. This has the following beneficial effects:

[0024] 1. Significant Nematicidal Activity: The Yunxue 39 tobacco leaf extract fraction exhibits strong nematicidal activity against southern root-knot nematodes. Indoor bioassay results showed that a 50 mg / ml dose of this fraction resulted in a 72-hour adjusted mortality rate of 70.79%. Its 24-, 48-, and 72-hour LC50 values were 60.868 mg / ml, 41.931 mg / ml, and 19.975 mg / ml, respectively. Potted plant control experiments demonstrated that increasing the concentration of the Yunxue 39 tobacco leaf extract fraction significantly increased its control efficacy against southern root-knot nematodes. At 40 mg / mL, this solution achieved a root knot inhibition rate of 74.59 ± 3.12% and a root knot control efficacy of 63.35 ± 9.56%. This solution effectively reduced soil population density, lowered the disease index, and ensured healthy root growth.

[0025] 2. High Safety: Developing a nematicide from the isolated components of Yunxue 39 tobacco leaf extract is safe for humans, livestock, natural enemies of pests, and other beneficial organisms. Compared to synthetic pesticides, this product avoids direct or indirect toxicity from chemicals to various organisms in the ecosystem, helping to maintain ecological balance, protect biodiversity, and reduce negative impacts on non-target organisms, thus playing a significant role in sustainable agricultural development.

[0026] 3. Good environmental compatibility: This nematicide is a biogenic pesticide with no residual toxicity after use. It can be naturally degraded in the soil and will not leave residues in the soil, water, or agricultural products like traditional chemical pesticides. It will not cause long-term pollution to the soil ecosystem and water environment, reducing the risk of pesticide residues in agricultural products, ensuring food safety and ecological safety, and meeting the development requirements of green agriculture and ecological protection.

[0027] 4. The preparation method is simple to operate. From drying and pulverizing Yunxue 39 tobacco leaves to cold soaking with organic solvents, extraction, concentration, and elution to separate components, the entire process requires no complex equipment or techniques, making it easy to implement on an industrial scale. Furthermore, the raw material, Yunxue 39 tobacco leaves, are widely available, resulting in low production costs, facilitating large-scale application and providing farmers with an affordable and effective nematicidal solution. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0029] The present invention provides a complete process for preparing a nematicide by separating components from a cigar tobacco leaf extract:

[0030] Material preparation

[0031] Cigar Tobacco Leaves: Yunxue 39 cigar tobacco leaves, grown in specific regions and reaching maturity during their growth cycle, are selected. After harvesting, they are naturally air-dried to remove surface impurities. They are then placed in a drying machine and dried at 50°C to a constant weight to ensure that the moisture content of the leaves remains within an appropriate range, avoiding the impact of excessive moisture content on subsequent extraction. The dried leaves are pulverized in a grinder to obtain a uniformly sized powder. The powder is then passed through a 40-mesh sieve to ensure a consistent particle size, ensuring a relatively stable contact area with the extraction solvent during the extraction process, which helps improve extraction efficiency.

[0032] Extraction Solvent: Analytical-grade ethanol with a purity of at least 95% should be used as the extraction solvent. Ethanol has excellent solubility and can effectively dissolve various chemical components in tobacco leaves. It is also relatively inexpensive and readily available, making it suitable for large-scale production. Before use, test the ethanol for purity to ensure it meets experimental requirements.

[0033] Chromatography columns and related reagents: Prepare a glass chromatography column with an inner diameter of 2.5 cm and a length of 30 cm for separating the different components in the extract. Silica gel (100-200 mesh) is selected as the stationary phase. It has a large specific surface area and a suitable pore size distribution, which can effectively separate the various components in the tobacco leaf extract. The eluent is a petroleum ether-ethyl acetate mixed solution, which is prepared according to different volume ratios (such as 3:1, 2:1, 1:1, etc.) to meet the elution requirements of different components. All reagents are purity tested and pretreated before use to ensure the accuracy and reliability of the experimental results.

[0034] Experimental instruments: equipped with an electronic balance (accuracy 0.0001g) for accurately weighing the mass of experimental materials such as tobacco leaf powder and reagents; a constant temperature water bath for controlling the temperature of the extraction process, with the temperature fluctuation range controlled within ±1°C; a rotary evaporator for concentrating the extract, with a vacuum degree that can be adjusted to 0.08-0.09MPa to ensure efficient concentration at a lower temperature and avoid the loss of heat-sensitive components; a centrifuge with an adjustable speed range of 2000-10000r / min for separating insoluble impurities in the extract to ensure the clarity of the extract.

[0035] Extraction process

[0036] Cold-infusion extraction: Accurately weigh 1000g of crushed Yunxue 39 cigar tobacco leaf powder and place it in a 5000mL glass container. Add 3000mL of analytical-grade ethanol, fully submerging the tobacco leaf powder in the ethanol solution. Seal the container and place it in a constant-temperature chamber at 25°C for 72 hours of cold-infusion extraction. Stir every 12 hours at a speed of 100 rpm to ensure full dissolution of the active ingredients in the tobacco leaf in the ethanol and ensure a uniform and complete extraction process.

[0037] Solid-Liquid Separation: After the cold extraction is complete, the mixed solution is filtered through a Buchner funnel. A vacuum pump is used to provide negative pressure, maintained at 0.06-0.07 MPa, to accelerate filtration and obtain the primary extract. The primary extract is transferred to a centrifuge tube and placed in a centrifuge. Centrifuge at 4000 rpm for 15 minutes to further remove small insoluble impurities in the extract and obtain a clarified extract.

[0038] Concentrate the extract: Transfer the clarified extract after centrifugation to a distillation flask on a rotary evaporator. Set the temperature of the rotary evaporator to 50°C and the vacuum degree to 0.08 MPa for reduced pressure concentration. During the concentration process, closely observe the volume change of the solution in the distillation flask. When the volume of the solution is concentrated to about 1 / 5 of the original volume, stop concentrating to obtain the concentrated extract.

[0039] Separation process

[0040] Column Packing: Secure the glass chromatography column vertically on an iron stand and pad the bottom with a layer of glass wool to prevent leakage of silica gel particles. Slowly add the treated silica gel to the column while gently tapping the column with an earwax bulb to ensure even filling of the silica gel to a height of 20 cm. Once filled, add a layer of quartz sand approximately 0.5 cm thick on top of the silica gel to ensure even distribution of the eluent through the silica gel layer.

[0041] Equilibrate the chromatography column: Slowly add a mixed eluent of petroleum ether and ethyl acetate (3:1) to the chromatography column at a flow rate of 1-2 mL / min to allow the eluent to fully infiltrate the silica gel. Equilibrate the chromatography column for 30 minutes to ensure that the silica gel reaches a stable state in the eluent environment, providing good conditions for the subsequent separation process.

[0042] Loading: Slowly add the concentrated extract to the top of the chromatography column using a dropper, taking care not to damage the smoothness of the silica gel surface. Load 10 mL. Once the extract has completely entered the silica gel layer, rinse the column wall with a small amount of eluent to ensure that all the sample has entered the column.

[0043] Elution and Collection: Elution was performed sequentially using different volume ratios of petroleum ether-ethyl acetate eluent at a constant flow rate (1-2 mL / min). First, 100 mL of the 3:1 mixture was used to elute and collect the eluate; then, 100 mL of the 2:1 mixture was used to elute and collect the eluate; finally, 100 mL of the 1:1 mixture was used to elute and collect the eluate. The collected eluates were labeled as eluate A, eluate B, and eluate C. Each eluate was concentrated using a rotary evaporator at 40°C and a vacuum of 0.08 MPa to a volume of approximately 10 mL to obtain the concentrated separated components.

[0044] Nematicidal activity test

[0045] Cultivation of southern root-knot nematodes: Select healthy, well-grown tomato seedlings as host plants for the southern root-knot nematode. Sow the tomato seeds in sterilized nutrient soil and cultivate the seedlings in a greenhouse at 25°C, 2000 lx of light intensity, and 16 hours of light per day. When the seedlings have 3-4 true leaves, inoculate the soil around the roots with southern root-knot nematode eggs, approximately 1000 eggs per plant. Continue incubation for 20 days to allow the nematodes to multiply and form root knots within the tomato roots.

[0046] Test sample preparation: The concentrated fractions obtained above (eluent A, eluent B, and eluent C) were diluted with sterile water to different concentrations, such as 5 mg / mL, 10 mg / mL, 20 mg / mL, and 50 mg / mL. Sterile water served as a blank control group, and a common chemical nematicide (e.g., avermectin, at a concentration of 1 mg / mL) served as a positive control group.

[0047] Indoor bioassay: The nematode killing activity test was performed using the insect immersion method. Take a clean culture dish and place a layer of moistened filter paper on the bottom of each culture dish to prevent the nematodes from drying out. Use a pipette to draw 100 μL of a suspension containing 100 second-instar larvae (J2) of southern root-knot nematodes and drop it evenly on the filter paper. Then add 1 mL of test sample solution of different concentrations to each culture dish so that the nematodes are completely immersed in the solution. Set up 3 replicates for each concentration. Place the culture dish in a constant temperature incubator at a temperature of 25°C, and observe and record the death of nematodes after 24h, 48h, and 72h. Use a dissecting microscope for observation, and use the stiffness of the nematode body and the lack of reaction when touched with a dissecting needle as the criterion for judging death. The corrected mortality rate of nematodes is calculated as follows:

[0048] Corrected mortality rate (%) = (mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group) × 100.

[0049] Example 1:

[0050] The extraction, extraction and separation methods of the ethyl acetate extract of the Yunxue 39 methanol extract of this example include:

[0051] A. Yunxue 39 tobacco leaves are dried and fully crushed into 1-5mm using a grinder to obtain coarsely ground material;

[0052] B. cold-extracting the coarse powder obtained in step A with methanol for 24 to 48 hours (the mass ratio of the coarse powder to methanol is 10 kg:1 kg), repeating 3 to 5 times to obtain a cigar tobacco leaf extract;

[0053] C. The extract obtained in step B is repeatedly extracted with an organic solvent to obtain an extract, and the extract is concentrated under pressure to 5 kg using a rotary evaporator.

[0054] D. The extract obtained in step C was eluted with 100-200 mesh silica gel as the stationary phase using a mobile phase of dichloromethane:ethyl acetate (7:3, V / V) for 10 column volumes. This eluate was discarded and then eluted with a mobile phase of dichloromethane:ethyl acetate (6:4, V / V) and the same fractions were combined to obtain 1 kg of separated components.

[0055] Indoor bioassay results of Yunxue 39 tobacco leaf extract against nematodes:

[0056] The nematode toxicity of Yunxue 39 tobacco leaf solution was determined by the drug immersion method. Healthy and uniformly sized nematodes were selected as test nematodes. The test was conducted in a 24-well cell culture plate. The nematode suspension (V1) was first injected into the sample well. The stock solution (V3) [calculated according to test (3)] was diluted to 100 mg / L with sterile water (V2), mixed evenly with the nematode suspension, and placed in a 25°C incubator. Each treatment was repeated three times, with 100 nematodes per repeat. Ethyl acetate treatment was used as a control. 24, 48, and 72 hours after treatment, the death and survival of the nematodes in each treatment were examined, and the nematode mortality and corrected mortality were calculated according to formula (1) and formula (2), respectively.

[0057] Method for judging whether nematodes are dead or alive: observe under a microscope. Those that are swimming and in an "S" shape are recorded as alive. In a static state, if the nematodes change shape significantly after being gently touched with an inoculation needle, they are recorded as alive; otherwise, they are recorded as dead.

[0058]

[0059] Where,

[0060] V = nematode suspension volume V1 + added sterile water volume V2 + drug stock solution volume V3; V = 400 μL; V1 = 100 μL;

[0061] In the experiment, C is pre-screened and set, in this example C = 100 mg / ml; C0 is known, in this example C0 = 300 mg / ml. The number of nematodes remains constant between treatments. After fixing V, the V2 and V3 values of different treatments can be obtained.

[0062] The nematode toxicity of the components of Yunxue 39 tobacco leaf extract was determined by the liquid immersion method, and the toxicity regression equation was calculated. The results are shown in Tables 1 and 2.

[0063] Table 1 Toxic effects of Yunxue 39 tobacco leaf extract fractions on incognita root-knot nematodes

[0064]

[0065] Table 2 Toxic activity and toxicity curve of Yunxue 39 tobacco leaf extract fractions against southern root-knot nematode

[0066]

[0067]

[0068] As can be seen from the above table, Yunxue 39 tobacco leaf extract has a strong toxic activity against southern root-knot nematodes. When treated with 50 mg / ml, the corrected mortality rate of southern root-knot nematodes was 70.79% after 72 hours. The LC50 values of southern root-knot nematodes at 24 hours, 48 hours and 72 hours were 60.868 mg / ml, 41.931 mg / ml and 19.975 mg / ml respectively.

[0069] Example 2:

[0070] This example describes the preparation of a 10% soluble liquid formulation of Yunxue 39 tobacco leaf extract fractions. Specifically, 1 kg of Yunxue 39 tobacco leaf extract concentrate was dissolved in 0.1 kg of methanol. 7.9 kg of sterile water and 1 kg of propylene glycol were added with stirring, and the mixture was thoroughly mixed to produce 10 kg of Yunxue 39 tobacco leaf soluble liquid formulation. The formulation's stability and appearance met the requirements for commercial preparations.

[0071] Example 3:

[0072] This example shows the results of an experimental study on the efficacy of Yunxue 39 tobacco leaf soluble solution in Example 2 against southern root-knot nematodes in potted plants.

[0073] 1. Test reagents

[0074] The Yunxue 39 tobacco leaf soluble liquid in Example 2;

[0075] 0.5% avermectin solution

[0076] 2. Test crops

[0077] Tobacco, variety "K326"

[0078] 3. Targets of prevention and control

[0079] Southern root-knot nematode (Meloidogyne incognita)

[0080] 4. Selection of experimental plots and experimental treatment

[0081] 4.1 Selection of experimental plots

[0082] This experiment was conducted in a greenhouse at Yunnan Agricultural University in Kunming, Yunnan Province. Soil with uniform and severe southern root-knot nematode infestation and mild other pests and diseases was selected as potting soil. The soil had high nutrient content and was managed with normal fertilizer and water management.

[0083] 4.2 Experimental treatment

[0084] The test agent was Yunxue 39 tobacco leaf extract soluble solution diluted to 40 mg / mL, 20 mg / mL, 10 mg / mL; 0.5% avermectin agent and blank control (water) were used in five different potted plants, with three replicates for each treatment.

[0085] 4.3 Application method

[0086] Before treatment, each potted plant was irrigated to ensure soil moisture reached 60% to 70% of its saturated water holding capacity. The pesticide was sprayed onto the soil at a rate of 50 L / mu (approximately 1 liter / mu) and thoroughly mixed with the soil before transplanting the tobacco plants. Treatments with 0.5% avermectin and 8% methanol in water served as positive and blank controls, respectively, with the same water and fertilizer management practices. Samples were collected and investigated two months later.

[0087] 4.4 Survey Methods

[0088] A comprehensive sampling method was used to check the number of root knots and disease stage of each tobacco plant according to the following standards. The number of root knots in each treatment and the number of root-knot nematodes in the rhizosphere soil were counted, and the root knot inhibition rate, disease index, root knot control effect, insect population density, and relative control effect of each treatment on susceptible tobacco plants were calculated.

[0089] Table 3 Root knot grading standards

[0090]

[0091] The control effect is calculated according to the following formula:

[0092] Disease index (%) = ∑ (number of plants at each level × number of levels) / (total number of surveys × number of the highest level) × 100%;

[0093] Root knot inhibition rate (%) = (number of root knots in control group - number of root knots in treatment group) / number of root knots in control group × 100%;

[0094] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100%;

[0095] Relative insect population control effect (%) = [(insect population density of soil sample in blank control group - insect population density of soil sample in pesticide treatment group) / insect population density of soil sample in blank control group] × 100%.

[0096] 5. Test results and statistics

[0097] The control effects of soluble liquid solutions of the separated components of Yunxue 39 tobacco leaf extract at concentrations of 40 mg / mL, 20 mg / mL, and 10 mg / mL against southern root-knot nematodes were determined. The results are shown in Table 4.

[0098] Table 4 Control effect of Yunxue 39 tobacco leaf extract soluble solution on southern root-knot nematode

[0099]

[0100]

[0101] As shown in Table 4, the three Yunxue 39 tobacco leaf preparations all had good control effects on southern root-knot nematodes. The control effects on southern root-knot nematodes increased significantly with increasing agent concentration.

[0102] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Use of a separated component of a cigar tobacco leaf extract for preparing a nematocide, comprising a cigar tobacco leaf extract, a separated component, and nematodes, characterized in that: The cigar tobacco leaf extract is an extract obtained by extracting cigar tobacco leaves with an organic solvent and then extracting with an extractant. The separated components are the same parts of the extract eluted with an eluent and combined. The nematode is the southern root-knot nematode of tobacco.

2. The use of the separated fraction of cigar tobacco leaf extract according to claim 1 for preparing a nematicide, characterized in that: The cigar tobacco leaf extract is a methanol extract of cigar tobacco leaves, the extract of the cigar tobacco leaf extract is an ethyl acetate extract of the cigar tobacco leaf extract, and the separation component is a cigar tobacco leaf extract using 100-200 mesh silica gel as a stationary phase, and the Yunxue 39 ethyl acetate extract is eluted for 10 column volumes with a mobile phase of dichloromethane:ethyl acetate (7:3, V / V), the eluent is discarded, and the same parts are eluted with a mobile phase of dichloromethane:ethyl acetate (6:4, V / V) and combined.

3. The use of the separated fraction of cigar tobacco leaf extract for preparing a nematicide according to claim 2, characterized in that: The method for preparing the separated components of the cigar tobacco leaf extract specifically comprises the following steps: S1. Yunxue 39 tobacco leaves were dried and crushed to 1-5 mm using a grinder to obtain a coarsely ground product; S2. The coarsely ground product obtained in step A is cold-soaked in an organic solvent for 24 to 48 hours, repeated 3 to 5 times to obtain a cigar tobacco extract; S3. The extract obtained in step B was repeatedly extracted with an organic solvent and concentrated under pressure by a rotary evaporator to obtain a cigar tobacco leaf extract; S4. The extract obtained in step C was washed with 100-200 mesh silica gel as the stationary phase, and the Yunxue 39 ethyl acetate extract was eluted for 10 column volumes with a mobile phase of dichloromethane:ethyl acetate (7:3, V / V). This eluate was discarded, and then eluted with a mobile phase of dichloromethane:ethyl acetate (6:4, V / V) and the same fractions were combined to obtain separated components.

4. A nematicide for cigar tobacco leaves, characterized by: The nematode is the southern root-knot nematode of tobacco, the active component of the nematode killer is a component separated from an extract of cigar tobacco leaf extract, and the preparation form of the nematocide is a soluble liquid.

5. The nematicide for cigar tobacco leaves according to claim 4, characterized in that: The nematicide contains 40% to 70% of separated components of the Yunxue 39 tobacco leaf extract; the separated components of the Yunxue 39 tobacco leaf extract are obtained by extracting 50 kg of dried Yunxue 39 tobacco leaves with 5 to 10 kg of organic solvent, extracting the extract with an extracting liquid, concentrating the extract to 5 kg, eluting the Yunxue 39 extract part with an eluent, and combining the same parts to obtain 1 kg of separated components.

6. A Yunxue 39 tobacco leaf nematicide, characterized by: The nematode is the southern root-knot nematode, and the raw materials for preparing the nematocide include the following components in percentage by mass: Yunxue 39 tobacco leaf extract separation component: 40% to 70%, organic solvent: 5% to 20%, antifreeze: 5% to 10%, and the sum of the percentages by mass of the above components is 100%; The Yunxue 39 tobacco leaf extract separation component is prepared by extracting 50 kg of dried Yunxue 39 tobacco leaves with 5 to 10 kg of organic solvent to obtain an extract, extracting the extract with an extracting solution, concentrating the extract to 5 kg, and then eluting with an eluent and combining the same parts to obtain 1 kg of separation components; The organic solvent is methanol; The extract is ethyl acetate; The eluent was dichloromethane:ethyl acetate (7:3, V / V), dichloromethane:ethyl acetate (6:4, V / V). The antifreeze agent is ethylene glycol, propylene glycol or glycerol.

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