A bacillus velezensis fungicide with multiple tobacco fungal disease prevention and its preparation method and application

Abstract

The application discloses a bacillus velezensis agent with multiple tobacco fungal disease prevention and preparation method and application thereof. The application discloses a bacillus velezensis strain with multiple tobacco fungal disease prevention, wherein the preservation name of the bacillus velezensis strain with multiple tobacco fungal disease prevention is Bacillus velezensis YC2177, and the preservation number is CCTCC NO: M20211635. The application can improve production efficiency and product quality, reduce the incidence of fungal diseases, thereby achieving good effects in the process of biological control, preventing and controlling multiple fungal diseases while applying base fertilizer, effectively replacing the use of chemical pesticides, being green and environment-friendly, reducing environmental pollution and improving crop safety.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, specifically to a Bacillus vesiculosus agent that can prevent multiple tobacco fungal diseases, its preparation method, and its application. Background Technology

[0002] Tobacco fungal diseases are caused by pathogens parasitizing tobacco plants under certain environmental conditions. They can occur during the seedling, field, and harvest stages, causing varying degrees of damage to the roots, stems, and leaves. Common tobacco fungal diseases include damping-off, root rot, frog-eye disease, anthracnose, and black shank. The causes are as follows: First, most tobacco pathogens can overwinter in the soil or in diseased plant debris, becoming the primary source of infection for the following year. During the tobacco growing season, they spread through wind, rain, insects, flowing water, compost, and agricultural operations, leading to reinfection. Second, high temperatures and rainfall in the seedbed and field, continuous cropping in humid conditions, overly dense planting, excessive nitrogen fertilization, and poor management also contribute to the frequent occurrence of tobacco fungal diseases. Furthermore, due to the relatively limited variety of flue-cured tobacco currently grown in my country, the high multiple cropping index of cultivated land, the difficulty in fallow rotation, and the long-term heavy application of chemical fertilizers and pesticides, soil compaction and changes in soil physicochemical properties have occurred. The population and number of beneficial microorganisms in the soil have decreased sharply, and the self-repair capacity has been reduced. As a result, the number of soil-borne pathogens and fungi has increased, the resistance of tobacco plants has decreased, and tobacco production has been seriously affected and restricted.

[0003] To prevent the occurrence of tobacco fungal diseases, on the one hand, it is necessary to apply fertilizer rationally, implement scientific crop rotation, select superior disease-resistant varieties, and strengthen plant cultivation management to avoid or reduce the infection of tobacco fungal diseases; on the other hand, by studying the disease characteristics and patterns of various tobacco fungal diseases, a Bacillus vesiculosus agent with the ability to control multiple tobacco fungal diseases and its supporting application methods can be developed for green control in tobacco planting, effectively replacing the use of chemical pesticides, reducing environmental pollution, increasing plant safety, and thus improving economic benefits.

[0004] Currently, commercially available microbial agents suffer from numerous problems, including poor efficacy, limited target pests, high prices, and environmental unfriendliness.

[0005] Currently, there is a lack of a green and environmentally friendly Bacillus vesiculosus agent that can prevent multiple tobacco fungal diseases, as well as its preparation method and application. Summary of the Invention

[0006] The purpose of this invention is to provide a green and environmentally friendly Bacillus vesiculosus agent that can prevent multiple tobacco fungal diseases, as well as its preparation method and application, in order to address the shortcomings of existing technologies.

[0007] To achieve the above objectives, the present invention employs the following technical solution: A strain of *Bacillus belye* that provides simultaneous prevention of multiple tobacco fungal diseases (…). Bacillus velezensis The preservation name of the *Bacillus belyssae* strain that has the ability to prevent multiple tobacco fungal diseases is... Bacillus velezensis YC2177 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. The deposit date is December 17, 2021. The accession number is CCTCC NO: M 20211635.

[0008] The present invention relates to a Bacillus vesiculosus agent that can prevent multiple tobacco fungal diseases.

[0009] Furthermore, its active ingredient is at least one of the following (a), (b), and (c): (a) The fermentation culture of Bacillus vesiculosus agent that has the effect of preventing multiple tobacco fungal diseases; (b) The spore suspension of Bacillus vesiculosus agent that has the effect of preventing multiple tobacco fungal diseases; (c) The ultrasonic lysis and precipitation of Bacillus vesiculosus cells that have the ability to prevent multiple tobacco fungal diseases.

[0010] The preparation method of the Bacillus vesiculosus agent with the ability to prevent multiple tobacco fungal diseases described in this invention includes the following steps: (1) Activation of the strain: Bacillus vesiculosus stored at -85℃ is activated on LB solid medium and cultured at 35℃-37℃ for 24-48h by streak culture. Single colonies are picked and streak cultured on solid medium for 24-48h at 35℃-37℃ to obtain the activated strain. (2) Seed culture preparation: The strain activated in the first step was scraped with a sterile inoculation loop and inoculated into 50 ml of LB liquid medium. The culture was carried out at 35℃ and 150 rpm for 24 h to obtain the seed culture of the strain. (3) Preparation of fermentation broth: The activated seed culture is inoculated into the fermentation culture medium in the fermenter at a ratio of 1‰-5‰, and cultured at a temperature of 35℃-38℃ and a rotation speed of 100-150 rpm / min for 48h-60h to obtain the fermentation broth; the effective viable count of the fermentation broth is greater than 2.0×10⁻⁶. 9 cfu / mL; (4) Preparation of bacterial powder: The fermentation broth is introduced into a centrifuge, and after centrifugation, the supernatant is removed to obtain a concentrated bacterial solution. The effective viable count of Bacillus vesiculosus YC2177 in the concentrated bacterial solution is not less than 1×10⁻⁶. 10CFU / mL; The process of spray drying the concentrated bacterial culture to obtain Bacillus powder includes the following steps: adding the concentrated bacterial culture to a mixing tank, adding 10%-13% excipients, and pressing the concentrated bacterial culture to a rotating nozzle under a pressure of 0.9 Pa, maintaining the nozzle at a rotation speed of 20000 r / min while spraying; simultaneously, introducing 150℃, 4000 m³ / min air 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 bacterial powder.

[0011] Further, in step (1), the LB solid culture medium is composed of the following components: 20g of bacteriological peptone, 10g of yeast extract powder, 10g of agar powder, and 2L of water.

[0012] Further, in step (3), the LB liquid culture medium is composed of the following components: 20g of bacteriological peptone, 10g of yeast extract powder, 10g of agar powder, and 2L of water.

[0013] Furthermore, in step (3), the fermentation culture medium comprises the following components by weight: 1-3 parts beef extract, 2-4 parts soybean flour, 0.5-1 part sucrose, 2-3 parts peptone, 2-3 parts corn flour, 0.8-1.5 parts calcium carbonate, 0.5-1 part ammonium sulfate, 0.5-1 part magnesium sulfate, 1-2 parts fish meal, 0.05-0.1 parts potassium dihydrogen phosphate, 0.05-0.1 parts dipotassium hydrogen phosphate, 1-5 parts sodium chloride, 0.1-0.3 parts defoamer, and 1000 parts water, with the pH adjusted to 7.0-7.5 using hydrochloric acid and sodium hydroxide.

[0014] Further, in step (4), the spray-drying excipients comprise the following components by weight: 55-65 parts diatomaceous earth, 15-25 parts calcium carbonate, and 15-25 parts starch.

[0015] The present invention relates to the application of the Bacillus vesiculosus strain, which has the ability to prevent multiple tobacco fungal diseases, in the production of preparations for the prevention and control of tobacco fungal diseases.

[0016] The present invention discloses a method for preparing bio-organic fertilizer by mixing a Bacillus vesiculosus agent that can prevent multiple tobacco fungal diseases with organic fertilizer, comprising the following steps: (1) Fermentation of organic fertilizer: Weigh 750 parts rapeseed cake, 50 parts chrysanthemum residue, 100 parts activated humic acid, and 5 parts composting agent according to the weight ratio. After mixing evenly, the C / N ratio of the fermentation material is 25-30:1. Adjust the mass percentage of the moisture content of the material to 45%-55% and the pH to 7.0-7.5. Pile the material for fermentation. When the fermentation temperature reaches 55℃, start turning the pile. Control the fermentation temperature to 55-65℃ and the fermentation time to 20 days. Gradually lower the temperature to room temperature to obtain well fermented organic fertilizer. (2) Post-fermentation of organic fertilizer: Transfer the fermented organic fertilizer from the first step to the post-fermentation workshop and allow it to mature for 10-15 days under ventilated and static conditions; (3) Addition of functional strains: After the organic fertilizer is fermented, the fermentation liquid of functional strains is added to the fertilizer and mixed evenly to obtain a semi-finished biological organic fertilizer. (4) After screening and packaging the mixture in the third step, the finished bio-organic fertilizer is obtained.

[0017] Beneficial effects: This invention can improve production efficiency and product quality, reduce the incidence of fungal diseases, and thus achieve good results in the process of biological control. It can control a variety of fungal diseases while applying base fertilizer, effectively replace the use of chemical pesticides, is green and environmentally friendly, reduces environmental pollution, and improves crop safety.

[0018] Compared with the prior art, the present invention has the following advantages: (1) The product of this invention can be used for disease control during the tobacco seedling stage and field stage, providing full-process control throughout the entire growth period of tobacco. It can effectively reduce the probability of pathogen infection and achieve a good effect of integrating disease resistance, growth promotion, yield increase and quality improvement. The product of this invention solves many problems of currently available inoculants, such as poor efficacy, single target of control, and environmental unfriendliness. It achieves the beneficial effect of controlling multiple tobacco fungal diseases and is the best choice for disease control in the process of tobacco industrialization.

[0019] (2) Using Bacillus vesiculus YC2177 as the inoculant, the effects were verified by four methods: root dipping and irrigation, foliar spraying, mixing the inoculant with seedling substrate, and making the inoculant into bio-organic fertilizer. This solved the above problems, improved production efficiency and product quality, reduced the incidence of fungal diseases, and achieved good results in the process of biological control. It also controlled a variety of fungal diseases while applying base fertilizer, effectively replacing the use of chemical pesticides, reducing environmental pollution, and improving crop safety.

[0020] (3) Bacillus velezensis YC2177, which inhibits major fungal diseases of tobacco, can be amplified by PCR using nine pairs of specific primers for antibiotic synthesis genes. Using the genomic DNA of strain YC2177 as a template, fragments of antibiotic synthesis genes such as fengycin, bacilysin, surfactantin, iturin A, and bacillomycin D can be amplified, which can produce the above five antibiotics. Through the review of the characteristics of the above antibiotics, it was found that these substances belong to the main active substances of Bacillus, which can inhibit fungal and bacterial diseases of crops. They have the characteristics of broad antibacterial spectrum, rapid and powerful action, and are not prone to drug resistance. They have broad application prospects in the prevention of various fungal diseases of tobacco. Attached Figure Description

[0021] The following will further explain with reference to the accompanying drawings, in which: Figure 1 This is a diagram of the antibiotic synthesis gene of the PCR amplification detection strain YC2177 of the present invention; Figure 2 This is a diagram illustrating the antagonistic effect of YC2177 of the present invention against the pathogen of red spot disease; Figure 3 This is a diagram illustrating the antagonistic effect of YC2177 of the present invention against root rot pathogens. Figure 4 This is a diagram illustrating the antagonistic effect of YC2177 of the present invention against the pathogen of black shank. Figure 5 This is a diagram illustrating the antagonistic effect of YC2177 of the present invention against the pathogen of gray mold. Figure 6 This is a diagram illustrating the antagonistic effect of YC2177 of the present invention against the pathogen of stem spot disease; Figure 7 This is a diagram illustrating the antagonistic effect of YC2177 of the present invention against damping-off pathogens. Figure 8 This is a diagram illustrating the antagonistic effect of YC2177 of the present invention against anthrax pathogens. Figure 9 This is a diagram illustrating the antagonistic effect of YC2177 of the present invention against the pathogen of frog eye disease. Detailed Implementation

[0022] The invention is further illustrated by the following test examples, but it should be noted that the scope of the invention is not limited by these test examples.

[0023] Example 1 This invention discloses a strain of Bacillus belye that also provides protection against multiple tobacco fungal diseases. Bacillus velezensis The preservation name of the *Bacillus belyssae* strain that has the ability to prevent multiple tobacco fungal diseases is... Bacillus velezensis YC2177 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. The deposit date is December 17, 2021. The accession number is CCTCC NO: M 20211635.

[0024] This invention relates to a Bacillus vesiculosus agent that also controls multiple tobacco fungal diseases. Its active ingredient is at least one of the following: (a), (b), and (c): (a) The fermentation culture of Bacillus vesiculosus agent that has the effect of preventing multiple tobacco fungal diseases; (b) The spore suspension of Bacillus vesiculosus agent that has the effect of preventing multiple tobacco fungal diseases; (c) The ultrasonic lysis and precipitation of Bacillus vesiculosus cells that have the ability to prevent multiple tobacco fungal diseases.

[0025] The present invention provides a method for preparing a Bacillus vesiculosus agent that can prevent multiple tobacco fungal diseases, comprising the following steps: (1) Activation of the strain: Bacillus vesiculosus stored at -85℃ is activated on LB solid medium by streak culture at 35℃ for 24h, and single colonies are picked and streak cultured on solid medium for 24-48h at 35℃-37℃ to obtain the activated strain; the LB solid medium is composed of the following components: 20g of bacteriological peptone, 10g of yeast extract powder, 10g of agar powder, and 2L of water.

[0026] (2) Seed culture preparation: The strain activated in the first step was scraped with a sterile inoculation loop and inoculated into 50 ml of LB liquid medium. The culture was carried out at 35℃ and 150 rpm for 24 h to obtain the seed culture of the strain. (3) Preparation of fermentation broth: The activated seed culture was inoculated into the fermentation culture broth in the fermenter at a ratio of 35‰, and cultured at 35℃ and 130 rpm / min for 48 h to obtain the fermentation broth; the effective viable count of the fermentation broth was greater than 2.0 × 10⁻⁶. 9 cfu / mL; The LB liquid culture medium consists of the following components: 20g bacteriological peptone, 10g yeast extract, 10g agar powder, and 2L water.

[0027] The fermentation broth comprises the following components by weight: 3 parts beef extract, 2 parts soybean flour, 0.8 parts sucrose, 3 parts peptone, 2 parts corn flour, 1.5 parts calcium carbonate, 0.8 parts ammonium sulfate, 0.5 parts magnesium sulfate, 1.5 parts fish meal, 0.05 parts potassium dihydrogen phosphate, 0.1 parts dipotassium hydrogen phosphate, 4 parts sodium chloride, 0.1 parts defoamer, and 1000 parts water. The pH is adjusted to 7.3 using hydrochloric acid and sodium hydroxide.

[0028] (4) Preparation of bacterial powder: The fermentation broth is introduced into a centrifuge, and after centrifugation, the supernatant is removed to obtain a concentrated bacterial solution. The effective viable count of Bacillus vesiculosus YC2177 in the concentrated bacterial solution is not less than 1×10⁻⁶. 10 CFU / mL; The process of spray drying the concentrated bacterial culture to obtain Bacillus powder includes the following steps: adding the concentrated bacterial culture to a mixing tank, adding 10% excipients, and pressing the concentrated culture to a rotating nozzle under a pressure of 0.9 Pa, maintaining the nozzle at a rotation speed of 20000 r / min while spraying; simultaneously, introducing 150℃, 4000 m³ / min air 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 bacterial powder.

[0029] The spray drying auxiliary materials consist of the following components by weight: 55-65 parts diatomaceous earth, 15 parts calcium carbonate, and 18 parts starch.

[0030] The present invention relates to the application of the Bacillus vesiculosus strain that can prevent multiple tobacco fungal diseases in the production of preparations for the prevention and control of tobacco fungal diseases.

[0031] This invention relates to a microbial agent that can prevent multiple tobacco fungal diseases, and its application method is as follows: 1. Microbial agents are directly applied to tobacco cultivation. The fungal agent can be used for root irrigation and root dipping during tobacco seedling cultivation and transplanting. When applying, dilute the fungal agent with water 1000 times and use it for root dipping and root irrigation. The application rate of the fungal agent is 1 kg / acre, which can control a variety of tobacco fungal diseases.

[0032] The microbial agent should be diluted with water and sprayed on tobacco plants 30, 40, and 50 days after transplanting. When applying, the microbial agent should be diluted with water 1500 times and the application rate should be 0.3 kg / mu. The above-ground parts should be sprayed on the leaves.

[0033] 2. Prepare seedling substrate for application. The inoculant is mixed with the seedling substrate and used for tobacco seedling cultivation. Functional microbial strains are colonized during the seedling stage to prevent the occurrence of various fungal diseases. The substrate and inoculant are mixed at a mass ratio of 1000:1 for seedling cultivation.

[0034] The preparation method of tobacco seedling substrate is as follows: Biogas residue, earthworm castings, coconut coir, perlite, and vermiculite are used as the main raw materials, with a weight ratio of biogas residue: earthworm castings: coconut coir: perlite: vermiculite = 4:2:2:1:1. The raw materials are mixed according to the ratio to obtain the tobacco seedling substrate. The production raw materials must meet the requirements for organic agricultural production materials and comply with the relevant provisions of GB38400-2019.

[0035] 3. Prepare bio-organic fertilizer for application. Microbial agents are mixed with organic fertilizer to make bio-organic fertilizer, which is used as base fertilizer during tobacco transplanting. Functional microbial strains are colonized when the tobacco seedlings enter the field to prevent various fungal diseases. Specifically, organic fertilizer and microbial agents are mixed at a mass ratio of 1000:1 to make bio-organic fertilizer. The application rate of bio-organic fertilizer is 50 kg per acre, which can be applied by hole application or broadcasting.

[0036] A method for preparing bio-organic fertilizer by mixing Bacillus vesiculosus inoculant, which has the ability to prevent multiple tobacco fungal diseases, with organic fertilizer includes the following steps: (1) Fermentation of organic fertilizer: Weigh 750 parts rapeseed cake, 50 parts chrysanthemum residue, 100 parts activated humic acid, and 5 parts composting agent according to the weight ratio. After mixing evenly, the C / N ratio of the fermentation material is 25-30:1. Adjust the mass percentage of the moisture content of the material to 45%-55% and the pH to 7.0-7.5. Pile the material for fermentation. When the fermentation temperature reaches 55℃, start turning the pile. Control the fermentation temperature to 55-65℃ and the fermentation time to 20 days. Gradually lower the temperature to room temperature to obtain well fermented organic fertilizer. (2) Post-fermentation of organic fertilizer. The fermented organic fertilizer from the first step is transferred to the post-fermentation workshop and allowed to mature for 10-15 days under ventilated and static conditions.

[0037] (3) Addition of functional microorganisms. After the organic fertilizer fermentation is completed, the fermentation liquid of functional microorganisms is added to the fertilizer and mixed evenly to obtain a semi-finished biological organic fertilizer.

[0038] (4) After screening and packaging the mixture in the third step, the finished bio-organic fertilizer is obtained.

[0039] Example 2 The difference between Example 2 and Example 1 is as follows: The preparation method of the Bacillus vesiculosus agent with the ability to prevent multiple tobacco fungal diseases according to the present invention includes the following steps: In step (1), the strain is activated by activating Bacillus vesiculosus stored at -85℃ on LB solid medium, using streak culture, and culturing at 37℃ for 48h, picking a single colony on solid medium and continuing streak culture, culturing at 35℃-37℃ for 24-48h to obtain the activated strain; In step (3), the fermentation broth is prepared by inoculating the activated seed culture into the fermentation culture medium in the fermenter at a ratio of 5‰, and culturing it at 38℃ and 100 rpm / min for 50 h to obtain the fermentation broth; the effective viable count of the fermentation broth is greater than 2.0 × 10⁻⁶. 9 cfu / mL; The LB liquid culture medium consists of the following components: 20g bacteriological peptone, 10g yeast extract, 10g agar powder, and 2L water.

[0040] The fermentation broth comprises the following components by weight: 1 part beef extract, 4 parts soybean flour, 1 part sucrose, 2 parts peptone, 3 parts corn flour, 0.8 parts calcium carbonate, 1 part ammonium sulfate, 1 part magnesium sulfate, 2 parts fish meal, 0.1 parts potassium dihydrogen phosphate, 0.07 parts dipotassium hydrogen phosphate, 1 part sodium chloride, 0.3 parts defoamer, and 1000 parts water. The pH is adjusted to 7.0 using hydrochloric acid and sodium hydroxide.

[0041] In step (4), the spray-drying excipients comprise the following components by weight: 55 parts diatomaceous earth, 20 parts calcium carbonate, and 15 parts starch.

[0042] Example 3 The difference between Example 3 and Example 1 is as follows: The preparation method of the Bacillus vesiculosus agent with the ability to prevent multiple tobacco fungal diseases according to the present invention includes the following steps: In step (1), the strain is activated by activating Bacillus vesiculosus stored at -85℃ on LB solid medium, using streak culture, and culturing at 36℃ for 40h, picking a single colony and continuing streak culture on solid medium, and culturing at 35℃-37℃ for 24-48h to obtain the activated strain; In step (3), the fermentation broth is prepared by inoculating the activated seed culture into the fermentation culture medium in the fermenter at a ratio of 1‰, and culturing it at 36℃ and 150 rpm / min for 60 h to obtain the fermentation broth; the effective viable count of the fermentation broth is greater than 2.0 × 10⁻⁶. 9 cfu / mL; The LB liquid culture medium consists of the following components: 20g bacteriological peptone, 10g yeast extract, 10g agar powder, and 2L water.

[0043] The fermentation broth comprises the following components by weight: 2 parts beef extract, 3 parts soybean flour, 0.5 parts sucrose, 2.5 parts peptone, 2.5 parts corn flour, 1.2 parts calcium carbonate, 0.5 parts ammonium sulfate, 0.8 parts magnesium sulfate, 1 part fish meal, 0.08 parts potassium dihydrogen phosphate, 0.05 parts dipotassium hydrogen phosphate, 5 parts sodium chloride, 0.2 parts defoamer, and 1000 parts water. The pH is adjusted to 7.5 using hydrochloric acid and sodium hydroxide.

[0044] In step (4), the spray-drying excipients comprise the following components by weight: 65 parts diatomaceous earth, 25 parts calcium carbonate, and 25 parts starch.

[0045] Experimental Example 1 The product in this test case was used for root dipping and root drenching to verify its effectiveness. In this experimental example, the inoculant can be used for root dipping and drenching during the tobacco seedling stage and after transplanting. It is used for the integrated control of various tobacco fungal diseases. The inoculant is diluted 1000 times with water and used for root dipping and drenching of tobacco seedlings at a rate of 1 kg / acre.

[0046] 1. Experimental Materials and Methods 1.1 Test Time and Location The experiment was conducted from March to September 2021 in Wanzi Village, Dazhuang Township, Malong County. The experimental site was a plot of land that had been continuously cropped for many years, with an altitude of 1894.5m and coordinates of (103.477205°E, 25.224440°N). Representative tobacco fields were selected where various fungal diseases of tobacco frequently occurred due to continuous cropping and environmental problems.

[0047] 1.2 Basic conditions of the test site, including soil and terrain 1.2.1 Test Site Conditions The experiment was conducted in Wanzi Village, Dazhuang Township, Malong County, at an altitude of 1894.5m and coordinates (103.477205°E, 25.224440°N). The soil fertility was moderate and uniform. The area was low-lying and prone to various fungal diseases of tobacco due to continuous cropping and extensive management over the years.

[0048] 1.2.2 Test Variety: Yunyan 87 1.3 Experimental Design The experiment adopted a randomized block design, with 3 treatments and 3 replicates, totaling 9 plots. The specific details are as follows: Treatment A (conventional fertilization): 40 kg / mu of tobacco-specific compound fertilizer + 5 kg / mu of potassium nitrate + 15 kg / mu of potassium sulfate; Treatment B: In addition to conventional fertilization, local conventional fungal disease control was applied; Treatment C: In addition to conventional fertilization, during the tobacco seedling stage and after transplanting, 1 kg / mu of a microbial agent diluted 1000 times with water was used for root dipping and drenching.

[0049] Root dipping and drenching treatments: Before transplanting floating tobacco seedlings into planting holes, their roots were immersed in a 1000-fold diluted solution of Bacillus vesiculosus YC2177 inoculant for 3-5 minutes. Afterward, the seedlings were transplanted into the holes, and the roots were covered with soil for seedling cultivation. On the 25th day after transplanting, the seedlings were drenched with the inoculant solution, with a 1000-fold diluted solution of Bacillus vesiculosus YC2177 inoculant evenly poured around the roots within a 5cm radius. All seedling cultivation measures were consistent with routine tobacco seedling management. The experimental treatment patterns are shown in Table 1 below. Table 1

[0050] 1.4 Land preparation, sowing, and field management measures The management shall be carried out in accordance with the high-quality tobacco production technology of Malong County, Qujing City.

[0051] 1.5 Survey Items and Methods 1.5.1 Records of the main reproductive period Record the transplanting period, seedling stage, vigorous growth stage, budding stage, topping stage, basal leaf maturity stage, top leaf maturity stage, and the entire growth period for each treatment.

[0052] 1.5.2 Survey of agronomic traits During the vigorous growth period and after the topping-out period, 25 representative tobacco plants were randomly selected from each plot to observe and measure plant height, stem circumference, number of leaves, internode distance, and leaf length and width of tobacco leaves in various parts.

[0053] 1.5.3 Investigation on the inhibitory effect on fungal diseases A systematic survey of common diseases was conducted in accordance with GB / T 23222-2008 Classification and Survey Methods for Tobacco Diseases and Pests.

[0054] 1.6 Data Processing and Analysis Microsoft Excel 2003 and SPSS 22.0 software were used for data processing and statistical analysis; 2 Results and Analysis 2.1 Effects of different treatments on the growth period of flue-cured tobacco The investigation, based on the tobacco growth period progress record tables, shows that the growth period data for the three treatments were basically consistent, indicating that the different treatment models had little impact on the tobacco growth period. Table 2 shows the growth period progress record tables for different treatments. Table 2

[0055] 2.2 Effects of different treatments on agronomic traits of flue-cured tobacco Table 3 shows the comparative analysis of field agronomic traits under different treatments: Table 3

[0056] Table 3 shows that treatments B and C are superior to treatment A in terms of plant height, stem circumference, number of effective leaves, maximum leaf length, and maximum leaf width, with treatment C > treatment B > treatment A. Furthermore, treatment C is significantly superior to treatment A in plant height, stem circumference, and maximum leaf length. In terms of internode distance, treatment B > A > C, while the differences among the three are not significant. In conclusion, treatment C exhibits the best performance in agronomic traits of flue-cured tobacco, followed by treatment B.

[0057] 2.3 Effects of different treatments on the inhibitory effect on major pathogens in flue-cured tobacco The results of the survey on the incidence of major pathogens are shown in Table 4: Table 4

[0058] Table 4 shows that treatments B and C were more effective than control group A in inhibiting the incidence of major tobacco pathogens than the control group A. Treatment B showed the best inhibitory effect on frog eye disease, i.e., treatment B > treatment C > treatment A. However, its control effect was limited to a single pathogen, and its inhibitory effect on other diseases was much lower than that of treatment C. Treatment C, which involved root dipping and drenching with YC2177, showed inhibitory effects on multiple major tobacco pathogens. Except for frog eye disease, where its inhibitory effect was slightly lower than that of treatment B, it maintained optimal inhibition levels against most other diseases. In conclusion, treatment C can effectively prevent multiple tobacco fungal diseases, promote healthy tobacco leaf growth, and improve tobacco leaf quality.

[0059] 3. Discussion and Conclusion The experimental results showed that, in terms of growth period, the various treatment modes had little impact on the growth period of flue-cured tobacco. Regarding agronomic traits, treatment C performed best overall, promoting plant growth and improving leaf quality, followed by treatment B. In terms of disease control, treatment C (conventional fertilization + 1 kg / mu of YC2177-based inoculant, diluted 1000 times with water, used for root dipping and drenching) showed the best inhibitory effect on major tobacco pathogens. Most pathogens parasitize and infect tobacco seedlings, and the root dipping during the seedling stage and drenching after transplanting allows for comprehensive pathogen control, achieving the effect of preventing multiple tobacco fungal diseases and reducing the incidence of disease.

[0060] Experimental Example 2 The effect of this experimental product was verified by foliar spraying. 1. Experimental Materials and Methods 1.1 Test Time and Location The experiment was conducted from February to October 2021 in Zhonglongtan Village, Dake Township, Shilin County, Kunming City, Yunnan Province. The previous crop was garlic. The soil type was red soil, with a pH of 6.71, an organic matter content of 25.6 g / kg, available nitrogen of 122 mg / kg, available phosphorus (P) of 51.7 mg / kg, and available potassium (K) of 152 mg / kg.

[0061] 1.2 Experimental Materials The tested flue-cured tobacco variety was Honghua Dajinyuan. The tested fertilizers were: tobacco-specific compound fertilizer (N-P2O5-K2O=12-10-24), potassium nitrate (N-P2O5-K2O=13.5-0-44.5), and farmyard manure (N-P2O5-K2O=2.56-0.44-1.72). The tested microbial agent was Bacillus belyceta var. baileyi YC2177 (hereinafter referred to as YC2177), with an effective viable count of 1×10⁻⁶. 11 cfu / g.

[0062] 1.3 Experimental Design The study employed a plot-based experimental model, with three treatments, each replicated three times. The plots were arranged in a randomized block design, with a plant spacing of 120cm × 50cm and a plot area of ​​144m². Two protective rows were established around the experimental plot. Field management followed local high-quality flue-cured tobacco production methods. Specifically: Treatment A: Conventional fertilization (600kg / hm² of tobacco-specific compound fertilizer + 150kg / hm² of potassium nitrate + 1500kg / hm² of farmyard manure), no pesticides, only water spraying; Treatment B: Conventional fertilization + foliar spraying of a commercially available fungicide; Treatment C: Conventional fertilization + foliar spraying of YC2177 inoculant (application rate 4.5kg / hm², diluted 1500 times with water and sprayed onto the entire above-ground part of the plant). 1.4 Survey Items and Methods 1.4.1 Investigation of Disease Occurrence A systematic survey of common diseases was conducted in accordance with GB / T 23222-2008 Classification and Survey Methods for Tobacco Diseases and Pests.

[0063] 1.4.2 Statistical Survey of Economic Characteristics In each treatment of the experiment, tobacco was harvested and dried individually in plots, and economic traits such as yield, output value, and the proportion of high-grade and medium-to-high-grade tobacco were statistically analyzed.

[0064] 1.5 Data Processing and Analysis Microsoft Excel 2003 and SPSS 22.0 software were used for data processing and statistical analysis; 2 Results and Analysis 2.1 Effects of different treatments on the inhibition of major pathogens in flue-cured tobacco Disease occurrence was statistically analyzed in tobacco plants within a unit area of ​​randomly selected treatments A, B, and C during their vigorous growth and topping stages, as shown in Tables 1 and 2. During the vigorous growth and topping stages of flue-cured tobacco, the incidence rates of common diseases in this tobacco-growing area—gray mold, red spot disease, and root rot—were lower in treatment C than in treatments B and A. This indicates that the inhibitory effect of different treatments on the main pathogens of flue-cured tobacco was: treatment C > treatment B > treatment A. Treatment A, which did not receive any pesticides or fungicides, had the highest incidence rate. Treatment B, which received commercially available fungicides, had a lower incidence rate of gray mold, reaching 0% by the topping stage. This suggests that this treatment had a certain inhibitory effect on gray mold, but its control effect on the disease was relatively limited, with poor inhibitory effects on red spot disease and root rot. Treatment C, on the other hand, had the lowest incidence rates of red spot disease, gray mold, and root rot, indicating that foliar spraying of Bacillus baileyi YC2177 could prevent multiple tobacco fungal diseases, reduce disease incidence, and promote healthy plant growth. Table 5 shows the incidence rates of major pathogens in flue-cured tobacco under different treatments during the vigorous growth period, and Table 6 shows the incidence rates of major pathogens in flue-cured tobacco under different treatments during the capping period. Table 5

[0065] Table 6

[0066] 2.2 Effects of different treatments on the economic traits of flue-cured tobacco The economic traits of tobacco leaves, such as yield per mu (667 square meters), output per mu, average price, and the proportion of high-grade tobacco, directly determine the economic income of tobacco farmers. The incidence of tobacco diseases also influences the economic traits of flue-cured tobacco to some extent. Table 3 shows that the yield per mu, ranked from highest to lowest, is C > B > A. Similarly, the output value per mu, average price, and the proportion of medium-to-high-grade and high-grade tobacco, ranked from highest to lowest, are C > B > A. Overall, treatment C exhibits the best economic traits, followed by treatment B, with treatment A being the worst. Table 7 shows the statistical tables of yield per mu and output value for each treatment. Table 7

[0067] 2.3 Conclusions and Discussion The results of the small-scale trial showed that different treatments had different inhibitory effects on the main pathogens of flue-cured tobacco. Whether during the vigorous growth stage or the topping stage of flue-cured tobacco, treatment C, which was sprayed with Bacillus baileyi YC2177, had the best control effect on multiple tobacco fungal diseases and could prevent multiple tobacco fungal diseases. In contrast, treatment B, which was sprayed with commercially available fungicides, had a relatively limited control effect on diseases, with only a significant inhibitory effect on gray mold. Treatment A, which was not sprayed with pesticides or fungicides, had a poor inhibitory effect on the main fungal diseases in this tobacco-growing area, which was not conducive to the healthy growth of tobacco plants.

[0068] The inhibitory effect of different treatments on the main fungal diseases of tobacco plants also determines the economic traits of flue-cured tobacco to a certain extent. The analysis shows that treatment C, which is sprayed with YC2177 on the leaves and can prevent multiple tobacco fungal diseases, has the highest economic traits, represented by the value per mu, yield per mu, average price and the proportion of high-grade tobacco. Treatment B is the second best, and treatment A is the worst.

[0069] Experimental Example 3 The effectiveness of the product in this experiment was verified by preparing a functional seedling substrate. 1. Experimental Materials and Methods 1.1 Test Time and Location The experiment was conducted on February 19, 2021, in a representative tobacco-growing area in Buga Township, Zhaoyang District, Zhaotong City, Yunnan Province.

[0070] 1.2 Experimental Materials The tested variety was Yunyan 87, a locally cultivated main variety, and the seeds were provided by the Zhaotong branch of Yunnan Tobacco Company.

[0071] The test substrate was prepared by mixing biogas residue fertilizer, earthworm castings, coconut coir, perlite, and vermiculite in a ratio of 4:2:2:1:1.

[0072] Test bacterial solutions: Bacillus subtilis, Bacillus belye YC2177 1.3 Experimental Design This experiment used a mixture of microbial agents and seedling substrate for seedling cultivation in 32-well (4×8-well) plastic trays. Three different formulations (treatments A, B, and C) were used, with each treatment replicated three times. Fertilizer preparation was carried out on the afternoon of February 18th and allowed to ferment for 24 hours. On February 19th, different microbial agents were added, and the mixture was then used for seedling cultivation in a representative tobacco-growing area of ​​Buga Township, Zhaoyang District, Zhaotong City, Yunnan Province. Treatment A: biogas residue + earthworm castings + coconut coir + perlite + vermiculite = 4:2:2:1:1; Treatment B: biogas residue + earthworm castings + coconut coir + perlite + vermiculite + Bacillus subtilis; Treatment C: biogas residue + earthworm castings + coconut coir + perlite + vermiculite + Bacillus subtilis YC2177. The mixing ratio of the seedling substrate to the two microbial agents was 1000:1. The treatment patterns are shown in Table 8. Table 8

[0073] 1.4 Survey Items and Methods 1.4.1 Survey of agronomic traits Agronomic traits of tobacco were investigated at the seedling stage, vigorous growth stage, and topping stage. Twenty-five representative tobacco plants were randomly selected from each plot to observe and measure plant height, stem circumference, number of leaves, internode distance, and leaf length and width of leaves at various parts of the plant.

[0074] 1.4.2 Investigation on the inhibitory effect on fungal diseases A systematic survey of common diseases was conducted in accordance with GB / T 23222-2008 Classification and Survey Methods for Tobacco Diseases and Pests.

[0075] 1.4.3 Data Processing and Analysis Microsoft Excel 2003 and SPSS 22.0 software were used for data processing and statistical analysis; 2 Results and Analysis 2.1 Survey of agronomic traits of flue-cured tobacco Table 9. Record of Survey on Agronomic Traits of Flue-cured Tobacco

[0076] Twenty-five tobacco plants from each of treatments A, B, and C were randomly selected and their agronomic traits were measured at different stages. The results showed that at all stages of tobacco growth, whether in the rosette stage, vigorous growth stage, or topping stage, treatment B showed better agronomic traits in stem circumference and internode distance than treatments C and A. However, treatment C performed best in plant height, number of effective leaves, maximum leaf length, and maximum leaf width, followed by treatment B, with treatment A performing the worst; that is, C > B > A. Overall, treatment C performed better.

[0077] 2.2 Investigation on the inhibitory effect on major fungal diseases in flue-cured tobacco The inhibitory effects of each treatment on major fungal diseases of flue-cured tobacco are shown in Table 10: Table 10

[0078] Table 9 shows that the inhibitory effects of each treatment on major fungal diseases of flue-cured tobacco varied. Treatment B, with the addition of Bacillus subtilis, showed the best inhibitory effect on damping-off, reaching 88.88%, while treatment C was slightly lower at 73.47%. However, treatment A did not show significant inhibitory effects on other fungal diseases. Treatment C, with the addition of Bacillus belycei YC2177, showed the best inhibitory effects on stem spot, Corynebacterium, and anthracnose, indicating that this treatment can control multiple fungal diseases of tobacco.

[0079] 2.3 Discussion and Conclusion This study investigated the effects of different microbial inoculants on the early rhizosphere colonization of tobacco plants and their influence on agronomic traits by introducing microbial inoculants into the substrate. Results showed that adding microbial inoculants to the seedling substrate optimized agronomic traits during the rosette, vigorous growth, and topping stages of tobacco plants, and improved their resistance to fungal diseases. Treatment C (with an effective viable count of 1×10¹¹ cfu / g) of Bacillus baileyi YC2177 in the seedling substrate showed the best effect, followed by treatment B. Regarding disease inhibition, treatment B, which incorporated Bacillus subtilis, showed the best inhibitory effect on damping-off disease, but its inhibitory effect on other diseases was significantly lower than that of treatment C. This indicates that Bacillus baileyi can control multiple fungal diseases in tobacco, possesses broad-spectrum antibacterial activity and strong stress resistance, grows rapidly, and has good stability, making it very suitable for large-scale production and application as a biocontrol agent.

[0080] Test Example 4 The product from this experiment was used to make bio-organic fertilizer for verification. 1. Experimental Materials and Methods 1.1 Test Time and Location The experiment was conducted on March 10, 2021, at Xinle Village, Longshu Town, Ludian County, Zhaotong City, at an altitude of 2149.8 m, N27.3626450, E103.439865.

[0081] 1.2 Experimental Materials Tested variety: Yunyan 99 The tested fertilizers were: tobacco-specific compound fertilizer (N-P20-K20=11-15-22) and potassium sulfate (K20≥50%), which were provided by the local tobacco company; and bio-organic fertilizer (containing phosphorus-solubilizing, potassium-solubilizing, growth-promoting and disease-resistant microorganisms, which were prepared by fermenting Bacillus vesiculosus YC2177 and organic fertilizers) which were provided by Yunnan Provincial Microbial Fermentation Engineering Research Center Co., Ltd.

[0082] 1.3 Experimental Design The study employed a small-plot experimental model, with two treatments, each replicated three times, arranged in a randomized block design. Treatment A served as the control group, receiving conventional local tobacco fertilization: tobacco-specific compound fertilizer (N-P20-K20=11-15-22) and potassium sulfate (K20≥50%). Treatment B served as the demonstration group, receiving tobacco-specific compound fertilizer (N-P20-K20=11-15-22), potassium sulfate (K20≥50%), and bio-organic fertilizer containing Bacillus belyssus YC2177. The bio-organic fertilizer was applied at a rate of 50 kg / mu, using hole application and broadcasting methods as basal fertilizer, root-setting fertilizer, and top dressing.

[0083] Except for the different fertilizer application methods, all other production management measures in the experiment were implemented in accordance with the local standards for high-quality tobacco production. The specific fertilizer application rates for each treatment are shown in Table 11 below: Table 11

[0084] 1.4 Survey Items and Methods 1.4.1 Survey of agronomic traits Agronomic traits of tobacco were investigated at the seedling stage, vigorous growth stage, and topping stage. Thirty representative tobacco plants were randomly selected from each plot to observe and measure plant height, stem circumference, number of leaves, internode distance, and leaf length and width of leaves at various parts of the plant.

[0085] 1.4.2 Economic Characteristics Survey The survey and statistics included economic characteristics such as output, output value, average price, proportion of high-grade tobacco, and proportion of medium-to-high-grade tobacco for each treatment process.

[0086] 1.4.3 Investigation on the inhibitory effect on fungal diseases A systematic survey of common diseases was conducted in accordance with GB / T 23222-2008 Classification and Survey Methods for Tobacco Diseases and Pests.

[0087] 1.4.4 Investigation of Chemical Composition of Tobacco Leaves Representative tobacco plants were selected for each treatment, and their middle and upper leaves were marked and retained. After harvesting and curing, the leaves were collected and sorted, and 5 kg of tobacco leaves were retained from each part for routine chemical composition analysis.

[0088] 1.4.5 Data Processing and Analysis Microsoft Excel 2003 and SPSS 22.0 software were used for data processing and statistical analysis; 2 Results and Analysis 2.1 Survey of agronomic traits of flue-cured tobacco Thirty plants from each of treatments A and B were randomly selected for agronomic trait testing. The results showed that the overall agronomic traits of tobacco plants in treatment B were superior to those in treatment A during the rosette stage, vigorous growth stage, and after topping. The agronomic trait survey record for flue-cured tobacco is shown in Table 12. Table 12

[0089] 2.2 Economic Characteristics Survey The yield, average price, and proportion of high-grade tobacco leaves per mu (a Chinese unit of area, approximately 0.067 hectares) are influenced by the health of the tobacco plants and directly determine the economic income of tobacco farmers by affecting the economic traits of tobacco. Table 3 shows that the tobacco plants in treatment B, which received bio-organic fertilizer (containing Bacillus berberis YC2177), yielded a high yield of 256.6 kg / mu, 55.8 kg / mu higher than the conventional treatment A without bio-organic fertilizer, with a yield value of 8262.52 yuan / mu. The proportion of high-grade and medium-to-high-grade tobacco was 28.9% and 16.7% higher than the control group A, respectively. Table 13 shows the statistics of flue-cured tobacco yield and value. Table 13

[0090] 2.3 Investigation on the Inhibition Effect of Flue-cured Tobacco Diseases Diseases severely affect tobacco growth, leading to reduced yield and quality, and diminishing the economic value of tobacco. Table 13 shows the main diseases occurring in the experimental field. As can be seen from the table, the main diseases occurring in the experimental field were damping-off, root rot, red spot disease, black shank disease, anthracnose, and frog eye disease, among which damping-off, root rot, and frog eye disease were more severe. Overall, treatment B had the mildest disease incidence, while treatment A had a higher incidence rate. This indicates that applying bio-organic fertilizer (containing Bacillus belycei YC2177) on top of conventional fertilization can improve the disease resistance of tobacco plants and has certain significance for disease control. Table 14 shows the results of the survey on the incidence rates of major diseases. Table 14

[0091] 2.4 Chemical Composition Analysis of Tobacco Leaves Total sugar, total nitrogen, nicotine, and reducing sugar are the main components determining the quality of tobacco leaves. Yunnan tobacco leaves are of the light aroma type, with high total sugar and reducing sugar content, while nicotine and total nitrogen content are negatively correlated, hence their relatively low nicotine and total nitrogen content. Table 14 shows that the total nitrogen and nicotine content in the middle and upper leaves of each treatment is low, and the chemical composition coordination is poor. Overall, treatment B has higher total sugar, total nitrogen, and nicotine content, and its chemical composition coordination is slightly better than treatment A. Chemical composition analysis is shown in Table 15. Table 15

[0092] 3. Conclusions and Discussion The results of a small-scale trial conducted in Xinle Village, Zhaotong City in 2021 showed that, in terms of agronomic traits, treatment B, which was treated with bio-organic fertilizer (containing Bacillus belye YC2177), performed better than treatment A, which was not treated with bio-organic fertilizer. This indicates that the application of bio-organic fertilizer can improve the absorption of nutrients by tobacco plants, thereby promoting the growth of tobacco plants and improving the agronomic traits of flue-cured tobacco.

[0093] In terms of economic conditions, treatment B, which adds bio-organic fertilizer, can increase the yield per mu, output value per mu, average price, and proportion of medium and high grade flue-cured tobacco, improve the economic characteristics of flue-cured tobacco, thereby determining the economic income of tobacco farmers and enabling them to increase production, increase income, and become wealthy.

[0094] Regarding the inhibitory effect on flue-cured tobacco diseases, treatment B, which added bio-organic fertilizer, improved the disease resistance of tobacco plants and reduced the occurrence of diseases. Compared with treatment A, which did not add bio-organic fertilizer, this treatment had a significant inhibitory effect on a variety of major fungal diseases in the experimental area and could prevent multiple diseases. In contrast, the incidence of disease in tobacco plants in treatment B was at a high level, and it did not have the special effect of preventing multiple fungal diseases in tobacco.

[0095] In terms of the chemical composition analysis of tobacco leaves, the coordination of chemical components in the middle and upper parts of tobacco leaves in each treatment was somewhat different from that of high-quality tobacco leaves. However, treatment B, which applied bio-organic fertilizer, was able to increase the total nitrogen and nicotine content in the middle tobacco leaves and the reducing sugar content in the upper tobacco leaves. To a certain extent, this improved the coordination of chemical components in the tobacco leaves, resulting in better plant growth and improved quality.

[0096] The bio-organic fertilizer in this experiment mainly consists of microbial inoculant Bacillus vesiculosus YC2177, which to some extent demonstrates that this inoculant can promote tobacco leaf growth, improve tobacco leaf quality, and prevent various tobacco fungal diseases, thereby achieving high-yield, high-quality, and intensive tobacco production.

[0097] The four experimental examples involved in this invention are all verifications of the efficacy of Bacillus vesiculosus YC2177, thereby obtaining a Bacillus vesiculosus agent with the ability to control multiple tobacco fungal diseases, as well as its preparation and application. Through analysis of the agronomic traits, economic traits, disease occurrence, and chemical composition of flue-cured tobacco, it can be seen that the application of this microbial agent can significantly improve the agronomic and economic traits of tobacco and enhance the quality of tobacco leaves. The above traits and tobacco leaf quality are affected by the health status of tobacco plants and are positively correlated with the disease control effect of tobacco plants. Therefore, for the control of tobacco leaf diseases and the promotion of healthy plant growth, it is crucial to develop a functional microbial agent with the ability to control multiple tobacco fungal diseases.

[0098] The Bacillus berberis YC2177 studied in this invention has a good inhibitory effect on various major tobacco pathogens involved in the test cases, including damping-off, gray mold, frog-eye, stem spot, root rot, black shank, anthracnose, red spot, and Corynebacterium. Its most distinctive feature compared with commercially available inoculants is that it can inhibit various fungal and bacterial diseases of tobacco, has a broad antibacterial spectrum, and can achieve the effect of preventing multiple fungal diseases of tobacco. It can be intensively produced as a biocontrol agent and applied to the prevention and control of tobacco diseases.

[0099] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above-described experimental examples. The experimental examples and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A strain of Bacillus velezensis YC2177 that can control multiple tobacco fungal diseases, characterized by: The Bacillus berreatus YC2177, which has the ability to prevent multiple tobacco fungal diseases, is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The deposit date is December 17, 2021; accession number: CCTCC NO: M 20211635.

2. The Bacillus vesiculosus agent according to claim 1, which has the effect of preventing multiple tobacco fungal diseases.

3. The Bacillus vesiculosus agent with the ability to control multiple tobacco fungal diseases according to claim 2, wherein the active ingredient is at least one of the following (a), (b), and (c): (a) The fermentation culture of Bacillus vesiculosus agent with anti-multiple tobacco fungal diseases as described in claim 1; (b) The spore suspension of Bacillus vesiculosus agent with anti-multiple tobacco fungal diseases obtained according to claim 1; (c) The ultrasonic lysis and precipitation of Bacillus vesiculosus cells obtained in claim 1, which has the effect of preventing multiple tobacco fungal diseases.

4. The method for preparing the Bacillus vesiculosus powder with anti-multiple tobacco fungal diseases as described in claim 1, characterized in that... The following steps are included: (1) Activation of the strain: Bacillus belye stored at -85℃ is activated on LB solid medium and cultured at 35℃-37℃ for 24-48h by streak culture. Single colonies are picked and streak cultured on solid medium for 24-48h at 35℃-37℃ to obtain activated strains. (2) Seed culture preparation: The strain activated in the first step was scraped with a sterile inoculation loop and inoculated into 50 ml of LB liquid medium. The culture was carried out at 35℃ and 150 rpm for 24 h to obtain the seed culture of the strain. (3) Preparation of fermentation broth: The activated seed culture is inoculated into the fermentation culture medium in the fermenter at a ratio of 1‰-5‰, and cultured at a temperature of 35℃-38℃ and a rotation speed of 100-150 rpm / min for 48h-60h to obtain the fermentation broth; the effective viable count of the fermentation broth is greater than 2.0×10⁻⁶. 9 cfu / mL; (4) Preparation of bacterial powder: The fermentation broth is introduced into a centrifuge, and after centrifugation, the supernatant is removed to obtain a concentrated bacterial solution. The effective viable count of Bacillus vesiculosus YC2177 in the concentrated bacterial solution is not less than 1×10⁻⁶. 10 CFU / mL; The process of spray drying the concentrated bacterial culture to obtain Bacillus powder includes the following steps: adding the concentrated bacterial culture to a mixing tank, adding 10%-13% excipients, and pressing the concentrated bacterial culture to a rotating nozzle under a pressure of 0.9 Pa, maintaining the nozzle at a rotation speed of 20000 r / min while spraying; simultaneously, introducing 150℃, 4000 m³ / min air 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 bacterial powder.

5. The method for preparing Bacillus vesiculosus powder with anti-multiple tobacco fungal diseases according to claim 4, characterized in that: In step (1), the LB solid culture medium is composed of the following components: 20g of bacteriological peptone, 10g of yeast extract, 10g of agar powder, and 2L of water; in step (2), the LB liquid culture medium is composed of the following components: 20g of bacteriological peptone, 10g of yeast extract, 10g of agar powder, and 2L of water.

6. The method for preparing Bacillus vesiculosus powder with antifungal properties against multiple tobacco fungal diseases according to claim 5, characterized in that: In step (3), the fermentation culture medium comprises the following components by weight: 1-3 parts beef extract, 2-4 parts soybean flour, 0.5-1 part sucrose, 2-3 parts peptone, 2-3 parts corn flour, 0.8-1.5 parts calcium carbonate, 0.5-1 part ammonium sulfate, 0.5-1 part magnesium sulfate, 1-2 parts fish meal, 0.05-0.1 parts potassium dihydrogen phosphate, 0.05-0.1 parts dipotassium hydrogen phosphate, 1-5 parts sodium chloride, 0.1-0.3 parts defoamer, and 1000 parts water; the pH is adjusted to 7.0-7.5 using hydrochloric acid and sodium hydroxide.

7. The method for preparing Bacillus vesiculosus powder with antifungal properties against multiple tobacco fungal diseases according to claim 6, characterized in that: In step (4), the spray-drying excipients comprise the following components by weight: 55-65 parts diatomaceous earth, 15-25 parts calcium carbonate, and 15-25 parts starch.

8. The method for preparing bio-organic fertilizer by mixing the Bacillus vesiculosus agent with multiple tobacco fungal diseases as described in claim 3 with organic fertilizer, characterized in that... Includes the following steps: (1) Fermentation of organic fertilizer: Weigh 750 parts rapeseed cake, 50 parts chrysanthemum residue, 100 parts activated humic acid, and 5 parts composting agent according to the weight ratio. After mixing evenly, the C / N ratio of the fermentation material is 25-30:

1. Adjust the mass percentage of the moisture content of the material to 45%-55% and the pH to 7.0-7.

5. Pile the material for fermentation. When the fermentation temperature reaches 55℃, start turning the pile. Control the fermentation temperature to 55-65℃ and the fermentation time to 20 days. Gradually lower the temperature to room temperature to obtain well fermented organic fertilizer. (2) Post-fermentation of organic fertilizer: Transfer the fermented organic fertilizer from the first step to the post-fermentation workshop and allow it to mature for 10-15 days under ventilated and static conditions; (3) Addition of functional strains: After the organic fertilizer is fermented, the active ingredients of the functional strains in claim 3 are added to the fertilizer and mixed evenly to obtain a semi-finished biological organic fertilizer. (4) After screening and packaging the mixture in the third step, the finished bio-organic fertilizer is obtained.

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