A composting agent with disease prevention and control function, and a preparation method and application thereof

Abstract

The application discloses a kind of rotten agent with disease prevention and control function and preparation method and application, belong to the cross field of agricultural microorganism technology and biological control.It is composed of compound bacteria powder and microcapsule fungicide by the rotten agent, the compound bacteria powder is made of bacillus subtilis ZKY01 bacteria powder and bacillus velezensis ZKY02 bacteria powder, and the microcapsule fungicide is bacillus siamensis ZKY03 bacteria powder embedded in microcapsule carrier;Wherein, the bacteria powder of three kinds of bacteria is the effective viable count not less than 1×10 11 CFU / g powder obtained by fermentation culture.The application can shorten the rotting period of agricultural organic waste such as straw, livestock and poultry manure to 12~15 days through the synergistic effect of three strains, and effectively prevent and control tobacco root black rot, black foot disease and other soil-borne diseases through the secretion of antibacterial substances, competition for nutrients and other mechanisms.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of agricultural microbial technology and biological control, specifically involving a composting agent with disease control function, its preparation method and application. Background Technology

[0002] With the development of ecological agriculture, the resource utilization of agricultural organic waste (such as straw and livestock manure) has become a key means to reduce non-point source pollution and improve soil fertility. However, traditional organic waste treatment faces two major problems: Low composting efficiency and high risk: The natural composting cycle takes more than 40 days, the degradation of cellulose and lignin is slow, and soil-borne pathogens (such as tobacco root black rot fungus and black shank fungus) are easy to grow during the composting process. After returning to the field, it will aggravate the occurrence of crop diseases, leading to reduced yield or even crop failure. Existing composting agents / compound microbial agents have limited functions: Most composting agents on the market focus on a single function—either they only contain cellulose-decomposing bacteria, which can only improve composting efficiency but cannot control diseases; or they only contain biocontrol strains, which lack the ability to degrade organic matter and cannot meet the needs of rapid composting.

[0003] Further analysis of existing technological shortcomings reveals the following: First, the combination of bacterial strains lacks scientific basis, and the synergistic mechanism between strains is not clearly defined, resulting in the inability to simultaneously maximize the functions of composting and biocontrol. For example, some compound composting agents simply mix 2-3 strains without verifying functional complementarity. Second, the antibacterial spectrum of biocontrol strains is narrow, with limited effectiveness in controlling specific soil-borne diseases of crops such as tobacco, and the types and mechanisms of action of antibacterial substances are not clearly defined. Third, the lack of standardized application parameters (such as material moisture content, application ratio, and turning frequency) leads to large fluctuations in effectiveness in actual applications, making large-scale promotion difficult.

[0004] Based on the aforementioned technical bottlenecks, there is an urgent need in this field to develop a composite microbial composting agent that combines "high-efficiency composting + precise biocontrol", has a clear strain mechanism, and has clear application parameters. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a composting agent with disease control functions, its preparation method, and its application. By screening Bacillus strains with specific degradation and biocontrol functions, and optimizing the compounding ratio, preparation method, and application conditions, the shortcomings of existing technologies are successfully solved, realizing the integration of agricultural waste resource utilization and disease control.

[0006] The technical solution provided by this invention is as follows:

[0007] This invention provides a composting agent with disease control function. The composting agent is composed of a compound bacterial powder and a microcapsule bacterial agent. The compound bacterial powder is a mixture of Bacillus subtilis ZKY01 bacterial powder and Bacillus belyss ZKY02 bacterial powder. The microcapsule bacterial agent is Bacillus sicca ZKY03 bacterial powder encapsulated in a microcapsule carrier. All three bacterial powders are obtained through fermentation culture with an effective viable count of not less than 1 × 10⁻⁶. 11 The powder contains CFU / g of Bacillus subtilis ZKY01, with accession number CCTCC NO:M 20252980; Bacillus belyss ZKY02, with accession number CCTCC NO:M20252981; and Bacillus sicca ZKY03, with accession number CCTCC NO:M 20252982.

[0008] Furthermore, the mass ratio of Bacillus subtilis ZKY01 powder to Bacillus vesiculosus ZKY02 powder in the compound bacterial powder is 1~3:1~3, and the number of effective viable bacteria in the compound bacterial powder is not less than 1.0×10⁻⁶. 11 CFU / g.

[0009] Furthermore, the microencapsulated bacterial agent uses maltodextrin and gum arabic as microcapsule carriers, the encapsulation rate of the microencapsulated bacterial agent is 60%~80%, and the number of effective viable bacteria in the microencapsulated bacterial agent is not less than 3.5×10⁻⁶. 10 CFU / g.

[0010] Furthermore, the mass ratio of compound bacterial powder to microencapsulated bacterial agent in the composting agent is 1:1~4, and the number of effective viable bacteria in the composting agent is not less than 7.0×10⁻⁶. 10 CFU / g.

[0011] This invention also provides a method for preparing the above-mentioned composting agent with disease control function, comprising the following steps: Bacillus subtilis ZKY01, Bacillus belyi ZKY02 and Bacillus sicca ZKY03 were prepared separately. The prepared Bacillus subtilis ZKY01 bacterial powder and Bacillus vesiculosus ZKY02 bacterial powder were mixed to prepare a compound bacterial powder. The prepared Bacillus sicca ZKY03 bacterial powder was mixed with a microcapsule carrier solution that had been filtered and purified, and the solid content was adjusted to 20%~30%. The mixture was then dried by hot air atomization to form a microencapsulated bacterial agent. The prepared compound bacterial powder is mixed with the prepared microencapsulated bacterial agent to obtain the composting agent.

[0012] Furthermore, the method for preparing the bacterial powders of Bacillus subtilis ZKY01, Bacillus belye ZKY02, and Bacillus sicca ZKY03 includes: The seed cultures of the three activated bacteria were inoculated into the corresponding fermentation broths at a ratio of 1-5‰, and cultured at 30-38℃ and 120-180 rpm / min for 40-60 h to obtain an effective viable count of not less than 2.0 × 10⁻⁶. 9 Fermentation broth with CFU / mL; The fermentation broth was transferred to a centrifuge, and after centrifugation, the supernatant bacterial suspension was removed to obtain an effective viable cell count of not less than 1×10⁻⁶. 10 CFU / mL bacterial culture concentrate; The concentrated microbial culture solution was added to a mixing tank, along with an auxiliary material comprising 10-13% of the concentrated culture solution by mass. The concentrated culture solution was then pressurized to a rotating nozzle under a pressure of 0.8-1.0 Pa, maintaining a spray speed of 18000-22000 r / min. Simultaneously, hot air at 140-160℃ and 3800-4200 m³ / h was introduced into a drying tower for drying, yielding an effective viable count of not less than 1×10⁻⁶. 11 CFU / g of bacterial powder.

[0013] Furthermore, the fermentation broth of Bacillus subtilis ZKY01 comprises, by weight: 10-20 parts corn flour, 1-4 parts sucrose, 2-6 parts calcium carbonate, 3-7 parts soybean flour, 1-4 parts peptone, 0.5-2 parts fish meal, 0.1-0.5 parts potassium dihydrogen phosphate, 0.1-0.3 parts manganese sulfate, 0.2-0.6 parts sodium chloride, 0.1-0.4 parts dipotassium hydrogen phosphate, 0.2-0.4 parts magnesium sulfate, 0.3-0.6 parts ammonium sulfate, 1000 parts water, with a pH of 7.2-7.8; The fermentation broth of *Bacillus belye* ZKY02 comprises, by weight: 15-25 parts soybean flour, 1-4 parts peptone, 3-9 parts corn flour, 4-8 parts sucrose, 0.2-0.5 parts sodium chloride, 0.1-0.4 parts potassium dihydrogen phosphate, 0.1-0.3 parts manganese sulfate, 0.2-0.4 parts magnesium sulfate, 1000 parts water, with a pH of 6.8-7.5. The fermentation broth of Bacillus sicca ZKY03 comprises, by weight: 8-16 parts glucose, 15-25 parts soybean meal, 2-6 parts peptone, 8-16 parts corn starch, 0.3-0.7 parts magnesium sulfate, 0.1-0.5 parts sodium chloride, 0.3-0.7 parts dipotassium hydrogen phosphate, 0.1-0.4 parts manganese sulfate, and 1000 parts water, with a pH of 7.0-7.5.

[0014] Furthermore, the microcapsule carrier solution is an aqueous solution with a mass concentration of 2-6% prepared by mixing maltodextrin and gum arabic at a mass ratio of 2-5:1; the excipient is a mixture of diatomaceous earth and calcium carbonate at a mass ratio of 25-45:55-75.

[0015] The present invention also provides a composting material for tobacco cultivation, which is obtained by composting organic materials with the composting agent with disease control function described above or the composting agent prepared by the preparation method described above; wherein the organic materials are selected from straw, livestock and poultry manure or a mixture of both.

[0016] The present invention also provides the application of the above-mentioned composted tobacco cultivation material in promoting tobacco growth and / or improving tobacco disease resistance, wherein promoting tobacco growth means increasing tobacco plant height, increasing the number of leaves and the weight of a single leaf; and improving tobacco disease resistance means improving the immunity of tobacco to the pathogen of tobacco black shank.

[0017] Beneficial effects

[0018] This invention utilizes the scientific combination and synergistic effect of Bacillus subtilis ZKY01, Bacillus belye ZKY02, and Bacillus sicca ZKY03 to achieve rapid and efficient decomposition of agricultural organic waste while significantly inhibiting the occurrence and spread of soil-borne diseases. The strains used in this invention have clearly defined sources, are easy to use, and are low-cost, possessing excellent prospects for widespread application. It is suitable for various large-scale planting areas and ecological agricultural scenarios, providing effective technical support for achieving green and sustainable agricultural development.

[0019] The composting agent of this invention, through a preferred combination of specific strains of Bacillus subtilis ZKY01, Bacillus belyeis ZKY02, and Bacillus sicca ZKY03, not only significantly improves the composting efficiency of agricultural organic waste such as straw and livestock manure, but also ensures the survival of biocontrol strains during the composting process. This effectively reduces crop diseases after returning the waste to the field, promoting the synergistic development of agricultural waste resource utilization and healthy plant cultivation. This invention is particularly suitable for modern ecological agriculture and organic farming systems, providing a new microbial technology pathway for reducing dependence on chemical pesticides and achieving green production.

[0020] The main objective of this invention is to provide a composite microbial composting agent that can significantly promote the rapid composting of agricultural organic materials (such as straw, livestock and poultry manure, etc.) and effectively control soil-borne crop diseases caused by the return of composted materials to the field. Its core lies in: constructing a composite system using three functionally complementary Bacillus strains, achieving simultaneous "rapid composting of organic waste" and "control of soil-borne diseases" through clear division of labor and synergistic effects: *Bacillus subtilis* ZKY01: As the core degrading strain, it secretes highly active cellulase and ligninase to rapidly decompose recalcitrant organic matter in straw and manure, providing a foundation for the composting process; *Bacillus vesiculosus* ZKY02: As the core degrading strain, it secretes highly active amylase and protease to accelerate organic matter degradation and improve composting efficiency; *Bacillus sicca* ZKY03: As the core biocontrol strain, it secretes lipopeptides (such as ituronin and fenbacin) antibacterial substances to precisely inhibit pathogens such as tobacco root black rot fungus and black shank fungus.

[0021] Another objective of this invention is to provide the application of the compound microbial composting agent in tobacco planting systems, particularly the method of application during the composting process of farmyard manure and straw, and its control effect on soil-borne diseases such as tobacco root black rot and black shank. Attached Figure Description

[0022] Figure 1 The images show the colony (A) and Gram staining results (B) of strain ZKY01.

[0023] Figure 2 This is a phylogenetic tree of strain ZKY01 constructed based on the 16S rDNA gene sequence;

[0024] Figure 3 The images show the colony (A) and Gram staining results (B) of strain ZKY02.

[0025] Figure 4 This is a phylogenetic tree of strain ZKY02 constructed based on the 16S rDNA gene sequence;

[0026] Figure 5 The images show the colony (A) and Gram staining results (B) of strain ZKY03.

[0027] Figure 6 This is a phylogenetic tree of strain ZKY03 constructed based on the 16S rDNA gene sequence. Detailed Implementation

[0028] To better understand the technical solution of this application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and culture media involved are all commercially available conventional reagents and culture media; unless otherwise specified, the experimental methods involved are all conventional methods.

[0030] The following are some of the reagent models and manufacturers used in this embodiment:

[0031] Composite excipients: diatomaceous earth (fineness 300 mesh, moisture content less than or equal to 1%), calcium carbonate (fineness ≥300 mesh, CaCO3 content ≥98%); microcapsule encapsulation composite wall material: maltodextrin (DE value: 15-20, item number MD20, purchased from Shandong Xiwang Sugar Industry Co., Ltd.), gum arabic (CAS: 9000-0105, purity 99.8%, purchased from Hebei Chuangzhiyuan Biotechnology Co., Ltd.).

[0032] This invention provides a composting agent with disease control function. The composting agent is composed of compound bacterial powder and microcapsule bacterial agent. The compound bacterial powder is a mixture of Bacillus subtilis ZKY01 bacterial powder and Bacillus belyss ZKY02 bacterial powder. The microcapsule bacterial agent is Bacillus sicca ZKY03 bacterial powder encapsulated in a microcapsule carrier. All three bacterial powders are obtained through fermentation culture with an effective viable count of not less than 1 × 10⁻⁶. 11 The powder contains CFU / g of Bacillus subtilis ZKY01, with accession number CCTCC NO:M 20252980; Bacillus belyss ZKY02, with accession number CCTCC NO:M 20252981; and Bacillus sicca ZKY03, with accession number CCTCC NO:M 20252982.

[0033] Specifically, the isolation and identification of Bacillus subtilis ZKY01

[0034] (1) Isolation of strain ZKY01: It was previously isolated from naturally decomposed farmyard manure in Ludian, Zhaotong. Strain ZKY01 was streaked onto LB solid medium and cultured at 37℃ for 3 days. The colonies were milky white, round, smooth, moist, and translucent. Figure 1 ).

[0035] (2) Identification of strain ZKY01: Gram staining and observation under an oil immersion microscope showed that the result was blue-purple, indicating that it was a Gram-positive bacterium. Figure 1 ).

[0036] (3) Molecular identification: Genomic DNA of strain ZKY01 was extracted using the Tiangen DP3032 kit. PCR amplification was performed using universal 16S rDNA primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.4)) and 1492R (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.5)). The amplification program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 30 cycles; after the cycles, a final extension at 72℃ for 5 min was performed. The amplified products were stored at 4℃. The amplified products were sent to Qingke Biotechnology Co., Ltd. for sequencing. After alignment with NCBI BLAST, a phylogenetic tree was constructed using MEGA v7.0.26 software. Phylogenetic analysis showed that the 16S rDNA sequence of strain ZKY01 was similar to that of strain ZKY01. Bacillus subtilis XeM20 clusters in the same evolutionary branch, with a sequence similarity of 98% (see...). Figure 2 Based on this, strain ZKY01 was identified as Bacillus subtilis.

[0037] Specifically, the isolation and identification of Bacillus belye ZKY02

[0038] (1) Isolation of strain ZKY02: It was previously isolated from naturally decomposed farmyard manure in Ludian, Zhaotong. Strain ZKY02 was streaked onto LB solid medium and incubated at 37℃ for 3 days. The colonies were milky white, round, smooth, moist, and opaque. Figure 3 ).

[0039] (2) Identification of strain ZKY02: Gram staining and observation under an oil immersion microscope showed that the result was blue-purple, indicating that it was a Gram-positive bacterium. Figure 3 ).

[0040] (3) Molecular identification: Genomic DNA of strain ZKY02 was extracted using the Tiangen DP3032 kit. PCR amplification was performed using universal 16S rDNA primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO.4)) and 1492R (5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO.5)). The amplification program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 30 cycles; after the cycles, a final extension at 72℃ for 5 min was performed. The amplified products were stored at 4℃. The amplified products were sent to Qingke Biotechnology Co., Ltd. for sequencing. After alignment with NCBI BLAST, a phylogenetic tree was constructed using MEGA v7.0.26 software. Phylogenetic analysis showed that the 16S rDNA sequence of strain ZKY02 was similar to that of strain ZKY02. Bacillus velezensis K-8 clusters in the same evolutionary branch, with a sequence similarity of 97% (see...). Figure 4 Based on this, strain ZKY02 was identified as Bacillus belye.

[0041] Specifically, the isolation and identification of Bacillus sicca ZKY03:

[0042] (1) Isolation of strain ZKY03: It was previously isolated from the roots of healthy tobacco plants in Ludian, Zhaotong. Strain ZKY03 was streaked onto LB solid medium and cultured at 37℃ for 3 days. The colonies were round, pale yellow, opaque, and slightly wrinkled on the surface. Figure 5 ).

[0043] (2) Identification of strain ZKY03: Gram staining and observation under an oil immersion microscope showed that it was a Gram-positive bacterium, appearing blue-purple. Figure 5 ).

[0044] (3) Molecular identification: Genomic DNA of strain ZKY03 was extracted using the Tiangen DP3032 kit. PCR amplification was performed using universal 16S rDNA primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO.4)) and 1492R (5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO.5)). The amplification program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 30 cycles; after the cycles, a final extension at 72℃ for 5 min was performed. The amplified products were stored at 4℃. The amplified products were sent to Qingke Biotechnology Co., Ltd. for sequencing. After alignment with NCBI BLAST, a phylogenetic tree was constructed using MEGA v7.0.26 software. Phylogenetic analysis showed that the 16S rDNA sequence of strain ZKY03 was similar to that of strain ZKY03. Bacillus siamensis PD-A10 clusters in the same evolutionary branch, with a sequence similarity of 95% (see...). Figure 6 Based on this, strain ZKY03 was identified as Bacillus sicca.

[0045] The Bacillus subtilis ZKY01 and Bacillus belye ZKY02 in the embodiments of this invention possess degradative enzyme activities such as cellulase, protease, and xylanase; Bacillus sicca ZKY03 can secrete lipopeptide antibacterial substances, which have significant inhibitory effects on a variety of soil-borne pathogenic fungi and bacteria, specifically:

[0046] Enzyme activity assays were performed on ZKY01 and ZKY02 according to NY 609-2002:

[0047] (1) Cellulase activity assay: The CMC-DNS reducing sugar method was used for detection. 1 mL of fermentation broth ZKY01 and ZKY02 were weighed and added to 50 mL of buffer solution (0.05 mol / L disodium hydrogen phosphate-citric acid buffer, pH=5.5). The mixture was vortexed for 20 min, centrifuged at 12000 r / min for 10 min, and the supernatant was collected and then serially diluted with buffer solution to obtain the crude enzyme solution.

[0048] Add 0.5 mL of diluted enzyme solution and 1.5 mL of 1% CMC substrate to the sample tube for enzymatic reaction. After the reaction, add 1.5 mL of DNS reagent to terminate and develop color. Vortex mix well, boil in a water bath for 10 min, cool under running water and bring the volume to 10 mL, and then measure the absorbance at a wavelength of 540 nm. In the control tube, first add DNS reagent to inactivate the enzyme, then add an equal amount of enzyme solution and substrate, followed by color development, volume adjustment and absorbance measurement.

[0049] (2) Protease activity assay: The casein-Folin-Ciocalteu method was used. 1 mL of fermentation broth ZKY01 and ZKY02 were weighed and added to 50 mL of buffer solution (0.05 mol / L Tris-HCl buffer solution, pH=7.5). The mixture was vortexed for 20 min, centrifuged at 12000 r / min for 10 min, and the supernatant was collected and then serially diluted with buffer solution to obtain the crude enzyme solution.

[0050] In the sample tube, 1 mL of diluted enzyme solution and 2 mL of 1% casein substrate were mixed and incubated. The enzymatic reaction was terminated with 3 mL of 10% trichloroacetic acid. After centrifugation to remove the precipitate, the supernatant was collected, and then 5 mL of 0.5 mol / L sodium carbonate and 1 mL of diluted Folin-Ciocalteu reagent were added and mixed thoroughly. After color development at 37℃ for 20 min, the absorbance was measured at 680 nm. In the control tube, trichloroacetic acid was added first to inactivate the protease, and then the enzyme solution and substrate were added. The remaining centrifugation, color development, and absorbance detection were performed simultaneously with the sample tube.

[0051] (3) Xylanase activity assay: The DNS reducing sugar method was used to determine the activity. 1 mL of fermentation broth ZKY01 and ZKY02 were weighed and added to 50 mL of buffer solution (0.1 mol / L acetate-sodium acetate buffer, pH=5.5). The mixture was vortexed for 20 min, centrifuged at 12000 r / min for 10 min, and the supernatant was collected. The supernatant was then serially diluted with buffer solution to obtain the crude enzyme solution.

[0052] Add 0.5 mL of diluted enzyme solution and 1.5 mL of 1% birch xylan substrate to the sample tube for enzymatic reaction. After the reaction, add 1.5 mL of DNS reagent to stop the color development. Vortex mix well, boil in a water bath for 10 min, cool under running water and bring the volume to 10 mL, and then measure the absorbance at a wavelength of 540 nm. In the control tube, first add DNS reagent to inactivate the enzyme, then add an equal amount of enzyme solution and substrate, followed by color development, volume adjustment and absorbance measurement.

[0053] The results of the enzyme activity assay are shown in the table below:

[0054] Table 1 Results of enzyme activity assay

[0055]

[0056] Detection of lipopeptides: Extraction was performed using acid precipitation. First, the fermentation broth of biocontrol bacteria ZKY03 was centrifuged at 4℃ and 10000 r / min for 15 min, and the supernatant was collected to remove bacterial impurities. Then, the pH of the supernatant was adjusted to 2.0 with 5 mol / L hydrochloric acid, and the mixture was placed in a 4℃ refrigerator for 14 h to allow for complete precipitation of lipopeptides. Next, it was centrifuged again at 4℃ and 10000 r / min for 15 min, and the supernatant was discarded, retaining the lipopeptide precipitate at the bottom. Methanol was added to the precipitate to dissolve it completely, and the pH of the system was adjusted to 7.2 with 1 mol / L NaOH. Extraction was performed at room temperature for 10 h, followed by centrifugation at 4℃ and 10000 r / min for 15 min to collect the supernatant. The above dissolution, neutralization, extraction, and centrifugation process was repeated three times, and all extracts were combined to obtain the crude lipopeptide extract.

[0057] Antibacterial activity test:

[0058] The Oxford cup method was used: Tobacco root black rot fungus (… Berkeleyomyces rouxiae Tobacco black shank bacterium ( Phytophthora parasitica var. nicotianae ) spore suspension (concentration 10) 6 The culture medium (CFU / mL) was evenly spread on a PDA plate; at a distance of 3 cm from the center of the plate, a 0.5 cm (5 mm) diameter sterile punch was used to prepare culture medium cavities (3 cavities per plate, distributed in an equilateral triangle); 80 μL of ZKY03 lipopeptide crude extract was added to the cavities, and 80 μL of sterile PBS buffer was added to the cavities as blank controls; the plates were placed in a 28℃ constant temperature incubator and incubated for 48-56 h (to avoid excessive mycelial growth covering the inhibition zone), the formation of the inhibition zone was observed, the diameter of the inhibition zone was measured, and the inhibition rate was calculated by combining the colony diameter of the control group. Each treatment was repeated 3 times, and the experimental results are shown in the table below.

[0059] Table 2. Antibacterial test results

[0060]

[0061] In this embodiment, the mass ratio of Bacillus subtilis ZKY01 powder to Bacillus vesiculosus ZKY02 powder in the compound bacterial powder is 1~3:1~3, and the number of effective viable bacteria in the compound bacterial powder is not less than 1.0×10⁻⁶. 11 CFU / g.

[0062] In this embodiment, the microencapsulated bacterial agent uses maltodextrin and gum arabic as microcapsule carriers, the encapsulation rate of the microencapsulated bacterial agent is 60%~80%, and the number of effective viable bacteria in the microencapsulated bacterial agent is not less than 3.5×10⁻⁶. 10 CFU / g.

[0063] In this embodiment, the mass ratio of compound bacterial powder to microencapsulated bacterial agent in the composting agent is 1:1~4, and the number of effective viable bacteria in the composting agent is not less than 7.0×10⁻⁶. 10 CFU / g.

[0064] This invention also provides a method for preparing the above-mentioned composting agent with disease control function, comprising the following steps: Bacillus subtilis ZKY01, Bacillus belyi ZKY02 and Bacillus sicca ZKY03 were prepared separately. The prepared Bacillus subtilis ZKY01 bacterial powder and Bacillus vesiculosus ZKY02 bacterial powder were mixed to prepare a compound bacterial powder. The prepared Bacillus sicca ZKY03 bacterial powder was mixed with a microcapsule carrier solution that had been filtered and purified, and the solid content was adjusted to 20%~30%. The mixture was then dried by hot air atomization to form a microencapsulated bacterial agent. The prepared compound bacterial powder is mixed with the prepared microencapsulated bacterial agent to obtain the composting agent.

[0065] In this embodiment, the method for preparing the mycelial powders of Bacillus subtilis ZKY01, Bacillus belyssus ZKY02, and Bacillus sicca ZKY03 includes: The seed cultures of the three activated bacteria were inoculated into the corresponding fermentation broths at a ratio of 1-5‰, and cultured at 30-38℃ and 120-180 rpm / min for 40-60 h to obtain an effective viable count of not less than 2.0 × 10⁻⁶. 9 Fermentation broth with CFU / mL; The fermentation broth was transferred to a centrifuge, and after centrifugation, the supernatant bacterial suspension was removed to obtain an effective viable cell count of not less than 1×10⁻⁶. 10 CFU / mL bacterial culture concentrate; The concentrated microbial culture solution was added to a mixing tank, along with an auxiliary material comprising 10-13% of the concentrated culture solution by mass. The concentrated culture solution was then pressurized to a rotating nozzle under a pressure of 0.8-1.0 Pa, maintaining a spray speed of 18000-22000 r / min. Simultaneously, hot air at 140-160℃ and 3800-4200 m³ / h was introduced into a drying tower for drying, yielding an effective viable count of not less than 1×10⁻⁶. 11 CFU / g of bacterial powder.

[0066] In this embodiment, the method for preparing the activated culture seed solution is as follows: Bacillus subtilis ZKY01, Bacillus belye ZKY02, and Bacillus sicca ZKY03, stored at -85℃, were activated on LB solid medium. They were then streaked at 35-37℃ for 24-48 hours. Single colonies were picked and streaked on solid medium for another 24-48 hours at 35-37℃ to obtain the activated strains.

[0067] The activated strain was scraped with a sterile inoculation loop and inoculated into 50 mL of LB liquid medium. The culture was carried out at 35°C and 150 rpm for 24 h to obtain the seed culture of the strain.

[0068] In this embodiment, the fermentation culture medium of Bacillus subtilis ZKY01 comprises, by weight: 10-20 parts corn flour, 1-4 parts sucrose, 2-6 parts calcium carbonate, 3-7 parts soybean flour, 1-4 parts peptone, 0.5-2 parts fish meal, 0.1-0.5 parts potassium dihydrogen phosphate, 0.1-0.3 parts manganese sulfate, 0.2-0.6 parts sodium chloride, 0.1-0.4 parts dipotassium hydrogen phosphate, 0.2-0.4 parts magnesium sulfate, 0.3-0.6 parts ammonium sulfate, 1000 parts water, and a pH of 7.2-7.8. The fermentation broth of *Bacillus belye* ZKY02 comprises, by weight: 15-25 parts soybean flour, 1-4 parts peptone, 3-9 parts corn flour, 4-8 parts sucrose, 0.2-0.5 parts sodium chloride, 0.1-0.4 parts potassium dihydrogen phosphate, 0.1-0.3 parts manganese sulfate, 0.2-0.4 parts magnesium sulfate, 1000 parts water, with a pH of 6.8-7.5. The fermentation broth of Bacillus sicca ZKY03 comprises, by weight: 8-16 parts glucose, 15-25 parts soybean meal, 2-6 parts peptone, 8-16 parts corn starch, 0.3-0.7 parts magnesium sulfate, 0.1-0.5 parts sodium chloride, 0.3-0.7 parts dipotassium hydrogen phosphate, 0.1-0.4 parts manganese sulfate, and 1000 parts water, with a pH of 7.0-7.5.

[0069] In this embodiment, the microcapsule carrier solution is an aqueous solution with a mass concentration of 2-6% prepared by mixing maltodextrin and gum arabic in a mass ratio of 2-5:1; the excipient is a mixture of diatomaceous earth and calcium carbonate in a mass ratio of 25-45:55-75.

[0070] This invention also provides a composting material for tobacco cultivation, obtained by composting organic materials with the aforementioned disease-controlling composting agent or a composting agent prepared by the aforementioned method; wherein the organic material is selected from straw, livestock and poultry manure, or a mixture of both. Specifically, straw includes crop straw such as corn straw, rice straw, and wheat straw, and livestock and poultry manure includes livestock and poultry manure such as cow manure, chicken manure, duck manure, and pig manure.

[0071] This invention also provides the application of the above-described composted tobacco cultivation material in promoting tobacco growth and / or improving tobacco disease resistance. The promotion of tobacco growth refers to increasing tobacco plant height, increasing the number of leaves and the weight of a single leaf; the improvement of tobacco disease resistance refers to improving the immunity of tobacco to the pathogen of tobacco black shank.

[0072] The following detailed description, in conjunction with embodiments, illustrates the present invention's method for preparing and applying a composting agent with disease prevention and control functions (Tables 3-8), but these should not be construed as limiting the scope of protection of the present invention.

[0073] Example 1

[0074] This invention provides a method for preparing a composting agent with disease control function, the specific steps of which are as follows:

[0075] Step 1, strain activation: Bacillus subtilis ZKY01, Bacillus belye ZKY02, and Bacillus sicca ZKY03, which were stored at -85℃, were activated on LB solid medium by streak culture at 37℃ for 24 hours. Single colonies were picked and streak cultured on solid medium for 48 hours at 35℃ to obtain activated strains.

[0076] The second step is seed culture preparation: the activated strain from the first step is scraped with a sterile inoculation loop and inoculated into 50 mL of LB liquid medium. The culture is carried out at 35°C and 150 rpm for 24 h to obtain the seed culture of the strain.

[0077] The third step is the preparation of the fermentation broth: the activated seed culture is inoculated into the fermentation culture medium in the fermenter at a ratio of 2‰, and cultured for 40 hours at a temperature of 37℃, an aeration ratio of 1:0.3, and a rotation speed of 180 rpm / min to obtain the fermentation broth; the effective viable count of the fermentation broth is greater than 2.0 × 10⁻⁶. 9 CFU / mL.

[0078] Step 4, Preparation of inoculum: The fermentation broth is introduced into a centrifuge, and after centrifugation, the supernatant is removed to obtain a concentrated inoculum solution, ensuring that the effective viable count in the concentrated inoculum solution is not less than 1×10⁻⁶. 10CFU / mL; Bacillus powder was prepared by spray drying of the concentrated bacterial culture, including the following steps: The concentrated bacterial culture was added to a mixing tank, along with 10%–13% of excipients (35 parts diatomaceous earth, 65 parts calcium carbonate). The concentrated bacterial culture was pressurized to a rotating nozzle under a pressure of 0.9 Pa, and the nozzle was maintained at a rotation speed of 20,000 r / min while spraying. Simultaneously, a 150°C, 4000 mL aeration system was introduced into a drying tower. 3 Drying with hot air at a rate of / h yields an effective viable bacteria count of not less than 1×10⁻⁶. 11 CFU / g of bacterial powder.

[0079] Step 5, Preparation of compound bacterial powder: The Bacillus subtilis ZKY01 and Bacillus belyeis ZKY02 bacterial powders obtained in step 4 are compounded at a mass ratio of 1:1 to prepare an effective viable count ≥1.0×10⁻⁶. 11 Compound bacterial powder with CFU / g.

[0080] Step 6, preparation of microcapsule bacterial agent: Maltodextrin and gum arabic are mixed at a mass ratio of 3:1 to prepare a 4% aqueous solution. Impurities are removed by filtration. The *Bacillus sicca* ZKY03 bacterial powder obtained in Step 4 is mixed with the maltodextrin and gum arabic aqueous solution, and the solid content is adjusted to 25%. The mixture containing biocontrol bacteria and carrier is atomized into tiny droplets using hot air and rapidly dried to form microcapsule bacterial agent. The encapsulation rate of the microcapsule bacterial agent is 70%, and the viable count of *Bacillus sicca* ZKY03 encapsulated in the microcapsule bacterial agent in the composting agent is 3.5 × 10⁻⁶. 10 CFU / g.

[0081] Step 7, Preparation of the composting agent: The compound bacterial powder and microcapsules are mixed at a mass ratio of 1:2 to obtain an effective viable bacteria count ≥7.0×10⁻⁶. 10 CFU / g composting agent.

[0082] In this embodiment, the culture medium, which is the component of the material in the preparation process of the composting agent, is as follows:

[0083] LB solid medium: 20g bacteriological peptone, 10g yeast extract, 10g agar powder, 2L water. For liquid medium, simply remove the agar powder.

[0084] Bacillus subtilis ZKY01 fermentation broth: 15 parts corn flour, 2 parts sucrose, 4 parts calcium carbonate, 5 parts soybean flour, 2 parts peptone, 1 part fish meal, 0.3 parts potassium dihydrogen phosphate, 0.2 parts manganese sulfate, 0.4 parts sodium chloride, 0.2 parts dipotassium hydrogen phosphate, 0.3 parts magnesium sulfate, 0.4 parts ammonium sulfate, and 1000 parts water. The pH is adjusted to 7.5 using hydrochloric acid or sodium hydroxide.

[0085] Bacillus vesiculosus ZKY02 fermentation broth: 20 parts soybean flour, 2 parts peptone, 6 parts corn flour, 6 parts sucrose, 0.3 parts sodium chloride, 0.2 parts potassium dihydrogen phosphate, 0.2 parts manganese sulfate, 0.3 parts magnesium sulfate, and 1000 parts water. The pH is adjusted to 7.2 using hydrochloric acid or sodium hydroxide.

[0086] Bacillus sicca ZKY03 fermentation broth: 12 parts glucose, 20 parts soybean meal powder, 4 parts peptone, 12 parts corn starch, 0.5 parts magnesium sulfate, 0.3 parts sodium chloride, 0.5 parts dipotassium hydrogen phosphate, 0.2 parts manganese sulfate, and 1000 parts water. The pH is adjusted to 7.3 using hydrochloric acid or sodium hydroxide.

[0087] The above-prepared composting agent is used to compost straw and prepare composted materials: Select fresh corn stalks free from mold and pests, dry them until the moisture content is 30%-35%, then crush them to 2-3 cm, remove impurities, and obtain clean crushed corn stalks for later use. Adjust the moisture content of the crushed corn stalks to about 50%, add the composting agent of this invention at a ratio of 0.4% of the dry weight of the stalks and mix well. After piling, place it in a ventilated environment at 20℃ for composting. Turn the pile every 3 days to ensure uniform oxygen supply until the stalks are completely decomposed.

[0088] Composting Results: Compared with natural composting, the straw composting time was shortened by 25 days after adding the composting agent, the seed germination index increased by 21 percentage points, the fecal coliform count decreased by 14 CFU / g, and the ascarid egg mortality rate increased by 8 percentage points. The effective viable count of Bacillus sicca ZKY03 in fully composted straw was 5.2 × 10⁻⁶. 7 CFU / g.

[0089] Table 3. Effects of composting agents on the composting of corn stalks.

[0090]

[0091] Application of the above-prepared decomposed material in tobacco cultivation: When fully decomposed corn stalks (800 kg / mu) are returned to the field for tobacco cultivation, and nitrogen fertilizer application is appropriately reduced (0.8 kg / mu of pure nitrogen is reduced), compared with conventional fertilization (without applying fully decomposed corn stalks in this example and without reducing the amount of pure nitrogen), the incidence of tobacco root black rot is reduced by 48.20%, the incidence of black shank is reduced by 56.19%, the tobacco plants grow vigorously, and the number of leaves and the weight of a single leaf are significantly increased.

[0092] Table 4. Application effects of decomposed corn stalks in tobacco cultivation

[0093]

[0094] The composting agent of this invention can efficiently compost corn stalks. The composted corn stalks can be returned to the field for tobacco cultivation, which can significantly improve the growth of tobacco, reduce the incidence of diseases, and reduce the application of nitrogen fertilizer, thus achieving both economic and ecological benefits.

[0095] Example 2: Application of composting agents in tobacco cultivation of livestock and poultry manure

[0096] This invention provides a method for preparing a composting agent with disease control function, the specific steps of which are as follows:

[0097] Step 1, strain activation: Bacillus subtilis ZKY01, Bacillus belye ZKY02, and Bacillus sicca ZKY03, which were stored at -85℃, were activated on LB solid medium by streak culture at 37℃ for 24 hours. Single colonies were picked and streak cultured on solid medium for 48 hours at 35℃ to obtain activated strains.

[0098] The second step is seed culture preparation: the activated strain from the first step is scraped with a sterile inoculation loop and inoculated into 50 mL of LB liquid medium. The culture is carried out at 35°C and 150 rpm for 24 h to obtain the seed culture of the strain.

[0099] The third step is the preparation of the fermentation broth: the activated seed culture is inoculated into the fermentation culture medium in the fermenter at a ratio of 2‰, and cultured for 40 hours at a temperature of 37℃, an aeration ratio of 1:0.3, and a rotation speed of 180 rpm / min to obtain the fermentation broth; the effective viable count of the fermentation broth is greater than 2.0 × 10⁻⁶. 9 CFU / mL.

[0100] Step 4, Preparation of inoculum: The fermentation broth is introduced into a centrifuge, and after centrifugation, the supernatant is removed to obtain a concentrated inoculum solution, ensuring that the effective viable count in the concentrated inoculum solution is not less than 1×10⁻⁶. 10 CFU / mL; The Bacillus powder is prepared by spray drying of the concentrated bacterial culture, including the following steps: The concentrated bacterial culture is added to a mixing tank, along with 10%–13% excipients (35 parts diatomaceous earth, 65 parts calcium carbonate). The concentrated bacterial culture is pressurized to a rotating nozzle under a pressure of 0.9 Pa, and the nozzle is maintained at a rotation speed of 20,000 r / min while spraying. Simultaneously, a 150°C, 4000 mL aeration system is introduced into the drying tower. 3 Drying with hot air at a rate of / h yields an effective viable bacteria count of not less than 1×10⁻⁶. 11 CFU / g of bacterial powder.

[0101] Step 5, Preparation of compound bacterial powder: The Bacillus subtilis ZKY01 and Bacillus belyssus ZKY02 bacterial powders obtained in step 4 are compounded at a mass ratio of 1:2 to prepare a mixture with an effective viable count ≥1.0 × 10⁻⁶.11 Compound bacterial powder with CFU / g.

[0102] Step 6, preparation of microcapsule bacterial agent: Maltodextrin and gum arabic are mixed at a mass ratio of 3:1 to prepare a 4% aqueous solution. Impurities are removed by filtration. The *Bacillus sicca* ZKY03 bacterial powder obtained in Step 4 is mixed with the maltodextrin and gum arabic aqueous solution, and the solid content is adjusted to 25%. The mixture containing biocontrol bacteria and carrier is atomized into tiny droplets using hot air and rapidly dried to form microcapsule bacterial agent. The encapsulation rate of the microcapsule bacterial agent is 70%, and the viable count of *Bacillus sicca* ZKY03 encapsulated in the microcapsule bacterial agent in the composting agent is 3.5 × 10⁻⁶. 10 CFU / g.

[0103] Step 7, Preparation of the composting agent: The compound bacterial powder and microcapsule bacterial agent are mixed at a mass ratio of 1:3 to obtain an effective viable bacteria count ≥7.0 × 10⁻⁶. 10 CFU / g composting agent.

[0104] Specifically, the composition of the culture medium in the preparation of the composting agent:

[0105] LB solid medium: 20g bacteriological peptone, 10g yeast extract, 10g agar powder, 2L water. For liquid medium, simply remove the agar powder.

[0106] Bacillus subtilis ZKY01 fermentation broth: 15 parts corn flour, 2 parts sucrose, 4 parts calcium carbonate, 5 parts soybean flour, 2 parts peptone, 1 part fish meal, 0.3 parts potassium dihydrogen phosphate, 0.2 parts manganese sulfate, 0.4 parts sodium chloride, 0.2 parts dipotassium hydrogen phosphate, 0.3 parts magnesium sulfate, 0.4 parts ammonium sulfate, and 1000 parts water. The pH is adjusted to 7.5 using hydrochloric acid or sodium hydroxide.

[0107] Bacillus vesiculosus ZKY02 fermentation broth: 20 parts soybean flour, 2 parts peptone, 6 parts corn flour, 6 parts sucrose, 0.3 parts sodium chloride, 0.2 parts potassium dihydrogen phosphate, 0.2 parts manganese sulfate, 0.3 parts magnesium sulfate, and 1000 parts water. The pH is adjusted to 7.2 using hydrochloric acid or sodium hydroxide.

[0108] Bacillus sicca ZKY03 fermentation broth: 12 parts glucose, 20 parts soybean meal powder, 4 parts peptone, 12 parts corn starch, 0.5 parts magnesium sulfate, 0.3 parts sodium chloride, 0.5 parts dipotassium hydrogen phosphate, 0.2 parts manganese sulfate, and 1000 parts water. The pH is adjusted to 7.3 using hydrochloric acid or sodium hydroxide.

[0109] The composting agent obtained above is used to compost livestock and poultry manure to prepare composted materials:

[0110] Select fresh, mold-free, odorless, and stone-free cow manure. Remove stones, weeds, plastic, and other impurities. Spread it out in a ventilated area for 1-2 days to initially reduce its moisture content. (Pre-treated cow manure will not clump, facilitating subsequent mixing.) Adjust the moisture content of the pre-treated cow manure to approximately 65%. Weigh out 0.2% of the dry weight of the manure to be fermented as a composting agent and add it to the manure, mixing thoroughly. Pile the manure and place it in a ventilated environment at 20℃ for composting. During composting, turn the pile every 2 days to ensure even oxygen supply until the manure is fully decomposed.

[0111] Composting Results: Compared with natural composting, the composting time of cow manure was shortened by 26 days after adding the composting agent, the seed germination index increased by 23 percentage points, the fecal coliform count decreased by 51 CFU / g, and the ascarid egg mortality rate increased by 9 percentage points. The effective viable count of Bacillus sicca ZKY03 in fully composted cow manure was 4.5 × 10⁻⁶. 7 CFU / g.

[0112] Table 5. Effects of composting agents on cow manure composting

[0113]

[0114] Application of the above-prepared decomposed material in tobacco cultivation: When fully decomposed cow manure (600 kg / mu) is returned to the field for tobacco cultivation, and nitrogen fertilizer application is appropriately reduced (pure nitrogen application is reduced by 1.2 kg / mu), compared with conventional fertilization (without applying fully decomposed corn stalks in this example and without reducing pure nitrogen application), the incidence of tobacco root black rot is reduced by 30.10%, the incidence of black shank is reduced by 50.76%, and the tobacco plants grow vigorously, with significant increases in plant height, number of leaves, and single leaf weight.

[0115] Table 6. Effects of well-rotted cow manure on tobacco cultivation

[0116]

[0117] In this embodiment of the invention, Bacillus subtilis ZKY01 and Bacillus belyss ZKY02 bacterial powders were compounded at a ratio of 1:2, and the compound bacterial powder was mixed with Bacillus sicca ZKY03 microcapsule bacterial agent at a ratio of 1:3, to obtain an effective viable count ≥7.0 × 10⁻⁶. 10 A composting agent with CFU / g. This composting agent can advance the composting process of cow manure by 26 days, improve the quality of composting, and after composting, the content of Bacillus sicca ZKY03 in the cow manure reaches 4.5×10⁻⁶. 7 With a concentration of CFU / g, applying 600 kg / mu of the composting agent to the field can reduce pure nitrogen by 1.2 kg / mu, significantly reducing the incidence of tobacco root black rot and black shank disease and improving plant growth. This expands the application scenarios of composting agents and has both economic and ecological benefits.

[0118] Example 3

[0119] This invention provides a method for preparing a composting agent with disease control function, the specific steps of which are as follows:

[0120] Step 1, strain activation: Bacillus subtilis ZKY01, Bacillus belye ZKY02, and Bacillus sicca ZKY03, which were stored at -85℃, were activated on LB solid medium by streak culture at 37℃ for 24 hours. Single colonies were picked and streak cultured on solid medium for 48 hours at 35℃ to obtain activated strains.

[0121] The second step is seed culture preparation: the activated strain from the first step is scraped with a sterile inoculation loop and inoculated into 50 mL of LB liquid medium. The culture is carried out at 35°C and 150 rpm for 24 h to obtain the seed culture of the strain.

[0122] The third step is the preparation of the fermentation broth: the activated seed culture is inoculated into the fermentation culture medium in the fermenter at a ratio of 2‰, and cultured for 40 hours at a temperature of 37℃, an aeration ratio of 1:0.3, and a rotation speed of 180 rpm / min to obtain the fermentation broth; the effective viable count of the fermentation broth is greater than 2.0 × 10⁻⁶. 9 CFU / mL.

[0123] Step 4, Preparation of inoculum: The fermentation broth is introduced into a centrifuge, and after centrifugation, the supernatant is removed to obtain a concentrated inoculum solution, ensuring that the effective viable count in the concentrated inoculum solution is not less than 1×10⁻⁶. 10 CFU / mL; The Bacillus powder is prepared by spray drying of the concentrated bacterial culture, including the following steps: The concentrated bacterial culture is added to a mixing tank, along with 10%-13% of excipients (35 parts diatomaceous earth, 65 parts calcium carbonate). The concentrated bacterial culture is pressurized to a rotating nozzle under a pressure of 0.9 Pa, and the nozzle is maintained at a rotation speed of 20000 r / min while spraying. Simultaneously, a 150℃, 4000m³ / min airflow is introduced into the drying tower. 3 Drying with hot air at a rate of / h yields an effective viable bacteria count of not less than 1×10⁻⁶. 11 CFU / g of bacterial powder.

[0124] Step 5, Preparation of compound bacterial powder: The Bacillus subtilis ZKY01 and Bacillus belyeis ZKY02 bacterial powders obtained in step 4 are compounded at a mass ratio of 1:1 to prepare a mixture with an effective viable count ≥1.0 × 10⁻⁶. 11 Compound bacterial powder with CFU / g.

[0125] Step 6, preparation of microcapsule bacterial agent: Maltodextrin and gum arabic are mixed at a mass ratio of 3:1 to prepare a 4% aqueous solution. Impurities are removed by filtration. The *Bacillus sicca* ZKY03 bacterial powder obtained in Step 4 is mixed with the maltodextrin and gum arabic aqueous solution, and the solid content is adjusted to 25%. The mixture containing biocontrol bacteria and carrier is atomized into tiny droplets using hot air and rapidly dried to form microcapsule bacterial agent. The encapsulation rate of the microcapsule bacterial agent is 70%, and the viable count of *Bacillus sicca* ZKY03 encapsulated in the microcapsule bacterial agent in the composting agent is 3.5 × 10⁻⁶. 10 CFU / g.

[0126] Step 7, Preparation of the composting agent: The compound bacterial powder and microcapsule bacterial agent are mixed at a mass ratio of 1:1 to obtain an effective viable bacteria count ≥ 8.0 × 10⁻⁶. 10 CFU / g composting agent.

[0127] Specifically, the composition of the culture medium in the preparation of the composting agent:

[0128] LB solid medium: 20g bacteriological peptone, 10g yeast extract, 10g agar powder, 2L water. For liquid medium, simply remove the agar powder.

[0129] Bacillus subtilis ZKY01 fermentation broth: 15 parts corn flour, 2 parts sucrose, 4 parts calcium carbonate, 5 parts soybean flour, 2 parts peptone, 1 part fish meal, 0.3 parts potassium dihydrogen phosphate, 0.2 parts manganese sulfate, 0.4 parts sodium chloride, 0.2 parts dipotassium hydrogen phosphate, 0.3 parts magnesium sulfate, 0.4 parts ammonium sulfate, and 1000 parts water. The pH is adjusted to 7.5 using hydrochloric acid or sodium hydroxide.

[0130] Bacillus vesiculosus ZKY02 fermentation broth: 20 parts soybean flour, 2 parts peptone, 6 parts corn flour, 6 parts sucrose, 0.3 parts sodium chloride, 0.2 parts potassium dihydrogen phosphate, 0.2 parts manganese sulfate, 0.3 parts magnesium sulfate, and 1000 parts water. The pH is adjusted to 7.2 using hydrochloric acid or sodium hydroxide.

[0131] Bacillus sicca ZKY03 fermentation broth: 12 parts glucose, 20 parts soybean meal powder, 4 parts peptone, 12 parts corn starch, 0.5 parts magnesium sulfate, 0.3 parts sodium chloride, 0.5 parts dipotassium hydrogen phosphate, 0.2 parts manganese sulfate, and 1000 parts water. The pH is adjusted to 7.3 using hydrochloric acid or sodium hydroxide.

[0132] The composting agent prepared above is used to compost a mixture of corn stalks and cow dung to prepare composted materials:

[0133] Select fresh, mold-free, odorless, and stone-free cow dung, remove stones, weeds, plastics, and other impurities, and spread it out in a ventilated place to air-dry for 1-2 days to initially reduce moisture. Simultaneously, select fresh, mold-free, and pest-free corn stalks, dry them until the moisture content is 30%-35%, then crush them to 2-3 cm and remove impurities. Mix the cow dung and corn stalks thoroughly at a mass ratio of 1:3 to obtain a ready-to-use mixture (the mixture should not have obvious lumps, facilitating subsequent mixing of the composting agent). Adjust the moisture content of the ready-to-use mixture to approximately 55%. Weigh 0.3% of the weight of the composting agent into the cow dung and corn stalk mixture and mix thoroughly. Compost at 20℃, turning the pile every 2 days during composting to ensure even oxygen supply.

[0134] Composting Results: Compared with natural composting, the composting time of the cow manure and corn stalk mixture was shortened by 28 days after adding the composting agent, the seed germination index increased by 28 percentage points, the fecal coliform count decreased by 65 CFU / g, and the ascarid egg mortality rate increased by 8 percentage points. The effective viable count of Bacillus sicca ZKY03 in the fully composted cow manure and corn stalk mixture was 4.9 × 10⁻⁶. 7 CFU / g.

[0135] Table 7. Effects of composting agents on the composting of cow manure and corn stalk mixtures.

[0136]

[0137] Application of the above-prepared decomposed material in tobacco cultivation: A mixture of fully decomposed cow manure and corn stalks (600 kg / mu) was returned to the field for tobacco cultivation. Under the condition of appropriately reducing nitrogen fertilizer application (reducing pure nitrogen application by 1.2 kg / mu), compared with conventional fertilization (not using fully decomposed corn stalks in this example and not reducing pure nitrogen application), the incidence of tobacco root black rot decreased by 43.77%, the incidence of black shank disease decreased by 55.09%, and the tobacco plants grew vigorously, with significant increases in plant height, number of leaves, and single leaf weight.

[0138] Table 8. Application effects of a mixture of well-rotted cow manure and corn stalks in tobacco cultivation.

[0139]

[0140] In this embodiment of the invention, Bacillus subtilis ZKY01 and Bacillus belye ZKY02 bacterial powders were compounded at a 1:1 ratio, and the compound bacterial powder was mixed with Bacillus sicca ZKY03 microcapsule bacterial agent at a 1:1 ratio to obtain an effective viable count ≥8.0 × 10⁻⁶. 10A composting agent with CFU / g. This composting agent, when used to compost a 1:3 mixture of cow manure and corn stalks, can complete the composting process 28 days earlier, increasing seed germination index by 28 percentage points, reducing fecal coliform count by 65 CFU / g, and increasing ascaris egg mortality by 8 percentage points. After composting, the ZKYO3 content reaches 4.9 × 10⁻⁶. 7 CFU / g; returning 600 kg / mu to the field can reduce pure nitrogen by 1.2 kg / mu, which reduces the incidence of tobacco root black rot and black shank disease by 43.77% and 55.09% respectively, and improves plant growth, expands application scenarios, and has both economic and ecological benefits.

[0141] The above description is merely a specific embodiment illustrative of the present invention and is not intended to limit the scope of protection of the present invention. Any equivalent changes and modifications made by those skilled in the art without departing from the concept and principles of the present invention shall fall within the scope of protection of the present invention.

[0142] The 16S rDNA sequence of Bacillus subtilis ZKY01 (SEQ ID NO.1):

[0143] The 16S rDNA sequence of Bacillus belyssus ZKY02 (SEQ ID NO.2):

[0144] The 16S rDNA sequence of Bacillus sicca ZKY03 (SEQ ID NO.3):

[0145] Information on the preservation of biological materials:

[0146] Bacillus subtilis ZKY01 was deposited at the China Center for Type Culture Collection on December 22, 2025, with accession number CCTCCNO:M 20252980. The deposit address is located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on the campus of Wuhan University (opposite to the First Affiliated Primary School of Wuhan University).

[0147] Bacillus velezensis ZKY02 was deposited on December 22, 2025 at the China Center for Type Culture Collection (CCTCCNO:M 20252981), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (opposite to the First Affiliated Primary School of Wuhan University).

[0148] Bacillus siamensis ZKY03 was deposited at the China Center for Type Culture Collection on December 22, 2025, with accession number CCTCCNO:M 20252982. The deposit address is located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on the campus of Wuhan University (opposite to the First Affiliated Primary School of Wuhan University).

Claims

1. A composting agent with disease control function, characterized in that, The composting agent consists of a compound bacterial powder and a microcapsule bacterial agent. The compound bacterial powder is a blend of Bacillus subtilis ZKY01 and Bacillus belyssus ZKY02 bacterial powders, and the microcapsule bacterial agent is Bacillus sicca ZKY03 bacterial powder encapsulated in a microcapsule carrier. All three bacterial powders are obtained through fermentation and culture, with an effective viable count of not less than 1 × 10⁻⁶. 11 The powder contains CFU / g of Bacillus subtilis ZKY01, with accession number CCTCC NO:M 20252980; Bacillus belyss ZKY02, with accession number CCTCC NO:M20252981; and Bacillus sicca ZKY03, with accession number CCTCC NO:M 20252982.

2. The composting agent with disease control function according to claim 1, characterized in that, The mass ratio of Bacillus subtilis ZKY01 powder to Bacillus vesiculosus ZKY02 powder in the compound bacterial powder is 1~3:1~3, and the number of effective viable bacteria in the compound bacterial powder is not less than 1.0×10⁻⁶. 11 CFU / g.

3. The composting agent with disease control function according to claim 1, characterized in that, The microencapsulated bacterial agent uses maltodextrin and gum arabic as microcapsule carriers, with an encapsulation rate of 60%~80% and an effective viable bacteria count of not less than 3.5×10⁻⁶. 10 CFU / g.

4. The composting agent with disease control function according to claim 1, characterized in that, The mass ratio of compound bacterial powder to microencapsulated bacterial agent in the composting agent is 1:1~4, and the number of effective viable bacteria in the composting agent is not less than 7.0×10⁻⁶. 10 CFU / g.

5. A method for preparing a composting agent with disease control function as described in any one of claims 1-4, characterized in that, Includes the following steps: Bacillus subtilis ZKY01, Bacillus belyi ZKY02 and Bacillus sicca ZKY03 were prepared separately. The prepared Bacillus subtilis ZKY01 bacterial powder and Bacillus vesiculosus ZKY02 bacterial powder were mixed to prepare a compound bacterial powder. The prepared Bacillus sicca ZKY03 bacterial powder was mixed with a microcapsule carrier solution that had been filtered and purified, and the solid content was adjusted to 20%~30%. The mixture was then atomized and dried with hot air to form a microencapsulated bacterial agent. The prepared compound bacterial powder is mixed with the prepared microencapsulated bacterial agent to obtain the composting agent.

6. The preparation method according to claim 5, characterized in that, The method for preparing the mycelial powders of Bacillus subtilis ZKY01, Bacillus belyi ZKY02, and Bacillus sicca ZKY03 includes: The seed cultures of the three activated bacteria were inoculated into the corresponding fermentation broths at a ratio of 1-5‰, and cultured at 30-38℃ and 120-180 rpm / min for 40-60 h to obtain an effective viable count of not less than 2.0 × 10⁻⁶. 9 Fermentation broth with CFU / mL; The fermentation broth was transferred to a centrifuge, and after centrifugation, the supernatant bacterial suspension was removed to obtain an effective viable cell count of not less than 1×10⁻⁶. 10 CFU / mL bacterial culture concentrate; Add the concentrated microbial culture solution to a mixing tank, along with an auxiliary material accounting for 10-13% of the culture solution's mass. Pressurize the concentrated culture solution under a pressure of 0.8-1.0 Pa, then spray it through a rotating nozzle at a speed of 18000-22000 r / min. Simultaneously, introduce a 140-160℃, 3800-4200m³ / min airflow into a drying tower. 3 Drying with hot air at a rate of / h yields an effective viable bacteria count of not less than 1×10⁻⁶. 11 CFU / g of bacterial powder.

7. The preparation method according to claim 6, characterized in that, The fermentation broth of Bacillus subtilis ZKY01 comprises, by weight: 10-20 parts corn flour, 1-4 parts sucrose, 2-6 parts calcium carbonate, 3-7 parts soybean flour, 1-4 parts peptone, 0.5-2 parts fish meal, 0.1-0.5 parts potassium dihydrogen phosphate, 0.1-0.3 parts manganese sulfate, 0.2-0.6 parts sodium chloride, 0.1-0.4 parts dipotassium hydrogen phosphate, 0.2-0.4 parts magnesium sulfate, 0.3-0.6 parts ammonium sulfate, 1000 parts water, with a pH of 7.2-7.

8. The fermentation broth of *Bacillus belye* ZKY02 comprises, by weight: 15-25 parts soybean flour, 1-4 parts peptone, 3-9 parts corn flour, 4-8 parts sucrose, 0.2-0.5 parts sodium chloride, 0.1-0.4 parts potassium dihydrogen phosphate, 0.1-0.3 parts manganese sulfate, 0.2-0.4 parts magnesium sulfate, 1000 parts water, with a pH of 6.8-7.

5. The fermentation broth of Bacillus sicca ZKY03 comprises, by weight: 8-16 parts glucose, 15-25 parts soybean meal, 2-6 parts peptone, 8-16 parts corn starch, 0.3-0.7 parts magnesium sulfate, 0.1-0.5 parts sodium chloride, 0.3-0.7 parts dipotassium hydrogen phosphate, 0.1-0.4 parts manganese sulfate, and 1000 parts water, with a pH of 7.0-7.

5.

8. The preparation method according to claim 5, characterized in that, The microcapsule carrier solution is an aqueous solution with a mass concentration of 2-6% prepared by mixing maltodextrin and gum arabic in a mass ratio of 2-5:1; the excipient is a mixture of diatomaceous earth and calcium carbonate in a mass ratio of 25-45:55-75.

9. A composted material for tobacco cultivation, characterized in that, The composting agent with disease control function as described in any one of claims 1-4 or the composting agent prepared by the preparation method described in any one of claims 5-8 is used to compost organic materials; wherein the organic materials are selected from straw, livestock and poultry manure or a mixture of both.

10. The application of the composted tobacco cultivation material according to claim 9 in promoting tobacco growth and / or improving tobacco disease resistance, characterized in that, The promotion of tobacco growth refers to increasing the height of tobacco plants, increasing the number of leaves and the weight of a single leaf; the improvement of tobacco disease resistance refers to improving the immunity of tobacco to the pathogen of tobacco black shank.

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