Isaria fumosorosea and its application in preventing and treating pine wood nematode disease

Abstract

This invention discloses a fungal encapsulation agent for *Ischemicula elais* and its application in the control of pine wilt disease, belonging to the field of microbial control technology. This invention uses *Ischemicula elais* as the functional microorganism, combined with carrier materials, additives, and active ingredients to prepare a solid microbial encapsulation agent. The shelf life of *Ischemicula elais* spores at room temperature is extended from 2-3 days for traditional spore solutions to over 3-6 months, and up to 12 months under 4°C refrigeration, solving the problem of inconvenient low-temperature storage in mountainous areas. The microencapsulation components form a slow-release protective layer, preventing activity loss caused by environmental factors such as high temperature and drought.

Description

Technical Field

[0001] This invention belongs to the field of microbial control technology, specifically relating to a fungal saccharin agent for killing Ischemicis nematodes and its application in the control of pine wilt disease. Background Technology

[0002] Pine wilt disease is a dangerous disease caused by the pine wood nematode (Bursaphelenchus xylophilus), resulting in the rapid withering and death of pine trees, leading to widespread destruction. Due to its severe damage and difficulty in control, it has received high attention from countries around the world and is listed as an important and dangerous forest disease.

[0003] Controlling pine wilt disease has become a global challenge, but effective control methods are currently lacking. The most common approach worldwide is to cut off the source and route of transmission to prevent the spread of pine wilt nematodes. Current methods include felling diseased trees to cut off the source; using traps to kill pine sawyer beetles; spraying chemical agents such as thiamethoxam and fenitrothion emulsions from the air and ground to control beetles; using predatory or parasitic natural enemies of beetles such as *Scleroderma spp.*, *Dastarcus longulus*, *Allecula fuliginosa*, and woodpeckers (*Dendrocops spp.*); using fungi such as *Beauveria bassiana* to control beetles; injecting chemical agents such as abamectin, nematicides, and plant extracts like malathion into tree trunks to control pine wilt nematodes; and breeding disease-resistant varieties through hybridization of *Pinus massoniana*, *Pinus thunbergii*, and *Pinus thunbergii*. Due to the insidious nature of the pine sawyer beetle's damage, chemical control is generally ineffective and can cause environmental pollution and toxic side effects on non-target organisms. In addition, due to the limitations of control methods such as trunk injection, it is urgent to develop a control method that is highly efficient, easy to operate, and environmentally friendly. Summary of the Invention

[0004] This invention addresses the problems existing in the prior art by using *Isodon esculenta* fungus as the functional microorganism, combined with various excipients and plant extracts. On the one hand, it solves the problem of poor storage stability of functional microbial spores, significantly extending shelf life. On the other hand, the combined action of various active ingredients greatly improves the control effect.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0006] A fungal sac-forming agent for killing nematodes, comprising the following raw materials: fungal spore liquid of killing nematodes, carrier material, additive components, and active ingredients.

[0007] Furthermore, the spore content of the *Echinococcus erinaceus* fungal spore solution is ≥3 × 10⁻⁶. 8 CFU / mL. The *Ischemicum elutifida* fungus of this invention can be a commercially available strain or a strain from existing patent literature.

[0008] Furthermore, 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%.

[0009] Furthermore, the additive ingredients include potassium penicillin G, streptomycin, and humic acid.

[0010] Furthermore, the active ingredient is an extract of sea buckthorn pomace, and the specific preparation method is as follows:

[0011] (1) Rinse the residue after juicing the sea buckthorn fruit with clean water to remove residual juice and seed fragments, drain the water, spread it evenly on a tray, dry it with hot air at 40-50℃ until the moisture content is ≤8%, crush it through a 40-mesh sieve, seal it and store it in the dark for later use.

[0012] (2) Degreasing treatment: The dried and pulverized sea buckthorn pomace in step (1) is mixed with petroleum ether at a material-liquid ratio of 1g:8mL, and extracted with sound-assisted extraction at 40-45℃ for 30-40 minutes, repeated 3 times; the oil-containing petroleum ether layer is removed by centrifugation; the solid obtained by centrifugation is dried in a ventilated environment at 50-60℃ to obtain degreased pomace.

[0013] (3) Mix the defatted fruit pomace with a 60% ethanol solution at a ratio of 1g:20mL, premix with magnetic stirring for 5 minutes, then treat in a constant temperature water bath at 50-55℃ for 30-45 minutes, stirring once every 10 minutes, centrifuge, collect supernatant 1, treat the filter residue again in a constant temperature water bath at 50-55℃ for 30-45 minutes, centrifuge, collect supernatant 2, combine the two supernatants, remove impurities by microfiltration membrane, and obtain a reddish-brown liquid;

[0014] (4) The liquid was rotary evaporated and concentrated to 1 / 10 of the original volume. 1% of the concentrated liquid volume of chitosan oligosaccharide was added. The pH of the system was adjusted to 6-7 using sodium citrate. After sterilization, the extract of sea buckthorn pomace was obtained.

[0015] A method for preparing a fungal saccharin agent for killing Ischemicis includes the following preparation steps:

[0016] (1) Preparation of fungal spores of *Ischemicula ebeni*: *Ischemicula ebeni* was inoculated onto PDA medium and cultured at 25°C for 2 weeks. Colonies were then picked and inoculated onto PDB medium and cultured on a shaker at 130 rpm at 25°C for 2 weeks. After culture, the culture was filtered through gauze to remove mycelia, and sterile water was added to adjust the concentration to 3 × 10⁻⁶. 8 CFU / mL was used to obtain fungal spores of *Echinococcus ileus*.

[0017] (2) Take 500 mL of fungal spore solution of Echinococcus ileiflora and 500 mL of carrier material and mix them evenly. Add potassium penicillin G and streptomycin to adjust the concentration of the two in the system to 100 ppm and 50 ppm respectively. Then add 3% humic acid by weight of the mixture and use a stirrer to mix the solution evenly.

[0018] (3) Preparation of active components;

[0019] (4) Add 3% of the active component by mass to the solution from step (2), mix well, and pour into a funnel with a micropipette tip. Slowly drop the solution into a 3 mol / L calcium chloride solution. The reaction temperature is 4±1℃ and the reaction time is 30 min to obtain round spore microspheres with a diameter of about 2-3 mm. Control the dropping rate with a pipette (20-30 drops / min).

[0020] (5) After collecting the microspheres, rinse them with sterile water (to remove residual CaCl2 and antibiotics), vacuum filter them, and dry them at low temperature (≤40℃) to obtain the capsule agent.

[0021] Application of a fungal saccharin for controlling pine wilt disease.

[0022] The fungal sac-forming agent for killing Erisia esculenta obtained in this invention is white to pale yellow spherical particles with a water activity ≤0.6, inhibiting the growth of non-target microorganisms; and has a survival rate ≥75% at room temperature for 3-6 months.

[0023] The fungal sac-forming agent for killing pine wilt disease obtained in this invention has two application methods: spraying in the forest to control pine wilt disease and injecting into boreholes to control pine wilt disease.

[0024] Spraying in forests to control pine wilt disease: Prepare fungal spore-forming agents with a spore count of 1×10⁶. 8 A CFU / mL dispersion was sprayed evenly using a stretcher sprayer (droplet diameter 200-300 μm), dosage: 50 mL / plant.

[0025] Drilling and injection for the control of pine wilt disease: The fungal spore-forming agent was prepared with a spore count of 2 × 10⁶. 8 For a CFU / mL dispersion, drill three holes about 1 cm in diameter and 10-15 cm deep in a pine tree at a height of 1-1.5 meters above the ground. Inject the dispersion at a dose of 5 mL per hole.

[0026] To address the following core deficiencies in existing fungal nematicides for Irwinia oleracea, this invention aims to provide a highly efficient and stable microbial control solution, specifically addressing the following issues:

[0027] 1. Poor spore storage stability: Existing spore concentrates can only be stored for 2-3 days at room temperature, and the shelf life is only 1 month under 4℃ refrigeration conditions. This cannot meet the long-term storage needs in non-cold chain environments such as mountainous areas, resulting in low transportation and application efficiency.

[0028] 2. Limited application scenarios: Liquid formulations rely on low-temperature storage and transportation, making them difficult to adapt to the large-scale application needs in complex environments such as mountainous and forest areas, thus restricting the actual promotion of biological control technologies.

[0029] Purpose of the invention: To develop microsphere formulations that greatly enhance the stress resistance of microbial agents and solve key technical bottlenecks in the storage, transportation, and application of Echinococcus erinaceus fungal spores.

[0030] Beneficial effects:

[0031] (1) This invention uses *Ichthyophthirius multifiliis* fungus as the functional microorganism, combined with carrier materials, additives, and active ingredients to prepare a solid microbial sac agent. Firstly, sodium alginate and clay serve as carrier materials, providing good mechanical strength and slow-release properties. Combined with antibiotics to inhibit harmful bacteria, the control effect is significantly improved. Furthermore, the introduction of humic acid further improves the surface properties of the microspheres, enhancing their adhesion to nematodes and plant tissues, resulting in increased retention of the microspheres on pine needles and bark after field spraying. Field trials show that the survival rate and control efficacy of pine trees sprayed with the microsphere preparation are significantly better than those of chemical agents and traditional biological control methods. A single application can maintain an effective control period of more than 6 months, reducing the frequency of manual re-application.

[0032] (2) Secondly, this invention adds a small amount of sea buckthorn pomace extract as a bioactive substance. The extract, after removing fat-soluble components, is rich in polyphenols, pectin polysaccharides, and other substances. Sea buckthorn polyphenols stabilize spore membrane lipids through hydrogen bonds, improving freeze-thaw tolerance and storage stability. Simultaneously, polyphenols inhibit nematode epidermal peroxidase, enhancing fungal penetration. Combined with 0.1% chitosan oligosaccharide, the stability of polyphenols is enhanced through hydrogen bonding, doubling the effect. Additionally, the addition of pectin polysaccharides forms a water-retaining film, prolonging spore survival time.

[0033] (3) Furthermore, the reagent's environmental adaptability is significantly enhanced. The microsphere formulation is resistant to high temperatures and compression, and outdoor storage and transportation in mountainous areas do not require low-temperature conditions, reducing logistics costs by more than 60%. Natural sodium alginate and clay components are biodegradable, avoiding chemical residue pollution of soil and water sources. It has strong compatibility: it can be used in conjunction with longhorn beetle trapping, disease-resistant variety cultivation, and other control measures to form a comprehensive prevention and control system.

[0034] (4) Finally, through microsphere formulation technology, the shelf life of *Echinococcus erinaceus* fungal spores under normal temperature conditions is extended from 2-3 days for traditional spore solutions to more than 3-6 months. They are also stable under 4°C refrigeration conditions, solving the problem of inconvenient low-temperature storage in mountainous areas. The microcapsule components form a sustained-release protective layer, avoiding activity loss caused by environmental factors such as high temperature and drought. Attached Figure Description

[0035] Figure 1 This refers to the spore germination rate of the fungal spore-forming agent in Example 1 of the present invention at different stages under low temperature and room temperature conditions;

[0036] Figure 2 A comparison of the killing activity of different treatment groups against pine wilt nematodes after 6 hours of treatment;

[0037] Figure 3 A comparison of the survival rates of pine trees in different treatment groups after inoculation with pine wood nematodes in Experiment 1;

[0038] Figure 4 This is a comparison of the survival rates of pine trees in different treatment groups under natural infection in Experiment 2. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0040] Example 1

[0041] A fungal sac-forming agent for killing nematodes, comprising the following raw materials: fungal spore liquid of killing nematodes, carrier material, additive components, and active ingredients.

[0042] The spore content of the *Isodon oryzae* fungal spore solution is ≥3×10⁻⁶. 8 CFU / mL. The *Ischemicum elutifida* fungus of this invention can be a commercially available strain or a strain from existing patent literature.

[0043] 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%.

[0044] The additives contain penicillin G potassium, streptomycin, and humic acid.

[0045] The active component is an extract of sea buckthorn pomace, and the specific preparation method is as follows:

[0046] (1) Rinse the residue after juicing the sea buckthorn fruit with clean water to remove residual juice and seed fragments, drain the water, spread it evenly on a tray, dry it with hot air at 40-50℃ until the moisture content is ≤8%, crush it through a 40-mesh sieve, seal it and store it in the dark for later use.

[0047] (2) Degreasing treatment: The dried and pulverized sea buckthorn pomace in step (1) is mixed with petroleum ether at a ratio of 1g:8mL, and extracted with sound at 40-45℃ for 30 minutes, repeated 3 times; the oil-containing petroleum ether layer is removed by centrifugation; the solid obtained by centrifugation is dried at 50-60℃ to obtain degreased pomace.

[0048] (2) Mix the defatted fruit pomace with a 60% ethanol solution at a ratio of 1g:20mL, premix with magnetic stirring for 5 minutes, then treat in a constant temperature water bath at 50-55℃ for 30 minutes, stirring once every 10 minutes, centrifuge, collect the supernatant 1, treat the filter residue again in a constant temperature water bath at 50-55℃ for 30 minutes, centrifuge, collect the supernatant 2, combine the two supernatants, remove impurities by microfiltration membrane, and obtain a reddish-brown liquid;

[0049] (4) The liquid is rotary evaporated and concentrated to 1 / 10 of the original volume. 1% of the concentrated liquid volume of chitosan oligosaccharide is added. The pH of the system is adjusted to 6-7 using sodium citrate. After sterilization, the extract of sea buckthorn pomace is obtained.

[0050] A method for preparing a fungal saccharin agent for killing Ischemicis includes the following preparation steps:

[0051] (1) Preparation of fungal spores of *Ischemicum oryzae*: *Ischemicum oryzae* was inoculated onto PDA medium and cultured at 25°C for 2 weeks. Then, colonies were picked and inoculated onto PDB medium and cultured on a shaker at 130 rpm and 25°C for 2 weeks. After the culture was completed, the mycelium was filtered out by gauze and sterile water was added to adjust the concentration to 3×10⁸ CFU / mL to obtain fungal spores of *Ischemicum oryzae*.

[0052] (2) Take 500 mL of fungal spore solution of Echinococcus oryzae and 500 mL of carrier material and mix them evenly. Add potassium penicillin G and streptomycin to adjust the concentration of the two in the system to 100 ppm and 50 ppm respectively. Then add 3% humic acid by weight of the mixture and use a stirrer to mix the solution evenly.

[0053] (3) Preparation of active components;

[0054] (4) Add 3% of the active component by mass to the solution from step (2), mix well, and pour into a funnel with a micropipette tip. Slowly drop the solution into a 3 mol / L calcium chloride solution. The reaction temperature is 4±1℃ and the reaction time is 30 min to obtain round spore microspheres with a diameter of about 2-3 mm. Control the dropping rate with a pipette (20-30 drops / min).

[0055] (5) After collecting the microspheres, rinse them with sterile water (to remove residual CaCl2 and antibiotics), vacuum filter them, and dry them at low temperature (≤40℃) to obtain the capsule agent.

[0056] Application of a fungal saccharin for controlling pine wilt disease.

[0057] The shelf life of the *Ichthyophthirius multifiliis* fungal sac agent obtained in this embodiment was tested, with the spore germination rate of the particles checked monthly. The activity of the conidial formulation was determined by measuring the germination capacity of conidia immediately after production and at 1, 2, 3, 4, 5, and 6 months later. Scenarios included room temperature, refrigeration (4°C), and slightly elevated room temperature (25°C) to simulate actual storage and transportation conditions. Ten random particles were added to a 15 mL test tube containing 10 mL of 0.1 M sodium citrate and shaken at 130 rpm for 1 hour to ensure complete spore dispersion. 0.2 mL of the suspension was spread onto water agar medium and incubated at 25°C for 2 days. The number of colonies formed by conidial germination was counted. Using the colony count immediately after production as a baseline, the survival rate at each time point was calculated, i.e., spore germination rate = (number of colonies formed after n months / initial colony count × 100%). All tests were performed five times to ensure the reliability of the results. Results are as follows... Figure 1 As shown, the fungal spore germination rate of the present invention is stable 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.

[0058] Example 2

[0059] A fungal sac-forming agent for killing nematodes, comprising the following raw materials: fungal spore liquid of killing nematodes, carrier material, additive components, and active ingredients.

[0060] The spore content of the *Isodon oryzae* fungal spore solution is ≥3×10⁻⁶. 8 CFU / mL. The *Ischemicum elutifida* fungus of this invention can be a commercially available strain or a strain from existing patent literature.

[0061] 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%.

[0062] The additives contain penicillin G potassium, streptomycin, and humic acid.

[0063] The active component is an extract of sea buckthorn pomace, and the specific preparation method is as follows:

[0064] (1) Rinse the residue after juicing the sea buckthorn fruit with clean water to remove residual juice and seed fragments, drain the water, spread it evenly on a tray, dry it with hot air at 40-50℃ until the moisture content is ≤8%, crush it through a 40-mesh sieve, seal it and store it in the dark for later use.

[0065] (2) Degreasing treatment: The dried and pulverized sea buckthorn pomace in step (1) is mixed with petroleum ether at a material-liquid ratio of 1g:8mL, and extracted with sound-assisted extraction at 40-45℃ for 30-40 minutes, repeated 3 times; the oil-containing petroleum ether layer is removed by centrifugation; the solid obtained by centrifugation is dried in a ventilated environment at 50-60℃ to obtain degreased pomace.

[0066] (3) Mix the defatted fruit pomace with a 60% ethanol solution at a ratio of 1g:20mL, premix with magnetic stirring for 5 minutes, then treat in a constant temperature water bath at 50-55℃ for 45 minutes, stirring once every 10 minutes, centrifuge, collect the supernatant 1, treat the filter residue again in a constant temperature water bath at 50-55℃ for 45 minutes, centrifuge, collect the supernatant 2, combine the two supernatants, remove impurities by microfiltration membrane, and obtain a reddish-brown liquid;

[0067] (4) The liquid was rotary evaporated and concentrated to 1 / 10 of the original volume. 1% of the concentrated liquid volume of chitosan oligosaccharide was added. The pH of the system was adjusted to 6-7 using sodium citrate. After sterilization, the extract of sea buckthorn pomace was obtained.

[0068] A method for preparing a fungal saccharin agent for killing Ischemicis includes the following preparation steps:

[0069] (1) Preparation of fungal spores of *Ischemicula ebeni*: *Ischemicula ebeni* was inoculated onto PDA medium and cultured at 25°C for 2 weeks. Colonies were then picked and inoculated onto PDB medium and cultured on a shaker at 130 rpm at 25°C for 2 weeks. After culture, the culture was filtered through gauze to remove mycelia, and sterile water was added to adjust the concentration to 3 × 10⁻⁶. 8 CFU / mL was used to obtain fungal spores of *Echinococcus ileus*.

[0070] (2) Take 500 mL of fungal spore solution of Echinococcus ileiflora and 500 mL of carrier material and mix them evenly. Add potassium penicillin G and streptomycin to adjust the concentration of the two in the system to 100 ppm and 50 ppm respectively. Then add 3% humic acid by weight of the mixture and use a stirrer to mix the solution evenly.

[0071] (3) Preparation of active components;

[0072] (4) Add 3% of the active component by mass to the solution from step (2), mix well, and pour into a funnel with a micropipette tip. Slowly drop the solution into a 3 mol / L calcium chloride solution. The reaction temperature is 4±1℃ and the reaction time is 30 min to obtain round spore microspheres with a diameter of about 2-3 mm. Control the dropping rate with a pipette (20-30 drops / min).

[0073] (5) After collecting the microspheres, rinse them with sterile water (to remove residual CaCl2 and antibiotics), vacuum filter them, and dry them at low temperature (≤40℃) to obtain the capsule agent.

[0074] Comparative Example 1

[0075] This comparative example is identical to Example 1 in all raw materials and process steps except for the absence of the active ingredient. That is:

[0076] A fungal sac-forming agent for killing Erisia esculenta comprises the following raw materials: fungal spore liquid of Erisia esculenta, carrier material, and additive components.

[0077] The spore content of the *Isodon oryzae* fungal spore solution is ≥3×10⁻⁶. 8 CFU / mL. The fungi used to kill E. elodea can be commercially available strains or strains described in existing patent literature.

[0078] 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%.

[0079] The additives contain penicillin G potassium, streptomycin, and humic acid.

[0080] A method for preparing a fungal saccharin agent for killing Ischemicis includes the following preparation steps:

[0081] (1) Preparation of fungal spores of *Ischemicum oryzae*: *Ischemicum oryzae* was inoculated onto PDA medium and cultured at 25°C for 2 weeks. Then, colonies were picked and inoculated onto PDB medium and cultured on a shaker at 130 rpm and 25°C for 2 weeks. After the culture was completed, the mycelium was filtered out by gauze and sterile water was added to adjust the concentration to 3×10⁸ CFU / mL to obtain fungal spores of *Ischemicum oryzae*.

[0082] (2) Take 500 mL of fungal spore solution of Echinococcus oryzae and 500 mL of carrier material and mix them evenly. Add potassium penicillin G and streptomycin to adjust the concentration of the two in the system to 100 ppm and 50 ppm respectively. Then add 3% humic acid by weight of the mixture and use a stirrer to mix the solution evenly.

[0083] (4) Pour the solution from step (2) into a funnel with a micropipette tip, and slowly drop the solution into a 3 mol / L calcium chloride solution. The reaction temperature is 4±1℃ and the reaction time is 30 min to obtain round spore microspheres with a diameter of about 2-3 mm. Control the dropping rate with a pipette (20-30 drops / min).

[0084] (5) After collecting the microspheres, rinse them with sterile water (to remove residual CaCl2 and antibiotics), vacuum filter them, and dry them at low temperature (≤40℃) to obtain the capsule agent.

[0085] Comparative Example 2

[0086] This comparative example is identical to Example 1 in all raw materials and process steps except that defatting is not performed during the extraction of the active components. That is:

[0087] A fungal sac-forming agent for killing nematodes, comprising the following raw materials: fungal spore liquid of killing nematodes, carrier material, additive components, and active ingredients.

[0088] The spore content of the *Isodon oryzae* fungal spore solution is ≥3×10⁻⁶. 8 CFU / mL. The fungi used to kill E. elodea can be commercially available strains or strains described in existing patent literature.

[0089] The active component is an extract of sea buckthorn pomace, and the specific preparation method is as follows:

[0090] (1) Rinse the residue after juicing the sea buckthorn fruit with clean water to remove residual juice and seed fragments, drain the water, spread it evenly on a tray, dry it with hot air at 40-50℃ until the moisture content is ≤8%, crush it through a 40-mesh sieve, seal it and store it in the dark for later use.

[0091] (2) Mix the fruit pomace with a 60% ethanol solution at a ratio of 1g:20mL, stir magnetically for 5 minutes, then treat in a constant temperature water bath at 50-55℃ for 30 minutes, stirring once every 10 minutes, centrifuge, collect the supernatant 1, treat the filter residue again in a constant temperature water bath at 50-55℃ for 30 minutes, centrifuge, collect the supernatant 2, combine the two supernatants, remove impurities by microfiltration membrane, and obtain a reddish-brown liquid;

[0092] (4) The liquid is rotary evaporated and concentrated to 1 / 10 of the original volume. 1% of the concentrated liquid volume of chitosan oligosaccharide is added. The pH of the system is adjusted to 6-7 using sodium citrate. After sterilization, the extract of sea buckthorn pomace is obtained.

[0093] Comparative Example 3

[0094] This comparative example is identical to Example 1 in all raw materials and process steps, except that chitosan oligosaccharide is not added during the extraction of the active components. That is:

[0095] The active component is an extract of sea buckthorn pomace, and the specific preparation method is as follows:

[0096] (1) Rinse the residue after juicing the sea buckthorn fruit with clean water to remove residual juice and seed fragments, drain the water, spread it evenly on a tray, dry it with hot air at 40-50℃ until the moisture content is ≤8%, crush it through a 40-mesh sieve, seal it and store it in the dark for later use.

[0097] (2) Degreasing treatment: The dried and pulverized sea buckthorn pomace in step (1) is mixed with petroleum ether at a ratio of 1g:8mL, and extracted with sound at 40-45℃ for 30 minutes, repeated 3 times; the oil-containing petroleum ether layer is removed by centrifugation; the solid obtained by centrifugation is dried at 50-60℃ to obtain degreased pomace.

[0098] (2) Mix the defatted fruit pomace with a 60% ethanol solution at a ratio of 1g:20mL, premix with magnetic stirring for 5 minutes, then treat in a constant temperature water bath at 50-55℃ for 30 minutes, stirring once every 10 minutes, centrifuge, collect the supernatant 1, treat the filter residue again in a constant temperature water bath at 50-55℃ for 30 minutes, centrifuge, collect the supernatant 2, combine the two supernatants, remove impurities by microfiltration membrane, and obtain a reddish-brown liquid;

[0099] (4) The liquid was rotary evaporated and concentrated to 1 / 10 of the original volume. The pH of the system was adjusted to 6-7 using sodium citrate. After sterilization, the extract of sea buckthorn pomace was obtained.

[0100] Comparative Example 4

[0101] This comparative example, except that defatting and the addition of chitosan oligosaccharides are omitted in the extraction of the active components, follows a conventional extraction method. All other raw materials and process steps are completely consistent with Example 1. That is:

[0102] The active component is an extract of sea buckthorn pomace, and the specific preparation method is as follows:

[0103] (1) Rinse the residue after juicing the sea buckthorn fruit with clean water to remove residual juice and seed fragments, drain the water, spread it evenly on a tray, dry it with hot air at 40-50℃ until the moisture content is ≤8%, crush it through a 40-mesh sieve, seal it and store it in the dark for later use.

[0104] (2) Mix the fruit pomace with a 60% ethanol solution at a ratio of 1g:20mL, stir magnetically for 5 minutes, then treat in a constant temperature water bath at 50-55℃ for 30 minutes, stirring once every 10 minutes, centrifuge, collect the supernatant 1, treat the filter residue again in a constant temperature water bath at 50-55℃ for 30 minutes, centrifuge, collect the supernatant 2, combine the two supernatants, remove impurities by microfiltration membrane, and obtain a reddish-brown liquid;

[0105] (4) The liquid was rotary evaporated and concentrated to 1 / 10 of the original volume. The pH of the system was adjusted to 6-7 using sodium citrate. After sterilization, the extract of sea buckthorn pomace was obtained.

[0106] Comparative Example 5

[0107] This comparative example is identical to Example 1 in all raw materials and process steps, except that humic acid is not used in the additive composition. That is:

[0108] A fungal sac-forming agent for killing nematodes, comprising the following raw materials: fungal spore liquid of killing nematodes, carrier material, additive components, and active ingredients.

[0109] The additives contain penicillin G potassium and streptomycin.

[0110] A method for preparing a fungal saccharin agent for killing Ischemicis includes the following preparation steps:

[0111] (1) Preparation of fungal spores of *Ischemicum oryzae*: *Ischemicum oryzae* was inoculated onto PDA medium and cultured at 25°C for 2 weeks. Then, colonies were picked and inoculated onto PDB medium and cultured on a shaker at 130 rpm and 25°C for 2 weeks. After the culture was completed, the mycelium was filtered out by gauze and sterile water was added to adjust the concentration to 3×10⁸ CFU / mL to obtain fungal spores of *Ischemicum oryzae*.

[0112] (2) Take 500 mL of fungal spore solution of Echinococcus oryzae and 500 mL of carrier material and mix them evenly. Add potassium penicillin G and streptomycin and adjust their concentrations in the system to 100 ppm and 50 ppm respectively. Use a stirrer to mix the solution evenly.

[0113] (3) Preparation of active components;

[0114] (4) Add 3% of the active component by mass to the solution from step (2), mix well, and pour into a funnel with a micropipette tip. Slowly drop the solution into a 3 mol / L calcium chloride solution. The reaction temperature is 4±1℃ and the reaction time is 30 min to obtain round spore microspheres with a diameter of about 2-3 mm. Control the dropping rate with a pipette (20-30 drops / min).

[0115] (5) After collecting the microspheres, rinse them with sterile water (to remove residual CaCl2 and antibiotics), vacuum filter them, and dry them at low temperature (≤40℃) to obtain the capsule agent.

[0116] Performance testing

[0117] The fungal saccharin for killing Nematodes was prepared according to the methods of the examples and comparative examples, and was formulated with a spore count of 1 × 10⁻⁶. 8 The nematicidal activity of the CFU / mL dispersion was determined by the immersion method.

[0118] The method is as follows: Take 1 mL of dispersion into a 24-well plate, and then add 100 μL of pine wood nematode suspension (100 nematodes·100 μL). -1Using sterile water as a control, the nematodes were incubated at 28℃ for 6 hours. After treatment, the nematodes were collected and revived with clean water for 4 hours. The nematodes' viability was confirmed and counted under a microscope using a needle prick method. Nematodes that did not move and remained motionless even after needle stimulation were considered dead. The mortality rate was calculated. Each treatment was repeated in 5 replicates, and the mean was taken. R = S2 / S1 × 100%, where R is the mortality rate, S1 is the total number of tested nematodes, and S2 is the number of dead nematodes. The experimental results are as follows: Figure 2 As shown, from Figure 2 It can be seen that the spore dispersion of the present invention showed significantly better killing effect on pine wood nematodes than the control group, with a mortality rate of nearly 100% after 6 hours, indicating that the spore-forming agent has highly efficient nematicidal activity. However, in Comparative Example 1, which lacked the active ingredient, the protective and penetration-enhancing effects on spores disappeared, resulting in weakened insecticidal activity. Comparative Examples 2-3, which altered the preparation process of the active ingredient, and Comparative Example 2 (which did not remove lipophilic components) and Comparative Example 3 (which did not add chitosan oligosaccharides), showed that the lipophilic components affected the activity of subsequent extracts, weakening the effect. Similarly, Comparative Example 3, lacking chitosan oligosaccharides, also experienced a decrease in efficacy and nematicidal effect due to the absence of chitosan oligosaccharides. Comparative Example 4, which only underwent ethanol extraction, produced complex active substances that were difficult to effectively help fungal spores penetrate the pine wood nematode body wall, significantly reducing the nematicidal effect. In conclusion, the active ingredient and its preparation process are crucial to the nematicidal activity of the spore-forming agent; the absence of either weakens the effect. While Comparative Example 5, which did not contain humic acid, showed improved nematicidal effect compared to the comparative example, the lack of humic acid reduced the adhesion of fungal spores to nematodes, resulting in a lower insecticidal effect than the example.

[0119] Practical application experiment

[0120] Experiment 1: Fuxing Forest Farm, Lai'an County, Anhui Province. At the beginning of the year, 50 20-25 year old (6-14 meters tall, 10-17 cm in diameter at breast height) Japanese black pines were selected as the drug treatment group (Example 1 group), and 6 groups of 50 Japanese black pines of the same size were selected as the control group. In the drug treatment group, three holes with a diameter of about 1 cm were drilled at a height of 1-1.5 meters above the ground, and each hole was injected with 2×10⁻⁶ microspheres of *Ischemicum oryzae* spores from Example 1. 8 (CFU / mL, 5mL / well). In the control group, three holes approximately 1 cm in diameter were drilled in the pine trees at a height of 1-1.5 meters above the ground, and each hole was filled with an equal amount of sterilized water. One month later, 20,000 nematodes were inoculated into the pine trees in both the treatment group and the control group. The survival rate of the pine trees was calculated in December as follows: Figure 3 .

[0121] Experiment 2: Fuxing Forest Farm, Lai'an County, Anhui Province. At the beginning of the year, 200 20-25 year old (6-14 meters tall, 10-17 cm diameter at breast height) Japanese black pines were selected as the treatment group (Example 1). 1200 Japanese black pines of the same size were selected as the control group 5 kilometers away. On cloudy days, the pine trees in the treatment group were sprayed with *Ischemicum oryzae* spore microspheres using a stretcher sprayer. For the prevention and control of pine wilt disease, the fungal spore microspheres were prepared with a spore count of 1×10⁻⁶. 8 A dispersion of CFU / mL was sprayed evenly using a stretcher sprayer (droplet diameter 200-300μm), dosage: 50 mL / plant.

[0122] The same amount of sterile water was sprayed onto the control group pine trees in the same manner. Both the treatment group and the control group were infected with pine sawyer beetles naturally, without artificial infection with pine wood nematodes. The survival rate of the pine trees was calculated in December as follows: Figure 4 .

[0123] In summary, whether by injection or spraying, the *Ischemicum oryzae* spore microencapsulation agent of this invention can effectively control pine wilt disease, with the survival rate of pine trees in the treated group being significantly higher than that in the control group. This achieves effective control of pine wilt disease.

[0124] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A fungal saccharin agent for killing Illicium erinaceus, characterized in that, The product comprises the following raw materials: *Isodon oryzae* fungal spore liquid, carrier material, additives, and active ingredients; the spore content of the *Isodon oryzae* fungal spore liquid is ≥3×10⁻⁶. 8 CFU / mL; 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%; the additive components include potassium penicillin G, streptomycin, and humic acid; the active component is an extract of sea buckthorn pomace, and the specific preparation method is as follows: (1) Rinse the residue after juicing the sea buckthorn fruit with clean water to remove residual juice and seed fragments, drain the water, spread it evenly on a tray, dry it with hot air at 40-50℃ until the moisture content is ≤8%, crush it through a 40-mesh sieve, seal it and store it in the dark for later use. (2) Degreasing treatment: The dried and pulverized sea buckthorn pomace in step (1) is mixed with petroleum ether at a material-liquid ratio of 1g:8mL, and extracted with sound-assisted extraction at 40-45℃ for 30-40 minutes, repeated 3 times; the oil-containing petroleum ether layer is removed by centrifugation; the solid obtained by centrifugation is dried in a ventilated environment at 50-60℃ to obtain degreased pomace. (3) Mix the defatted fruit pomace with a 60% ethanol solution at a ratio of 1g:20mL, premix with magnetic stirring for 5 minutes, then treat in a constant temperature water bath at 50-55℃ for 30-45 minutes, stirring once every 10 minutes, centrifuge, collect supernatant 1, treat the filter residue again in a constant temperature water bath at 50-55℃ for 30-45 minutes, centrifuge, collect supernatant 2, combine the two supernatants, remove impurities by microfiltration membrane, and obtain a reddish-brown liquid; (4) The liquid was rotary evaporated and concentrated to 1 / 10 of the original volume. 1% of the volume of chitosan oligosaccharide was added to the concentrated liquid. The pH of the system was adjusted to 6-7 using sodium citrate. After sterilization, the extract of sea buckthorn pomace was obtained.

2. A method for preparing the *Illicium erinaceus* fungal saccharifying agent according to claim 1, characterized in that, The preparation steps include the following: (1) Preparation of fungal spores of *Ischemicula ebeni*: *Ischemicula ebeni* was inoculated onto PDA medium and cultured at 25°C for 2 weeks. Colonies were then picked and inoculated onto PDB medium and cultured on a shaker at 130 rpm at 25°C for 2 weeks. After culture, the culture was filtered through gauze to remove mycelia, and sterile water was added to adjust the concentration to 3 × 10⁻⁶. 8 CFU / mL was used to obtain fungal spores of *Echinococcus ileus*. (2) Take 500 mL of fungal spore solution of Echinococcus ileiflora and 500 mL of carrier material and mix them evenly. Add potassium penicillin G and streptomycin to adjust the concentration of the two in the system to 100 ppm and 50 ppm respectively. Then add 3% humic acid by weight of the mixture and use a stirrer to mix the solution evenly. (3) Preparation of active components; (4) Add 3% of the active component by mass to the solution from step (2), mix well and pour into a funnel with a micropipette tip. Slowly drop the solution into a 3 mol / L calcium chloride solution to obtain round spore microspheres with a diameter of about 2-3 mm. (5) After collecting the microspheres, rinse them with sterile water and dry them to obtain the capsule agent.

3. The application of the E. elanis nematicide fungal saccharin of claim 1 in the prevention and control of pine wilt disease.

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