A method for controlling tobacco nematodes

Abstract

The present application relates to a method for preventing and treating tobacco nematodes, and relates to the technical field of tobacco root-knot nematode prevention and treatment. Through the combined treatment of soil preparation, lime nitrogen disinfection and root tip simulation prevention and treatment particles, the problem that the prevention and treatment effect of tobacco root-knot nematodes is not ideal after the root-knot nematodes drill into the host cells is solved.

Description

Technical Field

[0001] This invention belongs to the field of tobacco root-knot nematode control technology, and relates to a method for controlling tobacco nematodes. Background Technology

[0002] Tobacco root-knot nematode disease is caused by root-knot nematodes damaging the roots of tobacco plants, with lateral and fibrous roots being more susceptible. After infection, lateral roots thicken and form nodules. The wounds left after infection facilitate the invasion of soil-borne pathogens, leading to a complex disease that exacerbates the damage to tobacco, affecting leaf yield and quality. In recent years, tobacco root-knot nematode disease has occurred in all major tobacco-producing areas of China and has gradually risen from a secondary disease to a major one. Root-knot nematodes exhibit a vertical distribution pattern in the soil, with the highest numbers found in shallow soil layers, reaching 1000-5000 nematodes / 100 g. It is currently believed that the feeding and piercing actions of root-knot nematodes invading the host plant cause severe damage, affecting the absorption of water and nutrients by the roots, thus leading to poor plant development. Simultaneously, root-knot nematodes interact with other pathogens, inducing other diseases in tobacco. Because this nematode possesses a large esophageal gland, its secretions induce corresponding lesions in tobacco roots, disrupting the normal metabolic function of root cells and internal root tissues, causing deformation of the root surface. When second-instar larvae infect host plants, their mouthparts cause mechanical damage to the tobacco roots, while the hydrolytic enzymes they secrete degrade cellulose and pectin in the root tissue, thus aiding in the invasion of the tobacco plant roots.

[0003] Current methods for controlling root-knot nematodes include: crop rotation with gramineous crops in disease-prone tobacco fields, followed by tilling and sun-drying the soil, and soil disinfection; the use of chemical pesticides such as abamectin, chlorpyrifos, thiazophos, and carbofuran; or biological control using fungi such as Paecilomyces lilacinus and Verticillium chlamydosporium. Most current control methods employ integrated management, but due to the rapid reproduction rate of root-knot nematodes and their increasing resistance to pesticides, the overall control effectiveness has significantly declined. Excessive use of chemical pesticides not only produces pesticide residues but also further exacerbates nematode resistance. While some predatory or endoparasitic fungi can control root-knot nematodes through biological control, the short time root-knot nematode larvae spend in the soil and mostly burrow into host cells, resulting in limited effectiveness. Therefore, a new method for controlling tobacco root-knot nematodes is urgently needed. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for controlling tobacco nematodes, thereby solving the problem that the control effect of root-knot nematodes on tobacco root-knot nematodes is not ideal after they burrow into the host cells.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions: To achieve the above objectives, the present invention provides the following technical solutions:

[0006] A method for controlling tobacco nematodes, wherein the control measures are as follows:

[0007] (1) Land preparation: After applying organic fertilizer to the soil, plow it, disinfect it with lime nitrogen, deep plow and sun-dry the soil, and then cover it with film and let it sit for more than 28 days;

[0008] (2) Transplanting: Apply root tip mimicry control granules to the field before transplanting at a rate of 15-30 kg / hm. 2 Transplant tobacco seedlings 2-3 days after application, water them thoroughly, and maintain soil moisture content at 70-80% for 10 days after transplanting.

[0009] (3) Management: Apply root tip mimicry control granules during the tobacco seedling stage and vigorous growth stage; the application rate of root tip mimicry control granules during the seedling stage is 20-40 kg / hm. 2 The application rate during the peak growing season is 10-15 kg / hm. 2 The nematode control agent is applied to the roots of tobacco plants, with the application hole depth being 20-40 cm.

[0010] Furthermore, the soil moisture content should be maintained at 50-60% during the seedling stage and at 20-30% during the growing season.

[0011] Furthermore, the root tip mimicry control granules contain *Paecilomyces lilacinus* and *Bacillus subtilis*, and the effective viable count of *Paecilomyces lilacinus* in the root tip mimicry control granules is ≥1×10⁻⁶. 8 cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g.

[0012] In addition to the fungicide, the root tip mimicry control granules also contain the following raw materials:

[0013] Asparagus root powder, flax bast mix, 1,4-butanediamine, spermine, gelatin, sodium alginate.

[0014] Existing technologies have proven that *Paecilomyces lilacinus* and *Bacillus subtilis* can control root-knot nematodes. However, root-knot nematodes have a short activity time in the soil, and their control effect decreases significantly after burrowing into tobacco roots. Therefore, the root tip simulation control granules of this invention use *Paecilomyces lilacinus* and *Bacillus subtilis* as the main effective ingredients for killing nematodes. However, *Paecilomyces lilacinus* and *Bacillus subtilis* need a period of reproduction in the field before they become effective. Therefore, this invention also adds asparagus root powder to synergize with the microbial agent to kill root-knot nematodes. Asparagus roots contain a large amount of saponins, which have killing activity against root-knot nematodes, allowing them to kill nematodes before the microbial agent takes effect. In addition, since root-knot nematodes have parasitic characteristics and burrow into tobacco roots, the root tip simulation control granules can mimic the tobacco root tip, releasing signals to induce root-knot nematodes to burrow into the root tip simulation control granules for precise insecticidal treatment. This avoids the reduced killing effect caused by the short contact time between the mixed microbial agent and asparagus root powder and the root-knot nematodes.

[0015] Furthermore, the quality of the raw materials for the root tip mimicry control granules is as follows:

[0016] Asparagus root powder: flax bast mix: 1,4-butanediamine: spermine: gelatin: oxidized sodium alginate in the form of (2-4): (100-200): (1-2): (0.2-0.4): (10-20): (5-10).

[0017] Furthermore, the preparation method of the root tip mimicry control granules is as follows:

[0018] (1) Mix the flax bast mixture with water to make a 50-60% flax bast mixture solution, add asparagus root powder and stir evenly, let stand for 10-12 h, then add spermine and stir evenly to obtain the mixture.

[0019] (2) After preparing a 5wt% sodium alginate solution with water, add the mixture and dissolved gelatin and stir evenly. Mix 1,4-butanediamine with water, adjust the pH to 6-6.5 to prepare a 30wt% 1,4-butanediamine solution and stir and add it. Stir for 5-8 min and then add the mixed bacterial agent. Let stand to obtain the mixed gel.

[0020] (3) After the mixed gel is crushed into granules, it is dried at low temperature to obtain root tip mimicry control granules.

[0021] The control agent of this invention belongs to the gel particle type, using gelatin and sodium alginate oxide as the main gelling substances. Gelatin and sodium alginate oxide have a certain water absorption capacity, but the water absorption rate is moderate, which can prevent the root tip simulated control particles from dying due to excessive water absorption and the internal fungal agent being in a high water content environment. The flax bast mixture is made from young flax stems and contains a large amount of bast fiber, gum, and other substances. Mature flax stems have excessively strong bast fiber and lower gum content. The resulting root tip simulated control particles are too strong, making it difficult for nematodes to penetrate. At the same time, the bast fiber layers of young flax stems contain a large amount of gum with L-galactose side chains. During metabolism, this gum produces signaling molecules that can synergistically enhance the attraction to tobacco root-knot nematodes with 1,4-butanediamine, thereby attracting root-knot nematodes to penetrate into the gel particles for precise killing.

[0022] Controlling root-knot nematodes is a continuous process. Throughout the tobacco planting process, root-knot nematodes continuously hatch and form new nematodes. Therefore, the root tip mimicry control granules prepared in this invention also need to possess the characteristic of continuously attracting root-knot nematodes, causing them to mistakenly identify tobacco roots. Although the gum in flax fiber can attract root-knot nematodes, as the inoculant multiplies, the gum polysaccharide is consumed first. At this time, the released 1,4-butanediamine can further attract root-knot nematodes. After oxidative modification, sodium alginate contains aldehyde groups, which can react with the amino group of 1,4-butanediamine to form an imine bond. The imine bond hydrolyzes under weakly acidic conditions, releasing 1,4-butanediamine to continue attracting nematodes into the root tip mimicry control granules. As tobacco grows, the soil is generally weakly acidic, and its roots secrete organic acids. At this time, the pH of the soil around the tobacco plant roots is weakly acidic, which is conducive to the occurrence of hydrolysis reactions. In addition, the dried flax bast mixture and gelatin and sodium alginate are prone to cracking and separation due to their different chemical properties, while 1,4-butanediamine has certain cross-linking properties, which can improve the stability of gel particles.

[0023] Furthermore, the preparation method of the flax bast mixture is as follows:

[0024] A. Select tender flax stems, crush them, steam them for 5-8 minutes, cool them to room temperature, add water and cellulase, adjust the pH of the solution to 5-5.5, stir and treat at 40-45℃ for 10-12 hours, then heat to 90℃ for 1-2 minutes and remove them to obtain pretreated flax stems.

[0025] B. Mix the pretreated flax stems with a 0.5 wt% sodium hexametaphosphate aqueous solution and stir for 5-6 h. Then add a 5 wt% calcium chloride solution and continue stirring for 2-3 h. Add ammonium nitrate and continue stirring for 1-2 h. Remove the mixture and air dry it at low temperature to obtain a flax bast mixture.

[0026] Young flax stems contain bast fibers and a large amount of gum. When preparing control gel granules, it is necessary to disperse the fibers in the flax stems, which can serve as both a supporting structure for the control gel and a biomimetic structure mimicking root tips. The gum in flax stems contains L-galactose side chains that can attract nematodes; therefore, it is crucial to retain it as much as possible during treatment, controlling the concentration of cellulase and reaction time to disperse the fibers without excessive fiber degradation. To maximize the retention of gum in flax roots and stems, and to promote the growth of Paecilomyces lilacinus using a nitrogen source, this invention pre-treats flax stems by soaking them in sodium hexametaphosphate to chelate calcium ions. Then, the calcium ions adsorb ammonium ions and fix the gum, achieving the purpose of bridging calcium ions, ammonium ions, and flax stem gum with sodium hexametaphosphate. Simultaneously, this retains as much ammonium as possible within the root tip, mimicking the control granules and promoting the growth of the fungal agent.

[0027] Furthermore, in step B, the mass ratio of pretreated flax stems to sodium hexametaphosphate, calcium chloride, and ammonium nitrate is 1:0.5:0.05:0.2.

[0028] Furthermore, the cellulase added to the water in A is added at a rate of 10 U / g of young flax stems.

[0029] Furthermore, the asparagus root powder is obtained by pulverizing the dried asparagus rhizomes.

[0030] Furthermore, the oxidation degree of the oxidized sodium alginate is 30-35%.

[0031] The beneficial effects of this invention are as follows:

[0032] The root tip mimicking control granules prepared in this invention can simulate tobacco root tips, causing nematodes to mistakenly identify their parasitic location. This allows for precise and efficient killing of nematodes using biological agents and natural plant nematicides, reducing nematode numbers, inhibiting nematode reproduction, eliminating the impact of root-knot nematodes on tobacco growth, and improving tobacco planting efficiency. Simultaneously, this invention reduces the occurrence of tobacco root-knot nematodes by combining disinfection and land preparation with biological control, reducing the use of chemical agents and achieving the goal of green agricultural development. Detailed Implementation

[0033] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can understand other advantages and effects of the present invention from the content described in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used in the embodiments of this invention are commercially available.

[0035] The sodium alginate used in this invention is obtained by oxidizing sodium alginate solution with sodium periodate in the dark. Sodium alginate with an oxidation degree of 30-35% is acceptable; the sodium alginate used in the embodiments and comparative examples of this invention has an oxidation degree of 35%. Asparagus root powder is obtained by drying the asparagus rhizomes, pulverizing them into powder, and then passing them through a 200-mesh sieve.

[0036] Example 1: Preparation of root tip mimicry control granules

[0037] The preparation method of the flax bast mixture is as follows:

[0038] A. Select tender flax stems, crush them into 1-1.5 cm pieces, steam them for 5 minutes, cool them to room temperature, add water until the flax stems are submerged, add cellulase at a rate of 10 U per gram of tender flax stems, adjust the pH of the solution to 5, stir and treat at 40℃ for 12 hours, then heat to 90℃ for 2 minutes, and remove them to obtain pretreated flax stems.

[0039] B. The pretreated flax stems were mixed with a 0.5 wt% sodium hexametaphosphate aqueous solution and stirred for 5 h. Then, a 5 wt% calcium chloride solution was added and stirred for another 2 h. Ammonium nitrate was added and stirred for another 1 h. The mixture was then removed and air-dried at 35°C for 10 h to obtain a flax bast mixture. The mass ratio of the pretreated flax stems to sodium hexametaphosphate, calcium chloride, and ammonium nitrate was 1:0.5:0.05:0.2.

[0040] Preparation of root tip mimicry control granules:

[0041] (1) Mix 100 g of flax bast mixture with water to make a 50 wt% flax bast mixture, add 2 g of asparagus root powder, stir evenly and let stand for 10 h, then add 0.2 g of spermine, stir evenly and obtain a mixture;

[0042] (2) After preparing a 5wt% sodium alginate solution by mixing 10 g of sodium alginate with water, add the mixture and 5 g of dissolved gelatin, stir evenly, mix 1 g of 1,4-butanediamine with water, adjust the pH to 6 to prepare a 30wt% 1,4-butanediamine solution, stir and add it, stir for 5 min, then add 1 g each of Paecilomyces lilacinus and Bacillus subtilis powder, let stand to obtain a mixed gel;

[0043] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8 cfu / g, effective viable Bacillus subtilis count in root tip mimicry control granules ≥2×10 8 cfu / g.

[0044] Example 2: Preparation of root tip mimicry control granules

[0045] The preparation method of the flax bast mixture is as follows:

[0046] A. Select tender flax stems, crush them into 1-1.5 cm pieces, steam them for 8 minutes, cool them to room temperature, add water until the flax stems are submerged, add 10 U of cellulase per gram of tender flax stems, adjust the pH of the solution to 5.5, stir and treat at 45℃ for 10 hours, then heat to 90℃ for 2 minutes and remove to obtain pretreated flax stems.

[0047] B. The pretreated flax stems were mixed with a 0.5 wt% sodium hexametaphosphate aqueous solution and stirred for 6 h. Then, a 5 wt% calcium chloride solution was added and stirred for another 3 h. Ammonium nitrate was added and stirred for another 2 h. The mixture was then removed and air-dried at 35°C for 10 h to obtain a flax bast mixture. The mass ratio of the pretreated flax stems to sodium hexametaphosphate, calcium chloride, and ammonium nitrate was 1:0.5:0.05:0.2.

[0048] Preparation of root tip mimicry control granules:

[0049] (1) Mix 200 g of flax bast mixture with water to make a 60 wt% flax bast mixture, add 4 g of asparagus root powder, stir evenly and let stand for 12 h, then add 0.4 g of spermine, stir evenly and obtain a mixture;

[0050] (2) After preparing a 5wt% sodium alginate solution by mixing 20 g of sodium alginate with water, add the mixture and 10 g of dissolved gelatin, stir evenly, mix 2 g of 1,4-butanediamine with water, adjust the pH to 6.5 to prepare a 30wt% 1,4-butanediamine solution, stir and add it, stir for 8 min, then add 1.5 g each of Paecilomyces lilacinus and Bacillus subtilis powder, let stand to obtain a mixed gel;

[0051] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g.

[0052] Example 3:

[0053] (1) Land preparation: After applying organic fertilizer to the field soil, plow it. The amount of organic fertilizer used is about 1000 kg / mu. Then, use calcium cyanamide for disinfection, deep plow and sun-dry the soil. The amount of calcium cyanamide used is about 20 kg / mu. Then cover the soil with film and keep it for more than 28 days.

[0054] (2) Transplanting: Before transplanting, apply the prepared root tip mimicry control granules to the field at a rate of 15-30 kg / hm. 2 After applying the fertilizer for 2-3 days, transplant the disease-resistant tobacco seedlings at a planting density of 1200-1300 plants per acre. Water thoroughly to settle the roots and maintain soil moisture content at 70-80% for 10 days after transplanting.

[0055] (3) Management: Apply root tip mimicry control granules during the tobacco seedling stage and vigorous growth stage; the application rate of root tip mimicry control granules during the seedling stage is 20-40 kg / hm. 2 Apply once during the crowning stage, maintaining soil moisture content at 50-60%. During the vigorous growth stage, the application rate is 10-15 kg / hm². 2 During the vigorous growth period, apply only once and keep the soil moisture content at 20-30%. Nematode control agents are applied to the roots of tobacco plants by digging holes 20-40 cm deep and covering them with soil after application.

[0056] Comparative Example 1:

[0057] This comparative example is compared with the root tip mimicry control granules prepared in Example 1, the difference being that this comparative example does not use a flax bast mixture for preparation. The specific preparation method is as follows:

[0058] (1) Mix 2 g of Asparagus root powder with 0.2 g of spermine and stir until homogeneous to obtain a mixture;

[0059] (2) After preparing a 5wt% sodium alginate solution by mixing 10 g of sodium alginate with water, add the mixture and 5 g of dissolved gelatin, stir evenly, mix 1 g of 1,4-butanediamine with water, adjust the pH to 6 to prepare a 30wt% 1,4-butanediamine solution, stir and add it, stir for 5 min, then add 1 g each of Paecilomyces lilacinus and Bacillus subtilis powder, let stand to obtain a mixed gel;

[0060] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8cfu / g, effective viable Bacillus subtilis count in root tip mimicry control granules ≥2×10 8 cfu / g.

[0061] Comparative Example 2:

[0062] This comparative example is compared with the root tip mimicry control granules prepared in Example 1. The difference lies in the preparation method of the flax bast mixture in this comparative example. The specific preparation method is as follows:

[0063] The preparation method of the flax bast mixture is as follows:

[0064] A. Select tender flax stems, crush them into 1-1.5 cm pieces, steam them for 5 minutes, cool them to room temperature, add water (enough to cover the flax stems), add cellulase at a rate of 10 U per gram of tender flax stems, adjust the pH of the solution to 5, stir and treat at 40℃ for 12 hours, then raise the temperature to 90℃ and treat for 2 minutes before removing the mixture to obtain a flax bast mixture.

[0065] The preparation of root tip mimicry control granules was the same as in Example 1.

[0066] Comparative Example 3:

[0067] This comparative example is compared with the root tip mimicry control granules prepared in Example 1. The difference lies in the preparation method of the flax bast mixture in this comparative example. The specific preparation method is as follows:

[0068] The preparation method of the flax bast mixture is as follows:

[0069] A. Select tender flax stems, crush them into 1-1.5 cm pieces, steam them for 5 minutes, cool them to room temperature, add water until the flax stems are submerged, add cellulase at a rate of 10 U per gram of tender flax stems, adjust the pH of the solution to 5, stir and treat at 40℃ for 12 hours, then heat to 90℃ for 2 minutes, and remove them to obtain pretreated flax stems.

[0070] B. After soaking the pretreated flax stems in a mixture of 0.5 wt% ammonium nitrate for 5 h, remove them and air dry them at 35℃ for 10 h to obtain a flax bast mixture, wherein the mass ratio of the pretreated flax stems to ammonium nitrate is 1:0.2.

[0071] The preparation of root tip mimicry control granules was the same as in Example 1.

[0072] Comparative Example 4:

[0073] This comparative example is compared with the root tip mimicry control granules prepared in Example 1. The difference is that the flax bast mixture in this comparative example is prepared using mature flax stems and roots. The specific preparation method of the flax bast mixture is as follows:

[0074] A. Select mature flax stems, crush them into 1-1.5 cm pieces, steam them for 5 minutes, cool them to room temperature, add water (enough to cover the flax stems), add 10 U of cellulase per gram of young flax stems, adjust the pH of the solution to 5, stir and treat at 40℃ for 12 hours, then heat to 90℃ for 2 minutes, and remove them to obtain pretreated flax stems.

[0075] B. The pretreated flax stems were mixed with a 0.5 wt% sodium hexametaphosphate aqueous solution and stirred for 5 h. Then, a 5 wt% calcium chloride solution was added and stirred for another 2 h. Ammonium nitrate was added and stirred for another 1 h. The mixture was then removed and air-dried at 35°C for 10 h to obtain a flax bast mixture. The mass ratio of the pretreated flax stems to sodium hexametaphosphate, calcium chloride, and ammonium nitrate was 1:0.5:0.05:0.2.

[0076] The preparation method for root tip mimicry control granules is the same.

[0077] Comparative Example 5:

[0078] This comparative example is compared with the root tip mimicry control granules prepared in Example 1, the difference being that the flax bast mixture in this comparative example is replaced with flaxseed gum. The specific preparation method is as follows:

[0079] (1) Mix 100 g of flaxseed gum with water to make a 50 wt% flaxseed gum solution, add 2 g of asparagus root powder, stir evenly and let stand for 10 h, then add 0.2 g of spermine, stir evenly and obtain a mixture;

[0080] (2) After preparing a 5wt% sodium alginate solution by mixing 10 g of sodium alginate with water, add the mixture and 5 g of dissolved gelatin, stir evenly, mix 1 g of 1,4-butanediamine with water, adjust the pH to 6 to prepare a 30wt% 1,4-butanediamine solution, stir and add it, stir for 5 min, then add 1 g each of Paecilomyces lilacinus and Bacillus subtilis powder, let stand to obtain a mixed gel;

[0081] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8 cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g.

[0082] Comparative Example 6:

[0083] This comparative example is compared with the root tip mimicry control granules prepared in Example 1, the difference being that this comparative example does not contain 1,4-butanediamine. The specific preparation method is as follows:

[0084] (1) Mix 100 g of flax bast mixture with water to make a 50 wt% flax bast mixture, add 2 g of asparagus root powder, stir evenly and let stand for 10 h, then add 0.2 g of spermine, stir evenly and obtain a mixture;

[0085] (2) After preparing a 5wt% sodium alginate solution by mixing 10 g of sodium alginate with water, add the mixture and 5 g of dissolved gelatin, stir evenly, and then add 1 g each of Paecilomyces lilacinus and Bacillus subtilis powder, and let stand to obtain a mixed gel.

[0086] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8 cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g.

[0087] The preparation method of the flax bast mixture is the same as that in Example 1.

[0088] Comparative Example 7:

[0089] This comparative example is compared with the root tip mimicry control granules prepared in Example 1, the difference being that this comparative example does not contain sodium alginate oxide. The specific preparation method is as follows:

[0090] (1) Mix 100 g of flax bast mixture with water to make a 50 wt% flax bast mixture, add 2 g of asparagus root powder, stir evenly and let stand for 10 h, then add 0.2 g of spermine, stir evenly and obtain a mixture;

[0091] (2) Mix 1 g of 1,4-butanediamine with water, adjust the pH to 6 to make a 30 wt% 1,4-butanediamine solution, add it to 10 g of dissolved gelatin and stir evenly. After stirring for 5 min, add 1 g each of Paecilomyces lilacinus and Bacillus subtilis powder and let stand to obtain a mixed gel.

[0092] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g.

[0093] The preparation method of the flax bast mixture is the same as that in Example 1.

[0094] Comparative Example 8:

[0095] This comparative example is compared with the root tip mimicry control granules prepared in Example 1, the difference being that this comparative example does not contain gelatin. The specific preparation method is as follows:

[0096] (1) Mix 100 g of flax bast mixture with water to make a 50 wt% flax bast mixture, add 2 g of asparagus root powder, stir evenly and let stand for 10 h, then add 0.2 g of spermine, stir evenly and obtain a mixture;

[0097] (2) After preparing a 5wt% sodium alginate solution by mixing 10 g of sodium alginate with water, add it to the mixture and stir evenly. Mix 1 g of 1,4-butanediamine with water, adjust the pH to 6 to prepare a 30wt% 1,4-butanediamine solution, stir and add it. After stirring for 5 min, add 1 g each of Paecilomyces lilacinus and Bacillus subtilis powder, and let stand to obtain a mixed gel.

[0098] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8 cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g.

[0099] The preparation method of the flax bast mixture is the same as that in Example 1.

[0100] Comparative Example 9:

[0101] This comparative example is compared with the root tip mimicry control granules prepared in Example 1, the difference being that this comparative example does not contain asparagus root powder. The specific preparation method is as follows:

[0102] (1) Mix 100 g of flax bast mixture with water to prepare a 50 wt% flax bast mixture solution, let it stand for 10 h, then add 0.2 g of spermine, stir until homogeneous to obtain the mixture;

[0103] (2) After preparing a 5wt% sodium alginate solution by mixing 10 g of sodium alginate with water, add the mixture and 5 g of dissolved gelatin, stir evenly, mix 1 g of 1,4-butanediamine with water, adjust the pH to 6 to prepare a 30wt% 1,4-butanediamine solution, stir and add it, stir for 5 min, then add 1 g each of Paecilomyces lilacinus and Bacillus subtilis powder, let stand to obtain a mixed gel;

[0104] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8 cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g.

[0105] The preparation method of the flax bast mixture is the same as that in Example 1.

[0106] Comparative Example 10:

[0107] This comparative example is compared with the root tip mimicry control granules prepared in Example 1. The difference is that this comparative example adds conventional sodium alginate. The specific preparation method is as follows:

[0108] (1) Mix 100 g of flax bast mixture with water to make a 50 wt% flax bast mixture, add 2 g of asparagus root powder, stir evenly and let stand for 10 h, then add 0.2 g of spermine, stir evenly and obtain a mixture;

[0109] (2) After preparing a 5wt% sodium alginate solution by mixing 10 g of sodium alginate with water, add the mixture and 5 g of dissolved gelatin, stir evenly, mix 1 g of 1,4-butanediamine with water, adjust the pH to 6 to prepare a 30wt% 1,4-butanediamine solution, stir and add it, stir for 5 min, then add 1 g each of Paecilomyces lilacinus and Bacillus subtilis powder, let stand to obtain a mixed gel;

[0110] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8 cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g.

[0111] The preparation method of the flax bast mixture is the same as that in Example 1.

[0112] Comparative Example 11:

[0113] This comparative example is compared with the root tip mimicry control granules prepared in Example 1. The difference is that the mass of sodium alginate oxide and 1,4-butanediamine added in this comparative example is modified to 10 g and 2 g, respectively. The specific preparation method is as follows:

[0114] (1) Mix 100 g of flax bast mixture with water to make a 50 wt% flax bast mixture, add 2 g of asparagus root powder, stir evenly and let stand for 10 h, then add 0.2 g of spermine, stir evenly and obtain a mixture;

[0115] (2) After preparing a 5wt% sodium alginate solution by mixing 10 g of sodium alginate with water, add the mixture and 5 g of dissolved gelatin, stir evenly, mix 2 g of 1,4-butanediamine with water, adjust the pH to 6 to prepare a 30wt% 1,4-butanediamine solution, stir and add it, stir for 5 min, then add 1 g each of Paecilomyces lilacinus and Bacillus subtilis powder, let stand to obtain a mixed gel;

[0116] (3) After the mixed gel was crushed into granules, it was dried at 30℃ for 24 h to obtain root tip mimicry control granules. The final root tip mimicry control granules contained ≥1×10 effective viable bacteria of Paecilomyces lilacinus. 8 cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g.

[0117] The preparation method of the flax bast mixture is the same as that in Example 1.

[0118] Comparative Example 12:

[0119] This comparative example is compared with the root tip mimicry control granules prepared in Example 1. The difference is that the oxidation degree of sodium alginate added in this comparative example is 50%, while the rest of the preparation methods are the same.

[0120] Experiment: Indoor Nematode Control Experiment

[0121] 1. The test nematodes were second-instar larvae of the southern root-knot nematode, prepared into a suspension of 500 larvae / mL for later use; the tobacco variety used was Yunyan 87. Samples were root tip mimicking control granules prepared in Example 1 and Comparative Examples 1-12, respectively. The effective viable count of Paecilomyces lilacinus in all samples was approximately 1×10⁻⁶. 8 cfu / g, Bacillus subtilis effective viable count is approximately 2×10⁻⁶. 8 cfu / g.

[0122] 2. Potted plant nematode control experiment: Soil samples were taken from the Yunyan 87 planting field of a farmer, with a depth of 10-40 cm. The soil was passed through a 1 cm mesh sieve, and the river sand was passed through a 3.5 mm mesh sieve. The soil and river sand were mixed evenly in a ratio of 2:1, sterilized with high-temperature steam for 24 hours, and then transferred to a ventilated place to air dry for later use.

[0123] Four-week-old, uniformly growing Yunyan 87 seedlings were planted in seedling trays. Experimental group 1 used root tip mimicking control granules prepared in Example 1, while control groups 1-12 used root tip mimicking control granules prepared in Comparative Examples 1-12 respectively. The blank control was water. Each group contained 10 seedlings. After transplanting, the seedlings were further cultivated, and the tobacco root-knot nematode disease disease index and agronomic traits were investigated and detected.

[0124] The experimental cultivation process is as follows:

[0125] Transplanting: Before transplanting, apply 2 g of the prepared root tip mimicry control granules to the pot (1 / 2 depth), water thoroughly, and maintain soil moisture content at 70% for 10 days after transplanting; after transplanting, distribute nematode suspension around the tobacco plant, inoculating a total of about 1000 nematodes at a depth of 3 cm.

[0126] Management: Apply root tip mimicry control granules during the mid-stage of the tobacco crowning stage and the mid-stage of the vigorous growth stage. When using root tip mimicry control granules, apply 2.5 g / pot during the crowning stage, once during the crowning stage, and maintain a soil moisture content of 50%. When using root tip mimicry control granules, apply 1 g / pot, once during the vigorous growth stage, and maintain a soil moisture content of 20%. Nematode control agents are applied to the roots of the tobacco plants. When applying, dig a pit around the roots and fill it with the granules to a depth of 1 / 2 the height of the pot.

[0127] Control results: 60 days after tobacco transplanting, 3 plants from each group were randomly selected to count the nematode resistance. The plants were pulled out with soil, washed with clean water and dried. The number of root knots and eggs on the roots was counted, and the mean incidence rate and reproduction coefficient of root-knot nematodes were calculated. For mature tobacco plants, 3 plants from each group were randomly selected to count the plant height and number of effective leaves.

[0128] Table 1. Score Standards for Root-knot Nematode Infection Levels

[0129]

[0130] Mean disease incidence score = (sum of (number of diseased plants at each level × score)) / total number of plants;

[0131] Reproduction coefficient = number of eggs per plant / initial inoculation amount per plant;

[0132] The results are shown in Table 2:

[0133] Table 2

[0134]

[0135] Analysis of the data in Table 2 shows that:

[0136] 1. A reproduction coefficient > 1 is generally considered to be poor control effect. The root tip simulated control granules prepared by Example 1 of this invention in Experimental Group 1 can effectively kill nematodes and reduce the number of nematode eggs. At the same time, the fewer root knots on the tobacco roots will not inhibit the growth of tobacco plants. The plant height is 21 cm higher than the blank control, and the number of effective leaves is also significantly more than the blank control.

[0137] 2. In control groups 1-5, the control granules used were all replacements of the raw material flax fiber mixture. Data shows that without the flax fiber mixture, the control effect was significantly reduced, the nematode reproduction rate was significantly increased, the number of root knots was also greater, and tobacco growth was less than ideal. This is because flax fiber not only provides structural support in the control granules but also contains a large amount of mucilage secretion signaling molecules that attract nematodes, thus mimicking the biomimetic structure of root tips. However, when mature flax stems were used, the fiber structure was denser, the mucilage content was lower, making fiber dispersion difficult and mucilage harder to dissolve during pretreatment, resulting in poor attraction for nematodes and difficulty in burrowing. If the signaling molecules produced by the decomposition of flaxseed gum were used directly, the intensity would be too high, actually reducing the attraction for nematodes and resulting in an unsatisfactory insecticidal effect. Therefore, considering all factors, using young flax roots and stems to prepare the flax fiber mixture yielded the best overall effect. Treatment of flax roots and stems is also an essential step. Enzyme treatment alone can only disperse the fibers but is not conducive to the retention of the gum. Sodium hexametaphosphate is used to bridge the gum by chelating calcium ions and grafting ammonium ions. Nitrogen is used to promote the growth and reproduction of the microbial agent, while avoiding excessive loss of nitrogen source to the outside of the control gel.

[0138] 3. The control group 6-12 compares the raw materials of the control gel. 1,4-Butanediamine can attract nematode molecules, but high concentrations of 1,4-Butanediamine reduce its attractiveness to nematodes, thus reducing the control effect. Without the addition of 1,4-Butanediamine, the attraction of the flax fiber mixture alone is insufficient in duration, making it difficult to maintain a long-lasting effect and leading to more root knots. The aldehyde group of oxidized sodium alginate can react with the amino group of 1,4-Butanediamine to form an imine bond. In the soil environment, the imine bond hydrolyzes to release 1,4-Butanediamine. However, ordinary sodium alginate does not hydrolyze after cross-linking with 1,4-Butanediamine, resulting in insufficient release of 1,4-Butanediamine, affecting its attractiveness to nematodes and reducing the control effect. Gelatin can regulate the water absorption rate of the final control granules. A high water absorption rate hinders the reproduction of the fungicide. The degree of substitution of oxidized sodium alginate affects the number of aldehyde groups, and after reacting with gelatin, it affects the mechanical properties of the control granules, hindering nematode penetration.

[0139] experiment:

[0140] The control experiment was conducted from April to July 2024 in a tobacco field in Xinyan Village, Lixi Town, Huili City, Liangshan Prefecture, Sichuan Province. The experimental site, at an altitude of 1780 m, had been continuously planted with tobacco for five years and was a severely affected area with a history of persistent disease. Healthy tobacco seedlings of the Yunyan 87 variety, showing no symptoms of root-knot nematode infestation or other diseases and exhibiting uniform growth, were selected and transplanted in April. Normal fertilization and management were implemented, with a row spacing of 1.2 m × 0.5 m and each plot measuring 63 m. 2 Three plots were treated as one treatment, with five treatments per group. No chemical agents were applied to the entire experimental plot to control tobacco root-knot nematodes during the experimental year.

[0141] The experimental group used the root-knot nematode control granules prepared in Example 1; the control group 1 used 10% thiazophos GR; the blank control was treated with water, 2 L per plant.

[0142] The specific prevention and control process is as follows:

[0143] (1) Land preparation: After applying organic fertilizer to the field soil, plow it. The amount of organic fertilizer used is 1000 kg / mu. Then, use calcium cyanamide for disinfection, deep plow and sun-dry the soil. The amount of calcium cyanamide used is about 20 kg / mu. Then cover the soil with film and let it sit for 30 days.

[0144] (2) Transplanting: Seven days after removing the film, apply the prepared root tip mimicking control granules / thiazophos / water to the field. The dosage of root tip mimicking control granules / 10% thiazophos is 30 kg / hm. 2 Transplant disease-resistant tobacco seedlings 3 days later, water them thoroughly, and maintain soil moisture content at 70-80% for 10 days after transplanting.

[0145] (3) Management: Apply root tip mimicry control granules / thiazophos / water during the mid-stage of tobacco seedling emergence and mid-stage of vigorous growth; the application rate during the seedling emergence stage is 40 kg / hm. 2 Apply once during the crowning stage, maintaining soil moisture content at 50-60%. During the vigorous growth stage, the application rate is 15 kg / hm². 2 During the vigorous growth period, it should only be applied once, and the soil moisture content should be maintained at 20-30%. Root tip mimicry control granules / thiazophos are applied to the tobacco roots. When applying, dig holes 30 cm deep and cover with soil after application.

[0146] Ninety days after transplanting, the incidence rate of root-knot nematodes and the control effect were statistically analyzed. The incidence rate of root-knot nematodes is referenced in Table 1. The control effect is calculated as (control disease index - treatment disease index) / control disease index × 100%. The results are shown in Table 3.

[0147] Table 3 Field control experiment

[0148]

[0149] As shown in Table 3, the root tip mimicry control granules of the present invention have a good control effect and are superior to existing chemical control agents.

[0150] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for controlling tobacco nematodes, characterized in that, The prevention and control methods are as follows: (1) Land preparation: After applying organic fertilizer to the soil, plow it, disinfect it with lime nitrogen, deep plow and sun-dry the soil, and then cover it with film and let it sit for more than 28 days; (2) Transplanting: Apply root tip mimicry control granules to the field before transplanting at a rate of 15-30 kg / hm. 2 Transplant tobacco seedlings 2-3 days after application, water them thoroughly, and maintain soil moisture content at 70-80% for 10 days after transplanting. (3) Management: Apply root tip mimicry control granules during the tobacco seedling stage and vigorous growth stage; the application rate of root tip mimicry control granules during the seedling stage is 20-40 kg / hm. 2 The long-term usage rate in Wangchang is 10-15 kg / hm. 2 The root tip mimicry control granules are used on the roots of tobacco plants, with an application depth of 20-40 cm. The root tip simulation control granules contain *Paecilomyces lilacinus* and *Bacillus subtilis*; the effective viable count of *Paecilomyces lilacinus* in the root tip simulation control granules is ≥1×10⁻⁶. 8 cfu / g, the effective viable count of Bacillus subtilis in the root tip mimicry control granules is ≥2×10⁻⁶. 8 cfu / g; In addition to the fungicide, the root tip mimicry control granules also contain the following raw materials in the following mass ratios: Asparagus root powder: flax bast mix: 1,4-butanediamine: spermine: gelatin: oxidized sodium alginate in the form of (2-4): (100-200): (1-2): (0.2-0.4): (10-20): (5-10); The preparation method of the root tip mimicry control granules is as follows: (I) Mix the flax bast mixture with water to make a 50-60% flax bast mixture solution, add asparagus root powder, stir evenly and let stand for 10-12 h, then add spermine, stir evenly and get the mixture. (II) After preparing a 5wt% sodium alginate solution with water, add the mixture and dissolved gelatin and stir evenly. Mix 1,4-butanediamine with water, adjust the pH to 6-6.5 to prepare a 30wt% 1,4-butanediamine solution and add it with stirring. After stirring for 5-8 min, add the mixed bacterial agent of Paecilomyces lilacinus and Bacillus subtilis and let it stand to obtain a mixed gel. (III) After the mixed gel is crushed into granules, it is dried at low temperature to obtain root tip mimicry control granules; The preparation method of the flax bast mixture is as follows: A. Select tender flax stems, crush them, steam them for 5-8 minutes, cool them to room temperature, add water and cellulase, adjust the pH of the solution to 5-5.5, stir and treat at 40-45℃ for 10-12 hours, then heat to 90℃ for 1-2 minutes and remove them to obtain pretreated flax stems. B. Mix the pretreated flax stems with a 0.5 wt% sodium hexametaphosphate aqueous solution and stir for 5-6 h. Then add a 5 wt% calcium chloride solution made from calcium chloride and continue stirring for 2-3 h. Add ammonium nitrate and continue stirring for 1-2 h. Remove the mixture and air dry it at low temperature to obtain the flax bast mixture.

2. The method for controlling tobacco nematodes according to claim 1, characterized in that, Maintain soil moisture content at 50-60% during the tobacco bushing stage and at 20-30% during the vigorous growth stage.

3. The method for controlling tobacco nematodes according to claim 2, characterized in that, In step B, the mass ratio of pretreated flax stems to sodium hexametaphosphate, calcium chloride, and ammonium nitrate is 1:0.5:0.05:0.

2.

4. The method for controlling tobacco nematodes according to claim 3, characterized in that, In step A, the cellulase added to the water was added at a rate of 10 U / g of young flax stems.

5. The method for controlling tobacco nematodes according to claim 4, characterized in that, The oxidation degree of the oxidized sodium alginate is 30-35%.

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