A microbial preparation for controlling plant root-knot nematodes, its preparation method and application

Abstract

This application discloses a microbial preparation for controlling plant root-knot nematodes, its preparation method, and its application, belonging to the field of microbial preparation technology. The microbial preparation for controlling plant root-knot nematodes has a double-layered structure, including an inner layer and an outer layer. The inner layer includes fermentation broths of *Phaeodactylogyrus rubrum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus thuringiensis*, and *Beauveria bassiana*, and an inner carrier material. The outer layer includes fermentation broth of *Streptomyces niger* and an outer carrier material. This application utilizes a combination of fermentation broths of *Phaeodactylogyrus rubrum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus thuringiensis*, *Beauveria bassiana*, and *Streptomyces niger*, achieving synergistic effects and complementary functions. The double-layered structure, on the one hand, adapts to acidic soils, ensuring the initial activity of the strains and improving their survival efficiency; on the other hand, it extends the shelf life and functional period, achieving long-term pest control.

Description

Technical Field

[0001] This application relates to a microbial preparation for controlling plant root-knot nematodes, its preparation method, and its application, belonging to the field of microbial preparation technology. Background Technology

[0002] Root-knot nematode disease is one of the most destructive soil-borne diseases in global agricultural production, widely affecting more than 2,000 plant species, including vegetables, fruit trees, and cash crops. The pathogenic nematode invades the plant root system, forming giant cells that cause abnormal swelling of the root tissue, forming "root knots." This severely hinders the absorption of water and nutrients, causing plant wilting, stunted growth, and yield losses of 30%–80%, or even total crop failure.

[0003] Currently, the main methods for controlling root-knot nematodes include chemical control, physical control, and biological control. Chemical control commonly uses fumigants (such as methyl bromide) and non-fumigation chemical nematicides (such as abamectin and thiazophos). Although these methods are fast-acting, long-term use of a single chemical agent can easily lead to nematode resistance, and chemical residues can pollute soil, water bodies, and crops, threatening the ecological environment and the quality and safety of agricultural products, which does not meet the needs of green agriculture development. Physical control (such as soil fumigation and crop rotation) is complex to operate, costly, and limited by land resources and planting patterns, making it difficult to promote its application in large-scale production. Therefore, biological control technology, with microbial agents at its core, has become the mainstream development direction for root-knot nematode control due to its advantages such as environmental friendliness, no residue, and sustainable pest control.

[0004] Currently, *Paecilomyces lilacinus* has been found to have strong parasitic capabilities and is a relatively effective natural enemy fungus for controlling root-knot nematodes. However, single species are difficult to effectively kill root-knot nematodes, exhibiting relatively low activity and failing to leverage the ecological effects of microorganisms in the soil rhizosphere. Chinese Patent CN 114424776 B discloses a compound microbial agent for controlling southern root-knot nematodes and its preparation method. Specifically, it discloses that a compound microbial powder prepared from four species achieves a control rate of over 80% for southern root-knot nematode disease, significantly superior to the control effect of single microbial powders. This patent uses a combination of four bacterial strains, which is more effective at killing nematodes than a single strain. However, it still has many shortcomings: the optimal growth pH for the four bacterial strains in the patent is 6.5-7.5, while the soil in areas with severe root-knot nematode infestations is mostly acidic with a pH of 5.5-6.8. Under acidic conditions, the activity of the bacterial strains decreases, directly leading to a significant reduction in the efficiency of killing root-knot nematodes. In addition, the patent simply mixes the bacterial strains to obtain a compound strain, which makes it easy to lose and has a short shelf life. The control effect decreases over time, which in turn affects the control effect of root-knot nematodes and fails to meet the need for long-term pest control.

[0005] Therefore, providing a microbial agent that is suitable for the acidic soil environment of areas with severe root-knot nematode infestation and can effectively control the pest for a long time has become an urgent need for root-knot nematode control. Summary of the Invention

[0006] To address the aforementioned issues, a microbial preparation for controlling plant root-knot nematodes, along with its preparation method and application, is provided. This preparation utilizes a compound of six microbial strains: *Phaeodactylogyrus rubrum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus thuringiensis* fermentation broth, *Beauveria bassiana* fermentation broth, and *Streptomyces niger*. These six strains work synergistically to achieve complementary functions. The double-layered coating structure is adapted to acidic soils, ensuring the initial activity of the strains and improving their survival efficiency. Furthermore, it extends the shelf life and functional duration, achieving long-term pest control.

[0007] According to one aspect of this application, a microbial preparation for controlling plant root-knot nematodes is provided. The microbial preparation has a double-layered structure, comprising an inner layer and an outer layer. The inner layer comprises fermentation broth of *Phaeodactylogyrus rubrum*, fermentation broth of *Paecilomyces lilacinus*, fermentation broth of *Bacillus subtilis*, fermentation broth of *Bacillus thuringiensis*, fermentation broth of *Beauveria bassiana*, and an inner carrier material. The outer layer comprises fermentation broth of *Streptomyces nigra* and an outer carrier material. The volume-to-mass ratio of the total fermentation broth of the inner layer to the inner carrier material is 100 ml: (5-8) g, and the volume-to-mass ratio of the *Streptomyces nigra* fermentation broth to the outer carrier material is 100 ml: (1.5-3.5) g.

[0008] Specifically, this application utilizes a double-layered coating structure, with the fermentation broths of *Phaeophyte rubrum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus thuringiensis*, and *Beauveria bassiana* encapsulated in the inner layer, and *Streptomyces niger* fermentation broth placed in the outer layer. On the one hand, the inner layer of microorganisms achieves metabolic mutual supply, and the inner layer carrier material provides a pH buffer for the microorganisms, protecting them from direct inhibition by acidic soil. On the other hand, *Streptomyces niger*, placed in the outer layer, rapidly releases its metabolic products into the soil, completing pH adjustment and pathogen inhibition in advance, creating a suitable growth environment for the microorganisms in the inner layer, and also providing nitrogen and carbon sources for fungal growth, promoting mycelial expansion and spore proliferation. The synergistic effect of the inner and outer layer microorganisms effectively improves the survival rate of the strains.

[0009] Specifically, this application sets specific limits on the volume-to-mass ratio of the total fermentation broth of the inner layer to the inner carrier material, and the volume-to-mass ratio of the *Streptomyces niger* fermentation broth to the outer carrier material. Within these ratios, the mechanical strength and sphericity of the coating structure are guaranteed, while also considering the encapsulation rate and the rupture speed.

[0010] Optionally, the volume ratio of the fermentation broth of *Phaeophyte rubrum*, fermentation broth of *Paecilomyces lilacinus*, fermentation broth of *Bacillus subtilis*, fermentation broth of *Bacillus thuringiensis*, fermentation broth of *Beauveria bassiana*, and fermentation broth of *Streptomyces niger* is (2~3):(4~5):(1.5~2.5):1:1:1.

[0011] Specifically, this application sets specific limits on the volume ratios of the fermentation broths of *Phaeophyte rubrum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus thuringiensis*, *Beauveria bassiana*, and *Streptomyces niger*. This ensures the core function of killing root-knot nematodes while maximizing the synergistic effect of the strains and achieving a supply-demand balance in metabolic exchange.

[0012] Specifically, *Paecilomyces lilacinus* is the core strain for killing root-knot nematodes, occupying a dominant proportion; *Phaeodactylum rubrum*, as the core of microenvironment regulation and nutrient transformation, is second only to *Paecilomyces lilacinus* in proportion, requiring sufficient secretion of metabolic products such as glucose and amino acids to support the growth and functional expression of *Paecilomyces lilacinus* and *Bacillus subtilis*; *Bacillus subtilis* is a colonization and protection strain, not requiring an excessively high proportion, as its biofilm-forming ability is strong, and a small amount of the strain can encapsulate the other two fungi in the inner layer, while an excessive amount will lead to nutrient competition; *Bacillus thuringiensis* focuses on producing parasporal crystals to kill second-instar larvae of root-knot nematodes through contact and secreting chitinase to assist in the degradation of the nematode's body wall, while *Beauveria bassiana* can penetrate the body wall of adult nematodes and older larvae to parasitize and kill them. Synergistically killing root-knot nematodes with Paecilomyces lilacinus, achieving complete coverage of eggs, larvae, and adults; Streptomyces nigra is an environmentally optimized strain, whose metabolites, such as glucosamine and antibiotics, have a wide range of effects, enabling soil pH regulation and pathogen inhibition. Excessive alkalinity can affect the inner layer of microorganisms due to the accumulation of alkaline metabolites; the appropriate ratio of Paecilomyces lilacinus to Pharfovia rubescens achieves a balance between nutrient supply and demand; the appropriate ratio of Bacillus subtilis with Paecilomyces lilacinus and Pharfovia rubescens results in a suitable biofilm; the appropriate ratio of Streptomyces nigra to the inner layer of mixed microorganisms synergistically regulates pH, and the metabolites of Streptomyces nigra initially regulate soil pH to obtain a suitable rhizosphere pH, matching the optimal pH environment for the microorganisms.

[0013] Optionally, the inner carrier material comprises 1.5-1.8 parts sodium alginate, 2.0-2.5 parts chitin powder, 0.3-0.5 parts turmeric extract, 0.1-0.2 parts sophora flavescens extract, and 2.0-2.2 parts modified starch; the outer carrier material comprises 1.0-1.3 parts chitosan, 0.5-0.6 parts humic acid oligosaccharide, 0.2-0.5 parts cinnamon extract, 0.15-0.25 parts neem bark extract, and 0.2-0.3 parts rhizosphere adsorbent.

[0014] Specifically, sodium alginate serves as the core framework of the gel microspheres, uniformly encapsulating the fermentation broth of the inner layer of bacteria and reducing the rate of bacterial inactivation. The gel microspheres possess excellent hydrophilicity and porous structure, allowing them to slowly swell in the soil. Working in conjunction with modified starch, they enable the gradual release of the bacteria, avoiding the waste of function caused by a one-time release and extending the duration of effectiveness of the inner layer of bacteria. Chitin powder serves as a carbon source and degrades to produce chitin oligosaccharides, inducing Paecilomyces lilacinus to secrete chitinase, thereby enhancing the function of killing root-knot nematodes.

[0015] Specifically, the addition of Curcuma zedoaria extract and Sophora flavescens extract to the inner carrier material can, on the one hand, kill root-knot nematodes, and on the other hand, synergize with Paecilomyces lilacinus, Pharfovia rubescens, and Bacillus subtilis in the inner layer to have long-lasting antibacterial, growth-promoting, and stress-resistant effects on the microorganisms. It can be slowly released with the inner microspheres to achieve long-lasting synergistic effects.

[0016] Specifically, in the outer carrier material, chitosan forms a slow-release film on the surface of the inner microspheres, controlling the release rate, avoiding initial burst release, and prolonging the duration of effect. Humic acid oligosaccharides are beneficial for promoting root growth, can interact with chitosan, and enhance the stability of the outer film; they can also act as signaling molecules to improve rhizosphere targeting.

[0017] Specifically, cinnamon extract and neem bark extract are added to the outer carrier material. Cinnamon extract is volatile and has a fast-acting bactericidal and nematode-inhibiting effect, directly killing second-instar larvae of root-knot nematodes and rapidly inhibiting pathogens in the soil. Neem bark extract has a strong contact killing effect on soil-borne nematodes such as root-knot nematodes and root-rot nematodes, and inhibits egg hatching. When neem extract and cinnamon extract are combined with the outer layer components, they can be rapidly released along with the metabolites of Streptomyces niger, achieving rapid initial pest control and creating a favorable rhizosphere environment for the inner layer of fungi.

[0018] However, the combination of the inner and outer carrier materials mentioned above cannot regulate the pH of the inner microspheres and rhizosphere microdomains. The effect of killing root-knot nematodes is difficult to improve after reaching a certain level. In order to further improve the effect of killing root-knot nematodes, further improvements are needed to the inner and outer carrier materials.

[0019] Optionally, the inner carrier material further includes 0.8 to 1 part by weight of dolomite powder; The outer carrier material also includes 0.3 to 0.4 parts of chitinase inducer.

[0020] Specifically, dolomite powder can regulate the pH of the inner microspheres and rhizosphere microdomains to match the optimal growth pH of the inner microbial species, reducing the attenuation of the species in acidic soil. Chitinase inducers can specifically induce *Paecilomyces lilacinus* and *Streptomyces nigra* to secrete chitinase, efficiently degrading the chitin components of the root-knot nematode body wall and enhancing the nematode-killing effect. Simultaneously, chitinase inducers can serve as a nutrient source for microorganisms, promoting strain reproduction and further increasing rhizosphere colonization density. Dolomite powder regulates the rhizosphere pH, providing suitable survival conditions for *Paecilomyces lilacinus*, while chitinase inducers induce *Paecilomyces lilacinus* to secrete chitinase, strengthening the nematode-killing effect. Both work synergistically and are indispensable.

[0021] Under the action of the aforementioned inner and outer carrier materials, a good effect can be achieved in killing root-knot nematodes. However, it was also found that during the process of promoting the growth of dominant bacterial groups, the number of miscellaneous bacteria also increases in order to compete for nutrients and living space, thus limiting the colonization advantage of dominant bacterial groups.

[0022] Optionally, the inner carrier material may further include 0.4 parts of polylysine by weight.

[0023] Specifically, polylysine, as a natural antibacterial agent, prevents miscellaneous bacteria from competing with inner-layer bacteria for nutrients and living space, thus ensuring the colonization advantage of the dominant bacterial population.

[0024] Specifically, the antibacterial effect of polylysine can reduce the consumption of chitinase inducers by other bacteria, ensuring that the inducers can continuously induce the strain to secrete chitinase; while the chitinase inducers promote the reproduction of the strain, they can also enhance the strain's tolerance to polylysine, avoiding the inhibitory effect of excessively high polylysine concentrations on the strain. The two work synergistically to promote the aggregation of beneficial bacteria in the rhizosphere, further inhibiting the infection of root-knot nematodes.

[0025] Optionally, the modified starch is corn starch pretreated with α-amylase, and the pretreatment conditions are 60-65℃ for 30-40 min; and / or The rhizosphere adsorbent comprises sodium-modified bentonite and potassium humate, wherein the mass ratio of sodium-modified bentonite to potassium humate is 1:(0.9~1.1); and / or The chitinase inducer includes chitin oligosaccharides.

[0026] Specifically, chitin oligosaccharides were used to induce high expression of chitinase genes in Paecilomyces lilacinus, thereby enhancing the degradation ability of root-knot nematode eggshells. Chitin oligosaccharides are natural polysaccharide degradation products that can be metabolized and utilized by Pharrellis rubrum, without wasting resources, and can further promote the growth of the strain.

[0027] Specifically, the layered structure of sodium-modified bentonite possesses extremely strong cation exchange capacity and adsorption properties, specifically adsorbing organic acids such as citric acid and malic acid secreted by crop roots. This guides the formulation to actively migrate to the rhizosphere, solving the problem of strain loss due to water diffusion. Potassium humate serves two purposes: firstly, as a potassium source to promote crop growth; and secondly, by combining with the interlayer structure of bentonite, enhancing the stability of the adsorbent. Simultaneously, it provides nutrients for *Streptomyces nigra*, promoting its antibiotic secretion. The optimal mass ratio of sodium-modified bentonite to potassium humate ensures that these effects are maximized in the outer carrier material. Furthermore, it assists the inner carrier material in regulating rhizosphere pH, enhancing the activity of dominant bacterial communities, and further improving the efficacy against root-knot nematodes.

[0028] Optionally, in the microbial preparation, the viable count of the *Pharbitis erythropoiesis* fermentation broth is ≥5.0 × 10⁻⁶. 8 CFU / mL, viable count of *Paecilomyces lilacinus* fermentation broth ≥ 6.0 × 10⁻⁶ 8 CFU / mL, conidial concentration ≥5.0×10 8 The viable count of Bacillus subtilis fermentation broth is ≥8.0 × 10⁶ cells / mL. 8 CFU / mL, viable count of Bacillus thuringiensis fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, viable cell count of Beauveria bassiana fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, conidial concentration ≥4.0×10 8 The viable count of *Streptomyces cerevisiae* fermentation broth is ≥5.0 × 10⁻⁶ cells / mL. 8 CFU / mL.

[0029] According to another aspect of this application, a method for preparing the above-mentioned microbial agent for controlling plant root-knot nematodes is also provided, comprising the following steps: (1) Prepare fermentation broths of Rhodopseudomonas rubrum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Streptomyces niger according to volume ratios, and mix the fermentation broths of Rhodopseudomonas rubrum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana to obtain an inner mixed fermentation broth; (2) Weigh the inner layer carrier material according to the proportion, mix and dissolve, sterilize and cool to room temperature to obtain the inner layer carrier material liquid; mix the inner layer carrier material liquid with the inner layer mixed fermentation broth and stir to form a suspension; (3) The suspension was uniformly dropped into the calcium chloride curing solution and cured at a constant temperature. After rinsing and filtering, the inner microspheres were obtained. (4) After mixing and dissolving the outer carrier material, sterilize and cool to room temperature, mix with the fermentation broth of Streptomyces niger, coat the inner microspheres with a roller coating method, cure at a constant temperature, dry and sieve to obtain the microbial preparation.

[0030] Specifically, the preparation method of Rhodopseudomonas erythrosporum fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (30 g / L glucose, 20 g / L corn steep liquor, 5 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, pH 6.5) at an inoculation rate of 5% (v / v). Fermentation parameters: cultured at 28℃ and 200r / min for 48h with shaking. During the fermentation process, 1% (v / v) glucose solution was added every 12h to maintain sufficient carbon source. Endpoint indicator: viable cell count in fermentation broth ≥ 5.0 × 10⁻⁶ 8CFU / mL, polysaccharide content ≥2.0g / L.

[0031] The preparation method of Paecilomyces lilacinus fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (corn flour 30 g / L, shrimp meal 10 g / L, sucrose 5 g / L, potassium dihydrogen phosphate 0.8 g / L, magnesium sulfate 0.4 g / L, pH 6.5) at an inoculation rate of 8% (v / v). Fermentation parameters: 25℃, 180r / min shaking culture for 72h, the first 24h in the dark, the last 48h in the light (light intensity 2000 lux, 12h light / 12h dark) to promote spore formation; Endpoint indicator: viable cell count in fermentation broth ≥ 6.0 × 10⁻⁶ 8 CFU / mL, conidial concentration ≥5.0×10 8 Chitinase activity ≥15 U / mL.

[0032] The preparation method of Bacillus subtilis fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (soybean meal powder 25 g / L, corn starch 15 g / L, yeast extract 3 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.8 g / L, pH 7.2) at an inoculation rate of 3% (v / v). Fermentation parameters: 30℃, 220r / min shaking culture for 36h, add 0.5% (w / v) calcium carbonate at 24h of fermentation, adjust pH to stabilize at 7.0~7.2 to promote spore formation; Endpoint indicator: viable cell count in fermentation broth ≥ 8.0 × 10⁻⁶ 8 CFU / mL, spore rate ≥90%, extracellular polysaccharide content ≥1.5g / L.

[0033] The preparation method of Bacillus thuringiensis fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (soybean meal 35g / L, corn starch 20g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate 0.8g / L, calcium chloride 0.6g / L, manganese sulfate 0.05g / L, pH 7.3) at an inoculation rate of 4% (v / v). Fermentation parameters: 30℃, 230r / min shaking culture for 48h, 1% (w / v) corn starch solution was added at 36h of fermentation to maintain sufficient carbon source to promote the formation of spores and parasporal crystals; Endpoint indicator: viable cell count in fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, spore rate ≥90%, parasporal crystal content ≥1.2mg / mL.

[0034] The preparation method of Beauveria bassiana fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (corn flour 25 g / L, shrimp meal 8 g / L, sucrose 10 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, yeast extract 2 g / L, pH 6.6) at an inoculation rate of 8% (v / v). Fermentation parameters: 25℃, 180r / min shaking culture for 72h, the first 24h in the dark, the last 48h in the light (light intensity 1500 lux, 12h light / 12h dark) to promote conidia formation; Endpoint indicator: viable cell count in fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, conidial concentration ≥4.0×10 8 Cells / mL, chitinase activity ≥12U / mL, mycelial dry weight ≥8g / L.

[0035] The preparation method of Streptomyces fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (soluble starch 30 g / L, soybean meal 20 g / L, calcium carbonate 2 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, pH 7.2) at an inoculation rate of 10% (v / v). Fermentation parameters: cultured at 28℃ and 160r / min for 96h with shaking. During the fermentation process, pH was measured every 24h. If the pH was lower than 7.0, 0.1mol / L sodium hydroxide solution was added to maintain the pH at 7.0~7.2. Endpoint indicator: viable cell count in fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, antibiotic (streptomycin) content ≥100μg / mL.

[0036] Specifically, this application first prepares inner microspheres, and then uses a roller coating method to coat the outer carrier material to form a double-layer coating structure, which corresponds to the "outer layer releases first, inner layer releases later" sequence in field application, effectively extending the shelf life and matching acidic soils with recurrent root-knot nematodes.

[0037] Specifically, chitosan is a cationic polysaccharide that forms electrostatic adsorption with the surface of the inner microspheres, providing adhesion for subsequent coating. The inner microspheres continuously roll in the roller coating machine, and the sprayed coating liquid adheres evenly to the surface of the microspheres due to the adhesiveness and electrostatic adsorption of chitosan, allowing the outer material and Streptomyces to gradually form a dense and uniform coating, thereby achieving the slow release of the strain.

[0038] Optionally, the mass concentration of the calcium chloride curing liquid in step (3) is 1.8% to 2.0%, the curing temperature is 28 to 30°C, and the curing time is 10 to 12 hours; the curing temperature in step (4) is 25 to 27°C, the curing time is 8 to 9 hours; the drying temperature is 35 to 40°C, and the moisture content of the preparation after drying is ≤8%.

[0039] Specifically, the dropping rate of the suspension is 25-30 drops / min, and the dropping height is 15-20 cm.

[0040] Specifically, this application sets specific limits on parameters such as curing liquid, temperature, and time to ensure more complete cross-linking, thereby forming a stable three-dimensional network gel structure and obtaining sufficient mechanical strength.

[0041] Specifically, in step (3), the rotation speed of the roller coating machine is 30~35 r / min, and the spraying time is 15~18 min.

[0042] Specifically, during the encapsulation process, chitosan slowly undergoes intermolecular hydrogen bonding, while simultaneously forming electrostatic crosslinks with sodium alginate on the surface of the inner microspheres, allowing the outer coating to form a stable and dense film structure to achieve sustained release.

[0043] According to another aspect of this application, the application of the above-mentioned microbial preparation for controlling plant root-knot nematodes or the microbial preparation prepared by the above-mentioned method in controlling root-knot nematodes is also provided.

[0044] The beneficial effects of this application include, but are not limited to: 1. The microbial preparation for controlling plant root-knot nematodes according to this application uses a compound of six strains: Pharbitis erythrosporum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis fermentation broth, Beauveria bassiana fermentation broth, and Streptomyces niger. The six strains work synergistically to achieve complementary functions. The double-layer coating structure is adapted to acidic soils, ensuring the initial activity of the strains and improving their survival efficiency. On the other hand, it can extend the shelf life and functional period, achieving long-term pest control.

[0045] 2. According to the microbial preparation for controlling plant root-knot nematodes of this application, this application introduces acid-resistant Pharbitis erythrosporum, combined with the chemical buffering effect of dolomite powder in the inner carrier material and the alkaline metabolite regulation function of Streptomyces cyclohexane in the outer layer, to synergistically regulate the rhizosphere micro-pH of acidic soil in areas with severe root-knot nematode infestation, match the optimal growth environment of the strain, improve the retention rate of strain activity under acidic conditions, and enhance the control effect of root-knot nematodes.

[0046] 3. According to the preparation method of the microbial agent for controlling plant root-knot nematodes in this application, this application adopts a stepwise process of inner layer drip-addition and solidification and outer layer roll coating. No special equipment is required. The carrier materials are all natural biodegradable polymers and minerals, with no chemical residues and environmental friendliness, which meets the needs of green agricultural development. The raw materials are inexpensive and readily available, with high production efficiency and good batch stability, which has the conditions for large-scale industrial production and field promotion. The preparation process is scientific and controllable, taking into account both greenness and industrial application value. Detailed Implementation

[0047] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described in this patent are for illustrative purposes only.

[0049] The *Phaeophyra erythrorhizon*, *Paecilomyces lilacinus*, *Bacillus subtilis*, and *Streptomyces niger* used in the following examples are all existing strains available for purchase from the China Microbiological Culture Collection Center. This does not involve the development of new strains, but only the application of these existing strains. Furthermore, the *Phaeophyra erythrorhizon* used in the following examples is selected from strain CGMCC2.1557, *Paecilomyces lilacinus* from strain ACCC 32162, *Bacillus subtilis* from strain CGMCC No. 8546, *Bacillus thuringiensis* from strain CGMCC1.189, *Beauveria bassiana* from strain ACCC30006, and *Streptomyces niger* from strain CGMCC 4.1443.

[0050] The fermentation broths of *Phaeodactylogyrus rubrum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, and *Streptomyces niger* used in the following examples and comparative examples were prepared by the following methods.

[0051] The preparation method of Rhodopseudomonas erythrosporum fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (30 g / L glucose, 20 g / L corn steep liquor, 5 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, pH 6.5) at an inoculation rate of 5% (v / v). Fermentation parameters: cultured at 28℃ and 200r / min for 48h with shaking. During the fermentation process, 1% (v / v) glucose solution was added every 12h to maintain sufficient carbon source. Endpoint indicator: viable cell count in fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, polysaccharide content ≥2.0g / L.

[0052] The preparation method of Paecilomyces lilacinus fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (corn flour 30 g / L, shrimp meal 10 g / L, sucrose 5 g / L, potassium dihydrogen phosphate 0.8 g / L, magnesium sulfate 0.4 g / L, pH 6.5) at an inoculation rate of 8% (v / v). Fermentation parameters: 25℃, 180r / min shaking culture for 72h, the first 24h in the dark, the last 48h in the light (light intensity 2000 lux, 12h light / 12h dark) to promote spore formation; Endpoint indicator: viable cell count in fermentation broth ≥ 6.0 × 10⁻⁶ 8 CFU / mL, conidial concentration ≥5.0×10 8 Chitinase activity ≥15 U / mL.

[0053] The preparation method of Bacillus subtilis fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (soybean meal powder 25 g / L, corn starch 15 g / L, yeast extract 3 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.8 g / L, pH 7.2) at an inoculation rate of 3% (v / v). Fermentation parameters: 30℃, 220r / min shaking culture for 36h, add 0.5% (w / v) calcium carbonate at 24h of fermentation, adjust pH to stabilize at 7.0~7.2 to promote spore formation; Endpoint indicator: viable cell count in fermentation broth ≥ 8.0 × 10⁻⁶ 8 CFU / mL, spore rate ≥90%, extracellular polysaccharide content ≥1.5g / L.

[0054] The preparation method of Bacillus thuringiensis fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (soybean meal 35g / L, corn starch 20g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate 0.8g / L, calcium chloride 0.6g / L, manganese sulfate 0.05g / L, pH 7.3) at an inoculation rate of 4% (v / v). Fermentation parameters: 30℃, 230r / min shaking culture for 48h, 1% (w / v) corn starch solution was added at 36h of fermentation to maintain sufficient carbon source to promote the formation of spores and parasporal crystals; Endpoint indicator: viable cell count in fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, spore rate ≥90%, parasporal crystal content ≥1.2mg / mL.

[0055] The preparation method of Beauveria bassiana fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (corn flour 25 g / L, shrimp meal 8 g / L, sucrose 10 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, yeast extract 2 g / L, pH 6.6) at an inoculation rate of 8% (v / v). Fermentation parameters: 25℃, 180r / min shaking culture for 72h, the first 24h in the dark, the last 48h in the light (light intensity 1500 lux, 12h light / 12h dark) to promote conidia formation; Endpoint indicator: viable cell count in fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, conidial concentration ≥4.0×10 8 Cells / mL, chitinase activity ≥12U / mL, mycelial dry weight ≥8g / L.

[0056] The preparation method of Streptomyces fermentation broth is as follows: Seed culture inoculation: Inoculate the activated seed culture into the fermentation medium (soluble starch 30 g / L, soybean meal 20 g / L, calcium carbonate 2 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, pH 7.2) at an inoculation rate of 10% (v / v). Fermentation parameters: cultured at 28℃ and 160r / min for 96h with shaking. During the fermentation process, pH was measured every 24h. If the pH was lower than 7.0, 0.1mol / L sodium hydroxide solution was added to maintain the pH at 7.0~7.2. Endpoint indicator: viable cell count in fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, antibiotic (streptomycin) content ≥100μg / mL.

[0057] The modified starch was prepared as follows: corn starch was pretreated with α-amylase at 60℃ for 40 min.

[0058] Example 1 A method for preparing a microbial agent for controlling plant root-knot nematodes: (1) The fermentation broths of Rhodopseudomonas erythrosporum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Streptomyces spp. were prepared in a volume ratio of 2:4:1.5:1:1:1. The fermentation broths of Rhodopseudomonas erythrosporum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Streptomyces spp. were prepared separately. The fermentation broths of Rhodopseudomonas erythrosporum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, and Streptomyces spp. were then mixed to obtain an inner mixed fermentation broth. (2) Weigh out 1.5 parts of sodium alginate, 2.0 parts of chitin powder, 0.3 parts of turmeric extract, 0.1 parts of sophora flavescens extract, and 2.0 parts of modified starch according to the proportion. Mix and dissolve them, then sterilize and cool to room temperature to obtain the inner layer carrier material liquid. Mix the inner layer carrier material liquid with the inner layer mixed fermentation liquid. The volume-to-mass ratio of the inner layer mixed fermentation liquid to the inner layer carrier material is 100ml:5g. Stir to form a suspension. (3) The suspension was uniformly dropped into the calcium chloride curing solution and cured at a constant temperature. The mass concentration of the calcium chloride curing solution was 1.8%, the curing temperature was 28℃, and the curing time was 10h. The dropping rate of the suspension was 25 drops / min, the dropping height was 15cm, and the inner layer microspheres were obtained by rinsing and filtering. (4) Mix 1.0 part of chitosan, 0.5 part of humic oligosaccharide, 0.2 part of cinnamon extract, 0.15 part of neem bark extract, and 0.2 part of rhizosphere adsorbent, dissolve them, sterilize and cool to room temperature. The rhizosphere adsorbent includes sodium-modified bentonite and potassium humate, with a mass ratio of sodium-modified bentonite to potassium humate of 1:0.9. The volume mass ratio of the total fermentation broth of the inner layer to the outer carrier material is 100ml:1.5g. Then mix with the fermentation broth of Streptomyces niger and coat the inner microspheres with a roller coating method. After constant temperature curing, dry and sieve. The roller coating machine speed is 30r / min, the spraying time is 15min, the curing temperature is 25℃, the curing time is 8h, the drying temperature is 35℃, and the moisture content of the preparation after drying is ≤8%, thus obtaining the microbial preparation.

[0059] Example 2 A method for preparing a microbial agent for controlling plant root-knot nematodes: (1) The fermentation broths of Rhodopseudomonas erythrosporum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Streptomyces spp. were prepared in a volume ratio of 3:5:2.5:1:1:1. The fermentation broths of Rhodopseudomonas erythrosporum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Streptomyces spp. were prepared separately. The fermentation broths of Rhodopseudomonas erythrosporum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, and Streptomyces spp. were then mixed to obtain an inner mixed fermentation broth. (2) Weigh out 1.8 parts of sodium alginate, 2.5 parts of chitin powder, 0.5 parts of turmeric extract, 0.2 parts of sophora flavescens extract, and 2.2 parts of modified starch according to the proportion. Mix and dissolve them, then sterilize and cool to room temperature to obtain the inner layer carrier material liquid. Mix the inner layer carrier material liquid with the inner layer mixed fermentation liquid. The volume-to-mass ratio of the inner layer mixed fermentation liquid to the inner layer carrier material is 100ml:8g. Stir to form a suspension. (3) The suspension was uniformly dripped into the calcium chloride curing solution and cured at a constant temperature. The mass concentration of the calcium chloride curing solution was 2.0%, the curing temperature was 30℃, and the curing time was 12h. The dripping rate of the suspension was 30 drops / min, the dripping height was 20cm, and the inner layer microspheres were obtained by rinsing and filtering. (4) 1.3 parts of chitosan, 0.6 parts of humic oligosaccharide, 0.5 parts of cinnamon extract, 0.25 parts of neem bark extract, and 0.3 parts of rhizosphere adsorbent were mixed, dissolved, sterilized, and cooled to room temperature. The rhizosphere adsorbent included sodium-modified bentonite and potassium humate, with a mass ratio of sodium-modified bentonite to potassium humate of 1:1.1. The volume mass ratio of the total fermentation broth of the inner layer to the outer carrier material was 100 ml: 3.5 g. The mixture was then mixed with the fermentation broth of Streptomyces niger and coated onto the surface of the inner microspheres using a roller coating method. After constant temperature curing, the mixture was dried and sieved. The roller coating machine speed was 35 r / min, the spraying time was 18 min, the curing temperature was 27℃, the curing time was 9 h, and the drying temperature was 40℃. After drying, the moisture content of the preparation was ≤8%, thus obtaining the microbial preparation.

[0060] Example 3 A method for preparing a microbial agent for controlling plant root-knot nematodes: (1) The fermentation broths of Rhodopseudomonas rubrum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Streptomyces spp. were prepared in a volume ratio of 3:4:2:1:1:1. The fermentation broths of Rhodopseudomonas rubrum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Streptomyces spp. were prepared separately. The fermentation broths of Rhodopseudomonas rubrum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, and Streptomyces spp. were then mixed to obtain an inner mixed fermentation broth. (2) Weigh out 1.6 parts of sodium alginate, 2.2 parts of chitin powder, 0.4 parts of turmeric extract, 0.15 parts of sophora flavescens extract, and 2.1 parts of modified starch according to the proportion. Mix and dissolve them, then sterilize and cool to room temperature to obtain the inner layer carrier material liquid. Mix the inner layer carrier material liquid with the inner layer mixed fermentation liquid. The volume-to-mass ratio of the inner layer mixed fermentation liquid to the inner layer carrier material is 100ml:6g. Stir to form a suspension. (3) The suspension was uniformly dropped into the calcium chloride curing solution and cured at a constant temperature. The mass concentration of the calcium chloride curing solution was 2.0%, the curing temperature was 29℃, and the curing time was 11h. The dropping rate of the suspension was 28 drops / min, the dropping height was 20cm, and the inner layer microspheres were obtained by rinsing and filtering. (4) 1.2 parts of chitosan, 0.55 parts of humic oligosaccharide, 0.3 parts of cinnamon extract, 0.2 parts of neem bark extract, and 0.25 parts of rhizosphere adsorbent were mixed, dissolved, sterilized, and cooled to room temperature. The rhizosphere adsorbent included sodium-modified bentonite and potassium humate, with a mass ratio of sodium-modified bentonite to potassium humate of 1:1. The volume mass ratio of the total fermentation broth of the inner layer to the outer carrier material was 100 ml: 2.5 g. The mixture was then mixed with the fermentation broth of Streptomyces niger and coated onto the surface of the inner microspheres using a roller coating method. After constant temperature curing, the mixture was dried and sieved. The roller coating machine speed was 30 r / min, the spraying time was 16 min, the curing temperature was 26 ℃, the curing time was 9 h, and the drying temperature was 37 ℃. After drying, the moisture content of the preparation was ≤8%, and the microbial preparation was obtained.

[0061] Example 4 The difference between Example 4 and Example 3 is that the inner carrier material also includes 1 part of dolomite powder, and the outer carrier material also includes 0.3 parts of chitinase inducer; the rest are the same.

[0062] Example 5 The difference between Example 5 and Example 3 is that the inner carrier material also includes 0.8 parts of dolomite powder, and the outer carrier material also includes 0.4 parts of chitinase inducer; the rest are the same.

[0063] Example 6 The difference between Example 6 and Example 4 is that the inner carrier material also includes 0.4 parts of polylysine, while the rest are the same.

[0064] Example 7 The difference between Example 7 and Example 4 is that the outer carrier material does not include 0.4 parts of chitinase inducer, while the rest are the same.

[0065] Example 8 The difference between Example 8 and Example 3 is that the volume ratio of the fermentation broth of *Phaeophyte rubrum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus thuringiensis*, *Beauveria bassiana*, and *Streptomyces niger* is 1:1:1:1:1:1, while all other aspects are the same.

[0066] Example 9 The difference between Example 9 and Example 3 is that the rhizosphere adsorbent does not include potassium humate, but all other aspects are the same.

[0067] Example 10 The difference between Example 10 and Example 3 is that Example 10 does not include Curcuma zedoaria extract, Sophora flavescens extract, Cinnamomum cassia extract, and Melia azedarach bark extract; all other ingredients are the same.

[0068] Example 11 The difference between Example 11 and Example 3 is that Curcuma zedoaria extract and Sophora flavescens extract are placed in the outer carrier material, while Cinnamomum cassia extract and Melia toosendan bark extract are placed in the inner carrier material; all other aspects are the same.

[0069] Comparative Example 1 The difference between Comparative Example 1 and Example 3 is that Comparative Example 1 does not include the Rhodopseudomonas erythrosporum fermentation broth, but all other aspects are the same.

[0070] Comparative Example 2 The difference between Comparative Example 2 and Example 3 is that Trichoderma harzianum fermentation broth was used instead of Rhodopsinia rubra fermentation broth, while the rest were the same.

[0071] Comparative Example 3 The difference between Comparative Example 3 and Example 3 is that Comparative Example 3 is a single-layer coating structure, excluding the outer layer. All other aspects are the same. That is, the fermentation broth of Pharfia rubra, fermentation broth of Paecilomyces lilacinus, fermentation broth of Bacillus subtilis, fermentation broth of Bacillus thuringiensis, fermentation broth of Beauveria bassiana, and fermentation broth of Streptomyces niger are mixed in a certain proportion to form a mixed fermentation broth, which is then mixed with the inner carrier material liquid to form a suspension, and then added dropwise to solidify.

[0072] Comparative Example 4 The difference between Comparative Example 4 and Example 3 is that the volume-to-mass ratio of the inner layer mixed fermentation broth to the inner layer carrier material is 100ml:10g, while all other aspects are the same.

[0073] Comparative Example 5 The difference between Comparative Example 5 and Example 3 is that the total volume of the fermentation broth in the inner layer and the volume-to-mass ratio of the outer carrier material is 100ml:0.5g, while all other aspects are the same.

[0074] Comparative Example 6 The difference between Comparative Example 6 and Example 3 is that Comparative Example 6 does not include the fermentation broth of Bacillus thuringiensis and Beauveria bassiana. Instead, the volume ratio of the fermentation broths of Rhodopseudomonas erythrosporum, Paecilomyces lilacinus, Bacillus subtilis, and Streptomyces niger is 3:4:2:1. The fermentation broths of Rhodopseudomonas erythrosporum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana are mixed to obtain the inner mixed fermentation broth. The total amount of the inner mixed fermentation broth remains unchanged, and all other aspects are the same.

[0075] Experimental Example 1 Rhizosphere microdomain pH test The microbial agents of Examples 1-11 and Comparative Examples 1-6 of this application were applied to acidic soil at a dosage of 2 g / kg soil. The test crop was tomato, and the initial pH of the acidic soil was 5.8. Rhizosphere soil samples (0-2 mm) were collected using a rhizosphere soil sampler at 1, 3, 7, and 14 days after application. The soil pH was measured using a pH meter. Each group was repeated three times, and the average value was taken to obtain the stable value of rhizosphere pH. The test results are shown in Table 1.

[0076] Table 1. Results of pH test in the rhizosphere microsphere

[0077] Table 1 shows that in Examples 1-3 and 8-9, the pH remained stable at 5.9-6.1. This is attributed to the fact that the pH was adjusted solely by *Phaeophyte Rhodotorula* and *Streptomyces niger*, which provided some but limited adjustment. In Examples 4-6, the pH remained stable at 6.7-6.9, indicating that dolomite powder played a crucial role. Example 6 showed the optimal pH, attributed to the addition of polylysine, which prolonged the buffering time of the dolomite powder. Comparative Examples 1 and 2, which altered the bacterial strains, lacked pH adjustment. Comparative Example 3, with its monolayer structure, exhibited faster strain release but slightly weaker sustained pH adjustment. Comparative Example 4, with an excess of inner carrier, improved pH buffering capacity. Comparative Example 5, with an insufficient outer carrier, had a pH adjustment capacity similar to Comparative Example 3.

[0078] Experiment Example 2 Strain activity retention rate The microbial agents of Examples 1-11 and Comparative Examples 1-6 of this application were applied to acidic soil at a dosage of 2 g / kg soil. The test crop was tomato, and the initial pH of the acidic soil was 5.8. Seven days after soil application, the viable counts of *Phaeophyte rubrum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, and *Streptomyces niger* in the soil were determined by the dilution plate count method. The activity retention rate was calculated as (viable count after application / initial viable count) × 100%. The test results are shown in Table 2.

[0079] Table 2 Results of strain viability retention test

[0080] Table 2 shows that the activity retention rates of the strains in Examples 1-3 and Examples 8-9 were between 58% and 65%. The reason for this is that the acidic environment inhibits the activity of the strains. The ratio of the strains in Example 8 was 1:1:1:1, which showed poor synergy and resulted in the lowest retention rate. The retention rates of Examples 4-6 were all above 85%, with Example 6 being the best. Polylysine, as a natural antibacterial agent, prevented other bacteria from competing with the inner layer strains for nutrients and living space, thus ensuring the colonization advantage of the dominant bacterial group. The strains in Comparative Examples 1 and 2 were changed, resulting in activity retention rates below 50%. Comparative Example 3, with its single-layer structure, also had a low retention rate. In Comparative Example 4, the inner layer carrier was excessive and the encapsulation was too thick, preventing the strains from effectively breaking the capsule and inhibiting their metabolism, thus reducing the retention rate. In Comparative Example 5, the outer layer carrier was insufficient, failing to form effective protection and resulting in a decrease in activity retention rate.

[0081] Experimental Example 3 Prevention and control effect test One hundred nematode larvae (second instar) were evenly dispersed into 10 kg of soil (the soil was treated with high temperature to kill insects and was free of root-knot nematodes). After transplanting tomato seedlings, the microbial agents prepared in Examples 1-11 and Comparative Examples 1-6 of this invention were applied to the soil. The initial soil pH was 5.8, and the application rate was 2 g / kg of soil. Water was added to the soil to maintain a soil moisture content of 18-25%. Three replicate plots were set up for each group, with 20 tomato plants in each plot, and the average value was taken. Thirty days after the application of the microbial agent, the mortality rate of root-knot nematodes, the control effect, and the disease index were recorded. Sixty days after the application of the microbial agent, the control effect and the disease index were recorded. The test results are shown in Table 3.

[0082] Grading standards for root-knot nematode disease: Grade 0 (no root knots), Grade 1 (root knot rate ≤ 3%), Grade 2 (root knot rate 3%~25%), Grade 3 (root knot rate 25%~50%), Grade 4 (root knot rate 50%~75%), Grade 5 (root knot rate ≥ 75%). Disease index = [(Σ (number of disease-grade plants × representative grade)) / (total number of plants × highest representative grade value)] × 100; Prevention and control effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100%.

[0083] The control group, which did not receive any microbial agents, had a disease index of 78.5.

[0084] Table 3. Root-knot nematode mortality test results

[0085] Table 3 shows that Examples 4-6 exhibited the best root-knot nematode killing and sustained efficacy, with Example 6 being the most effective, achieving a nematode mortality rate of 90%, a control effect of 88% after 30 days, and 78.9% after 60 days. Example 10 showed a decrease in control efficacy compared to Examples 1-3, likely due to the absence of extracts such as Curcuma zedoaria extract, Sophora flavescens extract, Cinnamomum cassia extract, and Melia azedarach bark extract. Example 11, despite including the aforementioned extracts, also showed a decrease in control efficacy, likely due to the altered position of the extracts, affecting the synergistic effect between components. Comparative Examples 1 and 2 had poor control efficacy due to strain defects. Comparative Example 3, with its single-layer structure, resulted in poor initial release and sustained efficacy. Comparative Example 4, with its excessive inner carrier layer, hindered strain release, preventing timely action and resulting in the lowest root-knot nematode mortality rate and poor control efficacy. Comparative Example 5, with insufficient outer carrier layer, experienced rapid strain loss and activity decay, leading to poor sustained efficacy. Comparative Example 6, lacking Bacillus thuringiensis and Beauveria bassiana, showed a decreased root-knot nematode killing effect and reduced control efficacy.

[0086] Experiment Example 4 Shelf life stability test The microbial preparations prepared in Examples 1-11 and Comparative Examples 1-6 were sealed and stored in a cool, dry place at 25°C for 12 months. The viable count of each strain was measured and the viable count retention rate was calculated. The test results are shown in Table 4.

[0087] Table 4 Results of viable bacteria retention rate test

[0088] As shown in Table 4, the viable cell retention rates of all embodiments of this application can reach over 50%, with Example 6 being the best, reaching 86% and exhibiting the best shelf-life stability. Comparative Example 3, with its single-layer structure and lack of outer protection, experienced the fastest strain decay and a low retention rate. Comparative Example 4, with its excessive inner carrier, reduced the impact of the external environment on the strain to some extent, but the analysis showed poor compatibility with the strain, resulting in a slight decrease in retention rate. Comparative Example 5, with insufficient outer carrier protection, had a low retention rate and a short shelf life.

[0089] Experimental Example 5 Sustained-release performance test The microbial preparations prepared in Examples 1-7 and Comparative Examples 3-5 of this application were placed in dialysis bags with 1g of the preparations and immersed in sterile simulated soil extract (pH 6.0). The preparations were cultured in a constant temperature shaker at 28°C. The number of viable bacteria was measured at regular intervals, and the cumulative release rate over 30 days was calculated. The cumulative release rate = (cumulative number of viable bacteria released over 30 days / initial total number of viable bacteria in the preparation) × 100%. The test results are shown in Table 5.

[0090] Table 5 Results of sustained-release performance test

[0091] Table 5 shows that Examples 1-3 have a basic double-layer coating structure, with a cumulative release rate of 89%-90% over 30 days. In Examples 4 and 5, the cumulative release rate drops to 84%-85% over 30 days. The reason for this is that the particulate properties of dolomite powder fill the pores of the inner microspheres, and the chitinase inducer forms hydrogen bonds with chitosan to strengthen the outer membrane structure. The two work synergistically to reduce the release rate. In Example 6, the cumulative release rate over 30 days is the lowest. Comparative Example 3 has a single-layer structure, with the inner microspheres directly exposed to the extract, resulting in rapid dissolution of the strain and a cumulative release rate as high as 96% over 30 days. In Comparative Example 4, the inner layer structure hinders the release of the strain, resulting in a cumulative release rate of only 70% over 30 days. The release rate is low, but the strain cannot play its role in time. Comparative Example 5 has insufficient protection from the outer carrier, resulting in a cumulative release rate as high as 96% over 30 days.

[0092] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A microbial preparation for controlling plant root-knot nematodes, characterized in that, The microbial preparation has a double-layered structure, comprising an inner layer and an outer layer. The inner layer includes fermentation broths of *Phaeodactylogyrus rubrum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus thuringiensis*, and *Beauveria bassiana*, as well as an inner carrier material. The outer layer includes fermentation broth of *Streptomyces cyclohexane* and an outer carrier material. The volume-to-mass ratio of the total fermentation broth of the inner layer to the inner carrier material is 100 ml: (5-8) g, and the volume-to-mass ratio of the *Streptomyces cyclohexane* fermentation broth of the outer layer to the outer carrier material is 100 ml: (1.5-3.5) g. The volume ratio of the fermentation broth of *Pseudomonas erythrosporum*, *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus thuringiensis*, *Beauveria bassiana*, and *Streptomyces niger* is (2~3):(4~5):(1.5~2.5):1:1:1; The inner carrier material comprises 1.5-1.8 parts sodium alginate, 2.0-2.5 parts chitin powder, 0.3-0.5 parts turmeric extract, 0.1-0.2 parts sophora flavescens extract, and 2.0-2.2 parts modified starch; The outer carrier material includes 1.0 to 1.3 parts chitosan, 0.5 to 0.6 parts humic acid oligosaccharide, 0.2 to 0.5 parts cinnamon extract, 0.15 to 0.25 parts neem bark extract, and 0.2 to 0.3 parts rhizosphere adsorbent.

2. The microbial preparation for controlling plant root-knot nematodes according to claim 1, characterized in that, The inner carrier material also includes 0.8 to 1 part dolomite powder by weight. The outer carrier material also includes 0.3 to 0.4 parts of chitinase inducer.

3. The microbial preparation for controlling plant root-knot nematodes according to claim 2, characterized in that, The inner carrier material also includes 0.4 parts of polylysine by weight.

4. The microbial preparation for controlling plant root-knot nematodes according to claim 2, characterized in that, The modified starch is corn starch pretreated with α-amylase, and the pretreatment conditions are 60-65℃ for 30-40 min; and / or The rhizosphere adsorbent comprises sodium-modified bentonite and potassium humate, wherein the mass ratio of sodium-modified bentonite to potassium humate is 1:(0.9~1.1); and / or The chitinase inducer includes chitin oligosaccharides.

5. The microbial preparation for controlling plant root-knot nematodes according to claim 1, characterized in that, In the microbial preparation, the viable count of *Pharbitis purpureus* fermentation broth is ≥5.0 × 10⁻⁶. 8 CFU / mL, viable count of Paecilomyces lilacinus fermentation broth ≥ 6.0 × 10⁻⁶ 8 CFU / mL, conidial concentration ≥5.0×10 8 The viable count of Bacillus subtilis fermentation broth is ≥8.0 × 10⁶ cells / mL. 8 CFU / mL, viable count of Bacillus thuringiensis fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, viable count of Beauveria bassiana fermentation broth ≥ 5.0 × 10⁻⁶ 8 CFU / mL, conidial concentration ≥4.0×10 8 The viable count of *Streptomyces cerevisiae* fermentation broth is ≥5.0 × 10⁻⁶ cells / mL. 8 CFU / mL.

6. A method for preparing a microbial agent for controlling plant root-knot nematodes according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Prepare fermentation broths of Rhodopseudomonas rubrum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Streptomyces niger according to volume ratios, and mix the fermentation broths of Rhodopseudomonas rubrum, Paecilomyces lilacinus, Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana to obtain an inner mixed fermentation broth; (2) Weigh the inner layer carrier material according to the proportion, mix and dissolve, sterilize and cool to room temperature to obtain the inner layer carrier material liquid; mix the inner layer carrier material liquid with the inner layer mixed fermentation broth and stir to form a suspension; (3) The suspension was uniformly dropped into the calcium chloride curing solution and cured at a constant temperature. After rinsing and filtering, the inner microspheres were obtained. (4) After mixing and dissolving the outer carrier material, sterilize and cool to room temperature, mix with the fermentation broth of Streptomyces niger, coat the inner microspheres with a roller coating method, cure at a constant temperature, dry and sieve to obtain the microbial preparation.

7. The method for preparing a microbial agent for controlling plant root-knot nematodes according to claim 6, characterized in that, The mass concentration of the calcium chloride curing solution in step (3) is 1.8% to 2.0%, the curing temperature is 28 to 30°C, and the curing time is 10 to 12 hours; the curing temperature in step (4) is 25 to 27°C, the curing time is 8 to 9 hours; the drying temperature is 35 to 40°C, and the moisture content of the preparation after drying is ≤8%.

8. The application of the microbial preparation for controlling plant root-knot nematodes as described in any one of claims 1 to 5, or the microbial preparation prepared by any one of claims 6 to 7, in controlling root-knot nematodes.

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