Botanical preparation for preventing and treating root-knot nematode and application of botanical preparation

Abstract

The invention discloses a plant source preparation for preventing and treating root-knot nematode and application, and relates to the technical field of agricultural disease and insect pest prevention and treatment. The botanical preparation for preventing and treating the root-knot nematode comprises the following raw materials in percentage by weight: 1-4% of lycorine hydrochloride, 0.1-0.3% of a surfactant, 0.4-0.75% of a stabilizer, 0.2-0.4% of a preservative, 3-5% of an anti-freezing agent, 0.5-2% of a thickening agent and the balance of water. The preparation has a good poisoning effect on root-knot nematodes of crops, and the active components and the auxiliary components in the preparation can be matched with each other, so that the preparation is stable in physical property and chemical component, and meets the requirements of storage, transportation and use.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural pest control, and in particular to a plant-based preparation for controlling root-knot nematodes and its application. Background Art

[0002] Root-knot nematodes (Meloidogyne spp.) are important pathogenic organisms that harm crops. They can parasitize nearly 3,000 hosts, and new hosts are constantly being discovered. It is estimated that the damage caused by root-knot nematodes accounts for about 5% of the total agricultural production losses in the world. In recent years, greenhouse cultivation and continuous cropping of susceptible varieties have led to increasingly serious root-knot nematode diseases, generally causing a 10-20% reduction in production, and seriously up to 60% or more, which has seriously affected the sustainable development of related industries. The use of chemical nematicides for prevention and control is a common method, but it is easy to cause problems such as pesticide residues and environmental pollution. The secondary metabolites produced by plants not only have specific biological activities, but are also environmentally friendly, and nematodes are not easy to develop drug resistance. Therefore, screening plant-derived active substances that are toxic to root-knot nematodes, optimizing the formulation of preparations, testing the control effect of root-knot nematodes under field conditions, and detecting their effects on plant growth are of great significance to help the healthy and sustainable development of the agricultural industry.

[0003] Lycorine hydrochloride is a derivative of the isoquinoline alkaloid lycorine extracted from the Amaryllidaceae plant. It has bactericidal, insecticidal, anti-tumor, antiviral, and anti-inflammatory biological activities. Lycorine was first discovered to have anti-tumor activity in 1976 and is the first alkaloid in the Amaryllidaceae plant to be confirmed to inhibit cell growth. In subsequent studies, the research on lycorine and its derivatives in the medical field has made remarkable progress. Related studies have shown that lycorine and its derivatives may be a potential drug for the treatment of tumors, lung inflammation, diabetes, sepsis and other diseases.

[0004] In the field of plant protection, lycorine and its derivatives have been found to have strong biological activity against pathogens, insects, weeds, etc. Studies have found that the methanol extract of lycorine can inhibit the growth of rice blast mycelium, and it is speculated that lycorine may be one of the main active ingredients; lycorine has a significant control effect on apple post-harvest gray mold. In in vitro tests, lycorine strongly inhibits spore germination and the spread of mycelium in the culture medium. Studies using fluorescein diacetate and propidium iodide staining have shown that lycorine destroys the membrane integrity of Staphylococcus cinerea and impairs its cell viability; lycorine has good inhibitory activity against diamondback moth (LC50 = 10.6 mg / L), tobacco mosaic virus, and pepper phytophthora (EC50 = 7.76 mg / L); the IC50 of lycorine for weed root growth inhibition is 0.93 μM, which is equivalent to 0.11 mM herbicide chlorpromazine.

[0005] The research on lycorine and its derivatives in the field of plant protection mainly focuses on insects, fungi, viruses and weeds, and there are no reports on their biological activity against plant parasitic nematodes. Therefore, clarifying the toxicity of lycorine hydrochloride against root-knot nematodes, optimizing the formulation, and testing the field efficacy and its effect on cucumber growth can lay the foundation for the widespread application of the formulation. Summary of the invention

[0006] In view of the above shortcomings, the present invention provides a plant-derived preparation for controlling root-knot nematodes and its application, which has a good toxic effect on crop root-knot nematodes, and the active ingredients and auxiliary components in the preparation can cooperate with each other, so that its physical properties and chemical components are stable, which meets the requirements of storage, transportation and use. The specific technical scheme is as follows:

[0007] A plant-derived preparation for preventing and controlling root-knot nematodes comprises the following raw materials in percentage by weight: 1-4% of lycorine hydrochloride, 0.1-0.3% of a surfactant, 0.4-0.75% of a stabilizer, 0.2-0.4% of a preservative, 3-5% of an antifreeze agent, 0.5-2% of a thickener, and the balance is water.

[0008] Preferably, a plant-derived preparation for controlling root-knot nematodes comprises the following raw materials in percentage by weight: 2% lycorine hydrochloride, 0.2% surfactant, 0.5% stabilizer, 0.3% preservative, 4% antifreeze agent and 1% thickener, with the balance being water.

[0009] Preferably, the surfactant is prepared by mixing Tween 20 and Tween 80, and the weight ratio of Tween 20 to Tween 80 is 1:(1-3).

[0010] Preferably, the stabilizer is prepared by mixing citric acid and sodium citrate, and the weight ratio of the citric acid to sodium citrate is 1:(8-10).

[0011] Preferably, the preservative is a mixture of benzoic acid and sodium benzoate, and the weight ratio of benzoic acid to sodium benzoate is 1:(7-9).

[0012] Preferably, the antifreeze agent is propylene glycol.

[0013] Preferably, the thickener is polyvinyl alcohol.

[0014] The present invention also provides application of the plant-derived preparation, which is used for preventing and treating root-knot nematode disease in crops.

[0015] Preferably, the method for using the plant-derived preparation is: applying it by root irrigation on the day of crop transplanting, with a dosage of 200-300 mL / plant.

[0016] Preferably, the crop is cucumber.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention found that lycorine hydrochloride has good toxicity against the second-instar larvae of root-knot nematodes, with a median lethal concentration (LC) of 50 The plant-derived preparation made of lycorine hydrochloride has a control effect of about 47% on cucumber root-knot nematodes. After 30 days of application, the plant height is about 94 cm and the stem thickness is about 6 mm. The control effect, plant height and stem thickness are all higher than those of the existing plant-derived nematicide matrine. After 30 days of application, the insect population reduction rate can reach about 51%, and the fresh weight is about 44 g, which is equivalent to the effect of the existing plant-derived nematicide matrine. At the same time, since the lycorine hydrochloride used in the present invention has a simple structure, it can be industrialized, is safe to use, and has excellent toxicity, it is expected to be developed into a new preparation for controlling root-knot nematodes.

[0019] 2. The active ingredients and auxiliary components in the preparation of the present invention can cooperate with each other, so that its physical properties and chemical composition are stable, which meets the requirements of storage, transportation and use. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0021] Example 1

[0022] A plant-derived preparation for preventing and treating root-knot nematodes, comprising the following raw materials in percentage by weight: 2% of lycorine hydrochloride, 0.2% of a surfactant, 0.5% of a stabilizer, 0.3% of a preservative, 4% of propylene glycol and 1% of polyvinyl alcohol, with the remainder being water. The surfactant is prepared by mixing Tween 20 and Tween 80, wherein the weight ratio of Tween 20 to Tween 80 is 1:1. The stabilizer is prepared by mixing citric acid and sodium citrate, wherein the weight ratio of citric acid to sodium citrate is 1:9. The preservative is prepared by mixing benzoic acid and sodium benzoate, wherein the weight ratio of benzoic acid to sodium benzoate is 1:9.

[0023] Example 2

[0024] A plant-derived preparation for preventing and treating root-knot nematodes, comprising the following raw materials in percentage by weight: 1% of lycorine hydrochloride, 0.1% of a surfactant, 0.4% of a stabilizer, 0.2% of a preservative, 3% of propylene glycol and 0.5% of polyvinyl alcohol, with the remainder being water. The surfactant is prepared by mixing Tween 20 and Tween 80, wherein the weight ratio of Tween 20 to Tween 80 is 1:2. The stabilizer is prepared by mixing citric acid and sodium citrate, wherein the weight ratio of citric acid to sodium citrate is 1:8. The preservative is prepared by mixing benzoic acid and sodium benzoate, wherein the weight ratio of benzoic acid to sodium benzoate is 1:7.

[0025] Example 3

[0026] A plant-derived preparation for preventing and treating root-knot nematodes, comprising the following raw materials in percentage by weight: 4% of lycorine hydrochloride, 0.3% of a surfactant, 0.75% of a stabilizer, 0.4% of a preservative, 5% of propylene glycol and 2% of polyvinyl alcohol, with the remainder being water. The surfactant is prepared by mixing Tween 20 and Tween 80, wherein the weight ratio of Tween 20 to Tween 80 is 1:3. The stabilizer is prepared by mixing citric acid and sodium citrate, wherein the weight ratio of citric acid to sodium citrate is 1:10. The preservative is prepared by mixing benzoic acid and sodium benzoate, wherein the weight ratio of benzoic acid to sodium benzoate is 1:8.

[0027] Comparative Example 1: In this comparative example, Tween 80 is used as the surfactant, and other raw materials are the same as those in Example 1.

[0028] Comparative Example 2: In this comparative example, the stabilizer is sodium citrate, and the other raw materials are the same as those in Example 1.

[0029] Comparative Example 3: In this comparative example, sodium benzoate is used as the preservative, and other raw materials are the same as those in Example 1.

[0030] Test on the toxicity of lycorine hydrochloride to the second-instar larvae of root-knot nematodes:

[0031] Weigh 0.0500g of Lycorine Hydrochloride standard and dissolve it in a small amount of pure water, transfer to a 25mL volumetric flask, add pure water to the scale, and obtain 2000μg / mL Lycorine Hydrochloride standard solution. Add 0.5mL of second-instar larval suspension (about 100 worms) to a 12-well plate, and then add 0.04, 0.06, 0.08, 0.10, and 0.12mL of Lycorine Hydrochloride standard solution respectively, and make up to 1mL with pure water. Clean water is used as a control. The concentration gradient of Lycorine Hydrochloride is 80, 120, 160, 200, and 240μg / mL, and each treatment is repeated 3 times. Cultivate at 25℃, observe after 72h of treatment, and the immobile insect body is considered dead. Calculate the corrected mortality rate, and draw the toxicity curve based on this to calculate the half-lethal concentration LC 50 .

[0032]

[0033]

[0034] Table 1 Indoor toxicity of lycorine hydrochloride against second instar larvae of root-knot nematodes

[0035]

[0036] Note: The data in the table are mean ± standard error.

[0037] As can be seen from Table 1, with the increase of lycorine hydrochloride concentration and the extension of action time, the corrected mortality rate of the second-instar larvae of the root-knot nematode continued to increase. The experimental results showed that at 72 hours, the corrected mortality rate of lycorine hydrochloride at a concentration of 80-240 mg / L showed a good linear relationship with the concentration. The linear regression equation y=0.002 4x+0.0639, R 2 The LC50 of Lycorine Hydrochloride is 0.998 9. 50 The equivalent concentration of lycorine can be converted to 161.24 mg / L from the molecular weight of lycorine hydrochloride and lycorine.

[0038] Performance test of the preparation of the present invention:

[0039] The low temperature stability, heat storage stability, foaming property, viscosity, pH value, dilution stability and other properties of the preparations of Examples 1 to 3 were tested respectively, and the test methods were as follows:

[0040] For the low temperature stability test, refer to GB / T 19137-2003, store the preparation at 0±2℃, take it out after 7 days, bring it to room temperature, and check its precipitation.

[0041] For the thermal storage stability test, refer to GB / T 19136-2021, store in a constant temperature box at 54±2℃, take out after 14 days, cool to room temperature in a dry environment, and determine the active ingredients.

[0042] Foaming test: Measure 2.5mL of the sample into a 250mL beaker, add 200mL of 342mg / L standard hard water to dilute, stir evenly, transfer to a 250mL stoppered measuring cylinder, cover with the stopper, turn it upside down 30 times, and leave it for 1 minute. The liquid surface foam is less than 60mL and is qualified.

[0043] Viscosity determination: Determined by rotational viscometer.

[0044] pH value determination: refer to GB / T 1601-2023, take 1g of preparation, add 100mL of water and measure using a pH meter.

[0045] Dilution stability test: Weigh 5g of the preparation, place it in a 100mL measuring cylinder, dilute to the mark with standard hard water, and mix well. Place the measuring cylinder in a constant temperature water bath at 30±1℃ and let it stand for 1h. If the dilution is uniform and there is no precipitate, it is qualified. The test results are shown in Table 2:

[0046] Table 2 Comparison of performance of each group of preparations

[0047] Group Cold storage stability Thermal storage stability Decomposition rate % Viscosity mPa·s Foaming pH Dilution stability Example 1 qualified qualified 0.83 12 27.2 6.54 qualified Example 2 qualified qualified 0.86 11 27.6 6.53 qualified Example 3 qualified qualified 0.88 12 27.5 6.57 qualified Comparative Example 1 Failure Failure 0.60 11 32.9 6.76 Failure Comparative Example 2 Failure Failure 0.65 11 35.7 6.56 Failure Comparative Example 3 Failure Failure 0.82 11 38.0 6.89 Failure

[0048] Table 2 shows that the formula of the present invention has stable physical properties, stable chemical components, viscosity and foaming properties suitable for aqueous formulations, good pH and dilution stability, suitable for root irrigation, and a qualified formula in terms of physical and chemical properties. From the data of Example 1 and the comparative example, it can be seen that the surfactant is mixed with Tween 20 and Tween 80, the stabilizer is mixed with citric acid and sodium citrate, and the preservative is mixed with benzoic acid and sodium benzoate, which can make the physical properties of the preparation stable, the chemical components stable, and meet the storage, transportation and use requirements.

[0049] Evaluation of the control effect of the preparation of the present invention on cucumber root-knot nematode disease:

[0050] In 2023, a scientific research base was built at the Guangxi Zhuang Autonomous Region Academy of Agricultural Sciences in Wuming District, Nanning City, Guangxi Zhuang Autonomous Region, and a field test for the prevention and control of cucumber root-knot nematode disease was carried out using the preparation prepared in Example 1 to further verify the prevention and control effect.

[0051] Test agent: the preparation prepared in Example 1 (hereinafter referred to as 2% lycorine hydrochloride).

[0052] Control agent: 1.3% matrine aqueous solution (Tianjin Hengyuan Weiye Biotechnology Development Co., Ltd.)

[0053] Blank control: clean water.

[0054] The cucumber seeds were soaked in conventional warm water to germinate, and stored in a 30℃ incubator for 48 hours to keep them moist. When the buds broke through the seed shells, they were immediately sown in a seedling tray filled with seedling medium, placed in a place with sufficient sunlight, and watered once every two days. When the seedlings grew to four leaves and one heart, they were transplanted. On the day of transplanting, the roots were irrigated and the pesticides were applied (300mL / plant). The treatment agents were 13μg / mL of lycorine hydrochloride aqueous solution, 13μg / mL of matrine agent, and clean water. The control effects of each treatment on cucumber root-knot nematodes 30 days after transplanting are shown in Tables 3 to 5.

[0055] The root knot grading standard is divided into 0-5 levels according to Eisenback JD: Level 0, no root knots in the root system; Level 1, 0-10% of the root system has root knots; Level 2, 11%-30% of the root system has root knots; Level 3, 31%-50% of the root system has root knots; Level 4, 51%-75% of the root system has root knots; Level 5, more than 75% of the root system has root knots. Soil nematode population density: refer to the modified Baermann funnel method to separate soil nematodes, and calculate the number of second-instar larvae of root-knot nematodes in the soil. The stem diameter and the height of the whole plant at 2 cm above the ground are measured to analyze the growth.

[0056]

[0057]

[0058]

[0059]

[0060] Table 3 Relative control effect of each agent after treatment

[0061] deal with Average root knot index Control effect% 2% Lycorine hydrochloride 0.013mg ai / mL 26.67±2.72b 46.88±4.27a Matrine 0.013mg ai / mL 28.33±1.67b 43.30±2.56a Blank control 50.00±1.92a

[0062] The survey results showed that the disease index of each drug treatment group was lower than that of the blank control group, and the difference was significant; the control effect of the 2% lycorine hydrochloride treatment group was 46.88±4.27%, which was higher than that of the matrine treatment group (43.30±2.56%).

[0063] Table 4 shows the control effect of each treatment on cucumber root-knot nematodes 30 days after transplanting:

[0064] Table 4 Effects of various pesticide treatments on soil insect population density

[0065]

[0066] As can be seen from Table 4, the insect population of the blank control group did not decrease significantly after 30 days of the test, while the insect population reduction rates of the two drug treatment groups reached 50.75±1.86% and 51.14±1.09%, respectively, and the two drug treatment groups were significantly higher than the blank control. The insect population control efficiency of the two drug treatment groups reached 48.92±1.41% and 49.21±1.79%, respectively.

[0067] The effects of each treatment on the growth of the aboveground part of potted cucumbers 30 days after transplanting are shown in Table 5:

[0068] Table 5 Effects of each treatment on the growth of potted cucumber plants

[0069] deal with Stem diameter (mm) Plant height(cm) Fresh weight(g) Lycorine Hydrochloride 6.17±0.17a 94.25±4.38a 43.55±6.29a Matrine 6.08±0.46a 91.77±6.47a 47.97±8.63a Blank control 6.20±0.20a 71.33±6.12b 34.22±4.30b

[0070] The survey results showed that the plant heights of all treatment groups were significantly higher than those of the blank control group, among which the plants treated with 2% lycorine hydrochloride aqueous solution were the tallest, reaching 94.25±4.38cm, and the plant heights after matrine treatment were 91.77±6.47cm; the fresh weights of the two treatment groups were higher than those of the blank control group.

[0071] In summary, the present invention found that lycorine hydrochloride has good toxicity against the second-instar larvae of root-knot nematodes, and the half-lethal concentration LC 50 It is 181.70mg / L. The plant-based preparation made of lycorine hydrochloride has a control effect of about 47% on cucumber root-knot nematodes. After 30 days of application, the plant height is about 94cm and the stem thickness is about 6mm. The control effect, plant height and stem thickness are all higher than those of the existing plant-based nematicide matrine; after 30 days of application, the insect population reduction rate can reach about 51%, and the fresh weight is about 44g, which is equivalent to the effect of the existing plant-based nematicide matrine. At the same time, since the lycorine hydrochloride used in the present invention has a simple structure, it can be industrialized, is safe to use, and has excellent toxicity, it is expected to be developed into a new type of root-knot nematode control preparation. The active ingredients and auxiliary components in the preparation of the present invention can cooperate with each other, so that its physical properties are stable, the chemical composition is stable, and it meets the requirements of storage, transportation and use.

[0072] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A plant-derived preparation for controlling root-knot nematodes, characterized in that: The invention comprises the following raw materials in percentage by weight: 1-4% of lycorine hydrochloride, 0.1-0.3% of surfactant, 0.4-0.75% of stabilizer, 0.2-0.4% of preservative, 3-5% of antifreeze agent and 0.5-2% of thickener, and the balance is water.

2. A plant-derived preparation for controlling root-knot nematodes according to claim 1, characterized in that: The invention comprises the following raw materials in percentage by weight: 2% of lycorine hydrochloride, 0.2% of surfactant, 0.5% of stabilizer, 0.3% of preservative, 4% of antifreeze agent and 1% of thickener, and the balance is water.

3. A plant-derived preparation for controlling root-knot nematodes according to claim 1 or 2, characterized in that: The surfactant is prepared by mixing Tween 20 and Tween 80, and the weight ratio of Tween 20 to Tween 80 is 1:(1-3).

4. A plant-derived preparation for controlling root-knot nematodes according to claim 1 or 2, characterized in that: The stabilizer is prepared by mixing citric acid and sodium citrate, and the weight ratio of the citric acid to the sodium citrate is 1:(8-10).

5. A plant-derived preparation for controlling root-knot nematodes according to claim 1 or 2, characterized in that: The preservative is prepared by mixing benzoic acid and sodium benzoate, and the weight ratio of the benzoic acid to the sodium benzoate is 1:(7-9).

6. A plant-derived preparation for controlling root-knot nematodes according to claim 1 or 2, characterized in that: The antifreeze agent is propylene glycol.

7. A plant-derived preparation for controlling root-knot nematodes according to claim 1 or 2, characterized in that: The thickener is polyvinyl alcohol.

8. The use of the plant-derived preparation according to any one of claims 1 to 7, characterized in that: The application is for preventing and controlling root-knot nematode disease in crops.

9. The use of the plant-derived preparation according to claim 8, characterized in that: The method for using the plant-derived preparation is as follows: applying the preparation by root irrigation on the day of crop transplanting, with a dosage of 200-300 mL per plant.

10. The use of the plant-derived preparation according to claim 8, characterized in that: The crop is cucumber.

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