A microbial agent for the prevention and control of nematode diseases, its preparation method and its application

Abstract

This invention provides a microbial agent for controlling nematode diseases, its preparation method, and its application, relating to the field of organic fertilizer technology. The microbial agent, by weight percentage, is mainly composed of the following raw materials: 60%-75% microbial cells; 25%-40% additives; wherein the microbial cells are derived from *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus laterosporus*, *Bacillus mucilaginosus*, and *Lactobacillus spores*. Through the synergistic design of specific bacterial combinations and functional additives, the agent's resistance and colonization ability in complex soil environments can be effectively improved. Simultaneously, a highly efficient synergistic mechanism between fungi and bacteria is established, enabling it to adapt to complex soil types and effectively enhance nematode control. This invention fundamentally solves the industrialization obstacles of biological agents, such as poor environmental adaptability, short duration of action, and limited functionality, providing a scalable solution for replacing chemical pesticides.

Description

Technical Field

[0001] This invention relates to the field of organic fertilizer technology, and in particular to a microbial agent for the prevention and control of nematode diseases, its preparation method, and its application. Background Technology

[0002] Soil nematode diseases have become one of the biological stresses restricting agricultural production, especially root-knot nematodes, which can cause serious yield reductions in cash crops such as vegetables and fruit trees. Currently, control mainly relies on chemical nematicides (such as abamectin and thiazophos), but the long-term use of chemical pesticides leads to prominent problems such as excessive pesticide residues, soil microecological imbalance, and increased nematode resistance.

[0003] Biological control has emerged as an alternative approach due to its environmental compatibility. *Paecilomyces lilacinus*, a parasitic fungus for nematode eggs, has been widely used. However, single-strain preparations suffer from drawbacks such as unstable efficacy and poor environmental adaptability. While recent research has explored compound microbial agents (combining *Paecilomyces lilacinus* and *Bacillus subtilis*, or combining *Bacillus subtilis*, *Paecilomyces lilacinus*, and *Bacillus lilacinus*), such as the compound microbial agent for nematode control disclosed in Chinese invention patent application CN109355236A, which uses multiple strains including *Bacillus subtilis*, *Lactobacillus casei*, *Bacillus lilacinus*, and *Paecilomyces lilacinus*, improper strain compatibility can lead to insufficient synergistic effects. For example, *Bacillus subtilis* may rapidly consume nutrients and inhibit fungal growth, or the spore conversion rate may be low, resulting in a decrease in viable bacteria count exceeding 40% during storage. For soil nematode control, existing compound microbial agents perform poorly in complex soil environments, and the technological bottlenecks in nematode control remain to be overcome. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a microbial agent for the prevention and control of nematode diseases. Through the synergistic design of specific bacterial species combinations and functional additives, the agent can effectively improve its stress resistance and colonization ability in complex soil environments. At the same time, a highly efficient synergistic mechanism between fungi and bacteria is established, which can not only adapt to complex soil types but also effectively improve the nematode control effect.

[0005] To achieve the objectives of this invention, a microbial agent for the prevention and treatment of nematode diseases is provided, which is prepared from the following raw materials by weight percentage:

[0006] Microbial cell count 60%-75%;

[0007] Additives 25%-40%;

[0008] The microbial cells are prepared from *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus lateralis*, *Bacillus mucilaginosus*, and *Lactobacillus sporeans*; the additives are prepared from humic acid, sodium alginate, and chitosan.

[0009] Furthermore, the mass ratio of humic acid, sodium alginate and chitosan in the additive is (20-25):(1-3):(0.1-0.5).

[0010] Furthermore, the mass ratio of humic acid, sodium alginate, and chitosan in the additive is 25:2:0.3.

[0011] Furthermore, the humic acid is any one of mineral-derived humic acid, biochemical humic acid, or natural humic acid.

[0012] Furthermore, the total viable count of the microbial cells is ≥2×10⁻⁶. 10 CFU / g;

[0013] Furthermore, the total viable count of the microorganisms in the severely diseased area is ≥5×10⁻⁶. 10 CFU / g.

[0014] This invention also provides a method for preparing the above-mentioned microbial agent for controlling nematode diseases, comprising the following steps:

[0015] S1. Activate and culture *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus lateralis*, *Bacillus mucilaginosus* and *Lactobacillus spores*, respectively.

[0016] S2. Collect the spore suspensions of *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus lateralis*, *Bacillus mucilaginosus*, and *Lactobacillus spores*, respectively.

[0017] S3. The spore suspensions of *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus laterosporus*, *Bacillus mucilaginosus*, and *Lactobacillus spores* from step S2 are dried separately to obtain *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, *Bacillus laterosporus* spore powder, *Bacillus mucilaginosus* spore powder, and *Lactobacillus spores* spore powder, which are then mixed to prepare microbial cells.

[0018] S4. Humic acid, sodium alginate and chitosan are prepared into solutions and mixed sequentially, dried and granulated to obtain granular additives.

[0019] S5. Mix the microbial cells prepared in step S3 with the additives obtained in step S4 to obtain a microbial agent for preventing and controlling nematode diseases.

[0020] Furthermore, in step S3, the spore powder of *Paecilomyces lilacinus*, the spore powder of *Bacillus subtilis*, the spore powder of *Bacillus lateralis*, the spore powder of *Bacillus jellyoides*, and the spore powder of *Lactobacillus spores* are mixed in a mass ratio of (15-40):(15-20):(12-20):(1-8):(1-5).

[0021] Furthermore, the *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, *Bacillus lateralis* spore powder, *Bacillus mucilaginosus* spore powder, and *Lactobacillus spore powder* spore powder are mixed in a mass ratio of 30:16:16:5:3.

[0022] Furthermore, in step S4, the particle size of the additive is 0.5 mm to 1 mm.

[0023] Furthermore, in step S4, humic acid is dissolved in 1 wt% sodium hydroxide solution after passing through a 200-mesh sieve, and the pH is adjusted to 7.0; sodium alginate is dissolved in sterile water to prepare an aqueous solution with a concentration of 2 wt%; chitosan is prepared into a chitosan solution with a concentration of 1 wt% acetic acid; the chitosan solution is added last during the mixing process.

[0024] Furthermore, in step S3, the drying temperature is 15℃-35℃.

[0025] Furthermore, in step S5, the mixing and stirring time is ≥5h.

[0026] This invention also provides the application of the above-mentioned microbial agents for controlling nematode diseases in the control of soil nematodes.

[0027] Compared with the prior art, the present invention has the following technical effects:

[0028] Through the synergistic design of specific strain combinations and functional additives, the stress resistance and colonization ability of microbial agents in complex soil environments can be effectively improved. At the same time, a highly efficient synergistic mechanism between fungi and bacteria has been established, which can not only adapt to complex soil types, but also effectively improve the control effect on nematodes. This fundamentally solves the industrialization obstacles of biological agents such as poor environmental adaptability, short duration of effect, and single function, and provides a solution for large-scale application to replace chemical pesticides. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0030] In a first aspect, the present invention provides a microbial agent for the prevention and control of nematode diseases, which, by weight percentage, is mainly prepared from the following raw materials:

[0031] Microbial cell count 60%-75%;

[0032] Additives 25%-40%;

[0033] The microbial cells were prepared from *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus lateralis*, *Bacillus mucilaginosus*, and *Lactobacillus spores*.

[0034] In this invention, a composite system of fungi and four types of Bacillus is used to achieve synergistic control. The bacterial combination covers a triple mechanism of parasitism, inhibition, and growth promotion, overcoming the limitation of single-strain function. The synergistic effect of multiple bacterial species can significantly improve the control effect of nematodes and is suitable for sandy soil, clay soil, or other more complex soil types. Physical protection of the bacterial cells is achieved through additives.

[0035] In the microbial agent provided by this invention, the proportion of microbial cells exceeds 60%, which ensures the quantity of active microorganisms and meets the effective concentration in the field; while the proportion of additives exceeds 25%, which ensures the effective protection of microbial cells by the additives.

[0036] In some embodiments, the above-mentioned additive is prepared from humic acid, sodium alginate and chitosan in a mass ratio of (20-25):(1-3):(0.1-0.5).

[0037] In some embodiments, the mass ratio of humic acid, sodium alginate, and chitosan in the above-mentioned additives is 25:2:0.3;

[0038] Humic acid, as a carrier, possesses strong adsorption properties. Within the aforementioned ratio range, it can slowly release nutrients and buffer soil pH fluctuations while ensuring colonization space for the bacteria. Sodium alginate exhibits good film-forming properties and can encapsulate the bacteria, enabling them to resist drought or ultraviolet radiation and adapt to complex environments. Under the aforementioned mass ratio, it can form an effective gel layer. Excessive sodium alginate can lead to overly hard particles, thus affecting the release of bacteria. The cationic properties of chitosan enhance the adsorption of bacteria onto the nematode epidermis, thereby improving the parasitic efficiency of the bacteria and playing a supporting role in inhibiting nematodes. When selecting the ratio, the amount of chitosan should be minimal to prevent excessive chitosan from inhibiting fungal growth.

[0039] In some embodiments, the humic acid is any one of mineral-derived humic acid, biochemical humic acid, or natural humic acid. Humic acid itself contains carboxyl and phenolic hydroxyl groups, which can provide protection for the microbial cells. At the same time, mineral-derived humic acid is less expensive and more suitable for industrial applications.

[0040] In some embodiments, the total viable count of the microbial cells is ≥2×10⁻⁶. 10 CFU / g. In severely ill wards, the total viable count is required to be ≥5×10⁻⁶. 10 CFU / g.

[0041] Secondly, the present invention provides a method for preparing a microbial agent for preventing and controlling nematode diseases, comprising the following steps:

[0042] S1. Activate and culture *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus lateralis*, *Bacillus mucilaginosus* and *Lactobacillus spores*, respectively.

[0043] S2. Collect the spore suspensions of *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus lateralis*, *Bacillus mucilaginosus*, and *Lactobacillus spores*, respectively.

[0044] S3. The spore suspensions of *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus laterosporus*, *Bacillus mucilaginosus*, and *Lactobacillus spores* from step S2 are dried separately to obtain *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, *Bacillus laterosporus* spore powder, *Bacillus mucilaginosus* spore powder, and *Lactobacillus spores* spore powder, which are then mixed to prepare microbial cells.

[0045] S4. Humic acid, sodium alginate and chitosan are prepared into solutions and mixed sequentially, dried and granulated to obtain granular additives.

[0046] S5. Mix the microbial cells prepared in step S3 with the additives obtained in step S4 to obtain a microbial agent for preventing and controlling nematode diseases.

[0047] Culturing each strain separately avoids competitive inhibition between different strains during liquid mixing, ensuring the independent high activity of each strain. Collecting spore or bud suspensions separately during preparation effectively ensures the survival rate during subsequent drying. After drying, the strains are mixed in the form of dry powder, which avoids the secretion of antibiotics by bacteria and inhibition of fungi during liquid mixing. In addition, drying and mixing can eliminate physiological antagonism and retain their respective activities.

[0048] The interaction between humic acid, sodium alginate, and chitosan is not a simple physical mixture, but rather a functional composite structure formed through intermolecular forces, ultimately creating a carrier. Humic acid itself contains numerous carboxyl and hydroxyl groups, forming the final skeletal network. Sodium alginate can form a hydrogen bond network with the carboxyl and hydroxyl groups in humic acid, and upon release, it can gel with calcium ions in the soil to form "microcapsules." The amino groups in chitosan can electrostatically self-assemble with the carboxyl groups in sodium alginate, forming a positive charge on the particle surface and providing protection. In this process, the chitosan solution needs to be added last to prevent premature acidification of sodium alginate, ensuring the formation of a composite structure of "humic acid core - sodium alginate gel layer - chitosan shell." This structure enables the carrier to possess slow-release, stress-resistant, and targeted properties, thereby effectively slowing down and targeting nematodes, improving the killing effect on nematodes.

[0049] In fact, the bacterial cells and the carrier mix to form a "nucleus-shell-bacteria" structure, with the bacterial cells physically adsorbed into the gaps in the carrier.

[0050] In some embodiments, in step S3, *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, *Bacillus lateralis* spore powder, *Bacillus mucilaginosus* spore powder, and *Lactobacillus spore powder* spore powder are mixed in a mass ratio of (15-40):(15-20):(12-20):(1-8):(1-5).

[0051] In some embodiments, *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, *Bacillus lateralis* spore powder, *Bacillus mucilaginosus* spore powder, and *Lactobacillus spore powder* spore powder are mixed in a mass ratio of 30:16:16:5:3.

[0052] By limiting the proportions of each bacterial species, the dosage of each species should not be excessive to prevent competition for growth and squeezing out each other's living space, thereby reducing the killing effect on nematodes. In addition, the dosage of each bacterial species should also be considered to maximize the synergistic effect between them.

[0053] This formulation maximizes the synergistic effect of Bacillus subtilis and Bacillus brevis, effectively enhancing the suppression of nematodes. Simultaneously, Bacillus mucilaginosus and Lactobacillus spores are added in trace amounts as auxiliary bacteria. Bacillus mucilaginosus can solubilize phosphorus and fix nitrogen, promoting healthy root growth and indirectly enhancing crop resistance; however, excessive use of Bacillus mucilaginosus may compete for root colonization sites. Lactobacillus spores, on the other hand, can regulate soil pH, inhibit pathogenic bacteria, and create a favorable environment for functional bacteria; however, excessive use of Lactobacillus spores may lead to soil acidification.

[0054] In some embodiments, the drying temperature in step S3 is 15°C-35°C. Low-temperature drying can prevent high temperatures from killing spores or endospores, thus maximizing the survival rate of the fungi.

[0055] Thirdly, the present invention also provides the application of microbial agents for the prevention and control of nematode diseases in the control of soil nematodes.

[0056] The following is a detailed explanation using specific embodiments:

[0057] Example 1

[0058] a. Cultivation of *Paecilomyces lilacinus*

[0059] Using PDA medium (potato 200g / L, glucose 20g / L, agar 15g / L), the spores were cultured at a constant temperature of 25℃ and a humidity of 70%-80% for 6 days until they matured. The colonies were then scraped with sterile water and mycelia were removed by passing the culture through a 200-mesh sieve to obtain a spore suspension.

[0060] b. Culture of Bacillus subtilis and Bacillus lateralis

[0061] Bacillus subtilis and Bacillus lateralis were cultured separately in LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) at 37°C for 48 h at 180 rpm, and spores were collected.

[0062] c. Culture of Bacillus jellyii

[0063] The spores were cultured using a modified silicate medium (5 g / L sucrose, 0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, and 10 g / L silicate powder) at 30°C for 72 h at 150 rpm, and then collected.

[0064] d. Bacillus spores

[0065] Spores were collected by culturing in MRS medium (10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 5 g / L sodium acetate) at 37°C under anaerobic conditions (nitrogen gas to maintain oxygen concentration <0.5%) for 36 h.

[0066] e. Preparation of microbial cells

[0067] The collected spores and endospores were dried under low-temperature airflow at 30℃. After drying, 30g of *Paecilomyces lilacinus* spore powder, 16g of *Bacillus subtilis* spore powder, 16g of *Bacillus laterosporus* spore powder, 5g of *Bacillus mucilaginosus* spore powder, and 3g of *Lactobacillus spore powder* were weighed out. At this point, the mass ratio of *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, *Bacillus laterosporus* spore powder, *Bacillus mucilaginosus* spore powder, and *Lactobacillus spore powder* spore powder was 30:16:16:5:3. The above spore powder and endospore powder were mixed into a dry powder and stirred at 200 rpm for 3 hours to obtain a composite bacterial powder, which was used as the microbial cell.

[0068] f. Preparation of the carrier

[0069] Weigh out 27.5g of mineral humic acid, 2.2g of sodium alginate, and 0.3g of chitosan. The mass ratio of humic acid, sodium alginate, and chitosan is approximately 25:2:0.3. Dissolve the mineral humic acid in a 1wt% sodium hydroxide solution at 25℃, and gradually add hydrochloric acid to adjust the pH to 7.0. Dissolve the sodium alginate in a sterile aqueous solution to prepare a 2wt% sodium alginate aqueous solution. Prepare a 1wt% chitosan solution using a 1wt% acetic acid solution.

[0070] The mineral humic acid solution and sodium alginate solution were mixed and stirred until homogeneous to obtain a mixed solution. Then, chitosan solution was added to the mixed solution and stirred for 20 minutes. After that, spray drying was carried out (inlet 60℃ / outlet 40℃) to obtain granular additives. The prepared additives were sieved to obtain carriers with a particle size of 1 mm.

[0071] g. Preparation of compound microbial agents

[0072] The carrier obtained in step f and the microbial cells obtained in step e are mixed and stirred for 5 hours to obtain the microbial agent.

[0073] Example 2

[0074] Based on Example 1, the difference lies in changing the amount of each microbial cell in step e and the amount of each additive in step f. The remaining steps are consistent with Example 1, as follows:

[0075] Weigh out 25.6g of *Paecilomyces lilacinus* spore powder, 25.6g of *Bacillus subtilis* spore powder, 20.4g of *Bacillus lateralis* spore powder, 1.7g of *Bacillus mucilaginosus* spore powder, and 1.7g of *Lactobacillus spore powder*. At this point, the mass ratio of *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, *Bacillus lateralis* spore powder, *Bacillus mucilaginosus* spore powder, and *Lactobacillus spore powder* spore powder is approximately 15:15:12:1:1.

[0076] Weigh out 23.7g of mineral humic acid, 1.2g of sodium alginate, and 0.1g of chitosan. At this point, the mass ratio of mineral humic acid, sodium alginate, and chitosan is approximately 20:1:0.1.

[0077] Example 3

[0078] Based on Example 1, the difference lies in changing the amount of each microbial cell in step e and the amount of each additive in step f. The remaining steps are consistent with Example 1, as follows:

[0079] Weigh out 25.8g of *Paecilomyces lilacinus* spore powder, 12.9g of *Bacillus subtilis* spore powder, 12.9g of *Bacillus laterosporus* spore powder, 5.2g of *Bacillus mucilaginosus* spore powder, and 3.2g of *Lactobacillus spore powder*. At this point, the mass ratio of *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, *Bacillus laterosporus* spore powder, *Bacillus mucilaginosus* spore powder, and *Lactobacillus spore powder* spore powder is approximately 40:20:20:8:5.

[0080] Weigh out 35g of mineral humic acid, 4.2g of sodium alginate, and 0.8g of chitosan. At this point, the mass ratio of mineral humic acid, sodium alginate, and chitosan is approximately 25:3:0.5.

[0081] Comparative Example 1

[0082] Following the cultivation method of Example 1, *Paecilomyces lilacinus* was cultured and spore powder was collected. 70g of the spore powder was weighed out. At the same time, a carrier (additive) prepared in the same manner as in Example 1 was added, and the mass of the added carrier was the same as in Example 1.

[0083] Comparative Example 2

[0084] Based on Example 1, the difference is that steps c and d in Example 1 are not used, and the composition and amount of microbial cells in step e are changed. The remaining steps are consistent with Example 1, as follows:

[0085] Weigh out 33.8g of *Paecilomyces lilacinus* spore powder, 18.1g of *Bacillus subtilis* spore powder, and 18.1g of *Bacillus lateralis* spore powder. At this point, the mass ratio of *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, and *Bacillus lateralis* spore powder is approximately 30:16:16.

[0086] Comparative Example 3

[0087] Based on Example 1, the difference lies only in step f, i.e., changing the ratio between the various substances in the additive; the remaining steps remain consistent with Example 1, as follows:

[0088] Weigh out 22.5g of mineral humic acid, 6g of sodium alginate, and 1.5g of chitosan. At this point, the mass ratio of humic acid, sodium alginate, and chitosan is approximately 15:4:1.

[0089] Comparative Example 4

[0090] Based on Example 1, the difference is that step f is not used and the carrier prepared in step f of Example 1 is not added in step g, while the remaining steps are consistent with Example 1.

[0091] Experiment Example 1: Nematode Control Efficacy Test

[0092] Soil treatment: Take soil infected with nematodes (≥2000 root-knot nematode eggs / 100g) and divide it into pots (2kg / pot).

[0093] Group: A control group 1 was set up, which was treated with only water;

[0094] Experimental group: The microbial agents prepared in the examples and comparative examples were mixed into the soil at a mass ratio of 0.5% (5g / kg), tomato seedlings were planted, and the seedlings were cultured in a greenhouse at 25℃ for 30 days. The mortality rate of second-instar larvae, the reduction rate of egg masses, and the biomass of plants were tested. The experimental results are shown in Table 1.

[0095] Table 1. Detection of nematode control efficacy.

[0096]

[0097] Experiment Example 2: Environmental Adaptability and Slow-Release Performance Test

[0098] Soil preparation: Sandy soil (sand content > 85%, organic matter < 1%), clay soil (clay content > 40%, organic matter 2-3%), and diseased soil from continuous cropping (tomatoes planted for 3 years, pH 5.5-6.5, containing ≥ 2000 root-knot nematode eggs / 100g). Each type of soil is packaged in 1kg / pot.

[0099] Drought group: Normal soil, dried to a moisture content of 10%.

[0100] Ultraviolet group: UV-B irradiation (30W / m 2 (6 hours per day).

[0101] Control group 2: Soil without any added microbial agents.

[0102] Experimental group: The microbial inoculants prepared in the examples and comparative examples were added in equal amounts to the soils of sandy soil, clay soil, soil affected by continuous cropping, drought group and ultraviolet group, respectively;

[0103] Then, samples were taken on days 7, 14, and 30 to determine the number of viable bacteria and the residual amount of sodium alginate in the soil. The experimental results are shown in Tables 2-5.

[0104] Table 2. Number of viable bacteria and residual sodium alginate in sandy soil

[0105]

[0106] Table 3. Viable bacterial count and sodium alginate residue in clay.

[0107]

[0108] Table 4. Viable bacterial count and sodium alginate residue in diseased soil from continuous cropping.

[0109]

[0110] Table 5. Viable bacterial counts and sodium alginate residues in soils from the drought and ultraviolet groups.

[0111]

[0112] The experimental results above demonstrate that the microbial agent prepared by this invention overcomes the limitation of poor nematode control with single-species microorganisms. By using multiple microorganisms and a carrier (additive) in synergistic combination, the control effect against nematodes is greatly improved. The residual amount of sodium alginate shows the slow-release effect of the carrier (additive) structure of this invention, enabling the prepared microbial agent to adapt to different soil types and complex environments, thus improving its control performance against soil nematodes.

Claims

1. A microbial agent for controlling nematode disease, characterized in that, It is produced from the following raw materials by weight percentage: Microbial cell count 60%-75%; Additives 25%-40%; The microbial cells are prepared from *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus lateralis*, *Bacillus mucilaginosus*, and *Lactobacillus spores*; the additives are prepared from humic acid, sodium alginate, and chitosan. The mass ratio of humic acid, sodium alginate, and chitosan is (20-25):(1-3):(0.1-0.5). The preparation method of the microbial inoculant includes the following steps: S1. Activate and culture *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus lateralis*, *Bacillus mucilaginosus* and *Lactobacillus spores*, respectively. S2. Collect the spore suspensions of *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus lateralis*, *Bacillus mucilaginosus*, and *Lactobacillus spores*, respectively. S3. The spore suspensions of *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus laterosporus*, *Bacillus mucilaginosus*, and *Lactobacillus spores* from step S2 are dried separately to obtain *Paecilomyces lilacinus* spore powder, *Bacillus subtilis* spore powder, *Bacillus laterosporus* spore powder, *Bacillus mucilaginosus* spore powder, and *Lactobacillus spores* spore powder, which are then mixed to prepare microbial cells. S4. Humic acid, sodium alginate and chitosan are prepared into solutions and mixed sequentially, dried and granulated to obtain granular additives. S5. Mix and stir the microbial cells prepared in step S3 and the additives obtained in step S4 to obtain a microbial agent for preventing and controlling nematode diseases. In step S3, the spore powder of *Paecilomyces lilacinus*, the spore powder of *Bacillus subtilis*, the spore powder of *Bacillus lateralis*, the spore powder of *Bacillus mucilaginosus*, and the spore powder of *Lactobacillus spores* are mixed in a mass ratio of (15-40):(15-20):(12-20):(1-8):(1-5). In step S4, humic acid is dissolved in 1 wt% sodium hydroxide solution after passing through a 200-mesh sieve, and the pH is adjusted to 7.0; sodium alginate is dissolved in sterile water to prepare an aqueous solution with a concentration of 2 wt%; chitosan is prepared into a chitosan solution with a concentration of 1 wt% acetic acid; the chitosan solution is added last during the mixing process. The total viable count of the microbial cells is ≥2×10⁻⁶. 10 CFU / g; In step S3, the drying temperature is 15℃-35℃.

2. The microbial agent for controlling nematode disease according to claim 1, characterized in that, In the additive, the mass ratio of humic acid, sodium alginate, and chitosan is 25:2:0.

3.

3. The microbial agent for controlling nematode disease according to claim 2, characterized in that, The humic acid is either mineral-derived humic acid or biochemical humic acid.

4. The microbial agent for controlling nematode disease according to claim 1, characterized in that, The spore powders of *Paecilomyces lilacinus*, *Bacillus subtilis*, *Bacillus brevis*, *Bacillus mucilaginosus*, and *Lactobacillus spores* were mixed in a mass ratio of 30:16:16:5:

3.

5. The application of a microbial agent for controlling nematode diseases as described in any one of claims 1-4 in the control of tomato root-knot nematode disease.

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