A composition for preventing and treating pine wood nematode and application thereof

Abstract

The application discloses a composition for preventing and treating Bursaphelenchus xylophilus and application thereof, and belongs to the technical field of biological pesticides. The composition is compounded by taking a crude extract of Zanthoxylum bungeanum as a main active ingredient and a crude extract of Astragalus membranaceus as a synergistic ingredient. The composition has high Bursaphelenchus xylophilus killing activity. After Bursaphelenchus xylophilus is treated by 600ug / mL of the composition solution for 24 hours, the corrected mortality of the Bursaphelenchus xylophilus reaches 99.33%. The composition can effectively prevent the occurrence of the pine wilt disease in a forest, and the control effect reaches 79.59%. In addition, the preparation process of the composition is simple, the composition is easy to use, and the composition is suitable for popularization and use.

Description

Technical Field

[0001] This invention relates to a composition for controlling pine wilt disease and its application, belonging to the field of pine wilt disease control technology. Background Technology

[0002] Pine wilt disease, also known as pine wilt, is a highly devastating disease of pine trees. Once infected, it spreads rapidly, affecting dozens of species of Pinus, and is prevalent in many countries worldwide, including my country. It has become a serious global forest disease. The pine wilt nematode (Bursaphelenchus xylophilus) is the primary pathogen of pine wilt disease, and its control is crucial for managing the disease. Currently, the most direct and effective nematicides for controlling pine wilt are broad-spectrum synthetic agents. However, nematodes easily develop resistance, and these agents suffer from drawbacks such as high toxicity, high residue, environmental pollution, and threats to non-target beneficial organisms. This makes the development of eco-friendly natural nematicides particularly urgent. With abundant plant resources in nature, plant-derived nematicides have been a hot topic in pine wilt disease research in recent years. Improving stability and optimizing performance by mixing multiple bioactive natural plants is an effective approach to developing natural product-based biological agents.

[0003] Sichuan pepper (Zanthoxylum bungeanum Maxim.) is a deciduous small tree or perennial plant belonging to the Rutaceae family and the Zanthoxylum genus. It is a traditional Chinese medicinal and edible plant. The fruit of Sichuan pepper contains abundant active substances and possesses various activities such as analgesia and insecticidal properties. Patent CN101209059A discloses a novel biological agent for killing root-knot nematodes and its application method. It uses Stemona japonica powder as the main active ingredient and powders of several plant materials, including Sichuan pepper powder, as secondary active ingredients to form a wettable powder. While it can effectively inhibit the occurrence of tomato root-knot nematodes, its activity against root-knot nematodes has not been thoroughly studied; its effect on killing pine wilt nematodes has also not been studied. CN108925315A discloses a two-step method for biological fumigation control of root-knot nematodes in greenhouse vegetables. First, Sichuan pepper seed cake is applied at a rate of 1000 kg / mu by broadcasting and mixing it with the greenhouse soil, then covered with transparent mulch, and the greenhouse is sealed for about 30 days. Seven days before transplanting vegetables, evenly scatter inactivated Sichuan pepper seeds at a rate of 1000 kg / mu in the planting beds, mix them thoroughly with the soil, and then transplant the vegetables. The above method is not suitable for controlling pine root-knot nematodes. Summary of the Invention

[0004] This invention overcomes the shortcomings of the prior art and provides a composition for controlling pine wilt disease and its application. The composition is formulated with crude extract of Sichuan pepper as the main active ingredient and crude extract of Astragalus membranaceus as the synergistic ingredient. The composition can effectively control pine wilt disease, and the preparation process is simple and easy to use, making it suitable for widespread application.

[0005] The technical solution of the present invention is: a composition for controlling pine wood nematode, characterized in that the composition is composed of crude extracts of Sichuan pepper and crude extracts of Astragalus membranaceus, wherein the mass ratio of crude extracts of Sichuan pepper to crude extracts of Astragalus membranaceus is 24-25:1.

[0006] The crude extract of Sichuan pepper was obtained by soaking Sichuan pepper fruits in a 10% NH4Cl aqueous solution at 45-55℃, followed by extraction with xylene; the crude extract of Astragalus membranaceus was obtained by reflux extraction of Astragalus membranaceus roots with methanol or n-hexane.

[0007] Preferably, the crude extract of Astragalus membranaceus is obtained by extracting Astragalus membranaceus root by heating and reflux with methanol.

[0008] Preferably, both the pepper fruit and the astragalus root are pulverized into powder that passes through a 60-mesh sieve.

[0009] The above composition is prepared by the following method:

[0010] (1) The powder of Sichuan pepper fruit was extracted with 10% NH4Cl aqueous solution at 45-55℃, then extracted with xylene, concentrated to a viscous state, and then diluted with organic solvent to obtain crude extract solution of Sichuan pepper.

[0011] (2) Extract Astragalus root powder by heating and reflux with methanol or n-hexane, concentrate the extract until it becomes viscous, and then dilute it with an organic solvent to obtain a crude extract solution of Astragalus.

[0012] (3) Add the crude extract of Astragalus membranaceus to the crude extract of Zanthoxylum bungeanum to make a combination solution of Zanthoxylum bungeanum and Astragalus membranaceus.

[0013] Preferably, in step (1), the amount of 10% NH4Cl aqueous solution used is 8 to 12 times the mass of Sichuan pepper powder; the extraction time is 1.5 to 3 hours.

[0014] Preferably, step (2) involves heating and reflux extraction for 3–4 hours.

[0015] Preferably, the organic solvent used for dilution in steps (1) and (2) is DMF.

[0016] The present invention also provides the application of the above composition in the control of pine wilt disease.

[0017] This invention, through research on crude extracts of Sichuan pepper, has found that the crude extracts of Sichuan pepper have good killing activity against pine wilt nematodes; by adding crude extracts of Astragalus membranaceus, it was found that the crude extracts of Astragalus membranaceus have a significant synergistic effect on the nematode-killing effect of the crude extracts of Sichuan pepper, thus opening up a new direction for the research and development of novel biological agents for the control of pine wilt nematodes.

[0018] The beneficial effects of this invention are:

[0019] 1. Highly effective nematode killing. This composition exhibits high activity against pine wood nematodes. Treatment of pine wood nematodes with a 600 μg / mL solution for 24 hours resulted in a corrected mortality rate of 99.33%; 24-hour LC... 50 =67.67 μg / mL;

[0020] 2. This composition can effectively prevent the occurrence of pine wilt disease in the forest, with a control effect of 79.59%;

[0021] 3. This composition retains a variety of active ingredients from Sichuan pepper and astragalus, and the preparation method is simple, low-cost, and easy to mass-produce. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. In the following embodiments, the part of Sichuan pepper used is the mature fruit, and the part of Astragalus membranaceus used is the root.

[0023] Example 1: Acquisition of crude extracts of Sichuan pepper with different concentrations of NH4Cl and detection of nematicidal activity (detection concentration: based on crude drug, concentration approximately 1 mg / ml).

[0024] This embodiment provides a method for extracting crude extracts of Sichuan pepper and experiments on the killing effect of crude extracts of Sichuan pepper with different concentrations of NH4Cl on pine wilt nematodes.

[0025] 1. Obtaining crude extract of Sichuan pepper

[0026] (1) Grind fresh, dry, and clean pepper fruits in a plant pulverizer for 1-2 minutes and pass them through a 60-mesh sieve to obtain pepper powder;

[0027] (2) Add 10% (by weight) of Sichuan pepper powder to 5%, 10%, and 15% NH4Cl solutions, respectively, and extract at a constant temperature of 50°C for 2 hours, stirring once every 0.5 hours. The control was to use water instead of NH4Cl solution for extraction.

[0028] (3) After extraction, the coarse filtrate with different treatments is obtained by filtering through four layers of gauze.

[0029] (4) Mix the crude filtrates from different treatments with organic solvents (xylene or n-hexane) at a weight ratio of 1:1, and extract them in a separatory funnel for 24 hours. After extraction, the upper layer is the crude extract of Sichuan pepper extracted with different concentrations of NH4Cl and different organic solvents.

[0030] Based on this method, we initially obtained eight crude extracts of Sichuan pepper, namely: Sichuan pepper crude extract-CK-A, Sichuan pepper crude extract-5-A, Sichuan pepper crude extract-10-A, Sichuan pepper crude extract-15-A, Sichuan pepper crude extract-CK-B, Sichuan pepper crude extract-5-B, Sichuan pepper crude extract-10-B, and Sichuan pepper crude extract-15-B. Wherein: "CK" represents water, "5" represents 5% NH4Cl solution, "10" represents 10% NH4Cl solution, and "15" represents 15% NH4Cl solution; "A" represents xylene, an organic solvent, and "B" represents n-hexane, an organic solvent.

[0031] 2. Detection of nematicidal activity of different crude extracts of Sichuan pepper

[0032] The method for detecting the activity of the killing signal is as follows:

[0033] (1) Dilute 100 times: Add 8% Tween-80 surfactant to each of the 8 crude extracts of Sichuan pepper obtained above, and dilute with water 100 times for later use.

[0034] (2) Preparation of pine wood nematode solution. First, sterile pine wood nematodes (Bursaphelenchus xylophilus) were inoculated onto PDA plates contaminated with Botrytis cinerea and cultured at 25°C for approximately 5 days before isolating the nematodes using the Bellman funnel method. Then, the isolated nematode solution was centrifuged at 3500 rpm for 3–5 minutes to remove impurities, and diluted with sterile water; this process was repeated three times; the solution free of impurities was collected. Finally, a pine wood nematode solution with a concentration of approximately 2500 nematodes / mL was prepared using a dilution method.

[0035] (3) Detection steps (1) Killing effect of different crude extracts of Sichuan pepper on pine wilt nematodes. Different crude extracts of Sichuan pepper were diluted with nematode solutions at a volume ratio of 1:1; the total volume of the mixture was 200 μL, at which point the number of pine wilt nematodes in the mixture was stable at about 250. The blank control was sterile water instead of the crude extracts of Sichuan pepper to be tested. After mixing, the mixture was left to stand at 25℃ for 24 h. The mortality of pine wilt nematodes was observed under an optical microscope, and the corrected mortality rate of pine wilt nematodes treated with each crude extract was calculated and compared.

[0036] We determined death by nematode rigidity and lack of response to dissection needle stimulation.

[0037] Mortality rate (%) = (Number of nematodes that died in the treatment / Total number of nematodes in the treatment) × 100

[0038] Corrected mortality rate (%) = (Treatment mortality rate - Blank control mortality rate) / (1 - Blank control mortality rate) × 100

[0039] Microscopic examination method: After processing, centrifuge the mixture at 2000 r / min for 180-200s, remove the supernatant, resuspend in 200 μL of sterile water, take 50 μL of the resuspended solution on a glass slide, and examine under a microscope.

[0040] The results showed that the nematicidal effect of the crude extract of Sichuan pepper initially increased and then decreased with increasing NH4Cl solution concentration. The nematicidal effect of the crude extract obtained with 10% NH4Cl solution was significantly better than that of other concentrations (see Table 1). Further comparison revealed that the nematicidal effect of the crude extract obtained with xylene as the extractant was significantly better than that obtained with n-hexane (see Table 1). These results indicate that the crude extract of Sichuan pepper, obtained with 10% NH4Cl solution and then with xylene, has a significant effect against pine wilt nematodes.

[0041] Table 1. Statistical analysis of the killing effects of different crude extracts of Sichuan pepper on pine wilt nematode.

[0042]

[0043] Example 2: Study on the acquisition of crude extracts of Astragalus membranaceus in different organic solvents and their synergistic effect on the nematolytic activity of crude extract of Zanthoxylum bungeanum.

[0044] This embodiment provides an extraction method for crude Astragalus membranaceus extract and related experiments on the effect of different contents of crude Astragalus membranaceus extract on the nematolytic effect of crude Zanthoxylum bungeanum extract.

[0045] 1. Obtaining crude extract of Astragalus membranaceus

[0046] The components of Astragalus membranaceus were extracted using a Soxhlet extractor to obtain a crude extract. The specific method is as follows:

[0047] (1) Wash the Astragalus root with clean water and soak it for 5 minutes to remove impurities from the surface of the material, and then let it air dry naturally.

[0048] (2) Grind the powder using a grinder for 1-2 minutes and pass it through a 60-mesh sieve to obtain Astragalus powder;

[0049] (3) Take a clean 250mL Soxhlet extractor, add 20g of dried Astragalus powder, and place the Astragalus powder in a filter paper tube with sealed top and bottom and filled with defatted cotton; measure 100mL of methanol and n-hexane respectively and add them to the Soxhlet extractor; soak the sample thoroughly for 3-4 hours, and heat at 80℃ for reflux extraction for 3-4 hours.

[0050] (4) Cool and rotary evaporate the extract until it becomes viscous;

[0051] (5) After extraction, add DMF to make up to 100 mL to obtain different crude extracts of Astragalus membranaceus.

[0052] Using this method, we obtained two crude extracts of Astragalus membranaceus: Astragalus membranaceus crude extract-B and Astragalus membranaceus crude extract-C. Here, "B" represents the extractant n-hexane, and "C" represents the extractant methanol.

[0053] 2. Detection of nematicidal activity of different crude extracts of Astragalus membranaceus

[0054] The nematode-killing effects of different Astragalus crude extract solutions (without 100-fold dilution) were tested according to the method described in Example 1. The results showed that after 24 hours of treatment with Astragalus crude extract-B and Astragalus crude extract-C, the corrected mortality rates of pine wood nematodes were -0.53% and -0.59%, respectively (refer to Table 2); indicating that Astragalus crude extract has no killing activity against pine wood nematodes.

[0055] Table 2. Statistical analysis of the killing effects of different crude extracts of Astragalus membranaceus on pine wilt nematode.

[0056]

[0057] 3. Effects of different concentrations of Astragalus membranaceus crude extract on the nematicidal effect of Zanthoxylum bungeanum crude extract

[0058] (1) Preparation of standard crude extract solution of Sichuan pepper. The crude extract of Sichuan pepper was obtained by the optimal method described in Example 1 (extraction with 10% NH4Cl solution and xylene extraction). The extract was rotary evaporated to a viscous state and diluted with DMF to prepare a 1 mg / mL crude extract solution of Sichuan pepper (the concentration is the content of the active ingredient, crude extract of Sichuan pepper, the same below). This is the standard crude extract solution of Sichuan pepper.

[0059] (2) Preparation of standard Astragalus crude extract solution. Prepare Astragalus crude extract solution according to the method described in 1 above. After the extract solution is rotary evaporated to a viscous state, it is directly diluted with DMF to prepare 1 mg / mL Astragalus crude extract (the concentration is the content of the active ingredient Astragalus crude extract, the same below), which is the standard Astragalus crude extract solution. At this time, two standard Astragalus crude extract solutions are obtained.

[0060] (3) Preparation of crude extract composition solutions. Two standard Astragalus crude extract solutions were added to the standard Zanthoxylum bungeanum crude extract solution at volumes of 0.5%, 1%, 2%, 4%, and 8% of the standard Zanthoxylum bungeanum crude extract solution, respectively; and then the corresponding amounts of 7.5%, 7%, 6%, 4%, and 0% DMF were added to ensure that the Zanthoxylum bungeanum crude extract content was consistent (at this point, the effective component content of the Zanthoxylum bungeanum crude extract was 926 μg / mL, and the contents of the Astragalus crude extract were 4.63 μg / mL, 9.26 μg / mL, 18.5 μg / mL, 37.0 μg / mL, and 74.0 μg / mL, respectively), thus preparing different crude extract composition solutions.

[0061] (3) Detection of nematode killing effect of different crude extract compositions. The nematode killing effect of different crude extract compositions was detected according to the method described in Example 1. The difference was that it was not necessary to dilute 100 times, and the blank control 1 was a solution of crude extract of Sichuan pepper with added 8% DMF. After mixing, the mixture was left to stand at 25°C for 24 hours. The mortality of pine wood nematodes was observed under an optical microscope, and the corrected mortality rate of pine wood nematodes in each treatment was calculated and compared.

[0062] The results showed that regardless of whether methanol or n-hexane was used as the extraction solvent to prepare the crude extract of Astragalus membranaceus, mixing it with the standard crude extract of Zanthoxylum bungeanum enhanced the nematode-killing ability of the Zanthoxylum bungeanum extract, demonstrating a synergistic effect. Further analysis revealed that 4% and 8% methanol-based crude extracts of Astragalus membranaceus showed the most significant promoting effect on the nematode-killing effect of the Zanthoxylum bungeanum extract. After 24 hours of treatment with this mixture of crude extracts on pine wood nematodes, the corrected mortality rate of pine wood nematodes was 100%, an increase of 16.97% compared to the control; indicating that the 4% methanol-based crude extract of Astragalus membranaceus had reached saturation in its nematode-killing effect on the Zanthoxylum bungeanum extract. These results demonstrate that thoroughly mixing the crude extract of Astragalus membranaceus obtained by extracting Astragalus membranaceus with Zanthoxylum bungeanum extract at a concentration of 4% can effectively improve the nematode-killing effect of the Zanthoxylum bungeanum extract, with an effective component ratio of Zanthoxylum bungeanum extract to Astragalus membranaceus extract of 24–25:1.

[0063] Table 3. Statistics on the nematolytic effects of different crude extract compositions.

[0064]

[0065] Example 3: Determination of nematicidal toxicity of a crude extract combination of Sichuan pepper and astragalus.

[0066] This embodiment provides a method for preparing a crude extract composition of Sichuan pepper and astragalus, as well as the 24-hour toxicity equation and LC-12 of the composition. 50 Related experiments.

[0067] 1. Preparation of a crude extract composition of Sichuan pepper and Astragalus membranaceus

[0068] (1) Preparation of standard crude extract solution of Sichuan pepper. The crude extract of Sichuan pepper was obtained by the optimal method described in Example 1 (extraction with 10% NH4Cl solution and xylene extraction). The extract was rotary evaporated to a viscous state and diluted with DMF to prepare a 1 mg / mL crude extract solution of Sichuan pepper, which is the standard crude extract solution of Sichuan pepper.

[0069] (2) Preparation of standard Astragalus crude extract solution. After evaporating the extract to a viscous state according to the optimal method (methanol extraction) described in Example 2, the extract was directly diluted with DMF to prepare a 1 mg / mL Astragalus crude extract solution, which is the standard Astragalus methanol crude extract solution;

[0070] (3) Preparation of standard crude extract composition solution. Add 4% of the standard Astragalus methanol crude extract solution to the standard Zanthoxylum bungeanum crude extract solution to prepare a 1 mg / mL crude extract composition solution (the effective ingredient content of Zanthoxylum bungeanum crude extract: Astragalus membranaceus crude extract = 25:1), which is the standard crude extract composition solution.

[0071] 2. Determination of the nematicidal toxicity of the standard composition

[0072] The standard crude extract solution was diluted to 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, 1000 μg / mL, and 1200 μg / mL using a dilution method (DMF as solvent). The efficacy against pine wilt disease was tested according to the method described in Example 1. Pine wilt disease mortality was observed 24 hours after treatment, and the corrected mortality rate for each treatment was calculated. The final concentrations of the composition for each treatment were 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, and 600 μg / mL.

[0073] The results showed that treatment of pine wood nematodes with the 600 μg / mL composition for 24 h resulted in a corrected mortality rate of 99.33%. We further derived its toxicity regression equation and calculated the LC50 of the crude extract composition using this equation. 50 =67.67 μg / mL, indicating that the composition has high activity against pine wood nematodes (see Table 4).

[0074] Table 4. Statistical analysis of the killing effect of different concentrations of the composition on pine wilt nematode (treatment time 24 h).

[0075]

[0076] Example 4: Forest efficacy test of crude extracts of Sichuan pepper and astragalus.

[0077] This embodiment provides a field efficacy test of a formulation using a crude extract of Sichuan pepper and astragalus as the effective nematicidal component.

[0078] This experiment was conducted in June 2022 at the Huangshan Experimental Base in Anhui Province, an area historically plagued by severe pine wilt disease. The test agent was a DMF solution of crude extracts of Sichuan pepper and Astragalus membranaceus (active ingredient content 0.1 g / mL, mass ratio of the two crude extracts 25:1); diluted 300 times; the control was water. The application method was drone-based aerial spraying.

[0079] This experiment was divided into a treatment area and a control area. The treatment area covered 50 mu (approximately 3.3 hectares) with 2743 trees treated; the control area covered 50 mu (approximately 3.3 hectares) with 2805 trees treated. Aerial spraying was conducted at a dosage of 100 ml / mu (approximately 100 ml / hectare) of the tested pesticide. A second aerial spraying was conducted 15–20 days after the first application. After both applications, mortality rates of the Masson pine trees were statistically analyzed in the second year, and the corrected mortality rate for the treatment area was calculated.

[0080] The results showed that after two field sprays of the 300-fold diluted nematicide extract, the mortality rate of Masson pine decreased to 1.09‰; compared with the control, the mortality rate of Masson pine was significantly reduced. The results indicate that the nematicide extract can effectively prevent pine wilt disease in the forest, with a control efficacy of 79.59% (see Table 5). Furthermore, the application method is simple and easy to implement, making it suitable for widespread application.

[0081] Table 5. Statistics on Mortality of Masson Pine Trees

[0082]

Claims

1. A composition for controlling pine wood nematode, characterized in that, It is formulated with crude extract of Sichuan pepper as the main active ingredient and crude extract of Astragalus membranaceus as the synergistic ingredient; the mass ratio of crude extract of Sichuan pepper to crude extract of Astragalus membranaceus is 12.5~100:

1. The crude extract of Sichuan pepper was obtained by soaking Sichuan pepper fruits in a 10% NH4Cl aqueous solution at 45-55℃, followed by extraction with xylene; the crude extract of Astragalus membranaceus was obtained by reflux extraction of Astragalus membranaceus roots with methanol or n-hexane.

2. The composition for controlling pine wilt disease as described in claim 1, characterized in that, The mass ratio of crude extract of Sichuan pepper to crude extract of Astragalus membranaceus is 24-25:1; 3. The composition for controlling pine wilt disease as described in claim 1, characterized in that, The crude extract of Astragalus membranaceus was obtained by extracting Astragalus membranaceus root by heating and reflux with methanol.

4. The composition for controlling pine wood nematode as described in claim 1, characterized in that, Both the Sichuan pepper fruit and the astragalus root need to be pulverized into powder that passes through a 60-mesh sieve.

5. The method for preparing a composition for controlling pine wilt disease as described in claim 4, characterized in that, (1) The powder of Sichuan pepper fruit was extracted with 10% NH4Cl aqueous solution at 45~55℃, then extracted with xylene, concentrated to a viscous state, and then diluted with organic solvent to obtain crude extract solution of Sichuan pepper. (2) Extract the Astragalus root powder by heating and reflux with methanol or n-hexane, concentrate the extract until it becomes viscous, and then dilute it with an organic solvent to obtain a crude Astragalus extract solution; (3) Add the crude extract of Astragalus membranaceus to the crude extract of Zanthoxylum bungeanum to make a combination solution of Zanthoxylum bungeanum and Astragalus membranaceus.

6. The method for preparing a composition for controlling pine wilt disease as described in claim 5, characterized in that, In step (1), the amount of 10% NH4Cl aqueous solution used is 8 to 12 times the mass of Sichuan pepper powder; the extraction time is 1.5 to 3 hours.

7. The method for preparing a composition for controlling pine wood nematode as described in claim 5, characterized in that, Step (2) involves heating and reflux extraction for 3-4 hours.

8. The method for preparing a composition for controlling pine wilt disease as described in claim 5, characterized in that, The organic solvent used for dilution in steps (1) and (2) is DMF.

9. The use of the composition according to any one of claims 1-4 in the control of pine wilt disease.

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