Method for preparing nematicide from cigar fermented tobacco leaf extract and nematicide

Abstract

The invention discloses a method for preparing a nematicide from cigar fermented tobacco leaf extract and the nematicide. The method comprises the following steps: crushing dried cigar fermented tobacco leaves by using a crusher to obtain a coarse crushed material with the particle size of 1-5mm; performing cold soaking on the coarsely crushed material for 24-48 hours through an organic solvent, and repeating for 3-5 times to obtain cigar fermented tobacco leaf extract liquid; and repeatedly extracting the cigar fermented tobacco leaf extract liquid through an extraction liquid, pressurizing and concentrating the cigar fermented tobacco leaf extract liquid to 1 / 9-1 / 11 of the mass of the dried cigar fermented tobacco leaf through a rotary evaporator to obtain a cigar fermented tobacco leaf extract, adding an organic solvent, an anti-freezing agent and sterile water, and fully stirring to prepare the nematicide. The cigar fermented tobacco leaf nematicide with remarkable nematicidal activity is prepared, the nematicidal activity is remarkable, no chemical residue exists, the environment compatibility is good, the cost is reduced, and the problems that a plant source nematicide is weak in activity and high in cost are solved.

Description

Technical Field

[0001] The present invention relates to plants and application fields thereof, and in particular to a method for preparing a nematicide from a cigar fermented tobacco leaf extract and the nematicide. Background Art

[0002] Plant parasitic nematodes are one of the most destructive biological stress factors in global agricultural production. They infect the root system, leading to root knot formation, vascular tissue damage and nutrient absorption disorders, which can cause widespread crop yield reduction or even crop failure. For example, the southern root-knot nematode (Meloidogyne incognita) belongs to the order Meloidogyne, family Heterodera, and genus Meloidogyne. Its host is wide, covering vegetables, fruits, cash crops and some food crops. As one of the most serious plant parasitic nematodes in the world, it has caused damage to more than 3,000 plant species recorded worldwide. The insects mainly infect plant roots, stimulate the formation of root knots, destroy the root structure, and hinder the absorption of water and nutrients, which in turn causes slow plant growth, yellowing and wilting of leaves, and reduced fruit yield and quality. In severe cases, the entire plant dies. The wounds caused by the infection can easily become invasion channels for pathogenic fungi and bacteria such as wilt and root rot, aggravating the occurrence of complex diseases. When the disease occurs, it generally leads to a 20% to 50% reduction in yield. In severe cases, there will be no harvest (such as Solanaceae and Cucurbitaceae crops). Greenhouse vegetables and economic crops are particularly affected. In some areas, the losses of tomatoes, cucumbers and tobacco can reach 30% to 60%.

[0003] Currently, the mainstream approach relies on chemical nematicides such as avermectin. However, long-term use can lead to soil contamination from chemical residues and high lethality against natural enemies of pests, threatening ecological balance. The application of some botanical pesticides is still limited by insufficient activity or the complex and costly extraction of high-purity active ingredients.

[0004] Cigar-fermented tobacco leaves are rich in bioactive substances such as nicotine and chlorogenic acid, but have not previously been systematically developed as a resource for nematode control. Transforming cigar-fermented tobacco leaves into a highly effective, non-toxic, and low-cost new nematicide is a key path to solving this challenge. Summary of the Invention

[0005] In view of the above-mentioned defects or deficiencies in the prior art, the present invention provides a method for preparing a nematicide from a cigar fermented tobacco leaf extract and a nematicide. The cigar fermented tobacco leaves are subjected to an organic solvent extraction and an extract liquid extraction process to obtain a highly active nematicide extract, which is then prepared as a nematicide. The nematicide can effectively control nematodes, is environmentally friendly, and has low cost.

[0006] One aspect of the present invention provides a method for preparing a nematicide from a cigar fermented tobacco leaf extract, comprising:

[0007] The dried fermented cigar tobacco leaves are crushed by a crusher to obtain a coarse crushed product with a particle size of 1 to 5 mm;

[0008] cold soaking the coarsely ground material in an organic solvent for 24 to 48 hours, repeating this process 3 to 5 times to obtain a cigar fermented tobacco leaf extract;

[0009] Repeatedly extracting the fermented cigar tobacco leaf extract with an extraction liquid, and then pressurizing and concentrating the extract to 1 / 9 to 1 / 11 of the mass of the dried fermented cigar tobacco leaf using a rotary evaporator, to obtain a fermented cigar tobacco leaf extract;

[0010] The cigar fermented tobacco leaf extract is added to the organic solvent, antifreeze and sterile water and stirred thoroughly to prepare a nematocide.

[0011] Furthermore, the mass ratio of the components of the nematicide is:

[0012] Cigar fermented tobacco leaf extract: 10% to 50%, organic solvent: 1% to 10%, antifreeze: 5% to 10%, sterile water: 30% to 84%, and the sum of the mass percentages of the above components is 100%.

[0013] Furthermore, the mass ratio of the organic solvent to the coarsely pulverized material is 1:10 to 1:5.

[0014] Furthermore, the organic solvent includes one or more of methanol, ethanol and isopropanol.

[0015] Furthermore, the extract comprises one or more of ethyl acetate, methyl tert-butyl ether and n-butanol.

[0016] Furthermore, the antifreeze agent includes one or more of ethylene glycol, propylene glycol or glycerol.

[0017] Another aspect of the present invention provides a cigar fermented tobacco leaf nematicide prepared by any one of the methods described above.

[0018] The present invention provides a method for preparing a nematicide from a cigar fermented tobacco leaf extract and the nematicide. The method uses cigar fermented tobacco leaves as raw materials, performs cold-immersion extraction with an organic solvent, repeatedly extracts the extract, and pressurizes and concentrates it on a rotary evaporator to obtain a highly active nematicidal extract. The organic solvent, antifreeze, and sterile water are added to prepare the nematicide. The nematicide has significant nematicidal activity and can be used for preventing and controlling nematodes on crops such as tomatoes and tobacco. The method has no chemical residues, is safe for humans, livestock, natural enemies of pests, and other beneficial organisms, and has good environmental compatibility. The raw materials can be discarded fermented tobacco leaves from cigar processing, thereby reducing costs and improving resource utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Other features, objects and advantages of the present application will become more apparent upon reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0020] Figure 1 is a flowchart of a method for preparing a nematocide from a cigar fermented tobacco extract according to an embodiment of the present application. DETAILED DESCRIPTION

[0021] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0022] The terms used in the embodiments of the present application are only for the purpose of describing particular embodiments and are not intended to limit the present application. The singular forms "a," "an," and "the" used in the embodiments of the present application are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0023] It should be understood that although the terms first, second, third, etc. can be used in the embodiments of the present application to describe obtaining modules, these obtaining modules should not be limited by these terms. These terms are only used to distinguish the obtaining modules from each other.

[0024] Depending on the context, the word "if" as used herein can be interpreted to mean "when" or "while" or "in response to determining" or "in response to detecting." Similarly, the phrase "if it is determined" or "if (a stated condition or event) is detected" can be interpreted to mean "upon being determined" or "in response to determining" or "upon detecting (the stated condition or event)" or "in response to detecting (the stated condition or event)".

[0025] It should be noted that the terms "upper", "lower", "left", "right", and the like described in the embodiments of the present application are described from the perspective of the angle shown in the drawings, and should not be understood as a limitation on the embodiments of the present application. In addition, in the context, it should also be understood that when referring to an element being formed "on" or "under" another element, it can not only be directly formed "on" or "under" another element, but also indirectly formed "on" or "under" another element through an intermediate element.

[0026] Cigar is a kind of tobacco product, which is made of dried and fermented tobacco. Embodiments of the present application are illustrated by taking Yunxue 39 as an example, and are also applicable to other varieties of cigars. Yunxue 39 is one of the new cigar varieties cultivated by Yuxi World Tobacco Plantation in Yunnan, and is grown in a temperature range of 19 to 35 degrees Celsius, with a daily average temperature of more than 22 degrees Celsius for more than 95 days, and the soil is mostly red sandy soil rich in organic matter and good in drainage performance.

[0027] The embodiments of the present application provide a method for preparing a nematocide from a cigar fermented tobacco extract and the nematocide. The nematocide is prepared by adding a certain proportion of an organic solvent to the active substance of the cigar fermented tobacco extract of Yunxue 39 to obtain a soluble liquid agent. Those skilled in the art can optimize the related components and their contents within the scope of the present application to obtain a nematocide with ideal control effect.

[0028] Referring to Figure 1 The embodiments of the present application provide a method for preparing a nematocide from a cigar fermented tobacco extract, which comprises the following steps:

[0029] In step S101, the dried cigar fermented tobacco is crushed by a crusher to obtain a coarse powder with a particle size of 1-5 mm. In the present embodiment, the cigar is selected from Yunxue 39.

[0030] In step S102, preferably, to avoid degradation of active substances in the cigar fermented tobacco due to excessively high temperature, the coarse powder is cold-soaked in an organic solvent at room temperature of 25 degrees Celsius for 24-48 hours, and the process is repeated for 3-5 times to ensure that the active substances are fully dissolved in the organic solvent. During the cold-soaking process, the solution is stirred regularly, for example, every 4 hours, to improve the extraction efficiency and realize mild and efficient dissolution of the active substances, thereby obtaining a cigar fermented tobacco extract. The mass ratio of the organic solvent to the coarse powder is 1:10-1:5, for example, 5 kg of methanol is used to cold-soak 50 kg of the coarse powder of the dried cigar fermented tobacco.

[0031] In step S103, the cigar fermented tobacco extract is repeatedly extracted by an extraction liquid, and then concentrated by a rotary evaporator to 1 / 9-1 / 11 of the mass of the dried cigar fermented tobacco, thereby obtaining the cigar fermented tobacco extract. For example, after 50 kg of the coarse powder of the dried cigar fermented tobacco is cold-soaked to obtain the cigar fermented tobacco extract, the extract is extracted by ethyl acetate, and then concentrated by a rotary evaporator until the cigar fermented tobacco extract is concentrated to about 5 kg.

[0032] In step S104, the cigar fermented tobacco extract is added to the organic solvent, an antifreeze and sterile water, and then fully stirred to obtain the nematocide. The mass ratio of the components of the nematocide is as follows:

[0033] Cigar fermented tobacco leaf extract: 10% to 50%, organic solvent: 1% to 10%, antifreeze: 5% to 10%, sterile water: 30% to 84%, and the sum of the mass percentages of the above components is 100%.

[0034] Wherein, the organic solvent includes one or more of methanol, ethanol and isopropanol;

[0035] The extract comprises one or more of ethyl acetate, methyl tert-butyl ether and n-butanol;

[0036] The antifreeze agent includes one or more of ethylene glycol, propylene glycol or glycerin.

[0037] Another embodiment of the present invention provides a cigar fermented tobacco leaf nematicide prepared by any one of the methods described above.

[0038] The embodiments of the present invention are described using the southern root-knot nematode as an example, but the method of the present invention is also applicable to other types of nematodes.

[0039] Example 1

[0040] The extraction and extraction method of the ethyl acetate extract of the Yunxue 39 methanol extract of this embodiment include:

[0041] The dried Yunxue 39 cigar fermented tobacco leaves were fully crushed with a grinder to obtain a coarse powder with a particle size of about 3 mm;

[0042] At room temperature (25°C), 50 kg of the coarsely ground material was cold-soaked and extracted with 5 kg of methanol for 36 hours, repeated 4 times to obtain a cigar fermented tobacco leaf extract;

[0043] The obtained cigar fermented tobacco leaf extract was repeatedly extracted with ethyl acetate to obtain an extract, and the extract was concentrated under pressure to about 5 kg using a rotary evaporator to obtain a cigar fermented tobacco leaf extract.

[0044] Indoor bioassay of Yunxue 39 fermented tobacco leaf extract against nematodes using the liquid immersion method:

[0045] In this example, healthy, uniformly sized southern root-knot nematodes were selected as test nematodes to determine the toxicity of a test agent prepared from Yunxue 39 fermented tobacco leaf extract. The test was conducted in a 24-well cell culture plate. First, a nematode suspension was injected into the sample wells. The volume of the test agent stock solution and the volume of sterile water to be added were calculated using the following formula:

[0046]

[0047] V=V1+V2+V3

[0048] Wherein, C is the concentration of the test agent, C0 is the concentration of the test agent stock solution, V3 is the volume of the test agent stock solution, V is the total volume of the solution in each well, V = 400 μL, V1 is the volume of the nematode suspension, V1 = 100 μL, and V2 is the volume of sterile water.

[0049] The concentration of the test agent in the experiment 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 treated nematodes remains constant, and after fixing V, the V2 and V3 values ​​of different treatments can be obtained.

[0050] Add the test agent stock solution to the desired test agent concentration of 100 mg / mL and dilute with sterile water to the sample well. Mix thoroughly with the nematode suspension and place in a 25°C incubator. Perform three replicates per treatment, with 100 nematodes per replicate. Ethyl acetate treatment served as a control. 24, 48, and 72 hours after treatment, examine the nematode mortality and survival for each treatment. Calculate the nematode mortality and adjusted mortality using the following formulas:

[0051]

[0052] Method for judging the death and survival of nematodes: When observed under a microscope, nematodes that are swimming and in an "S" shape are recorded as alive. In a static state, nematodes that change shape significantly after being gently touched with an inoculation needle are recorded as alive, otherwise they are recorded as dead.

[0053] The nematode toxicity of Yunxue 39 fermented 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.

[0054] Table 1 Toxic effect of Yunxue 39 fermented tobacco leaf extract on southern root-knot nematode (100 mg / ml)

[0055]

[0056] Table 2 Toxicity curve of Yunxue 39 fermented tobacco leaf extract against southern root-knot nematode

[0057]

[0058] From the above table, we can see that the Yunxue 39 fermented tobacco leaf test agent at a concentration of 100 mg / ml treated southern root-knot nematode, the corrected mortality rate at 72 hours was 89.7%, and the LC values ​​at 24 hours, 48 ​​hours and 72 hours were 50 The (median lethal concentration) were 80.3 mg / ml, 66.4 mg / ml and 39.9 mg / ml respectively. The Yunxue 39 fermented tobacco leaf extract has a strong and sustained toxic activity against southern root-knot nematode.

[0059] Example 2

[0060] This example describes the preparation of a 10% soluble liquid formulation of Yunxue 39 fermented tobacco leaf extract. Specifically, 1 kg of Yunxue 39 fermented tobacco leaf 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 the Yunxue 39 fermented tobacco leaf extract soluble liquid formulation. The formulation's stability and appearance met the requirements for commercial preparations.

[0061] Example 3

[0062] This example shows the results of an experimental study on the efficacy of a soluble solution of Yunxue 39 fermented tobacco leaf extract against southern root-knot nematodes in potted plants in Example 2.

[0063] 1. Test reagents

[0064] The soluble liquid of Yunxue 39 fermented tobacco leaf extract in Example 2; 0.5% avermectin agent.

[0065] 2. Test crops

[0066] Tobacco, variety "K326".

[0067] 3. Targets of prevention and control

[0068] Southern root-knot nematode.

[0069] 4. Selection of experimental plots and experimental treatment

[0070] 4.1 Selection of experimental plots

[0071] 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.

[0072] 4.2 Experimental treatment

[0073] The test agent was Yunxue 39 fermented 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 pots, with three replicates for each treatment.

[0074] 4.3 Application method

[0075] 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 1.5 liters per mu) and thoroughly mixed with the soil before transplanting the tobacco plants. Treatments with 0.5% avermectin and water served as positive and blank controls, respectively, with the same water and fertilizer management practices. Samples were collected and investigated two months later.

[0076] 4.4 Survey Methods

[0077] 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 root soil were counted. 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.

[0078] Table 3 Root knot grading standards

[0079]

[0080] The control effect is calculated according to the following formula:

[0081] Disease index (%) = ∑ (number of plants at each level × number of levels) / (total number of surveys × number of the highest level) × 100%;

[0082] 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%;

[0083] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100%;

[0084] 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%.

[0085] 5. Test results and statistics

[0086] The control effects of 40 mg / mL, 20 mg / mL, and 10 mg / mL soluble liquid extracts of Yunxue 39 fermented tobacco leaves on southern root-knot nematodes were determined. The results are shown in Table 4.

[0087] Table 4 Control effect of Yunxue 39 fermented tobacco leaf extract soluble solution on southern root-knot nematode

[0088]

[0089]

[0090] As shown in Table 4, the three Yunxue 39 fermented tobacco leaf preparations all had good control effects against southern root-knot nematodes. The control effects against southern root-knot nematodes increased significantly with increasing concentrations of the preparations.

[0091] The present invention provides a method for preparing a nematicide from a cigar fermented tobacco leaf extract and the nematicide. The cigar fermented tobacco leaf is used as a raw material, and a highly active nematicide extract is prepared by cold-immersion extraction with an organic solvent, repeated extraction of the extract, and pressure concentration using a rotary evaporator. The organic solvent, antifreeze, and sterile water are added to prepare the nematicide. Taking the southern root-knot nematode as an example, the extract has a corrected mortality rate of nearly 90% for the southern root-knot nematode at a concentration of 100 mg / mL, and the 72-hour median lethal concentration for the southern root-knot nematode is as low as 39.9 mg / mL. The nematicide has significant nematicidal activity and can be used for the prevention and control of nematodes on crops such as tomatoes and tobacco. The nematicide has no chemical residues, is safe for humans, livestock, natural enemies of pests, and other beneficial organisms, and has good environmental compatibility. The raw material can be discarded fermented tobacco leaves from cigar processing, thereby reducing costs, improving resource utilization, and solving the problems of weak activity and high cost of plant-derived nematicides.

[0092] The above description is merely a preferred embodiment of the present invention. Those skilled in the art should understand that the scope of disclosure involved in the present invention is not limited to the technical solutions formed by the specific combination of the above-mentioned technical features, but also includes other technical solutions formed by any combination of the above-mentioned technical features or their equivalents without departing from the above-mentioned disclosed concepts. For example, a technical solution formed by replacing the above-mentioned features with (but not limited to) technical features with similar functions disclosed in the present invention.

Claims

1. A method for preparing a nematicide from a cigar fermented tobacco leaf extract, characterized in that: include: The dried fermented cigar tobacco leaves are crushed by a crusher to obtain a coarse crushed product with a particle size of 1 to 5 mm; cold soaking the coarsely ground material in an organic solvent for 24 to 48 hours, repeating this process 3 to 5 times to obtain a cigar fermented tobacco leaf extract; Repeatedly extracting the fermented cigar tobacco leaf extract with an extraction liquid, and then pressurizing and concentrating the extract to 1 / 9 to 1 / 11 of the mass of the dried fermented cigar tobacco leaf using a rotary evaporator, to obtain a fermented cigar tobacco leaf extract; The cigar fermented tobacco leaf extract is added to the organic solvent, antifreeze and sterile water and stirred thoroughly to prepare a nematocide.

2. The method for preparing a nematicide from a cigar fermented tobacco leaf extract according to claim 1, characterized in that: The mass ratio of each component of the nematicide is: Cigar fermented tobacco leaf extract: 10% to 50%, organic solvent: 1% to 10%, antifreeze: 5% to 10%, sterile water: 30% to 84%, and the sum of the mass percentages of the above components is 100%.

3. The method for preparing a nematicide from a cigar fermented tobacco leaf extract according to claim 2, characterized in that: The mass ratio of the organic solvent to the coarsely pulverized material is 1:10 to 1:

5.

4. The method for preparing a nematicide from a cigar fermented tobacco leaf extract according to claim 3, characterized in that: The organic solvent includes one or more of methanol, ethanol and isopropanol.

5. The method for preparing a nematicide from a cigar fermented tobacco leaf extract according to claim 4, characterized in that: The extract comprises one or more of ethyl acetate, methyl tert-butyl ether and n-butanol.

6. The method for preparing a nematicide from a cigar fermented tobacco leaf extract according to claim 5, characterized in that: The antifreeze agent includes one or more of ethylene glycol, propylene glycol or glycerol.

7. A nematicide for fermented cigar tobacco leaves prepared by the method according to any one of claims 1 to 6.

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