Nematode killing fungus balloon agent and application thereof in prevention and treatment of pine wood nematode disease

Abstract

The invention discloses a nematode killing fungus balloon agent and application thereof in prevention and treatment of pine wood nematode diseases, and belongs to the technical field of microbial prevention and treatment. The solid microorganism balloon agent is prepared by taking nematicidal ilobster fungi as a functional microorganism and matching with a carrier material, an additive component and an active component. The shelf life of the nematicidal ivani fungal spores under the normal temperature condition is prolonged to 3-6 months or above from 2-3 days of traditional spore liquid, the shelf life can reach 12 months under the 4 DEG C refrigeration condition, and the problem that low-temperature storage in mountainous areas is inconvenient is solved. The micro-capsule components form a slow-release protective layer, so that activity loss caused by environmental factors such as high temperature and drought is avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial control, and particularly relates to a nematicidal fungal spherical agent and application thereof in preventing and controlling pine wood nematode disease. Background Art

[0002] Pine wilt disease is a dangerous disease caused by the pine wood nematode (Bursaphelenchusxylophilus) that causes rapid withering and death of pine trees, and even large-scale destruction. Due to its serious damage and difficulty in prevention and control, it has been highly valued by countries around the world and is listed as an important dangerous forest disease.

[0003] The prevention and control of pine wood nematode disease has become a global problem, but there is currently a lack of effective prevention and control methods. The measures generally taken in the world are to cut off the transmission source and transmission route to prevent the spread of pine wood nematodes. The methods currently used include felling diseased trees to cut off the transmission source; using traps to kill pine brown beetles, spraying chemicals such as thiacloprid and cypermethrin emulsion from the air and on the ground to control longhorn beetles, using predatory or parasitic natural enemies of longhorn beetles such as Scleroderma spp., Dastarcus longulus, Allecula fuliginosa and woodpeckers (Dendrocops spp.) to control longhorn beetles, and using fungi such as Beauveria bassiana to control longhorn beetles; injecting chemical agents such as avermectin, nematode nematodes, and plant extracts such as alopecurine into tree trunks to control pine wood nematodes; hybridizing Masson pine, loblolly pine and Japanese black pine to breed disease-resistant varieties, etc. Due to the hidden nature of the damage caused by pine brown alternating beetles, chemical control is generally difficult to be effective, and it will cause environmental pollution and toxic side effects to non-target organisms. At the same time, due to the limitations of control methods such as trunk injection, it has become an urgent need to develop a control method with high control efficiency, strong operability and green environmental protection. Summary of the invention

[0004] The present invention aims at the problems existing in the prior art. The present invention uses the nematicidal fungus of I. erythroxylum bungeana as the functional microorganism and compound a variety of auxiliary ingredients and plant extract ingredients. On the one hand, it solves the problem of poor storage stability of functional microbial spores and greatly extends the shelf life. On the other hand, it cooperates with various active ingredients to work together to greatly improve the prevention and control effect.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is: A nematode-killing fungal spherical preparation comprises the following raw materials: nematode-killing fungal spore liquid, carrier materials, additive components and active components.

[0006] Furthermore, the spore content of the nematode-killing fungus spore liquid is ≥3×108 CFU / mL. The nematode-killing fungus of the present invention can use commercially available strains or strains in the patent literature of the prior art.

[0007] Furthermore, the carrier material is a mixed dispersion of sodium alginate and clay, where the mass concentration of sodium alginate is 4% and the mass concentration of clay is 10%.

[0008] Furthermore, the additive components include potassium penicillin G, streptomycin, and humic acid.

[0009] Furthermore, the active component is an extract of seabuckthorn pomace, and the specific preparation method is as follows: (1) Wash the residue after squeezing the seabuckthorn fruit with clean water, remove the remaining juice and seed fragments, drain the water, spread it flat on a tray, dry it at 40 - 50 °C with hot air until the water content ≤ 8%, crush it through a 40-mesh sieve, and store it in a sealed and light-proof manner for later use; (2) Degreasing treatment: Mix the seabuckthorn pomace after drying and crushing in step (1) with petroleum ether at a material-liquid ratio of 1 g:8 mL, perform ultrasonic-assisted extraction at 40 - 45 °C for 30 - 40 minutes, repeat 3 times; centrifuge to remove the petroleum ether layer containing grease; dry the solid obtained by centrifugation at 50 - 60 °C under ventilation to obtain degreased pomace; (3) Mix the degreased pomace with an ethanol solution with a mass concentration of 60% at a material-liquid ratio of 1 g:20 mL, magnetically stir and premix for 5 minutes, then treat it in a constant temperature water bath at 50 - 55 °C for 30 - 45 min, stir once every 10 minutes, perform centrifugation treatment, collect the supernatant 1, filter the residue again in a constant temperature water bath at 50 - 55 °C for 30 - 45 min, perform centrifugation treatment, collect the supernatant 2, combine the two supernatants, and remove impurities with a microfiltration membrane to obtain a reddish-brown liquid; (4) Rotate and evaporate the liquid, concentrate it to 1 / 10 of the original volume, add 1% of chitosan oligosaccharide based on the volume of the concentrated liquid, adjust the pH of the system to 6 - 7 with sodium citrate, and obtain the extract of seabuckthorn pomace after sterilization treatment.

[0010] A preparation method of a nematode-killing fungus globule of the nematode-killing fungus of the present invention includes the following preparation steps: (1) Prepare the spore liquid of the nematode-killing fungus of the present invention: Inoculate the nematode-killing fungus onto a PDA medium and culture it at 25 °C for 2 weeks, then pick the colony and inoculate it into a PDB medium, culture it on a shaker at 130 revolutions per minute and 25 °C for 2 weeks, filter it with a gauze after culturing, filter out the mycelium, and adjust the concentration to 3×10 8 CFU / mL to obtain the spore liquid of the nematode-killing fungus of the present invention; (2) Mix 500 mL of the nematode-killing fungus spore solution of Isaria and 500 mL of the carrier material evenly, add potassium penicillin G and streptomycin, adjust their concentrations in the system to 100 ppm and 50 ppm, then add 3% of humic acid based on the mass of the mixed solution, and use a stirrer to mix the solution evenly; (3) Prepare the active ingredient; (4) Add the solution from step (2) to 3% of the active ingredient based on the mass of the solution, mix evenly, pour it into a funnel with a micropipette tip, and slowly drip the solution into 3 mol / L calcium chloride solution. The reaction temperature is 4 ± 1 °C, and the reaction time is 30 min, then spherical spore microspheres can be obtained. The diameter of the spore microspheres is about 2 - 3 mm; The micropipette controls the dripping rate (20 - 30 drops / minute); (5) After collecting the microspheres, rinse them with sterilized water (to remove residual CaCl2 and antibiotics), perform vacuum filtration, and then dry them at low temperature (≤40 °C) to obtain the balloon agent.

[0011] Application of an Isaria nematode-killing fungus balloon agent in controlling pine wilt disease.

[0012] The Isaria nematode-killing fungus balloon agent obtained in the present invention has an appearance of white to light yellow spherical particles, with a water activity ≤ 0.6, inhibiting the growth of non-target microorganisms; The survival rate is ≥ 75% at normal temperature for 3 - 6 months.

[0013] There are two methods for using the Isaria nematode-killing fungus balloon agent obtained in the present invention: Spraying in the forest to control pine wilt disease and drilling injection to control pine wilt disease.

[0014] Spraying in the forest to control pine wilt disease: Configure the fungus balloon agent into a dispersion with a spore count of 1×10 8 CFU / mL, and evenly spray it with a stretcher sprayer (the droplet diameter is 200 - 300 μm), dosage: 50 mL / plant.

[0015] Drilling injection to control pine wilt disease: Configure the fungus balloon agent into a dispersion with a spore count of 2×10 8 CFU / mL. At a position 1 - 1.5 meters above the ground of the pine tree, use an electric drill to drill three holes with a diameter of about 1 cm and a depth of 10 - 15 cm, inject the dispersion, dosage: 5 mL / hole.

[0016] Aiming at the following core defects of the existing Isaria nematode-killing fungus preparation in the prior art, the present invention aims to provide an efficient and stable microbial control solution to solve the following problems: 1. Poor storage stability of spores: The existing spore concentrate can only be stored for 2 - 3 days at normal temperature, and the shelf life is only 1 month under refrigerated conditions at 4 °C, which cannot meet the long-term storage requirements in non-cold chain environments such as mountainous areas, resulting in low transportation and application efficiency.

[0017] 2. Limited application scenarios: Liquid formulations rely on low-temperature storage and transportation, making it difficult to meet the large-scale application requirements in complex environments such as mountainous areas and forest areas, which restricts the actual promotion of biological control technologies.

[0018] Objective of the invention: To develop a microsphere formulation to greatly improve the stress resistance of microbial agents and solve the key technical bottlenecks in the storage, transportation, and application of spores of nematode-killing fungi of the genus Hirsutella.

[0019] Beneficial effects: (1) The present invention uses nematode-killing fungi of the genus Hirsutella as functional microorganisms, and prepares a solid microbial balloon agent in combination with a carrier material, an additive component, and an active component. First of all, sodium alginate and clay are used as carrier materials, providing good mechanical strength and slow-release performance, and cooperating with antibiotics to inhibit miscellaneous bacteria, greatly improving the control effect. In addition, the introduction of humic acid further improves the surface characteristics of the microspheres, enhances their adhesion to nematodes and plant tissues, and increases the retention rate of the microspheres on the surfaces of pine tree leaves and bark after field spraying. Field tests show that the survival rate control effect of pine trees sprayed with the microsphere preparation is significantly better than that of chemical agents and traditional biological control methods. A single application can maintain an effective prevention and control period of more than 6 months, reducing the frequency of manual supplementary prevention.

[0020] (2) Secondly, the present invention adds a small amount of seabuckthorn pomace extract as a bioactive substance, using the extract from which the fat-soluble components are removed, which is rich in substances such as polyphenols, pectin polysaccharides, etc. Seabuckthorn polyphenols stabilize the lipid of the spore membrane through hydrogen bonds, improving the freeze-thaw tolerance and storage stability. At the same time, polyphenols inhibit the epidermal peroxidase of nematodes, enhancing the fungal penetration ability. When combined with 0.1% chitosan oligosaccharide, the stability of polyphenols can be enhanced through hydrogen bond binding, doubling the effect. In addition, the addition of pectin polysaccharides forms a water-retaining film, prolonging the survival time of spores.

[0021] (3) Thirdly, the adaptability of the reagent to the environment is significantly enhanced. The microsphere formulation is resistant to high temperature and extrusion. Low-temperature conditions are not required for outdoor storage and transportation in mountainous areas, reducing the logistics cost by more than 60%. The natural sodium alginate and clay components are biodegradable, avoiding chemical residue pollution of the soil and water sources. Strong compatibility: It can be used in combination with prevention and control means such as longhorn beetle trapping and disease-resistant variety cultivation to form an integrated prevention and control system.

[0022] (4) Finally, through the microsphere formulation technology, the shelf life of spores of nematode-killing fungi of the genus Hirsutella at room temperature is extended from 2-3 days of traditional spore liquid to more than 3-6 months, and it is stable for storage under refrigeration conditions at 4°C, solving the problem of inconvenient low-temperature storage in mountainous areas. The microcapsule components form a slow-release protective layer, avoiding activity loss caused by environmental factors such as high temperature and drought. Description of the drawings

[0023] Figure 1Sporulation rates of the fungal mycorrhizal agent of Example 1 of the present invention at different times under low temperature and room temperature conditions; Figure 2 Comparison chart of the killing activity against Bursaphelenchus xylophilus after 6-hour treatment of different treatment groups; Figure 3 Comparison chart of the survival rates of pine trees in different treatment groups after inoculation with Bursaphelenchus xylophilus in Experiment 1; Figure 4 Comparison chart of the survival rates of pine trees in different treatment groups under natural infection in Experiment 2. Detailed implementation manners

[0024] The technical solutions of the present invention will be further described below in conjunction with specific embodiments, but not limited thereto.

[0025] Example 1 A nematode-killing fungal mycorrhizal agent of Isaria includes the following raw materials: Isaria nematode-killing fungal spore liquid, carrier material, additive components, and active components.

[0026] The spore content of the Isaria nematode-killing fungal spore liquid ≥ 3×10 8 CFU / mL. The Isaria nematode-killing fungus of the present invention can use commercially available strains or strains in the prior art patent literature.

[0027] The carrier material is a mixed dispersion of sodium alginate and clay, where the mass concentration of sodium alginate is 4% and the mass concentration of clay is 10%.

[0028] The additive components include potassium penicillin G, streptomycin, and humic acid.

[0029] The active component is an extract of sea buckthorn pomace. The specific preparation method is as follows: (1) The residue after juicing the sea buckthorn fruit is rinsed with clean water to remove residual juice and seed fragments, drained, spread out on a tray, and dried at 40 - 50 °C with hot air until the water content ≤ 8%, crushed through a 40-mesh sieve, and stored in a sealed and light-proof manner for later use; (2) Degreasing treatment: The sea buckthorn pomace after drying and crushing in step (1) is mixed with petroleum ether at a material-liquid ratio of 1 g:8 mL, sonicated and extracted at 40 - 45 °C for 30 minutes, and repeated 3 times; centrifuge to remove the petroleum ether layer containing oil; the solid obtained by centrifugation is dried at 50 - 60 °C under ventilation to obtain defatted pomace; (2) The defatted pomace is mixed with an ethanol solution with a mass concentration of 60% at a material-liquid ratio of 1 g:20 mL, magnetically stirred and premixed for 5 minutes, then treated in a constant temperature water bath at 50 - 55 °C for 30 min, stirred once every 10 minutes, centrifuged, the supernatant 1 is collected, the filter residue is again treated in a constant temperature water bath at 50 - 55 °C for 30 min, centrifuged, the supernatant 2 is collected, the two supernatants are combined, and impurities are removed with a microfiltration membrane to obtain a reddish-brown liquid; (4) Rotate and evaporate the liquid, concentrate it to 1 / 10 of the original volume, add chitosan oligosaccharide accounting for 1% of the volume of the concentrated liquid, adjust the pH of the system to 6 - 7 using sodium citrate, and obtain the extract of sea buckthorn pomace after sterilization treatment.

[0030] A preparation method of an Isaria nematocida fungal balloon agent, comprising the following preparation steps: (1) Prepare the Isaria nematocida fungal spore liquid: Inoculate Isaria nematocida on a PDA medium and culture it at 25°C for 2 weeks. Then pick the colony and inoculate it into a PDB medium, and culture it on a shaker at 130 revolutions per minute and 25°C for 2 weeks. After the culture is completed, filter it with a gauze to filter out the mycelium, and adjust the concentration to 3×108 CFU / mL with sterile water to obtain the Isaria nematocida fungal spore liquid; (2) Take 500 mL of the Isaria nematocida fungal spore liquid and 500 mL of the carrier material, mix them evenly, add potassium penicillin G and streptomycin, adjust their concentrations in the system to 100 ppm and 50 ppm, then add humic acid accounting for 3% of the mass of the mixed solution, and use a stirrer to mix the solution evenly; (3) Prepare the active ingredient; (4) Add the solution from step (2) to the active ingredient accounting for 3% of the mass of the solution, mix evenly, then pour the solution into a funnel with a micropipette tip, and slowly drip the solution into 3 mol / L calcium chloride solution. The reaction temperature is 4 ± 1°C, and the reaction time is 30 min to obtain round spore microspheres with a diameter of about 2 - 3 mm; control the dropping rate of the liquid with a pipette (20 - 30 drops per minute); (5) After collecting the microspheres, rinse them with sterile water (to remove residual CaCl2 and antibiotics), vacuum filter them, and then dry them at a low temperature (≤40°C) to obtain the balloon agent.

[0031] An application of an Isaria nematocida fungal balloon agent in controlling pine wilt disease.

[0032] Conduct a shelf - life test on the Isaria nematocida fungal balloon agent obtained in this example, and check the spore germination rate of the particles every month. The activity of the conidia preparation is determined by measuring the germination ability of conidia immediately after production and after 1, 2, 3, 4, 5, and 6 months. Include normal temperature, refrigerated (4°C), and slightly higher normal temperature (25°C) scenarios to simulate actual storage and transportation conditions. Take 10 random particles and add them to a 15 mL test tube containing 10 mL of 0.1 M sodium citrate, shake at 130 revolutions per minute for 1 hour to fully disperse the spores. Pipette 0.2 mL of the suspension and spread it on a water agar medium, culture it at a constant temperature of 25°C for 2 days, and count the number of colonies formed by the germination of conidia. Taking the number of colonies immediately after production as the reference value, calculate the survival rate at each time point, that is, the spore germination rate = (the number of colonies formed after n months / the initial number of colonies × 100%). All tests are repeated five times to ensure the reliability of the results. The results are asFigure 1 As shown, the fungal balloon agent of the present invention has a stable spore germination rate under low-temperature conditions, and the spore germination rate is not less than 50% under low temperature for six months, making it suitable for use in extreme environments such as mountainous areas.

[0033] Example 2 An Isaria nematocida fungal balloon agent, comprising the following raw materials: Isaria nematocida fungal spore liquid, carrier material, additive components, and active components.

[0034] The spore content of the Isaria nematocida fungal spore liquid ≥ 3×10 8 CFU / mL. The Isaria nematocida fungus of the present invention can use commercially available strains or strains in the prior art patent literature.

[0035] The carrier material is a mixed dispersion of sodium alginate and clay, wherein the mass concentration of sodium alginate is 4% and the mass concentration of clay is 10%.

[0036] The additive components include potassium penicillin G, streptomycin, and humic acid.

[0037] The active component is an extract of seabuckthorn pomace, and the specific preparation method is as follows: (1) The residue after squeezing the seabuckthorn fruit is rinsed with clean water, the residual juice and seed fragments are removed, the water is drained, spread out on a tray, and dried at 40 - 50°C with hot air until the water content ≤ 8%, pulverized through a 40-mesh sieve, and stored in a sealed and light-proof manner for later use; (2) Degreasing treatment: Mix the dried and pulverized seabuckthorn pomace obtained in step (1) with petroleum ether at a solid-liquid ratio of 1 g:8 mL, perform ultrasonic-assisted extraction at 40 - 45°C for 30 - 40 minutes, repeat 3 times; centrifuge to remove the petroleum ether layer containing oil; the solid obtained by centrifugation is dried at 50 - 60°C under ventilation to obtain defatted pomace; (3) Mix the defatted pomace with an ethanol solution with a mass concentration of 60% at a solid-liquid ratio of 1 g:20 mL, magnetically stir and premix for 5 minutes, then treat at 50 - 55°C in a constant temperature water bath for 45 min, stir once every 10 minutes, perform centrifugation treatment, collect supernatant 1, filter residue is treated again at 50 - 55°C in a constant temperature water bath for 45 min, perform centrifugation treatment, collect supernatant 2, combine the two supernatants, and remove impurities with a microfiltration membrane to obtain a reddish-brown liquid; (4) Rotate and evaporate the liquid, concentrate it to 1 / 10 of the original volume, add 1% of chitosan oligosaccharide based on the volume of the concentrated liquid, adjust the pH of the system to 6 - 7 with sodium citrate, and obtain an extract of seabuckthorn pomace after sterilization treatment.

[0038] A preparation method of an Isaria nematocida fungal balloon agent, comprising the following preparation steps: (1) Preparation of the nematode-killing fungus spores solution of **Ilyonectria**: Inoculate **Ilyonectria** on PDA medium and culture at 25 °C for 2 weeks. Then pick the colonies and inoculate them into PDB medium, and culture on a shaker at 130 rpm and 25 °C for 2 weeks. After the culture is completed, filter with gauze to remove the mycelium, and add sterilized water to adjust the concentration to 3×10 8 CFU / mL to obtain the nematode-killing fungus spores solution of **Ilyonectria**; (2) Take 500 mL of the nematode-killing fungus spores solution of **Ilyonectria** and 500 mL of the carrier material, mix them evenly, add potassium penicillin G and streptomycin, and adjust their concentrations in the system to 100 ppm and 50 ppm. Then add 3% of humic acid based on the mass of the mixed solution, and use a stirrer to mix the solution evenly; (3) Prepare the active ingredient; (4) Add 3% of the active ingredient based on the mass of the solution in step (2) to the solution in step (2), mix evenly, then pour the solution into a funnel with a micropipette tip, and slowly drip the solution into 3 mol / L calcium chloride solution. The reaction temperature is 4 ± 1 °C, and the reaction time is 30 min to obtain spherical spores microspheres with a diameter of about 2 - 3 mm. Control the dropping rate of the liquid with a pipette (20 - 30 drops / min); (5) Collect the microspheres, rinse them with sterilized water (to remove residual CaCl₂ and antibiotics), and then vacuum filter and dry them at low temperature (≤40 °C) to obtain the balloon agent.

[0039] Comparative Example 1 In this comparative example, except for not adding the active ingredient, the other raw materials and process steps are exactly the same as those in Example 1. That is: A nematode-killing fungus balloon agent of **Ilyonectria** comprises the following raw materials: nematode-killing fungus spores solution of **Ilyonectria**, carrier material, and additive components.

[0040] The spore content of the nematode-killing fungus spores solution of **Ilyonectria** ≥ 3×10 8 CFU / mL. The nematode-killing fungus of **Ilyonectria** can use commercially available strains or strains in the prior art patent documents.

[0041] The carrier material is a mixed dispersion of sodium alginate and clay, where the mass concentration of sodium alginate is 4% and the mass concentration of clay is 10%.

[0042] The additive components include potassium penicillin G, streptomycin, and humic acid.

[0043] A preparation method of a nematode-killing fungus balloon agent of **Ilyonectria** comprises the following preparation steps: (1)Preparation of the nematode-killing fungus spore solution of Isaria: Inoculate Isaria nematocida on PDA medium and culture at 25 °C for 2 weeks. Then pick the colonies and inoculate them into PDB medium, and culture on a shaker at 130 rpm and 25 °C for 2 weeks. After the culture is completed, filter with gauze to remove the mycelia, and adjust the concentration to 3×108 CFU / mL with sterilized water to obtain the nematode-killing fungus spore solution of Isaria; (2)Take 500 mL of the nematode-killing fungus spore solution of Isaria and 500 mL of the carrier material, mix them evenly, add potassium penicillin G and streptomycin, and adjust their concentrations in the system to 100 ppm and 50 ppm. Then add 3% of humic acid based on the mass of the mixed solution, and use a stirrer to mix the solution evenly; (4)Pour the solution in step (2) into a funnel with a micropipette tip, and slowly drip the solution into 3 mol / L calcium chloride solution. The reaction temperature is 4±1 °C, and the reaction time is 30 min to obtain round spore microspheres with a diameter of about 2-3 mm; Control the dropping rate of the liquid with a pipette (20-30 drops / min); (5)Collect the microspheres, rinse them with sterilized water (to remove residual CaCl2 and antibiotics), and then dry them at low temperature (≤40 °C) by vacuum filtration to obtain the balloon agent.

[0044] Comparative Example 2 In this comparative example, except that degreasing treatment is not carried out during the extraction of the active component, the remaining raw materials and process steps are exactly the same as those in Example 1. That is: An Isaria nematode-killing fungus balloon agent, comprising the following raw materials: the nematode-killing fungus spore solution of Isaria, a carrier material, an additive component, and an active component.

[0045] The spore content of the nematode-killing fungus spore solution of Isaria ≥ 3×10 8 CFU / mL. The Isaria nematode-killing fungus can be a commercially available strain or a strain in the prior art patent literature.

[0046] The active component is an extract of seabuckthorn pomace, and the specific preparation method is as follows: (1)Rinse the residue after squeezing the seabuckthorn fruit with clean water to remove the residual fruit juice and seed fragments, drain the water, spread it flat on a tray, and dry it at 40-50 °C with hot air until the water content ≤ 8%. Then crush it through a 40-mesh sieve, and store it in a sealed and light-proof manner for later use; (2)Mix the pomace with an ethanol solution with a mass concentration of 60% according to a solid-liquid ratio of 1 g:20 mL, premix it with magnetic stirring for 5 minutes, then treat it in a constant temperature water bath at 50-55 °C for 30 min, stir it once every 10 minutes, perform centrifugation, collect the supernatant 1, filter the residue again in a constant temperature water bath at 50-55 °C for 30 min, perform centrifugation, collect the supernatant 2, combine the two supernatants, and remove impurities with a microfiltration membrane to obtain a reddish-brown liquid; (4) Rotate and evaporate the liquid, concentrate it to 1 / 10 of the original volume, add chitosan oligosaccharide accounting for 1% of the volume of the concentrated liquid, adjust the pH of the system to 6 - 7 using sodium citrate, and obtain the extract of sea buckthorn pomace after sterilization treatment.

[0047] Comparative Example 3 In this comparative example, except that no chitosan oligosaccharide is added during the extraction of the active ingredient, the other raw materials and process steps are exactly the same as those in Example 1. That is: The active ingredient is the extract of sea buckthorn pomace, and the specific preparation method is as follows: (1) Rinse the residue after squeezing the sea buckthorn fruit with clean water, remove the residual juice and seed fragments, drain the water, spread it flat on a tray, dry it with hot air at 40 - 50 °C until the water content ≤ 8%, crush it through a 40 - mesh sieve, and store it in a sealed and light - proof manner for later use; (2) Degreasing treatment: Mix the sea buckthorn pomace after drying and crushing in step (1) with petroleum ether at a solid - liquid ratio of 1 g:8 mL, perform ultrasonic - assisted extraction at 40 - 45 °C for 30 minutes, repeat 3 times; centrifuge to remove the petroleum ether layer containing grease; dry the solid obtained by centrifugation at 50 - 60 °C under ventilation to obtain degreased pomace; (2) Mix the degreased pomace with an ethanol solution with a mass concentration of 60% at a solid - liquid ratio of 1 g:20 mL, magnetically stir and premix for 5 minutes, then treat it in a constant - temperature water bath at 50 - 55 °C for 30 min, stir once every 10 minutes, perform centrifugation treatment, collect the supernatant 1, filter the residue again in a constant - temperature water bath at 50 - 55 °C for 30 min, perform centrifugation treatment, collect the supernatant 2, combine the two supernatants, and remove impurities with a microfiltration membrane to obtain a reddish - brown liquid; (4) Rotate and evaporate the liquid, concentrate it to 1 / 10 of the original volume, adjust the pH of the system to 6 - 7 using sodium citrate, and obtain the extract of sea buckthorn pomace after sterilization treatment.

[0048] Comparative Example 4 In this comparative example, except that no degreasing treatment is carried out and no chitosan oligosaccharide is added during the extraction of the active ingredient, which is a conventional extraction method, the other raw materials and process steps are exactly the same as those in Example 1. That is: The active ingredient is the extract of sea buckthorn pomace, and the specific preparation method is as follows: (1) Rinse the residue after squeezing the sea buckthorn fruit with clean water, remove the residual juice and seed fragments, drain the water, spread it flat on a tray, dry it with hot air at 40 - 50 °C until the water content ≤ 8%, crush it through a 40 - mesh sieve, and store it in a sealed and light - proof manner for later use; (2) Mix the pomace with an ethanol solution at a mass concentration of 60% according to a solid-liquid ratio of 1 g:20 mL, magnetically stir for premixing for 5 minutes, then treat it in a constant temperature water bath at 50 - 55 °C for 30 min, stir once every 10 minutes, perform centrifugation, collect supernatant 1, filter residue is treated again in a constant temperature water bath at 50 - 55 °C for 30 min, perform centrifugation, collect supernatant 2, combine the two supernatants, and remove impurities with a microfiltration membrane to obtain a reddish-brown liquid; (4) Rotate evaporate the liquid, concentrate it to 1 / 10 of the original volume, adjust the pH of the system to 6 - 7 using sodium citrate, and obtain the extract of seabuckthorn pomace after sterilization treatment.

[0049] Comparative Example 5 In this comparative example, except for not using humic acid in the additive components, the rest of the raw materials and process steps are exactly the same as those in Example 1. That is: An Isaria nematocida fungal pellet agent, comprising the following raw materials: Isaria nematocida fungal spore solution, carrier material, additive components, and active components.

[0050] The additive components include potassium penicillin G and streptomycin.

[0051] A preparation method of an Isaria nematocida fungal pellet agent, comprising the following preparation steps: (1) Prepare Isaria nematocida fungal spore solution: Inoculate Isaria nematocida on a PDA medium and culture it at 25 °C for 2 weeks, then pick the colonies and inoculate them into a PDB medium, culture them on a shaker at 130 revolutions per minute and 25 °C for 2 weeks. After the culture is completed, filter with gauze, filter out the mycelia, add sterilized water to adjust the concentration to 3×108 CFU / mL to obtain Isaria nematocida fungal spore solution; (2) Take 500 mL of Isaria nematocida fungal spore solution and 500 mL of carrier material, mix them evenly, add potassium penicillin G and streptomycin, adjust their concentrations in the system to 100 ppm and 50 ppm, and use a stirrer to mix the solution evenly; (3) Prepare active components; (4) Add the solution from step (2) to 3% of the active components by the mass of the solution, mix evenly, then pour the solution into a funnel with a micropipette tip, and slowly drip the solution into 3 mol / L calcium chloride solution. The reaction temperature is 4 ± 1 °C, and the reaction time is 30 min to obtain round spore microspheres with a diameter of about 2 - 3 mm; Control the dropping rate of the pipette (20 - 30 drops per minute); (5) After collecting the microspheres, rinse them with sterilized water (to remove residual CaCl2 and antibiotics), perform vacuum filtration, and then dry them at low temperature (≤40 °C) to obtain the pellet agent.

[0052] Performance Test The preparation of the nematocidal fungal sclerotium of *Isaria* was carried out according to the methods of the examples and comparative examples, and it was configured into a dispersion with a spore count of 1×10 8 CFU / mL, and the nematocidal activity was determined by the impregnation method.

[0053] The method was as follows: Take 1 mL of the dispersion in a 24-well plate, and then add 100 μL of the suspension of *Bursaphelenchus xylophilus* (100 nematodes·100 μL -1 ). Use sterile water as a control, place it in an incubator at 28 °C. After 6 h of treatment, collect the nematodes and resuscitate them with clear water for 4 h. Use the needle-pricking method under a microscope to confirm the dead and alive state of the nematodes and count them. Nematodes that do not wriggle and still do not move when stimulated with a dissecting needle are regarded as dead. Finally, calculate the mortality rate. Set 5 replicates for each treatment, and the results are averaged. R = S2 / S1×100%, where R is the mortality rate, S1 is the total number of test nematodes, and S2 is the number of dead nematodes. The experimental results are as Figure 2 shown. It can be seen from Figure 2 that the spore dispersion of the example group of the present invention has a significantly better killing effect on *Bursaphelenchus xylophilus* than the control group, and the mortality rate at 6 h is close to 100%, indicating that the sclerotium has high nematocidal activity. For Comparative Example 1 lacking the active ingredient, the protective and penetration-enhancing effects on spores disappear, and its insecticidal power weakens. For Comparative Examples 2-3 with the preparation process of the active ingredient changed, Comparative Example 2 without removing the fat-soluble ingredients and Comparative Example 3 without adding chitosan, their fat-soluble ingredients will affect the activity of the subsequent extracts, and the effect weakens. For Comparative Example 3 lacking chitosan, similarly, the lack of chitosan leads to a decrease in the effect and a reduction in the nematocidal effect. For Comparative Example 4 with simple ethanol extraction, the extracted active substances are complex and it is difficult to effectively help the fungal spores penetrate the body wall of *Bursaphelenchus xylophilus*, and the nematocidal effect is significantly reduced. In summary, the active ingredient and its preparation process are crucial for the nematocidal activity of the sclerotium, and the effect weakens without either. For Comparative Example 5 without adding humic acid, although its nematocidal effect is improved compared with the comparative examples, the lack of humic acid leads to a decrease in the adhesion effect between fungal spores and nematodes, and its insecticidal effect is also lower than that of the examples.

[0054] Practical application experiment Experiment 1, Fuxing Forest Farm, Laian County, Anhui Province. At the beginning of the year, 50 black pines aged 20-25 years (plant height 6-14 m, diameter at breast height 10-17 cm) were selected as the administration group (Example 1 group), and 6 groups of 50 black pines of the same size were selected as the control group. For the pine trees in the administration group, at a position 1-1.5 m above the ground, drill three holes with a diameter of about 1 cm with an electric drill, and inject the *Isaria* fungal spore microspheres of Example 1 (2×10 8CFU / mL, 5 mL / well). For the pine trees in the control group, drill three holes with a diameter of about 1 cm at a position 1 - 1.5 meters above the ground, and inject sterilized water in equal amounts into each hole. One month later, inoculate 20,000 nematodes into the pine trees in the administration group and the control group. At the end of December, count the survival rate of the pine trees as follows Figure 3 .

[0055] Experiment 2: In Fuxing Forest Farm, Laian County, Anhui Province, at the beginning of the year, select 200 20 - 25-year-old black pine trees (tree height 6 - 14 meters, breast diameter 10 - 17 cm) as the administration group (Example 1). Select 1200 black pine trees of the same size 5 kilometers away as the control group. On a cloudy day, use a stretcher sprayer to spray the administration group of pine trees with the microspheres of Ips fungus spores for controlling pine wilt disease in the forest: configure the fungal balloon agent into a dispersion with a spore count of 1×10 8 CFU / mL, and evenly spray with a stretcher sprayer (droplet diameter 200 - 300 μm), dosage: 50 mL / tree.

[0056] Spray the control group of pine trees with an equal amount of sterile water in the same way. For the administration group and the control group, adopt the method of natural infection of nematodes by Monochamus alternatus, and do not artificially infect pine wilt nematodes. At the end of December, count the survival rate of the pine trees as follows Figure 4 .

[0057] In summary, whether by injection or spraying, the microsphere agent of Ips fungus spores of the present invention can effectively control pine wilt disease, and the survival rate of the pine trees in the administration group is significantly higher than that in the control group. It realizes the effective prevention and control of pine wilt disease.

[0058] It should be noted that the above embodiments are only some of the preferred embodiments for implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Claims

1. A nematode-killing fungal sclerotium of **Ilyonectria**, characterized in that, It includes the following raw materials: spores solution of **Hirsutella rhossiliensis**, carrier material, additive components and active components.

2. The nematode-killing fungal sclerotium agent of claim 1, characterized in that, The spore content of the nematode-killing fungus spores liquid of Y. is ≥ 3×10 8 CFU / mL.

3. The nematode-killing fungal mycelium pellet of Y. eudermatum according to claim 1, characterized in that The carrier material is a mixed dispersion of sodium alginate and clay, where the mass concentration of sodium alginate is 4% and the mass concentration of clay is 10%.

4. The nematode-killing fungal sclerotium agent of claim 1, characterized in that, The additive components include potassium penicillin G, streptomycin and humic acid.

5. The nematode-killing fungal sphaeroplast agent according to claim 1, characterized in that, The active component is the extract of sea buckthorn pomace, and the specific preparation method is as follows: (1) Rinse the residue after juicing the sea buckthorn fruits with clean water to remove residual fruit juice and seed fragments, drain the water, spread it out on a tray, and dry it with hot air at 40 - 50 °C until the water content ≤ 8%, then crush it through a 40-mesh sieve, and store it in a sealed and light-proof manner for later use; (2) Degreasing treatment: Mix the sea buckthorn pomace after drying and crushing in step (1) with petroleum ether at a material-liquid ratio of 1 g:8 mL, and perform ultrasonic-assisted extraction at 40 - 45 °C for 30 - 40 minutes, repeating 3 times; centrifuge to remove the petroleum ether layer containing oil; dry the solid obtained by centrifugation at 50 - 60 °C in ventilation to obtain degreased pomace; (3) Mix the degreased pomace with an ethanol solution with a mass concentration of 60% at a material-liquid ratio of 1 g:20 mL, magnetically stir and premix for 5 minutes, then treat it in a constant temperature water bath at 50 - 55 °C for 30 - 45 min, stirring once every 10 minutes, perform centrifugation treatment, collect supernatant 1, filter residue is again treated in a constant temperature water bath at 50 - 55 °C for 30 - 45 min, perform centrifugation treatment, collect supernatant 2, combine the two supernatants, and remove impurities with a microfiltration membrane to obtain a reddish-brown liquid; (4) Rotate and evaporate the liquid to concentrate it to 1 / 10 of the original volume, add 1% of chitosan oligosaccharide based on the volume of the concentrated liquid, adjust the pH of the system to 6 - 7 with sodium citrate, and obtain the extract of sea buckthorn pomace after sterilization treatment.

6. A preparation method of the nematode-killing fungal sclerotium agent of the genus Hirsutella according to any one of claims 1-5, characterized in that, It includes the following preparation steps: (1) Preparation of nematode-killing fungus spores of **Ilyonectria** spp.: Inoculate **Ilyonectria** spp. on PDA medium and culture at 25 °C for 2 weeks. Then pick the colonies and inoculate them into PDB medium, and culture on a shaker at 130 rpm and 25 °C for 2 weeks. After the culture is completed, filter with gauze to remove the mycelium, and add sterilized water to adjust the concentration to 3×10 8 CFU / mL to obtain the nematode-killing fungus spores of **Ilyonectria** spp.; (2) Take 500 mL of spores solution of **Hirsutella rhossiliensis** and 500 mL of carrier material, mix them evenly, add potassium penicillin G and streptomycin, adjust their concentrations in the system to 100 ppm and 50 ppm, then add 3% of humic acid based on the mass of the mixed solution, and mix the solution evenly with a stirrer; (3) Prepare the active component; (4) Add 3% of the active component based on the mass of the solution in step (2) to the solution, mix it evenly, then pour the solution into a funnel with a micropipette tip, and slowly drip the solution into 3 mol / L calcium chloride solution to obtain round spore microspheres, and the diameter of the spore microspheres is about 2 - 3 mm; (5) Collect the microspheres, rinse them with sterilized water, and dry them to obtain the balloon agent.

7. Use of the **Hirsutella rhossiliensis** balloon agent according to any one of claims 1 - 5 in the prevention and control of pine wilt disease.

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