Complex microbial inoculant BAA and application thereof

Abstract

The invention relates to a complex microbial inoculant BAA and application thereof, the active ingredient of the complex microbial inoculant BAA comprises at least one strain of bacillus amyloliquefaciens CJ-796 and bacillus altitudinis CJ-365, the viable bacterium concentration of the complex microbial inoculant BAA is 1 * 10 < 8 > CFU / mL, the active ingredient of the complex microbial inoculant BAA is at least one strain of bacillus amyloliquefaciens CJ-796 and bacillus altitudinis CJ-365, and the viable bacterium concentration of the complex microbial inoculant BAA is 1 * 10 < 8 > CFU / mL. By performing root irrigation treatment on plants, the tobacco black shank can be effectively prevented and treated, tobacco plant growth can be remarkably promoted, the tobacco black shank prevention effect is 75% or above, the plant growth promoting effect can reach 50% or above, and the composition has the advantages of being safe, free of residues and environmentally friendly, meets the requirement for pesticide safety and has a wide application prospect. The potential application value is realized in the aspect of biological prevention and control of the tobacco black shank.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural biological control, and in particular to a composite bacterial agent BAA and application thereof. Background Art

[0002] Tobacco black shank is a devastating soil-borne disease caused by the tobacco fungus Phytophthora parasitica var. nicotianae. The pathogen belongs to the Oomycetes, Peronosporales, Pythiaceae, and Phytophthora genus, and exhibits strong host specificity and environmental adaptability. Chlamydospores and dormant hyphae in the soil are the primary source of tobacco black shank infection, while zoospores are the secondary source of infection. Chlamydospores and dormant hyphae are primarily found in the 0-5 cm surface soil, primarily infecting the rhizomes through root wounds. Zoospores can swim rapidly in water and can spread rapidly with the help of rainfall and running water. They tend to accumulate on the surface of crop roots, invading through wounds or orifices, causing browning and necrosis of vascular bundles and wilting and lodging of the plants.

[0003] Black shank disease rapidly develops under high temperature and high humidity conditions, and is particularly severe in tobacco fields with continuous cropping or in low-lying areas. Typical symptoms include stunted plants, withering and yellowing of leaves from the bottom up, and root and stem shrinkage and necrosis, often manifesting as "black shank," "black plaster," "wearing a long gown," "loose waist," and "bamboo shoot knots." According to a report from the International Tobacco Scientific Research Cooperation Center, major tobacco-growing countries worldwide (such as China, Brazil, India, and the United States) suffer direct economic losses exceeding $1.5 billion annually due to black shank. In my country's major tobacco-producing regions, such as Yunnan, Guizhou, and Sichuan, the disease consistently affects 20% to 40% of the total planted area, with severe diseased fields experiencing a yield loss rate of up to 60% to 80%.

[0004] Currently, chemical control remains the primary means of preventing and controlling tobacco black shank disease. Commonly used pesticides include phenylamides and triazine compounds such as metalaxyl and dimethomorph. However, the long-term, monopolistic use of chemical pesticides has led to a significant increase in pathogen resistance. For example, strains of Phytophthora nicotianae with high metalaxyl resistance (EC50 values ​​>100 μg / mL) have been detected in some tobacco-growing areas in Yunnan, my country. Recent molecular analysis has revealed that some strains of Phytophthora nicotianae in tobacco-growing areas in my country have undergone adaptive evolution of virulence genes (such as RxLR effectors), significantly enhancing their ability to infect traditionally resistant varieties. The pathogen can damage plant tissues by secreting cell wall-degrading enzymes (such as pectinases and cellulases) and toxins (such as phytorenolic acid), suppressing the host immune response and leading to a year-on-year decline in the effectiveness of chemical pesticides. Furthermore, chemical pesticides have broad-spectrum bactericidal properties. Their extensive use not only leads to resistance in pathogens but also damages soil microbial diversity and pollutes air and water. Flue-cured tobacco is a cash leaf crop. The use of chemical pesticides can lead to excessive pesticide residues in tobacco leaves, compromising tobacco safety. Therefore, although the application of chemical pesticides is convenient and has quick short-term effects, it pollutes the environment and is not in line with the development trend of green tobacco and green agriculture.

[0005] Biological control, with its environmentally friendly, residue-free, and low plant-damaging properties, has become a safer and more effective alternative to chemical pesticides for plant disease control. The mechanisms of biological control primarily include competitive exclusion, hyperparasitism, the production of antimicrobial substances, the induction of plant resistance, and the promotion of plant growth. Biocontrol agents can inhibit the growth of pathogens by leveraging competition for nutrients and ecological niches, thereby achieving disease prevention. Currently, biocontrol agents used to control tobacco black shank disease include bacteria, fungi, and actinomycetes. For example, Bacillus, Pseudomonas, Trichoderma, and actinomycetes all have strong inhibitory effects against tobacco black shank pathogens.

[0006] However, at present, the control effect of biocontrol agents on tobacco black shank pathogen is still not ideal, and there are problems such as cumbersome application of pesticides. Although some biocontrol agents can control tobacco black shank pathogen to a certain extent, they also have a certain impact on the growth of tobacco plants. There are few reports on biocontrol agents that can both control tobacco black shank pathogen and promote tobacco plant growth. Summary of the Invention

[0007] The purpose of the present invention is to address at least one of the shortcomings of the prior art and provide a composite bacterial agent BAA and its application, using biological control means to prevent and control tobacco soil-borne black shank disease. Through antagonism tests, greenhouse growth promotion and disease prevention tests, etc., it was screened that the composite bacterial agent BAA has a prevention effect of more than 75% on tobacco black shank disease and a growth promotion effect of more than 50% on plants.

[0008] In order to overcome the defects in the prior art, the present invention provides a biocontrol agent for preventing and controlling tobacco black shank disease and promoting tobacco growth and its application, which can effectively prevent and control tobacco black shank disease and promote the growth of tobacco plants.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention is to provide a composite bacterial agent, the active ingredient of which contains at least one of Bacillus amyloliquefaciens and Bacillus altitudinis; wherein the Bacillus amyloliquefaciens is named Bacillus amyloliquefaciens CJ-796, and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on June 6, 2025, with a deposit number of CGMCC NO.34788; the Bacillus altitudinis is named Bacillus altitudinis CJ-365, and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on June 6, 2025, with a deposit number of CGMCC NO.34787.

[0011] Furthermore, the viable bacterial concentration of the Bacillus amyloliquefaciens CJ-796 in the composite bacterial agent is 1×10 6 ~1×10 9 CFU / mL, the viable bacterial concentration of the Bacillus subtilis CJ-365 is 1×10 6 ~1×10 9 CFU / mL.

[0012] Furthermore, the viable bacterial concentration of the Bacillus amyloliquefaciens CJ-796 in the composite bacterial agent is 5×10 7 CFU / mL, the viable bacterial concentration of the Bacillus subtilis CJ-365 is 5×10 7 CFU / mL.

[0013] Furthermore, the active ingredients of the composite bacterial agent include the bacterial bodies, fermentation broth and / or metabolites produced by Bacillus amyloliquefaciens CJ-796 and Bacillus subtilis CJ-365.

[0014] Furthermore, the Bacillus amyloliquefaciens CJ-796 has protease activity, β-1,3-glucanase activity, cellulase activity, and siderophore-producing activity; the Bacillus altaica CJ-365 has protease activity, β-1,3-glucanase activity, and cellulase activity.

[0015] Furthermore, the 16s rDNA of the Bacillus amyloliquefaciens CJ-796 is shown as SEQ ID NO: 1, and the 16s rDNA of the Bacillus altaica CJ-365 is shown as SEQ ID NO: 2.

[0016] Furthermore, the formulation of the composite bacterial agent includes tablets, dry suspensions, water dispersible granules or wettable powders.

[0017] The second aspect of the present invention is to provide a method for preparing the composite bacterial agent as described in the first aspect, comprising the following steps:

[0018] S1. Inoculate Bacillus amyloliquefaciens CJ-796 and Bacillus subtilis CJ-365 onto LB solid medium, culture in a biochemical incubator until single colonies grow, then inoculate into liquid LB medium and culture in a shaking incubator to prepare seed solution:

[0019] S2. Inoculate the seed solution into LB culture medium at a 1% inoculum volume. After shaking culture for 14-16 hours, centrifuge and resuspend the two bacterial solutions respectively, and adjust the concentration of live bacteria in the bacterial solution to 1×10 6 ~1×10 9 CFU / mL, to obtain Bacillus amyloliquefaciens and Bacillus subtilis bacterial solutions;

[0020] S3. Mix the Bacillus amyloliquefaciens bacterial solution and the Bacillus subtilis bacterial solution in a volume ratio of (1-2):(1-2) to obtain a composite bacterial agent.

[0021] Furthermore, in step S1, the biochemical incubator culture is an inverted culture in a biochemical incubator at 26-30°C for 14-16 hours; preferably, the culture is at 28°C.

[0022] Furthermore, in step S1, the shaking culture is cultured on a shaking table at 26-30°C and 150-300 rpm / min for 14-16 hours; preferably, the culture is cultured on a shaking table at 28°C and 200 rpm / min for 14-16 hours.

[0023] Furthermore, in step S2, the shaking culture is carried out on a shaking platform at 26-30°C and 150-300 rpm / min for 14-16 hours; preferably, the culture is carried out on a shaking platform at 28°C and 200 rpm / min for 14-16 hours.

[0024] Furthermore, in step S2, the centrifugation is performed at 7000-9000 rpm for 4-6 min; preferably, at 8000 rpm for 5 min.

[0025] Furthermore, in step S2, the resuspending is performed using sterile distilled water.

[0026] Furthermore, in step S2, the viable bacteria concentration is 1×10 8 CFU / mL.

[0027] Furthermore, in step S3, the volume mixing ratio of the Bacillus amyloliquefaciens bacterial solution and the Bacillus subtilis bacterial solution is 1:1.

[0028] The third aspect of the present invention is to provide a biocontrol agent, the active ingredient of which comprises the composite agent as described in the first aspect or the composite agent prepared by the method described in the second aspect, and the plant diseases controlled by the biocontrol agent include tobacco black shank.

[0029] Furthermore, the biocontrol agent has a control efficiency of 75.71% against tobacco black shank.

[0030] The fourth aspect of the present invention is to provide a growth agent, the active ingredient of which includes the composite bacterial agent as described in the first aspect or the composite bacterial agent prepared by the method described in the second aspect, and the promoting effect of the growth agent includes promoting leaf area growth and increasing leaf dry weight.

[0031] Furthermore, the growth promoting effect of the growth agent on the dry weight of tobacco plant leaves reaches 50.68%.

[0032] Furthermore, the growth promoting effect of the growth agent on the leaf area of ​​tobacco plants reaches 34.32%.

[0033] The fifth aspect of the present invention is to provide the use of the composite bacterial agent as described in the first aspect, the biocontrol bacterial agent as described in the third aspect, or the growth agent as described in the fourth aspect, wherein the use includes at least one of the following applications: use in the preparation of a preparation for preventing and controlling tobacco black shank disease, and use in the preparation of a preparation for promoting tobacco growth.

[0034] Furthermore, the application method is to irrigate the roots of tobacco plants with the composite bacterial agent, the biocontrol bacterial agent or the growth agent when the tobacco plants have 4 to 5 leaves.

[0035] Furthermore, the root irrigation treatment is 50 mL / plant, and the root irrigation is done 1 to 2 times.

[0036] In the present invention, the composite bacterial agent is named composite bacterial agent BAA, and is abbreviated as BAA in the subsequent description.

[0037] Compared with the prior art, the above technical solution of the present invention has the following technical effects:

[0038] The present invention obtains Bacillus amyloliquefaciens and Bacillus subtilis by isolating and screening from rhizosphere soil collected from fields with serious tobacco disease, and cultured and mixed them in a certain proportion to prepare a composite bacterial agent BAA. After the mixed bacterial agent BAA is used to irrigate the roots of tobacco plants, the biocontrol effect of tobacco black shank disease reaches 75.71%, and the growth-promoting effects on the leaf dry weight and leaf area of ​​tobacco plants reach 50.68% and 34.32%, respectively. The mixed bacterial agent BAA has the significant advantages of being environmentally friendly, highly safe, and highly sustainable, promotes ecological balance and healthy crop growth, meets the requirements for drug safety in tobacco production, has potential application value in the biological control of tobacco black shank disease, solves the problems of poor results or residues of other methods such as chemical agents, can protect the environment, and promote sustainable agricultural development.

[0039] The classification name of the Bacillus amyloliquefaciens involved in the present invention is Bacillus amyloliquefaciens, which is referred to as Bacillus amyloliquefaciens CJ-796 in the subsequent description. It was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on June 6, 2025, with a deposit number of CGMCC NO.34788; the classification name of the Bacillus altitudinis involved in the present invention is Bacillus altitudinis, which is referred to as Bacillus altitudinis CJ-365 in the subsequent description. It was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on June 6, 2025, with a deposit number of CGMCC NO.34787; the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0041] Figure 1 This is a morphological diagram and plate antagonistic effect diagram of Bacillus amyloliquefaciens CJ-796 and Bacillus altaica CJ-365 in one embodiment of the present invention;

[0042] Figure 2 This is a graph showing the results of enzyme activity testing of CJ-796 and CJ-365 in one embodiment of the present invention; wherein: A is the control water treatment; B is the biocontrol bacteria treatment;

[0043] Figure 3 This is a graph showing the compatibility test results of the CJ-796 and CJ-365 strains in one embodiment of the present invention;

[0044] Figure 4This is a diagram showing the control effect of the composite bacterial agent BAA, the single bacteria CJ-796 and CJ-365 on tobacco black shank in greenhouse potted plants in one embodiment of the present invention;

[0045] Figure 5 This is a diagram showing the effect of the composite bacterial agent BAA, single bacteria CJ-796 and CJ-365 on the growth of tobacco in greenhouse pots in one embodiment of the present invention. DETAILED DESCRIPTION

[0046] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. It is obvious that the described embodiments are only some of the embodiments of the present invention, and are not all of the embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally measured in accordance with national standards. The experimental materials in the following examples, for which the sources are not specified, are all commercially available raw materials. The equipment used in each step of the following examples is conventional equipment. If there are no corresponding national standards, the steps are carried out in accordance with general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise specified, all parts are by weight, and all percentages are by mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention.

[0047] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0048] Other materials not specifically marked with manufacturers in the embodiments of the present invention can be obtained through conventional commercial purchases.

[0049] The present invention is described below by way of example.

[0050] Example 1 - Isolation, screening and identification of strains

[0051] The Bacillus amyloliquefaciens CJ-796 and Bacillus altitudinis CJ-365 involved in this embodiment were isolated and screened from rhizosphere soil. The specific steps are as follows:

[0052] 1. Sampling

[0053] In July 2024, rhizosphere soil was collected from healthy tobacco plants in a severely diseased field in Chengjiang County, Yuxi City, Yunnan Province, China. During sampling, loose soil and leaves around the roots were removed. The tobacco plants, including their roots, were removed with a shovel, ensuring a wide and deep shovel to avoid damage. Large clumps of soil were shaken off, and soil tightly attached to the roots was collected and placed in sterile bags for later use.

[0054] 2. Separation

[0055] Isolate potential biocontrol bacteria from collected samples. Weigh 5g of rhizosphere soil and pour it into a beaded flask. Add 45mL of sterile water and incubate in a shaker at 28°C, 150rpm, for 30 minutes. After thorough shaking, let it rest for 5 minutes. Make a gradient dilution with sterile water and spread the 10-4, 10-5, and 10-6 dilutions on plates. Pipette 100μL of each of these three dilutions onto pre-injected NA plates and spread the plates using a disposable spreader. Incubate the spread plates in a 28°C incubator for 48 hours. Select colonies of different morphology and color from the plates, purify them, and store them in an ultra-low temperature freezer with glycerol.

[0056] 3. Screening

[0057] Antagonistic bacteria were screened for by plate antagonism. A single colony of the purified strain was picked up with a sterile toothpick and placed in LB liquid medium. Cultured in a shaker at 28°C and 200 rpm for 16 hours to obtain a bacterial suspension. Using the plate standoff method, inoculate tobacco black shank pathogens onto the center of an OA plate using a 5mm borer. A line was drawn 2.5 cm from each end of the pathogen, and the plate was incubated in a 28°C incubator for 5 days. A blank control was provided. The standoff experiment was repeated three times for each isolated strain. Strains with obvious inhibition zones and stable inhibitory effects were selected for purification. The purified strains were then cultured again in a standoff culture to verify their antagonistic effects. The diameters of the inhibition zones of the antagonistic bacterial colonies were measured, and their inhibition rates were calculated.

[0058] Inhibition rate (%) = (colony diameter on control plate - colony diameter on treated plate) / colony diameter on control plate × 100.

[0059] According to the results of relative inhibition rate, the two strains with the best inhibitory effect on tobacco black shank pathogen were obtained: Bacillus amyloliquefaciens CJ-796 and Bacillus altaica CJ-365. Figure 1 and as shown in Table 1.

[0060] from Figure 1 It can be seen that the colonies of strain CJ-796 are round, white, with a wet surface, irregular edges, protrusions and wrinkles, and are opaque; the colonies of strain CJ-796 are milky white and round, with a smooth and moist surface and neat edges; Bacillus amyloliquefaciens CJ-796 and Bacillus highlandii CJ-365 have antagonistic effects on tobacco black shank pathogen.

[0061] Table 1 Inhibitory effect of biocontrol strains on tobacco phytophthora

[0062]

[0063] 4. Identification

[0064] Preparation of bacteria: Use a pipette to pick up a single colony and place it in LB liquid medium. Incubate overnight at 28°C in a shaker (200 rpm) for 12-16 h. Transfer the bacterial solution to a 2 mL centrifuge tube and centrifuge at 10,000 rpm for 1 min. Remove the supernatant.

[0065] Genomic DNA extraction: The genomic DNA of the strain was extracted using the TIANGEN plasmid mini-extraction kit (spin column type, Beijing Tiangen). The specific operation steps were carried out according to the instructions, and the DNA product was stored at -20°C.

[0066] 16S rDNA PCR amplification: PCR amplification was performed using 16S rDNA universal amplification primers. The amplification system is shown in Table 2.

[0067] Table 2 PCR reaction system and reaction conditions

[0068]

[0069] PCR product detection: 5 μL of PCR product was taken for 1% agarose gel electrophoresis to detect bands, and the obtained PCR product was sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing.

[0070] Sequence alignment on the NCBI website revealed that CJ-796 was identified as Bacillus amyloliquefaciens and CJ-365 as Bacillus altitudinis. The 16s rDNA sequence of Bacillus amyloliquefaciens CJ-796 is shown in SEQ ID NO: 1, and the 16s rDNA sequence of Bacillus altitudinis CJ-365 is shown in SEQ ID NO: 2. Both strains have been deposited, and the deposit information is as follows:

[0071] Bacillus amyloliquefaciens was named Bacillus amyloliquefaciens CJ-796 and was deposited in the General Microbiology Center of the China Culture Collection Administration on June 6, 2025, with the deposit number CGMCC NO.34788; the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0072] Bacillus altitudinis was named Bacillus altitudinis CJ-365 and was deposited in the General Microbiology Center of the China Culture Collection Administration on June 6, 2025, with the deposit number CGMCC NO. 34787. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0073] The 16s rDNA sequence of Bacillus amyloliquefaciens CJ-796 is shown in SEQ ID NO: 1:

[0074]

[0075] The 16s rDNA sequence of Bacillus subtilis CJ-365 is shown in SEQ ID NO: 2:

[0076]

[0077] Example 2 - Determination of enzyme activity of strains

[0078] This example is based on the enzyme activity assay of Bacillus amyloliquefaciens CJ-796 and Bacillus altaica CJ-365 isolated, screened and preserved in Example 1.

[0079] 1. Protease Activity Assay

[0080] Use a toothpick to pick a strain in the vigorous growth stage and spot it on the protein culture medium (A: 8g skim milk powder, dissolved in 300mL water, sterilized at 121℃ for 10min; B: 8g agar, dilute to 300mL, sterilized at 121℃ for 20min, A and B are sterilized separately and then mixed). After inoculation, culture at 30℃ for 3 days to observe whether there is a transparent zone, and record the inner and outer diameters of the transparent zone respectively. The results are as follows: Figure 2 and shown in Table 3.

[0081] 2. Detection of β-1,3-glucanase activity

[0082] After inoculation on a glucan plate (β-1,3-glucan 0.1 g, TSB 0.4 g, agar 1.6 g, 4 g / L Congo red 1 mL, dilute to 100 mL), culture at 30°C for 48 h, observe and measure the inner and outer diameters of the transparent zone. The results are as follows: Figure 2 and shown in Table 3.

[0083] 3. Cellulase Activity Assay

[0084] The prepared strain was inoculated onto a cellulase activity assay plate (10 g peptone, 10 g yeast powder, 10 g sodium carboxymethyl cellulose, 5 g sodium chloride, 1 g potassium dihydrogen phosphate, 18 g agar, fixed to 1000 mL, pH = 7.0), cultured at 30°C for 48 h, stained with 1 g / L Congo red for 1 h, discarded the stain, and soaked in 1 M NaCl for 1 h. The presence of a transparent zone was then detected and its inner and outer diameters were measured. The results were as follows: Figure 2 and as shown in Table 3.

[0085] 4. Siderophore Production Activity Assay

[0086] Preparation of siderophore detection medium:

[0087] Solution A: Dissolve 60.5 mg of chrome azurol S in 50 mL of ultrapure water; 10 mL of ferric iron solution (1 mM FeCl₃·6H₂O, 10 mM hydrochloric acid); and 72.9 mg of HDTMA in 40 mL of ultrapure water. Mix all three solutions and dilute to 100 mL with ultrapure water. Adjust the pH to 7.0 with 1 mol / L NaOH and sterilize at 121°C for 20 min.

[0088] B: Dissolve 30.24 g of Pipes in 900 mL of WA medium, adjust the pH to 6.8 with 1 mol / L NaOH, and sterilize at 121°C for 20 min.

[0089] When using, mix liquid A and liquid B and pour into the plate. After inoculation, culture in a 28℃ incubator for 3 days. Observe and record the size of the transparent circle. The results are as follows: Figure 2 and shown in Table 3.

[0090] Table 3 Results of enzyme activity assays of biocontrol strains

[0091]

[0092] Note: Different lowercase letters indicate significant differences at the 0.05 level.

[0093] Depend on Figure 2 It can be seen that strains CJ-796 and CJ-365 both have protease, cellulase and β-1,3-glucanase activities, among which strain CJ-796 has weak siderophore-producing activity, and strain CJ-365 has no siderophore-producing activity.

[0094] As shown in Table 3, the protease activity of strain CJ-796 was the highest, with a transparent zone diameter of 4.32 cm, while the β-1,3-glucan hydrolysis activity of strain CJ-365 was the weakest, with a transparent zone diameter of only 0.99 cm.

[0095] Example 3-Preparation of composite bacterial agent BAA

[0096] This embodiment relates to a preferred preparation method, which is based on the preparation of the composite bacterial agent BAA by compounding the Bacillus amyloliquefaciens CJ-796 and Bacillus altaica CJ-365 isolated, screened and preserved in Example 1.

[0097] 1. Strain compatibility testing

[0098] Bacillus amyloliquefaciens CJ-796 and Bacillus altaica CJ-365 were inoculated into 5 mL of NB medium and cultured overnight in a thermostatic shaker at 28°C and 150 rpm / min. The bacterial suspensions were measured using a microplate reader to an OD600 of 1.0–1.2 and then diluted with ultrapure water to maintain an OD600 of 0.1 ± 0.02. Using CJ-796 as the test strain, 200 μL of the single bacterial suspension was evenly spread on a NA plate. After drying, 5 μL of the test strain CJ-365 was inoculated onto a sterile dry filter paper sheet, which was then placed in the center of the NA plate inoculated with CJ-796. Conversely, the CJ-365 strain was used as the test strain and inoculated onto a filter paper sheet, which was then placed in the center of the NA plate. Three replicates were used for each treatment. The treated NA culture medium was placed in a 28°C incubator for 2 days and the results were observed.

[0099] Through strain compatibility testing, strains CJ-796 and CJ-365, as the tested and tested strains, respectively, had no antagonistic effects (e.g. Figure 3 As shown), the two strains can be used in combination.

[0100] 2. Preparation of composite bacterial agent BAA

[0101] Strains CJ-796 and CJ-365 were streaked onto LB solid medium, incubated upside down in a 28°C biochemical incubator for 14-16 h, and inoculated into a test tube containing 5 mL of liquid LB medium after a single colony grew. The tubes were then cultured in a shaker at 28°C and 200 rpm / min to prepare seed solution: the seed solution was inoculated into a conical flask containing 200 mL of LB culture medium at a 1% inoculum volume, and cultured in a shaker at 28°C and 200 rpm / min for 14-16 h. The two bacterial solutions were centrifuged at 8000 rpm for 5 min, resuspended in sterile distilled water, and the concentration was adjusted to 1×10 6 ~1×10 9 After the mixture was purified by centrifugation, CFU / mL was added and mixed in a volume ratio of 1:1 for use, which was the prepared biocontrol compound agent BAA.

[0102] Example 4 - Verification of the efficacy of the composite bacterial agent BAA and a single bacterial agent against tobacco black shank in greenhouse potted plants

[0103] This example is based on the composite bacterial agent BAA prepared in Example 3, and verifies its disease prevention effect on tobacco black shank in greenhouse potted plants.

[0104] 1. Preparation of Blackleg Pathogen Suspension

[0105] Pour the sterilized 10% V8 culture medium into a sterile culture dish, add 15-20 mL of activated Phytophthora to each dish, place 8-10 bacterial plates on each dish, and culture in the dark at 26°C for 2-3 days. Pour out the supernatant after mycelium is formed, add 15-20 mL of sterile water and resuspend. Change the sterile water every 12-24 hours. Change the water 3-4 times and then pick the mycelium to observe whether a large number of sporangia are produced. If a large number of sporangia are produced, place the culture dish in a 4°C refrigerator, induce at low temperature for 30 minutes, take it out and place it at room temperature for 30 minutes, that is, a large number of zoospores are released. Take it out and add 1% glucose solution to adjust the zoospore concentration to 1×10 8 / mL, and prepare a suspension of black leg pathogenic bacteria for later use.

[0106] 2. Experimental Group Setup and Treatment

[0107] Select tobacco seedlings that have fully grown 4 to 5 leaves and are strong and uniform, and transplant them into small pots filled with 125g of substrate, one plant per pot. For each treatment, 10 pots are placed in a bread box, and 3g of tobacco seedling fertilizer and 3L of tap water are added to the box.

[0108] Preparation of single bacterial inoculum: strains CJ-796 and CJ-365 were streaked onto LB solid medium, incubated upside down in a 28°C biochemical incubator for 14-16 hours, and after a single colony grew, the cells were inoculated into a test tube containing 5 mL of liquid LB medium and cultured in a shaker at 28°C and 200 rpm / min. Seed solution was prepared by inoculating the seed solution into a conical flask containing 500 mL of LB culture medium at a 1% inoculum volume, incubated in a shaker at 28°C and 200 rpm / min for 14-16 hours, and the two bacterial solutions were centrifuged at 8000 rpm for 5 minutes, resuspended in sterile distilled water, and adjusted to a concentration of 1×10 8 CFU / mL is reserved.

[0109] Compound bacterial agent: the above concentration is 1×10 8 The two single bacterial agents with the same CFU / mL were mixed in a volume ratio of 1:1 to prepare the biocontrol compound agent BAA for later use.

[0110] On the day of transplanting, roots were irrigated with 50 mL of the inoculant per plant, while the control was treated with plain water. Twenty-four hours after transplanting, 20 mL of a spore suspension of the black shank pathogen were applied per plant, repeated three times. Seven days after inoculation with the pathogen, the incidence of black shank in each treatment was recorded and the disease index was calculated per plant.

[0111] The classification of tobacco black shank disease refers to the tobacco industry standard of the People's Republic of China - Grading and Investigation Methods of Tobacco Diseases and Insects GB / T23222-2008. The classification standards are:

[0112] Level 0, the whole plant is disease-free;

[0113] Level 1: stem lesions do not exceed 1 / 3 of the stem circumference or less than 1 / 3 of the leaves wilt;

[0114] Level 3: lesions on the stem surround 1 / 3 to 1 / 2 of the stem circumference or 1 / 3 to 1 / 2 of the leaves are slightly wilted, or lesions appear on a few leaves at the bottom;

[0115] Level 5: The lesions on the stem exceed 1 / 2 of the stem circumference, but do not completely surround the stem circumference or 1 / 2 to 2 / 3 of the leaves are withered;

[0116] Level 7: The stem lesions completely surround the stem or more than 2 / 3 of the leaves wilt;

[0117] Level 9: diseased plants are basically dead.

[0118] Disease severity % = [Σ(number of diseased plants × representative level) / total number of plants × highest representative level] × 100%;

[0119] Relative control efficacy (%) = (control incidence rate - treatment prevention rate) / control incidence rate × 100%.

[0120] Table 4 Control effect of composite bacterial agents and single bacteria on tobacco black shank

[0121]

[0122] The greenhouse pot experiment showed that when tobacco leaves were 4 to 5, the concentration of 1×10 8 CFU / mL of the compound bacterial agent BAA was irrigated with 50mL / plant to effectively prevent and control the occurrence of black shank disease. Compared with the blank control group, the prevention and control effect reached 75.71%. The prevention effects of single bacteria CJ-796 and CJ-365 were 56.52% and 65.22%, respectively. Therefore, the compound bacterial agent BAA has a better prevention and control effect on tobacco black shank disease than the single bacterial agent.

[0123] Example 5 - Effects of the composite bacterial agent BAA and a single bacterial strain on tobacco growth

[0124] This example is based on the composite bacterial agent BAA prepared in Example 3 to verify its promoting effect on tobacco growth.

[0125] 1. Experimental Group Setup and Treatment

[0126] Select healthy, uniform tobacco seedlings with 4-5 leaves fully developed and transplanted into small pots containing 125g of substrate, one plant per pot. Ten pots per treatment were placed in a bread box with 3g of tobacco seedling-enhancing fertilizer and 3L of tap water. Root irrigation was performed on the day of transplanting, with 50mL of a microbial agent applied to the roots per plant. A control was treated with plain water. Seven days after transplanting, root irrigation was repeated, for a total of two irrigations. Seven days after the second root irrigation, seedling growth was observed and recorded, including plant height, stem girth, number of effective leaves, maximum leaf length, maximum leaf width, leaf fresh weight, and dry weight.

[0127] 2. Experimental Results

[0128] The results of seedling growth in different experimental groups are shown in Table 5.

[0129] Table 5 Growth-promoting effects of composite bacterial agent BAA and single bacteria on tobacco

[0130]

[0131] CJ-796 22.48±0.69a 7.89±0.42ab 33.32±0.92a 11.38±0.26a 25.47±1.61c 1.53±0.12c 6.67±0.52b

[0132] CJ-365 22.83±0.99a 7.53±0.32b 33.9±0.85a 11.17±0.31a 29.10±3.14b1.98±0.33b 7.50±0.55a

[0133] CK 20.94 ± 0.97b 7.5 ± 0.28b 31.13 ± 0.94b 9.30 ± 0.7b 20.10 ± 3.59d 1.48 ± 0.11c 6.0 ± 0c

[0134] Note: Different letters indicate significant differences at the P<0.05 level.

[0135] As shown in Table 5, the agronomic traits of the compound bacterial agent BAA were significantly higher than those of the control and single bacterial agent treatment groups. Among them, the compound bacterial agent BAA promoted the growth of tobacco plant leaf dry weight and leaf area by 50.68% and 34.32%, respectively.

[0136] In summary, the composite bacterial agent BAA prepared by the present invention not only has the effect of preventing and controlling tobacco black shank disease, but also can significantly promote the growth of tobacco plants. Among them, after the tobacco plants are treated with root irrigation with the composite bacterial agent BAA, the biocontrol effect of tobacco black shank disease reaches 75.71%, and the growth-promoting effect on tobacco plant leaf dry weight and leaf area reaches 50.68% and 34.32%, respectively; it has the significant advantages of being environmentally friendly, highly safe and highly sustainable, and can significantly promote the healthy growth of crops, meeting the requirements for drug safety in tobacco production, and has important application significance and value in the biological control of tobacco black shank disease and promoting tobacco plant growth.

[0137] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The active ingredient of the composite bacterial agent comprises at least one strain of Bacillus amyloliquefaciens and Bacillus altitudinis; wherein the Bacillus amyloliquefaciens is named Bacillus amyloliquefaciens CJ-796, and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on June 6, 2025, with a deposit number of CGMCC NO.34788; the Bacillus altitudinis is named Bacillus altitudinis CJ-365, and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on June 6, 2025, with a deposit number of CGMCC NO.34787.

2. The composite bacterial agent according to claim 1, characterized in that The live bacterial concentration of Bacillus amyloliquefaciens CJ-796 in the composite bacterial agent is 1×10 6 ~1×10 9 CFU / mL, the viable bacterial concentration of the Bacillus subtilis CJ-365 is 1×10 6 ~1×10 9 CFU / mL.

3. The composite bacterial agent according to claim 1, characterized in that The active ingredients of the composite bacterial agent include bacterial bodies, fermentation liquid and / or metabolites produced by Bacillus amyloliquefaciens CJ-796 and Bacillus subtilis CJ-365.

4. The composite bacterial agent according to claim 1, characterized in that The Bacillus amyloliquefaciens CJ-796 has protease activity, β-1,3-glucanase activity, cellulase activity, and siderophore-producing activity; and the Bacillus altaica CJ-365 has protease activity, β-1,3-glucanase activity, and cellulase activity.

5. A method for preparing the composite bacterial agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Inoculate Bacillus amyloliquefaciens CJ-796 and Bacillus subtilis CJ-365 onto LB solid medium respectively and culture in a biochemical incubator until single colonies grow. Then inoculate into liquid LB medium and culture on a shaking platform to prepare seed solution: S2. Inoculate the seed solution into LB culture medium at a 1% inoculum volume. After shaking culture for 14-16 hours, centrifuge and resuspend the two bacterial solutions respectively, and adjust the viable bacterial concentration of the bacterial solution to 1×10 6 ~1×10 9 CFU / mL, to obtain Bacillus amyloliquefaciens and Bacillus subtilis bacterial solutions; S3. Mix the Bacillus amyloliquefaciens bacterial solution and the Bacillus subtilis bacterial solution in a volume ratio of (1-2): (1-2) to obtain a composite bacterial agent.

6. The method according to claim 5, characterized in that In step S1, the biochemical incubator culture is carried out by inverted culture in a biochemical incubator at 26 to 30° C. for 14 to 16 hours; and / or In step S1, the shaking culture is carried out by placing the culture on a shaking table at 26-30° C. and 150-300 rpm / min for 14-16 hours; and / or In step S2, the shaking incubation is performed on a shaking incubator at 26-30° C. and 150-300 rpm / min for 14-16 hours; and / or In step S2, the centrifugation is performed at 7000-9000 rpm for 4-6 minutes; and / or In step S2, the resuspending is performed using sterile distilled water.

7. A biocontrol agent, characterized in that The active ingredient of the biocontrol agent comprises the composite bacterial agent according to any one of claims 1 to 4 or the composite bacterial agent prepared by the method according to any one of claims 5 to 6, and the plant diseases controlled by the biocontrol agent include tobacco black shank.

8. A growth agent, characterized in that The active ingredient of the growth agent comprises the composite bacterial agent according to any one of claims 1 to 4 or the composite bacterial agent prepared by the method according to any one of claims 5 to 6, and the promoting effect of the growth agent includes promoting leaf area growth and increasing leaf dry weight.

9. Use of the composite bacterial agent according to any one of claims 1 to 4, the biocontrol agent according to claim 7, or the growth agent according to claim 8, characterized in that: The application includes at least one of the following applications: application in preparing a preparation for preventing and treating tobacco black shank disease, and application in preparing a preparation for promoting tobacco growth.

10. The use according to claim 9, characterized in that The application method is to irrigate the roots of tobacco plants with the composite bacterial agent, the biocontrol bacterial agent or the growth agent when the tobacco plants have 4 to 5 leaves.

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