Bacillus microbial agent for preventing and controlling tobacco diseases, and preparation method and application thereof

Abstract

This invention discloses a Bacillus microbial agent for controlling tobacco diseases, its preparation method, and its application. The microbial agent is prepared by compounding Bacillus amyloliquefaciens and Bacillus bellis in a volume ratio of 0.8-1.2:1, resulting in a total effective viable count ≥2×10¹⁰ CFU / mL. This invention utilizes independently isolated specific Bacillus amyloliquefaciens and Bacillus bellis; the compounding of these two agents creates a synergistic effect. Bacillus amyloliquefaciens secretes antibacterial substances that directly inhibit the growth of pathogens, while Bacillus bellis induces resistance in tobacco plants and enhances rhizosphere colonization, synergistically improving the control effect against tobacco bacterial wilt and black shank. Field trials show that this microbial agent achieves a control efficacy of over 75% against tobacco bacterial wilt and over 85% against black shank, significantly higher than single-strain agents. The strains used in this invention are isolated from the rhizosphere soil of tobacco plants in different tobacco-growing areas of Baoshan. The addition of a special stabilizer after compounding enhances the strains' resistance, extends shelf life at room temperature, and maintains stable efficacy.

Description

Technical Field

[0001] This invention relates to the field of microbial control technology, specifically to a Bacillus microbial agent for controlling tobacco diseases, its preparation method, and its application. Background Technology

[0002] In agricultural production, plant pathogenic fungi and bacteria easily infect plants, causing tens of millions of yuan in economic losses annually. How to control diseases and maintain normal plant growth has always been a hot topic for scientific researchers. Chemical pesticides can effectively control fungal and bacterial diseases, playing a significant role in plant disease prevention and control. However, with increased awareness of chemical pesticides—such as their pollution of soil, water, and air, increased pesticide residues in agricultural products, direct harm to human health and survival, and the development of pesticide resistance in pathogens—the use of many pesticides has been restricted. This has accelerated the search for new, safe, and effective methods for plant disease control. Utilizing antagonistic microorganisms in the natural environment to prevent plant pathogen invasion, commonly known as biological control, not only avoids the pesticide resistance problems caused by chemical pesticides but is also safe for both the environment and human health.

[0003] Existing microbial agents generally suffer from problems such as unclear synergistic mechanisms, significant susceptibility to environmental factors in their efficacy, and weak adaptability to continuously cropped tobacco areas, making it difficult to meet the needs of tobacco production for efficient, stable, and green control technologies. Therefore, developing a Bacillus microbial agent with strong synergistic effects, stable efficacy, and broad adaptability, capable of simultaneously and efficiently controlling both bacterial wilt and black shank in tobacco, has significant practical implications and application value. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus microbial agent for the prevention and control of tobacco diseases, its preparation method, and its application, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a Bacillus microbial agent for the prevention and control of tobacco diseases, wherein the microbial agent is prepared by compounding Bacillus amyloliquefaciens bacterial solution and Bacillus belye bacterial solution at a volume ratio of 0.8~1.2:1, and the total effective viable count of the agent after compounding is ≥2×10¹⁰ CFU / mL;

[0006] The Bacillus amyloliquefaciens strain described is an independently isolated strain, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M20252418 and deposit date: November 10, 2025.

[0007] The *Bacillus velezensis* strain described is an independently isolated strain, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M20252419 and deposit date: November 10, 2025.

[0008] A method for preparing a Bacillus microbial agent for the prevention and control of tobacco diseases includes the following steps:

[0009] S1. Strain activation: Bacillus amyloliquefaciens CCTCCNO:M20252418 and Bacillus bereaves CCTCCNO:M20252419 were inoculated onto LB solid medium and cultured at 35°C for 24 h to obtain single colonies; then, the single colonies were picked and inoculated into LB liquid medium and cultured with shaking for a short time to obtain activated bacterial solution.

[0010] S2. Seed culture: Inoculate the activated bacterial solution obtained in step S1 into the seed culture medium at an inoculation rate of 2%–3% by volume, and culture with shaking for a short period until the viable bacterial count is ≥1×10⁻⁶. 8 CFU / mL, to obtain Bacillus amyloliquefaciens seed culture and Bacillus vesicle seed culture;

[0011] S3. Fermentation Culture: The seed culture obtained in step S2 is inoculated into the fermentation medium at an inoculation rate of 5%–8% by volume, and cultured with shaking for a long time, controlling the pH at 7.0–7.5, until the viable count of Bacillus amyloliquefaciens in the culture is ≥1×10¹. 0 CFU / mL, viable count of Bacillus belysinus in bacterial suspension ≥1×10¹ 0 CFU / mL was used to obtain fermentation broths of two strains;

[0012] S4. Compound preparation: Mix the two fermentation liquids obtained in step S3 at a volume ratio of 0.8 to 1.2:1, stir evenly, add a stabilizer with a volume fraction of 8.2%, and let stand at room temperature for 1 to 2 hours to obtain the microbial agent.

[0013] Preferably, the seed culture medium consists of: 20 g / L glucose, 15 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, pH 7.0–7.2, and is sterilized at 121°C for 20 min.

[0014] Preferably, the fermentation medium consists of: 8 g / L corn flour, 18 g / L soybean flour, 4 g / L fish meal, 2 g / L peptone, 6 g / L white sugar, 5 g / L calcium carbonate, 0.2 g / L manganese sulfate, 0.3 g / L sodium chloride, 0.5 g / L magnesium sulfate, 0.3 g / L potassium dihydrogen phosphate, 0.3 g / L dipotassium hydrogen phosphate, 0.5 g / L ammonium sulfate, and 0.25 g / L sodium hydroxide.

[0015] Preferably, the stabilizer comprises the following components: methylparaben 0.1%, glycerol 3%, sodium dodecyl sulfate 2%, xanthan gum 0.2%, azone 0.5%, and OP-10 2%.

[0016] Preferably, the brief shaking culture is carried out at 32-37℃ and 150-200 r / min for 14-20 h.

[0017] Preferably, the long-term shaking culture is carried out at 30-32℃ and 180-200 r / min for 36-48 hours.

[0018] Preferably, the total number of viable bacteria in the bacterial agent in S4 is ≥2×10¹. 0 CFU / mL, pH 7.0–7.5.

[0019] Application of a Bacillus microbial agent for controlling tobacco diseases in the control of soil-borne diseases.

[0020] Preferably, the soil-borne diseases include tobacco bacterial wilt and black shank, and the control methods are as follows:

[0021] Seedling application: Dilute 2 liters of microbial agent with water 50-100 times to obtain a diluted solution. Mix the diluted solution thoroughly with 100 kg of substrate and then fill the trays.

[0022] Application during transplanting: Dilute the microbial agent with water 300 times to obtain a diluted solution; when transplanting tobacco, use the root dipping method to immerse the roots of the tobacco seedlings in the diluted solution for 3-5 minutes, or use the root irrigation method to irrigate each tobacco seedling with 200-300 mL of the diluted solution, and cover with soil after planting;

[0023] Application during the seedling stage: 25-30 days after tobacco transplanting (seedling stage), dilute the microbial agent with water 200-500 times and drench the roots of each tobacco seedling with 300-900 mL. If the soil moisture is low, the amount of diluted solution can be increased appropriately.

[0024] Preferably, this microbial agent can be used in conjunction with organic fertilizer. The amount of organic fertilizer should be 60-100 kg / mu. After mixing with the diluted microbial agent, it can be applied to enhance the rhizosphere colonization ability of the strain and improve the control effect.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. Significant synergistic effect and excellent control effect: This invention selects independently isolated specific Bacillus amyloliquefaciens and Bacillus belye. The two combine to form a synergistic effect. The antibacterial substances secreted by Bacillus amyloliquefaciens directly inhibit the growth of pathogens, while Bacillus belye induces resistance in tobacco plants and enhances rhizosphere colonization ability, thus synergistically improving the control effect on tobacco bacterial wilt and black shank. Field trials show that the control efficacy of this microbial agent against tobacco bacterial wilt is over 75% and against black shank is over 85%, which is significantly higher than that of single-strain agents.

[0027] 2. Wide adaptability and strong stability: The strain of this invention was isolated from the rhizosphere soil of tobacco in different tobacco-growing areas of Baoshan. After domestication, it has strong adaptability to the soil environment of different tobacco-growing areas in Baoshan. After compounding, the addition of special stabilizers can enhance the stress resistance of the strain. It has a long shelf life at room temperature and is less affected by temperature, humidity and soil texture when applied in the field, with stable control effect.

[0028] 3. Promotes growth and improves soil, green and environmentally friendly: This microbial agent can not only control diseases, but also secrete growth-promoting substances, promote tobacco root development and increase biomass. At the same time, it optimizes the soil microbial community structure, increases the number of beneficial microorganisms, improves the physical and chemical properties of the soil, and avoids the residue and ecological damage caused by chemical agents, which meets the needs of green tobacco production.

[0029] 4. Simple preparation process and controllable cost: This invention can achieve large-scale production using conventional fermentation equipment. The fermentation medium mainly uses inexpensive raw materials such as corn flour and soybean meal, resulting in low cost. The preparation process is simple and easy to industrialize. The field application method is simple and farmers can operate directly, making it a promising technology for promotion and application. Attached Figure Description

[0030] Figure 1 The diagram shows the antagonistic effect of the strain of this invention against the pathogen of black shank (B7 and B9 from left to right).

[0031] Figure 2 The diagram shows the antagonistic effect of the strain of the present invention against the pathogen of bacterial wilt (B7 and B9 from left to right).

[0032] Figure 3 This is a layout diagram of the residential area according to the present invention;

[0033] Figure 4 This is a comparison diagram of root conditions under different treatments according to the present invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Please see Figures 1-4 This invention provides a technical solution: a highly efficient and synergistic Bacillus microbial agent for the prevention and control of tobacco bacterial wilt and black shank.

[0036] This microbial agent is prepared by mixing Bacillus amyloliquefaciens and Bacillus belye bacterial solutions at a volume ratio of 0.8~1.2:1. After mixing, the total effective viable count of the agent is ≥2×10¹⁰ CFU / mL.

[0037] The *Bacillus amyloliquefaciens* strain described is an independently isolated strain, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20252418 and deposit date: November 10, 2025. This strain was isolated from the rhizosphere soil of healthy plants in severely diseased fields with serious root and stem diseases in different tobacco-growing areas of Baoshan, Yunnan Province. It was identified as *Bacillus amyloliquefaciens*, exhibiting inhibition zones ≥15 mm in diameter against *Ralstonia solanacearum* and *Phytophthora indicum*, and demonstrating strong colonization ability in the tobacco rhizosphere. It can secrete lipopeptide antibacterial substances such as surfactants and iturin.

[0038] The *Bacillus velezensis* strain described is an independently isolated strain, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20252419 and deposit date: November 10, 2025. This strain was isolated from the rhizosphere soil of healthy plants in severely diseased fields with serious root and stem diseases in different tobacco-growing areas of Baoshan, Yunnan Province. It was identified as *Bacillus velezensis*, exhibiting inhibition zones ≥15 mm in diameter against *Ralstonia solanacearum* and *Phytophthora indicum*. It can efficiently induce tobacco plants to produce resistance-related enzymes such as peroxidase (POD) and polyphenol oxidase (PPO), thereby enhancing the plant's disease resistance.

[0039] The above-mentioned method for preparing microbial agents

[0040] Includes the following steps:

[0041] S1. Activation of bacterial strains: Bacillus amyloliquefaciens CCTCC NO: M20252418 and Bacillus bereaves CCTCC NO: M20252419 were inoculated onto LB solid medium and cultured at 35℃ for 24 h to obtain single colonies; single colonies were picked and inoculated onto LB liquid medium and cultured at 32~37℃ and 150~200 r / min for 14~20 h to obtain activated bacterial solution.

[0042] S2. Seed culture: Inoculate the activated Bacillus amyloliquefaciens and Bacillus belye bacterial cultures into the seed culture medium at an inoculation rate of 2%–3% (v / v), and culture at 32–37°C with shaking at 150–200 rpm for 14–20 h, until the viable bacterial count is ≥1×10⁻⁶. 8 CFU / mL was used to obtain seed culture of two strains; the seed culture medium consisted of: glucose 20 g / L, peptone 15 g / L, yeast extract 5 g / L, NaCl 5 g / L, pH 7.0~7.2, sterilized at 121℃ for 20 min.

[0043] S3. Fermentation Culture: Seed cultures of the two strains were inoculated into the fermentation medium at a volume fraction of 5%–8%, and cultured at 30–32℃ and 180–200 r / min for 36–48 h with shaking. During fermentation, the pH was controlled at 7.0–7.5 until the viable count of Bacillus amyloliquefaciens in the culture was ≥10¹⁰ CFU / mL and the viable count of Bacillus bellis in the culture was ≥10¹⁰ CFU / mL, thus obtaining the fermentation cultures of the two strains. The fermentation medium consisted of: 8 g / L corn flour, 18 g / L soybean flour, 4 g / L fish meal, 2 g / L peptone, 6 g / L white sugar, 5 g / L calcium carbonate, 0.2 g / L manganese sulfate, 0.3 g / L sodium chloride, 0.5 g / L magnesium sulfate, 0.3 g / L potassium dihydrogen phosphate, 0.3 g / L dipotassium hydrogen phosphate, 0.5 g / L ammonium sulfate, and 0.25 g / L sodium hydroxide.

[0044] S4. Compound preparation: Mix the fermented Bacillus amyloliquefaciens and Bacillus vesicles at a volume ratio of 0.8~1.2:1, stir evenly, add 8.2% by volume of stabilizer (0.1% methylparaben, 3% glycerol, 2% sodium dodecyl sulfate, 0.2% xanthan gum, 0.5% azone and 2% OP-10), and let stand at room temperature for 1~2 hours to obtain the microbial agent; after compounding, the total effective viable count of the agent is ≥2×10¹⁰ CFU / mL, pH 7.0~7.5, and the shelf life can reach more than 6 months at room temperature.

[0045] Application of the above-mentioned microbial agents

[0046] This microbial inoculant is used to control bacterial wilt and black shank in tobacco, and is especially suitable for controlling soil-borne diseases in tobacco in continuously cropped areas. It can be applied by root drenching, mixing with substrate, or root dipping. Specific application methods are as follows:

[0047] Seedling application: Dilute 2 liters of microbial agent with water 50-100 times to obtain a diluted solution. Mix the diluted solution thoroughly with 100 kg of substrate and then fill the trays.

[0048] Application during transplanting: Dilute the microbial agent with water 300 times to obtain a diluted solution; when transplanting tobacco, dip the roots of the seedlings into the diluted solution for 3-5 minutes, or irrigate each seedling with 200-300 mL of the diluted solution, and cover with soil after planting.

[0049] Application during the seedling stage: 25-30 days after tobacco transplanting (seedling stage), dilute the microbial agent with water 200-500 times and drench the roots of each tobacco seedling with 300-900 mL. If the soil moisture is low, the amount of diluted solution can be increased appropriately.

[0050] This microbial agent can be used in conjunction with organic fertilizer. The amount of organic fertilizer is 60-100 kg / mu. After mixing with the diluted microbial agent, it can enhance the rhizosphere colonization ability of the strain and improve the control effect.

[0051] Example 1

[0052] Screening of biocontrol strains resistant to tobacco black shank and bacterial wilt in Baoshan tobacco-growing area

[0053] 1. Materials and Methods

[0054] 1.1 Test Materials

[0055] Rhizosphere soil from healthy plants in severely diseased fields with serious root and stem diseases in Baoshan, Yunnan Province, and hot spring mud from Tengchong hot springs were collected.

[0056] 1.2 Culture medium

[0057] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.4; sterilized at 120°C for 20 min.

[0058] PDA medium: 200 g potato, 20 g glucose, 15-20 g agar, 1000 mL water.

[0059] Oat culture medium: 38 g oats, 14 g agar, 1000 mL water.

[0060] NA medium: 5g beef extract; 10g peptone; 5g NaCl; pH 7.0-7.2.

[0061] PDA medium: 200 g potato; 20 g sucrose.

[0062] 1.3 Methods

[0063] 1.3.1 Isolation of antagonistic strains

[0064] On a sterile operating table, weigh 10 g of soil sample and add it to an Erlenmeyer flask containing 990 mL of sterile water and sterile glass beads. Shake on a shaker for 30 min to evenly disperse the soil sample in the diluent, preparing a 10⁻² soil suspension. Using a sterile pipette tip, inject 1 mL of the soil suspension into a test tube containing 9 mL of sterile water, shake well to prepare a 10⁻³ soil suspension. Repeat the same method to prepare a 10⁻⁴ soil suspension. Prepare sterile LB agar plates. After cooling, spread the 10⁻⁴ soil bacterial suspension onto the plates for bacterial morphology observation and culture. Incubate upside down in a 37℃ incubator for 24 h, observing colony growth. Select single colonies for isolation and purification 3-4 times to obtain pure bacterial cultures, which are then stored at 4℃. Similarly, isolate fungi using PDA agar. Incubate upside down in a 28℃ incubator for 48 h. Select single hyphae for isolation and purification 3-4 times to obtain pure fungal cultures, which are then stored at 4℃.

[0065] 1.3.2 Screening of antagonistic strains

[0066] (1) The pathogen of tobacco black shank was activated on oat medium. After growing to the edge of the plate, holes were punched in the edge area of ​​the colony to form a bacterial block. The block was then transferred to the center of the oat medium and cultured at 28°C for 24 h. The isolated bacterial strains were then spot-inoculated at a distance of 20 mm from the bacterial block using the confrontation method. Four strains were inoculated per plate. A blank control (without inoculation) was also set up. The process was repeated three times and the plates were incubated at 28°C for 7 days. Colonies with significant antibacterial effects were selected for purification, preservation, and measurement of the diameter of the inhibition zone.

[0067] (2) The pathogen of tobacco bacterial wilt was inoculated into NB medium and cultured at 28°C with shaking at 170 r / min for 36 h. The bacterial cells were collected by centrifugation and resuspended in sterile water. The bacterial cells were placed in a sterile spray bottle sterilized with 70% alcohol and 10% hydrogen peroxide, and then sprayed evenly onto NA plates inoculated with 3 strains (the strains were spotted 20 mm from the edge of the NA plate). About 0.2 mL of the spray was sprayed on each plate to prepare bacterial plates. A blank control (without bacterial inoculation) was set up at the same time. The process was repeated three times and incubated at 28°C for 2 days. Colonies with antagonistic zones were picked.

[0068] 1.3.3 Identification of Antagonistic Bacteria

[0069] (1) Take 50 μL of Lysis Buffer for Microorganism to Direct PCR into a sterile Microtube;

[0070] (2) Use a sterile toothpick or pipette tip to pick up a single colony and stir it a few times in the Microtube before removing it;

[0071] (3) After denaturation at 80℃ for 15 min, centrifuge at low speed and take 1-5 μL of the supernatant after lysis as a template for PCR reaction;

[0072] (4) Set up the reaction system and conditions according to the instructions;

[0073] (5) Agarose gel electrophoresis and sequencing.

[0074] 1.3.4 Obtaining Drug-Resistant Strains

[0075] After activating the various functional bacteria stored at -70℃ by streaking on selective plates, single colonies of antagonistic bacteria were picked and transferred to 3 mL of LB liquid medium. The culture was incubated for 12 h at 37℃ and 180 rpm in a constant temperature shaker. 100 μL of each colony was then transferred to LB plates containing antibiotics at concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL. These plates were then incubated at 28℃ for 3 days, with daily observation of colony growth. If single colonies grew on LB plates containing 100 μg / mL antibiotics, single colonies were picked and alternately subcultured several times in LB solid medium (without antibiotics) and LB liquid medium (without antibiotics) to ensure the stability of the mutant strain against antibiotics. If no single colony grows on a 100 μg / mL LB agar plate, pick a single colony from a lower concentration plate where single colonies can grow and streak it onto a plate of the next lower concentration to acclimate and enhance the strain's resistance to the antibiotic, until a 100 μg / mL mutant strain is obtained. Then, repeat the same steps to verify and ensure the stability of the mutant strain's resistance. If no mutant strain is obtained even at the lowest antibiotic concentration plate, it indicates that the antibiotic is lethal to the strain, and other antibiotics need to be used instead. The same methods are then used to screen, acclimate, and assess the strain's stability against a specific antibiotic and insecticide.

[0076] 1.3.5 Antagonism test of functional strains

[0077] The cross-streaking method was used to streak different functional strains in pairs. Each bacterium, preserved in an ultra-low temperature freezer at -70℃, was activated on its corresponding selective plate. Then, two corresponding single colonies were picked and streaked on YN solid medium. The growth of bacteria on the plates was observed daily.

[0078] 2 Results and Analysis

[0079] 2.1 Results of Plate Inhibition Measurement

[0080] A total of 64 bacterial strains and 11 fungal strains were screened by plate plating. The inhibitory effects of these 75 strains were determined, and 49 strains with certain antagonistic effects were obtained.

[0081] Of these, 10 strains (7 bacterial strains and 3 fungal strains) showed good inhibitory effects against tobacco black shank, with inhibition zones exceeding 6 mm in diameter. Strains B7 (Base Area No. 4) and B9 (Base Area No. 3) exhibited particularly strong antagonistic effects against black shank, with inhibition zones exceeding 15 mm in diameter. Simultaneously, 6 bacterial strains showed good inhibitory effects against tobacco bacterial wilt, with inhibition zones exceeding 6 mm in diameter. Strains B7 and B9 also showed particularly strong antagonistic effects against bacterial wilt, with inhibition zones exceeding 15 mm in diameter. Strains B7 (Base Area No. 4) and B9 (Base Area No. 3) showed significant inhibitory effects against both tobacco black shank and bacterial wilt.

[0082] 2.2 Identification of antagonistic strains

[0083] The results of PCR amplification and BLAST comparison with NCBI showed that strain B7 had more than 99% similarity to the reported Bacillus belysinus sequence, and strain B9 had high homology with Bacillus amyloliquefaciens.

[0084] Table 1. Sequence alignment results of antagonistic strains

[0085]

[0086] 3. Discussion and Conclusion

[0087] This experiment used indoor plate confrontation culture to screen 49 bacterial strains that showed some antagonistic effects against tobacco black shank and bacterial wilt. Among them, 12 strains showed strong antagonistic effects against black shank, with strains B7 and B9 exhibiting particularly prominent antagonistic effects. Simultaneously, 6 bacterial strains with good inhibitory effects against tobacco bacterial wilt were obtained, with strains B7 and B9 showing the best antagonistic effects against bacterial wilt. Strains B7 (Base Area No. 4) and B9 (Base Area No. 3) showed significant inhibitory effects against both tobacco black shank and bacterial wilt.

[0088] The PCR amplification and sequencing comparison results of the strains showed that strain B7 had more than 99% similarity to the reported Bacillus belyssus sequence, while strain B9 had high homology with Bacillus amyloliquefaciens.

[0089] Example 2

[0090] The efficacy of different antagonistic bacterial combinations in preventing black shank and bacterial wilt

[0091] 1.1 Test Materials

[0092] The pathogens of tobacco black shank and tobacco bacterial wilt were preserved by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences. Two strains with good antagonistic properties were isolated by the laboratory through antagonistic screening and used as antagonistic strains.

[0093] The tested tobacco plant was Yunyan 87.

[0094] 1.2 Test Methods

[0095] Preparation of antagonistic bacterial suspension: Two antagonistic bacterial strains were inoculated into LB liquid medium using an inoculation loop and fermented at 28°C and 140 rpm for 24 h with shaking. The fermentation broth was then diluted with sterile water to 10¹⁰ CFU / mL.

[0096] Preparation of Ralstonia solanacearum inoculum: The Ralstonia solanacearum strain was inoculated into NB liquid medium with an inoculation loop and fermented at 28℃ and 140 rpm for 48 h. The fermentation broth was then diluted with sterile water to 10¹⁰ cfu / mL.

[0097] 1.2.1 Preparation of bacterial suspension

[0098] 1.2.2 Treatment methods for potted plant control efficacy against black shank disease

[0099] Sow Yunyan 87 in seedling trays. When the seedlings grow to about 10-15cm (4-5 leaves), transplant them into flowerpots filled with sterile soil. When the tobacco plants have grown to 8-10 true leaves, lightly scratch the base of the stem with a blade and inoculate the wound with black shank fungus (black shank fungus is cultured in cooked rice at 28℃; after 5 days, the fungus mycelium will have covered the area around the rice). The experiment included a control group and treatment groups. The control group consisted of a blank control CK1 (no inoculation), a disease control CK2 (inoculated only with *Tobacco Black Shank*), and a positive control CK3 (treated with 58% metalaxyl-mancozeb wettable powder). The treatment groups had three different combinations: ①B1, ②B9, and ③B7+B9. In the greenhouse efficacy test, the treatment groups were inoculated twice with antagonistic bacterial solutions. The first inoculation was performed 24 hours after *Tobacco Black Shank* inoculation, followed by a second inoculation 7 days later. The control groups CK1 and CK2 were treated with sterile water, while CK3 was treated with 58% metalaxyl-mancozeb wettable powder. Two days after the second inoculation, *Tobacco Black Shank* suspension was inoculated into the rhizosphere soil of the tobacco seedlings in each treatment group. CK1 was treated with sterile water, while CK2 and CK3 were treated with *Tobacco Black Shank* suspension. According to Table 2, 10 mL of bacterial solution was used per plant, with a total of 6 treatments, each treatment replicated 3 times, and each replicate consisting of 8 tobacco seedlings. The disease is managed using conventional methods, without the application of pesticides. The plants are kept warm and moist in a greenhouse at around 30°C. Disease incidence is observed, and the incidence and disease index are investigated to calculate the disease prevention effect.

[0100] Table 2. Design of pot experiment for the control efficacy of antagonistic strains

[0101]

[0102] 1.2.3 Treatment methods for potted plant control efficacy against bacterial wilt

[0103] Yunyan 87 tobacco was sown in seedling trays. When the seedlings reached 10-15cm (4-5 leaves), they were transplanted into pots filled with sterile soil. The experiment included a control group and treatment groups. The control group consisted of a blank control CK1 (no inoculation), a disease control CK2 (inoculated only with Ralstonia solanacearum), and a positive control CK3 (72% agricultural streptomycin). The treatment groups had three different combinations: ①B1, ②B9, and ③B7+B9. In the greenhouse efficacy test, the treatment groups were inoculated twice with antagonistic bacterial solutions. The first inoculation was done by root drenching with the antagonistic bacterial solution at the time of transplanting, followed by a second inoculation 5 days later. The control groups CK1 and CK2 were treated with water without seedlings, while CK3 was treated with agricultural streptomycin. Two days after the second root drenching, a suspension of Ralstonia solanacearum was inoculated into the rhizosphere soil of the seedlings in each treatment group. CK1 was treated with sterile water, while CK2 and CK3 were treated with Ralstonia solanacearum suspension. According to Table 3, a total of 6 treatments were set up, with each treatment replicated 3 times, and each replicate consisting of 8 seedlings. The disease is managed using conventional methods, without the application of pesticides. The plants are kept warm and moist in a greenhouse at around 30°C. Disease incidence is observed, and the incidence and disease index are investigated to calculate the disease prevention effect.

[0104] Table 3. Design of pot experiment for the control efficacy of antagonistic strains

[0105]

[0106] 2 Results and Analysis

[0107] 2.1 Effect of antagonistic bacteria on greenhouse control of tobacco black shank disease

[0108] Using antagonistic bacteria B7 and B9 as test strains, single-strain bacterial suspensions and mixed-strain bacterial suspensions were prepared respectively. Field trials were conducted to determine their control efficacy against tobacco black shank. The results are shown in Table 4. The control efficacy of single-strain B7 and B9 bacterial suspensions against tobacco black shank was 78.90% and 77.98%, respectively, while the control efficacy of the chemical control CK3 (58% metalaxyl-mancozeb) treatment was 72.48%. The mixed-strain bacterial suspension of B7 and B9 showed significantly better control efficacy against tobacco black shank than the single-strain and chemical control treatments, reaching 87.16%, making it the best among all treatments.

[0109] Table 4. Effects of biocontrol bacteria on the control of tobacco black shank in greenhouses.

[0110]

[0111] 2.2 Effect of antagonistic bacteria on greenhouse control of tobacco bacterial wilt

[0112] Using antagonistic bacteria B7 and B9 as test strains, single-strain bacterial suspensions and mixed-strain bacterial suspensions were prepared respectively. Field trials were conducted to determine their control efficacy against tobacco bacterial wilt. The results are shown in Table 5. The control efficacy of single-strain B7 and B9 bacterial suspensions against tobacco bacterial wilt was 61.98% and 56.20%, respectively, while the control efficacy of the chemical control CK3 (72% agricultural streptomycin) was 51.24%. The mixed-strain bacterial suspension of B7 and B9 showed significantly better control efficacy against tobacco bacterial wilt than the single-strain and chemical control treatments, reaching 75.21%, making it the best among all treatments.

[0113] Table 5. Effects of biocontrol bacteria on greenhouse control of tobacco bacterial wilt.

[0114]

[0115] 3 Results and Analysis

[0116] This study used antagonistic bacteria B7 and B9 as test strains, preparing single-strain and dual-strain mixed bacterial suspensions to determine their field control efficacy against tobacco black shank and bacterial wilt. The results showed that single-strain B7 and B9 bacterial suspensions had certain control effects on both diseases: efficacy against tobacco black shank was 78.90% and 77.98%, respectively, higher than the chemical control 58% metalaxyl-mancozeb (72.48%); efficacy against tobacco bacterial wilt was 61.98% and 56.20%, respectively, also better than the chemical control 72% agricultural streptomycin (51.24%). The mixed bacterial suspension of B7 and B9 showed even better control efficacy, with efficacy against tobacco black shank and bacterial wilt reaching 87.16% and 75.21%, respectively, both significantly higher than the single-strain treatment and the corresponding chemical control, making it the best among all treatments. In conclusion, the combination of antagonistic bacteria B7 and B9 has good field control potential against both tobacco black shank and bacterial wilt. In tobacco-growing areas where soil-borne diseases (bacterial wilt, black shank) occur, it can be mixed and used in a ratio of 1:1 for Bacillus amyloliquefaciens (10¹⁰ CFU / mL) and Bacillus belyss (10¹⁰ CFU / mL).

[0117] Example 3

[0118] Study on the biocontrol effect of substrate-mixed microorganisms on soil-borne diseases in flue-cured tobacco

[0119] 1. Experimental Materials

[0120] 1.1 Test bacterial agent

[0121] According to the requirements of the No. 3 microbial agent (effective viable count ≥ 1×10¹⁰ CFU / mL, effective strain: Bacillus amyloliquefaciens B9, other indicators meet the requirements of GB20287-2006 standard) and the No. 4 microbial agent (effective viable count ≥ 1×10¹⁰ CFU / mL, effective strain: Bacillus belyssus B7, other indicators meet the requirements of GB20287-2006 standard).

[0122] 1.2 Seedling substrate tested

[0123] Perlite, vermiculite, and peat moss are mixed in a ratio of 3:3:4.

[0124] 1.3 Tobacco Varieties Tested

[0125] The tobacco variety is K326.

[0126] 2. Test site

[0127] The demonstration area in Jietou Town, Tengchong City, Baoshan City, Yunnan Province, selected plots with a high incidence of bacterial wilt / black shank disease due to continuous cropping.

[0128] 3. Test Methods

[0129] 3.1 Experimental Design

[0130] The experiment consisted of 6 treatments, each replicated 3 times, for a total of 18 plots (60 tobacco plants per plot), with a plot area of ​​approximately 36 m². The plots were arranged in a randomized block design with a row spacing of 1.2 m and a plant spacing of 0.5 m. A protection row was included, resulting in a total experimental area of ​​648 m². Microbial inoculants No. 3 and No. 4 were mixed thoroughly at a 1:1 volume ratio, and then mixed with the seedling substrate according to the proportions in Table 6. The plot layout is shown in Figure 3.

[0131] Table 6. Division of Experimental Groups

[0132]

[0133] 3.2 Survey of agronomic traits of tobacco plants

[0134] Fifteen representative tobacco plants from each treatment were selected and tagged. Agronomic traits of the plants were measured at fixed locations and on fixed plants during the early and mature stages of the tobacco plant growth, according to the YC / T142-2010 standard "Methods for Surveying and Measuring Agronomic Traits of Tobacco". These traits mainly included plant height, stem circumference, number of effective leaves, maximum leaf length, and maximum leaf width. The maximum leaf area was calculated using formula (1):

[0135] Leaf area (cm²) = 0.6345 × leaf length (cm) × leaf width (cm) (1)

[0136] 3.3 Disease Investigation

[0137] The occurrence of tobacco diseases was investigated according to the national standard GB / T 23222-2008, "Classification and Investigation Methods for Tobacco Diseases and Pests". Based on local disease characteristics, a systematic investigation was conducted primarily on bacterial wilt and black shank, investigating the number of infected plants and the severity level in each plot, and calculating the incidence rate. Disease investigation could be conducted concurrently with the determination of tobacco agronomic traits. Depending on the occurrence of bacterial wilt or black shank, the investigation began at the early stage of vigorous growth, with three fixed investigation points per treatment, and five plants investigated at each point, for a total of 15 plants. Investigations were conducted every five days for five consecutive times.

[0138] 3.3.1 Grading of the severity of tobacco bacterial wilt and black shank disease

[0139] a) Bacterial wilt (per plant):

[0140] Grade 0: The entire plant is disease-free;

[0141] Grade 1: Occasionally, there are chlorotic spots on the stem, or the leaves on the diseased side below 1 / 2 wither;

[0142] Grade 3: Black streaks on the stem, but not exceeding 1 / 2 of the stem height, or 1 / 2 to 2 / 3 of the leaves on the diseased side wither;

[0143] Grade 5: Black streaks at the base of the stem extend beyond 1 / 2 of the stem height but do not reach the top of the stem, or more than 2 / 3 of the leaves on the diseased side wither;

[0144] Level 7: Black streaks on the stem extend to the top of the stem, or all the leaves of the diseased plant wither.

[0145] Level 9: The diseased plants are basically dead.

[0146] b) Blackleg disease (per plant):

[0147] Grade 0: The entire plant is disease-free;

[0148] Grade 1: Stem lesions do not exceed 1 / 3 of the stem circumference, or less than 1 / 3 of the leaves wither;

[0149] Grade 3: Stem lesions surround 1 / 3 to 1 / 2 of the stem circumference, or 1 / 3 to 1 / 2 of the leaves are slightly wilted, or a few leaves at the bottom have lesions;

[0150] Grade 5: Stem lesions extend beyond 1 / 2 of the stem circumference, but do not completely encircle the stem circumference, or 1 / 2 to 2 / 3 of the leaves wither;

[0151] Level 7: Stem lesions completely surround the stem circumference, or more than 2 / 3 of the leaves wither;

[0152] Level 9: The diseased plants are basically dead.

[0153] 3.3.2 Statistical methods for disease incidence

[0154] Incidence rate (%) = (Number of infected plants / Total number of plants surveyed) × 100 (2)

[0155] Disease index = [∑(number of diseased plants at each level × disease severity value at each level)] / (total number of plants surveyed × highest severity value) × 100 (3)

[0156] Prevention efficacy (%) = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100 (4)

[0157] 3.4 Research on Rhizosphere Microecology

[0158] 3.4.1 Rhizosphere soil sampling

[0159] Six tobacco plants were randomly selected from each treatment before ridging, during the occurrence of bacterial wilt or black shank disease, respectively. The top 0-2 cm layer of soil was removed, and larger soil clumps were removed. 50 g of soil adhering to the root surface of the tobacco plants (0-4 mm) was collected, mixed well, sealed in a self-sealing bag, and brought back to the laboratory for storage at 4℃.

[0160] 3.4.2 Determination of Rhizosphere Microbial Structural Diversity

[0161] Soil microbial structural diversity was assessed using 16S rRNA and rDNA-ITS sequence analysis. Differences in rhizosphere microorganisms under the regulation of microbial agents were analyzed to identify key microorganisms influencing the occurrence of bacterial wilt or black shank.

[0162] 3.5 Sampling and yield measurement

[0163] The economic traits of tobacco leaves were graded according to the national standard for flue-cured tobacco (G2635-92). Tobacco yield, output value, proportion of high-grade tobacco, and average price were recorded, and statistics on tobacco yield and economic benefits were compiled. Data processing and statistical analysis were performed using Excel 2013 and SPSS 24.0.

[0164] 4 Results and Analysis

[0165] 4.1 Root system comparison

[0166] The bacterial solution and substrate were mixed evenly at concentrations of 0.5 L / 100 kg, 1 L / 100 kg, 2 L / 100 kg, 4 L / 100 kg, 8 L / 100 kg, and 0 L / 100 kg, respectively. During the seedling stage, it was observed that tobacco seedlings grown in substrates with added bacterial solution had more developed and longer root systems than those without, as shown in Figure 4.

[0167] 4.2 Statistical Analysis of Agronomic Traits

[0168] Analysis of agronomic traits such as plant height, number of effective leaves, stem circumference, and maximum leaf area revealed significant differences in the regulatory effects of different application ratios on tobacco growth and development. Table 7 shows that low ratios (0.5, 1 L / 100kg) had limited effects, while medium to high ratios (2, 4 L / 100kg) significantly promoted plant height, stem thickening, increase in the number of effective leaves, and expansion of leaf area, with the 4 L / 100kg ratio showing the best overall performance. Excessively high ratios (8 L / 100kg) weakened the synergistic effect. In production, a suitable application ratio of 2–4 L / 100kg is recommended to achieve optimal improvement in tobacco agronomic traits.

[0169] Table 7. Analysis of agronomic traits under different treatments at different time periods.

[0170]

[0171] 4.3 Results of Disease Investigation

[0172] Because the experimental field had experienced bacterial wilt outbreaks for several consecutive years, black shank disease was not found during the investigation. The focus was on bacterial wilt, and the field control effects of the fifth investigation are shown in Table 8. Different application ratios significantly affected the occurrence of tobacco diseases. The blank control had an incidence rate of 4.44% and a disease index of 3.46. Treatments with ratios of 0.5 and 8 both had an incidence rate of 4.44%, disease indices of 3.95 and 3.81 respectively, and control efficacy of -14.16% and -10.20% respectively, indicating no control effect and a slightly higher disease severity than the control. Treatments with ratios 1 and 2 had an incidence rate and disease index of 0, achieving 100% control efficacy and completely controlling the disease. Treatment with ratio 4 had an incidence rate of 1.27%, a disease index of 1.22, and a control efficacy of 64.74%, showing some control effect, but significantly lower than ratios 1 and 2. Overall, the optimal application ratio for disease control is 1–2 L / 100kg. Low and high doses are unlikely to achieve the desired control effect, and it is recommended to apply within this suitable range in production.

[0173] Table 8. Effects of different concentrations of substrate-mixed bacteria on the control of bacterial wilt.

[0174]

[0175] 4. Conclusion

[0176] The results of the comprehensive analysis of the effects of different application ratios on the main agronomic traits and disease control of tobacco showed that the application ratio can significantly affect the growth and development of tobacco and the degree of disease occurrence, and there is a clear dose effect.

[0177] In terms of agronomic traits, during the vigorous growth and maturity stages, plant height, number of effective leaves, stem circumference, and maximum leaf area generally showed a pattern of limited improvement with low ratios (0.5, 1 L / 100kg), significant optimization with medium and high ratios (2, 4 L / 100kg), and weakened improvement with high ratios (8 L / 100kg). The ratios of 2 and 4 L / 100kg effectively promoted the growth of tobacco plant height, thickening of stems, increase in the number of effective leaves, and expansion of leaf area, resulting in the best agronomic traits.

[0178] In terms of disease control, the incidence rate of the blank control was 4.44%, and the disease index was 3.46; the 0.5 and 8 L / 100kg ratios had no control effect, the disease index was slightly higher than the control, and the control efficacy was negative; the 4 L / 100kg ratio had a control efficacy of only 64.74%, and the control effect was limited; the 1 and 2 L / 100kg ratios could completely suppress the occurrence of the disease, with both the incidence rate and the disease index being 0, and the control efficacy reaching 100%, which was the best control effect.

[0179] Considering the dual effects of tobacco growth traits and disease control, 2 L / 100kg is the optimal application ratio. This ratio can significantly improve tobacco agronomic traits and promote robust plant growth, while also achieving complete disease control, thus balancing the dual effects of growth promotion and disease prevention. A low ratio (0.5 L / 100kg) is ineffective in disease prevention and has poor growth promotion effect, while an excessively high ratio (8 L / 100kg) can easily lead to a simultaneous decline in growth traits and disease prevention effect, neither of which is suitable for production application.

[0180] Example 4

[0181] The effects of organic fertilizer mixed with microorganisms on the control of soil-borne diseases in flue-cured tobacco and its yield and quality.

[0182] 1. Experimental Materials

[0183] Fertilizers tested: Based on the No. 3 microbial inoculant (effective viable count ≥ 1 × 10¹⁰ CFU / mL, effective strain: Bacillus amyloliquefaciens B9, other indicators meet the requirements of GB20287-2006); Based on the No. 4 microbial inoculant (effective viable count ≥ 1 × 10¹⁰ CFU / mL, effective strain: Bacillus belyssus B7, other indicators meet the requirements of GB20287-2006); organic fertilizer (organic matter ≥ 55%); conventional tobacco-specific compound fertilizer N-P₂O₅-K₂O = 8-16-26.

[0184] Tobacco variety tested: K326

[0185] 2. Test site

[0186] The demonstration area in Jietou Town, Tengchong City, Baoshan City, Yunnan Province, selected plots with a high incidence of bacterial wilt / black shank disease due to continuous cropping.

[0187] 3. Test Methods

[0188] 3.1 Experimental Design

[0189] This experiment included four treatments, each with three replicates, for a total of 12 plots. Each plot was approximately 40 square meters, with 66 tobacco plants planted per plot. Plants were arranged in a randomized block design with a row spacing of 1.2 meters and a plant spacing of 0.5 meters. A protective row was established around the perimeter, resulting in a total experimental area of ​​480 square meters excluding the protective rows. Except for the application of fermented bacterial solution mixed with organic fertilizer as required by the protocol, other management practices were the same as in typical tobacco field production. *Bacillus amyloliquefaciens* and *Bacillus belyceae* were mixed at a volume ratio of 1:1 and then used for the following treatments:

[0190] Treatment 1: 4L mixed fermentation broth + 50kg / 667㎡ of tested organic fertilizer;

[0191] Treatment 2: 6L mixed fermentation broth + 50kg of tested organic fertilizer / 667㎡;

[0192] Treatment 3: 8L mixed fermentation liquid + 50kg / 667㎡ of tested organic fertilizer.

[0193] Treatment 4: 50 kg of organic fertilizer was applied to 667 m², with a blank control.

[0194] 3.2 Survey of agronomic traits of tobacco plants

[0195] Fifteen representative tobacco plants from each treatment were selected and tagged. Agronomic traits of the plants were measured at fixed locations and on fixed plants during the early and mature stages of the tobacco plant growth, according to the YC / T142-2010 standard "Methods for Surveying and Measuring Agronomic Traits of Tobacco". These traits mainly included plant height, stem circumference, number of effective leaves, maximum leaf length, and maximum leaf width. The maximum leaf area was calculated using formula (1):

[0196] 3.3 Disease Assessment

[0197] The occurrence of tobacco diseases was investigated according to the national standard GB / 23222-2008 "Classification and Investigation Methods of Tobacco Diseases and Pests". Based on the local disease occurrence characteristics, a systematic investigation was conducted mainly on bacterial wilt and black shank, investigating the number of diseased plants and the disease severity level in each plot, and calculating the incidence rate. Disease investigation can be carried out simultaneously with the determination of tobacco agronomic traits. According to the occurrence of bacterial wilt or black shank, the investigation began at the early stage of vigorous growth, with 3 fixed points for each treatment, 5 plants investigated at each point, for a total of 15 plants. The investigation was conducted once every 5 days, for a total of 5 investigations. The disease statistics were calculated according to formulas (2) to (3).

[0198] 3.4 Data Analysis and Processing

[0199] The occurrence of soil-borne diseases, agronomic traits, and flue-cured tobacco quality were statistically analyzed using Excel 2016 and SPSS 24.0.

[0200] 4 Results and Analysis

[0201] 4.1 Statistical Analysis of Agronomic Traits

[0202] Different treatments had varying effects on tobacco agronomic traits, with the overall effect being a slight inhibition of growth, and only a few indicators showing positive effects. During the vigorous growth phase, treatment 3 showed the best performance in plant height and leaf area, approaching or slightly exceeding the control; at maturity, treatment 1 had the highest number of effective leaves, significantly higher than the control; at other stages, plant height, stem circumference, and leaf area were mostly lower than the control. In summary, treatment 3 was relatively favorable for the vigorous growth phase of tobacco, while treatment 1 had a significant advantage in the number of effective leaves at maturity. Further selection of suitable treatments can be based on subsequent disease control, yield, and quality indicators. The results are shown in Table 9.

[0203] Table 9. Analysis of variance of agronomic traits under different treatments during the vigorous growth and maturity stages.

[0204]

[0205] 4.3 Results of Disease Investigation

[0206] The field control effects of the fifth survey are shown in Table 10. The control effects of different treatments on tobacco diseases varied significantly. Treatment 1 completely suppressed disease occurrence, with both the incidence rate and disease index at 0, achieving a control efficacy of 100%, demonstrating the best control effect. Treatment 2 had a control efficacy of only 5.24%, essentially offering no control effect. Treatment 3 had a disease index higher than the control, with a control efficacy of -15.07%, exhibiting a certain disease-promoting effect. In summary, only treatment 1 possesses good application value for disease control; treatments 2 and 3 are unlikely to achieve effective disease control.

[0207] 5. Conclusion

[0208] Analysis of the combined agronomic traits and disease control effects revealed significant differences in the impact of different treatments on tobacco growth, development, and disease control. Overall, each treatment slightly inhibited tobacco agronomic traits, with only a few showing positive effects. Treatment 3 (8L mixed fermentation broth + 50kg / 667㎡ of tested organic fertilizer) showed relatively superior performance in vegetative growth indicators such as plant height and leaf area during the vigorous growth stage, while Treatment 1 (4L mixed fermentation broth + 50kg / 667㎡ of tested organic fertilizer) demonstrated a significant advantage in the number of effective leaves at maturity. Regarding disease control, Treatment 1 (4L mixed fermentation broth + 50kg / 667㎡ of tested organic fertilizer) completely suppressed disease occurrence with a 100% control efficacy, demonstrating excellent control. Treatment 2 (6L mixed fermentation broth + 50kg / 667㎡ of tested organic fertilizer) showed virtually no control effect, while Treatment 3 (8L mixed fermentation broth + 50kg / 667㎡ of tested organic fertilizer) not only showed no control effect but also exhibited a trend of promoting disease.

[0209] Considering both growth characteristics and disease control effects, Treatment 1 (4L mixed fermentation liquid + 50kg / 667㎡ of tested organic fertilizer) exhibits excellent disease control and a superior number of effective leaves at maturity, making it the best overall treatment and possessing potential for practical production application. Although Treatment 3 (8L mixed fermentation liquid + 50kg / 667㎡ of tested organic fertilizer) showed good growth during its vigorous growth period, it carries a risk of promoting disease. Treatment 2 (6L mixed fermentation liquid + 50kg / 667㎡ of tested organic fertilizer) showed no outstanding advantages in either growth or disease control, and neither of these treatments is suitable as the preferred treatment for tobacco production.

[0210] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A Bacillus microbial agent for the prevention and control of tobacco diseases, characterized in that: This microbial agent is prepared by mixing Bacillus amyloliquefaciens bacterial solution and Bacillus belye bacterial solution at a volume ratio of 0.8~1.2:

1. After mixing, the total effective viable count of the agent is ≥2×10¹⁰ CFU / mL. The Bacillus amyloliquefaciens strain described is an independently isolated strain, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M20252418 and deposit date: November 10, 2025. The *Bacillus velezensis* strain described is an independently isolated strain, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M20252419 and deposit date: November 10, 2025.

2. A method for preparing a Bacillus microbial agent for the prevention and control of tobacco diseases, characterized in that: Includes the following steps: S1. Strain activation: Bacillus amyloliquefaciens CCTCCNO:M20252418 and Bacillus bereaves CCTCCNO:M20252419 were inoculated onto LB solid medium and cultured at 35°C for 24 h to obtain single colonies; then, the single colonies were picked and inoculated into LB liquid medium and cultured with shaking for a short time to obtain activated bacterial solution. S2. Seed culture: Inoculate the activated bacterial solution obtained in step S1 into the seed culture medium at an inoculation rate of 2%–3% by volume, and culture with shaking for a short period until the viable bacterial count is ≥1×10⁻⁶. 8 CFU / mL, to obtain Bacillus amyloliquefaciens seed culture and Bacillus vesicle seed culture; S3. Fermentation Culture: The seed culture obtained in step S2 is inoculated into the fermentation medium at an inoculation rate of 5%–8% by volume, and cultured with shaking for a long time, controlling the pH at 7.0–7.5, until the viable count of Bacillus amyloliquefaciens in the culture is ≥1×10¹. 0 CFU / mL, viable count of Bacillus bellis in bacterial suspension ≥1×10¹ 0 CFU / mL was used to obtain fermentation broths of two strains; S4. Compound preparation: Mix the two fermentation liquids obtained in step S3 at a volume ratio of 0.8 to 1.2:1, stir evenly, add a stabilizer with a volume fraction of 8.2%, and let stand at room temperature for 1 to 2 hours to obtain the microbial agent.

3. The method for preparing a Bacillus microbial agent for controlling tobacco diseases according to claim 1, characterized in that: The seed culture medium consisted of 20 g / L glucose, 15 g / L peptone, 5 g / L yeast extract, and 5 g / L NaCl, with a pH of 7.0–7.2, and was sterilized at 121°C for 20 min.

4. The method for preparing a Bacillus microbial agent for controlling tobacco diseases according to claim 1, characterized in that: The fermentation medium consists of: 8 g / L corn flour, 18 g / L soybean flour, 4 g / L fish meal, 2 g / L peptone, 6 g / L white sugar, 5 g / L calcium carbonate, 0.2 g / L manganese sulfate, 0.3 g / L sodium chloride, 0.5 g / L magnesium sulfate, 0.3 g / L potassium dihydrogen phosphate, 0.3 g / L dipotassium hydrogen phosphate, 0.5 g / L ammonium sulfate, and 0.25 g / L sodium hydroxide.

5. The method for preparing a Bacillus microbial agent for controlling tobacco diseases according to claim 1, characterized in that: The stabilizer comprises the following components: methylparaben 0.1%, glycerol 3%, sodium lauryl sulfate 2%, xanthan gum 0.2%, azone 0.5%, and OP-10 2%.

6. The method for preparing a Bacillus microbial agent for controlling tobacco diseases according to claim 1, characterized in that: The brief shaking culture was carried out at 32–37℃ and 150–200 r / min for 14–20 h.

7. The method for preparing a Bacillus microbial agent for controlling tobacco diseases according to claim 1, characterized in that: The long-term shaking culture refers to shaking culture at 30-32℃ and 180-200 r / min for 36-48 hours.

8. The method for preparing a Bacillus microbial agent for controlling tobacco diseases according to claim 1, characterized in that: The total number of viable bacteria in the S4 microbial agent is ≥2×10¹ 0 CFU / mL, pH 7.0–7.

5.

9. The application of the Bacillus microbial agent for controlling tobacco diseases according to any one of claims 1-8 in the control of soil-borne diseases.

10. The application of the Bacillus microbial agent for controlling tobacco diseases according to claim 9, characterized in that: The soil-borne diseases mentioned include tobacco bacterial wilt and black shank, and the control methods are as follows: Seedling application: Dilute 2 liters of microbial agent with water 50-100 times to obtain a diluted solution. Mix the diluted solution thoroughly with 100 kg of substrate and then fill the trays. Application during transplanting: Dilute the microbial agent with water 300 times to obtain a diluted solution; when transplanting tobacco, use the root dipping method to immerse the roots of the tobacco seedlings in the diluted solution for 3-5 minutes, or use the root irrigation method to irrigate each tobacco seedling with 200-300 mL of the diluted solution, and cover with soil after planting; Application during the seedling stage: 25-30 days after tobacco transplanting (seedling stage), dilute the microbial agent with water 200-500 times and drench the roots of each tobacco seedling with 300-900 mL. If the soil moisture is low, the amount of diluted solution can be increased appropriately.

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