A compound microbial agent BAA and its application

Abstract

This invention relates to a compound microbial agent BAA and its application. The active ingredient of the compound microbial agent BAA contains at least one strain selected from Bacillus amyloliquefaciens CJ-796 and Bacillus altitudinis CJ-365. The viable bacterial concentration of the compound microbial agent BAA is 1×10⁻⁶. 8 CFU / mL; By applying this solution to the roots of the plants, it can effectively prevent the occurrence of tobacco black shank disease and significantly promote the growth of tobacco plants. The control efficacy against tobacco black shank disease is over 75%, and the growth promotion effect on plants can reach over 50%. It is safe, residue-free, and environmentally friendly, meeting the requirements for safe use of pesticides. It has potential application value in the biological control of tobacco black shank disease.

Description

Technical Field

[0001] This invention relates to the field of agricultural biological control technology, specifically to a compound microbial agent BAA and its application. Background Technology

[0002] Tobacco black shank is a devastating soil-borne disease caused by *Phytophthora parasitica* var. *nicotianae*. The pathogen belongs to the class Oomycetes, order Peronosporales, family Pythiaceae, and genus *Phytophthora*, exhibiting strong host specificity and environmental adaptability. Chlamydospores and dormant mycelia in the soil are the primary sources of infection for tobacco black shank, while zoospores are the secondary source of infection. Chlamydospores and dormant mycelia are mainly distributed in the top 0–5 cm of soil, primarily infecting through root injuries at the base of the tobacco plant. Zoospores can swim rapidly in water and spread quickly with the help of rainfall and flowing water, tending to aggregate on the surface of the crop roots, entering through wounds or orifices, leading to browning and necrosis of the vascular bundles, and plant wilting and lodging.

[0003] Black shank disease breaks out rapidly under hot and humid conditions, and is particularly severe in tobacco fields with continuous cropping or in low-lying areas. Typical symptoms include stunted growth, yellowing and wilting of leaves from the bottom up, and shrinkage and necrosis of the roots and stems, mainly manifested as "black shank," "black plaster," "wearing a coat," "leaky waist," and "shoot nodes." According to a report by 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 US$1.5 billion annually due to black shank disease. In my country, the main producing areas such as Yunnan, Guizhou, and Sichuan provinces consistently account for 20%–40% of the total planted area affected by the disease, with severely affected fields experiencing crop failure rates as high as 60%–80%.

[0004] Currently, chemical control remains the primary method for controlling tobacco black shank disease, with commonly used agents including phenylamides and triazine compounds such as metalaxyl and dimethomorph. However, long-term use of chemical pesticides alone has led to a significant increase in pathogen resistance. For example, strains of *Phytophthora indicum* with high resistance to metalaxyl (EC50 value > 100 μg / mL) have been detected in some tobacco-growing areas of Yunnan, my country. Recent molecular testing has revealed that some strains of *Phytophthora indicum* in tobacco-growing areas of my country have undergone adaptive evolution in their virulence genes (such as RxLR effector factors), significantly enhancing their ability to infect traditionally resistant varieties. Pathogens can damage plant tissues and suppress host immune responses by secreting cell wall-degrading enzymes (such as pectinase and cellulase) and toxins (such as phytoalexin), leading to a gradual decline in the efficacy of chemical pesticides. Furthermore, chemical pesticides have broad-spectrum bactericidal activity; excessive application not only leads to pathogen resistance but also damages soil microbial diversity and pollutes air and water. Flue-cured tobacco is a leaf-based economic crop, and the application of chemical pesticides results in excessive pesticide residues in tobacco leaves, reducing their safety. Therefore, although the application of chemical pesticides is convenient and has a quick effect in the short term, it pollutes the environment and does not conform to the development trend of green tobacco and green agriculture.

[0005] Biological control, with its advantages of being environmentally friendly, leaving no residue, and causing less damage to plants, has become a safer and more effective alternative to chemical pesticides for controlling plant diseases. The mechanisms of action of biological control mainly include competitive exclusion, hyperparasitism, production of antimicrobial substances, induction of plant resistance, and promotion of plant growth. Biocontrol agents can inhibit the growth of pathogens by competing for nutrients and ecological niches, thereby achieving disease control. Currently, biocontrol bacteria, fungi, and actinomycetes are used to control tobacco black shank. For example, Bacillus, Pseudomonas, Trichoderma, and actinomycetes all have strong inhibitory effects on the tobacco black shank pathogen.

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

[0007] The purpose of this invention is to address at least one deficiency in the existing technology by providing a compound microbial agent BAA and its application, which uses biological control methods to prevent and control soil-borne black shank disease in tobacco. Through antagonism tests, greenhouse growth promotion and disease prevention tests, the compound microbial agent BAA was screened and found to have a control efficacy of more than 75% against 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 and its application for preventing and controlling tobacco black shank disease and promoting tobacco growth, which can effectively prevent and control tobacco black shank disease and promote the growth of tobacco plants.

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

[0010] The first aspect of the present invention is to provide a compound microbial agent, wherein the effective component of the compound microbial agent comprises at least one of Bacillus amyloliquefaciens and Bacillus altitudinis; wherein the Bacillus amyloliquefaciens is named Bacillus amyloliquefaciens CJ-796 and was deposited at the China General Microbiological Culture Collection Center on June 6, 2025, with accession number CGMCC NO.34788; and the Bacillus altitudinis is named Bacillus altitudinis CJ-365 and was deposited at the China General Microbiological Culture Collection Center on June 6, 2025, with accession number CGMCC NO.34787.

[0011] Furthermore, the viable bacterial concentration of Bacillus amyloliquefaciens CJ-796 in the compound bacterial agent is 1×10⁻⁶. 6 ~1×10 9 The viable bacterial concentration of the *Bacillus hygroscopicus* CJ-365 was 1 × 10⁻⁶ CFU / mL. 6 ~1×10 9 CFU / mL.

[0012] Furthermore, the viable bacterial concentration of Bacillus amyloliquefaciens CJ-796 in the compound bacterial agent is 5 × 10⁻⁶. 7 The viable bacterial concentration of the *Bacillus hygroscopicus* CJ-365 was 5 × 10⁻⁶ CFU / mL. 7 CFU / mL.

[0013] Furthermore, the effective components of the compound microbial agent include the cells, fermentation broth, and / or metabolites of Bacillus amyloliquefaciens CJ-796 and Bacillus glacialis CJ-365.

[0014] Furthermore, the *Bacillus amyloliquefaciens* CJ-796 possesses protease activity, β-1,3-glucanase activity, cellulase activity, and heparin-producing activity; the *Bacillus hygroscopicus* CJ-365 possesses protease activity, β-1,3-glucanase activity, and cellulase activity.

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

[0016] Furthermore, the dosage form of the compound microbial agent includes tablets, dry suspensions, water-dispersible granules, or wettable powders.

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

[0018] S1. Inoculate Bacillus amyloliquefaciens CJ-796 and Bacillus hygroscopicus CJ-365 onto LB solid medium, culture them in a biochemical incubator until single colonies appear, then inoculate them into liquid LB medium and culture them in a shaker to prepare seed culture.

[0019] S2. Inoculate the seed culture into LB culture medium at an inoculation rate of 1%, and culture on a shaker for 14-16 hours. Then, centrifuge and resuspend both bacterial cultures separately, and adjust the viable cell concentration of the bacterial cultures to 1×10⁻⁶. 6 ~1×10 9 CFU / mL was used to obtain Bacillus amyloliquefaciens and Bacillus hygroscopicus bacterial suspensions;

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

[0021] Further, in step S1, the biochemical incubator culture is performed by inverting the incubator at 26-30℃ for 14-16 hours; preferably, it is performed at 28℃.

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

[0023] Further, in step S2, the shaking culture is carried out at 26-30℃ and 150-300 rpm / min for 14-16 hours; preferably, it is carried out at 28℃ and 200 rpm / min for 14-16 hours.

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

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

[0026] Further, in step S2, the concentration of live bacteria is 1×10⁻⁶. 8 CFU / mL.

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

[0028] A third aspect of the present invention is to provide a biocontrol agent, wherein the active ingredient of the biocontrol agent comprises a compound microbial agent as described in the first aspect or a compound microbial 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 achieves a control efficiency of 75.71% against tobacco black shank.

[0030] A fourth aspect of the present invention is to provide a growth agent, wherein the effective component of the growth agent comprises a compound microbial agent as described in the first aspect or a compound microbial 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 agent has a growth-promoting effect of 50.68% on the dry weight of tobacco leaves.

[0032] Furthermore, the growth agent increased the leaf area of ​​tobacco plants by 34.32%.

[0033] The fifth aspect of the invention is to provide the use of the compound microbial agent as described in the first aspect, or the biocontrol agent as described in the third aspect, or the growth agent as described in the fourth aspect, the use comprising at least one of the following uses: use in the preparation of preparations for the prevention and control of tobacco black shank, use in the preparation of preparations for promoting tobacco growth.

[0034] Furthermore, the application method involves applying the compound microbial agent, the biocontrol agent, or the growth agent to the roots of the tobacco plants when they have 4-5 leaves.

[0035] Furthermore, the root drenching treatment is 50 mL per plant, drenched 1 to 2 times.

[0036] In this invention, the compound microbial agent is named compound microbial agent BAA, and will be referred to as BAA in the following description.

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

[0038] This invention isolates and screens *Bacillus amyloliquefaciens* and *Bacillus hygroscopicus* from rhizosphere soil collected from severely diseased tobacco fields, and then mixes them in a certain proportion to prepare a compound microbial agent BAA. After root irrigation treatment of tobacco plants, the mixed microbial agent BAA achieves a biocontrol effect of 75.71% against tobacco black shank disease, and promotes leaf dry weight and leaf area growth by 50.68% and 34.32%, respectively. It has significant advantages of being environmentally friendly, highly safe, and sustainable, promoting ecological balance and healthy crop growth, meeting the safety requirements for pesticide use in tobacco production, and has potential application value in the biological control of tobacco black shank disease. It solves the problems of poor efficacy or residues associated with other methods such as chemical agents, protects the environment, and promotes sustainable agricultural development.

[0039] The *Bacillus amyloliquefaciens* involved in this invention is classified as *Bacillus amyloliquefaciens*, and will be referred to as *Bacillus amyloliquefaciens* CJ-796 in the following description. It was deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34788. The *Bacillus altitudinis* involved in this invention is classified as *Bacillus altitudinis*, and will be referred to as *Bacillus altitudinis* CJ-365 in the following description. It was also deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34787. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0040] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings:

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

[0042] Figure 2 This is a graph showing the enzyme activity detection results 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 CJ-796 and CJ-365 strains in one embodiment of the present invention;

[0044] Figure 4This is a diagram illustrating the control effect of compound microbial agents BAA, single bacteria CJ-796, and CJ-365 on tobacco black shank in greenhouse potted plants according to an embodiment of the present invention.

[0045] Figure 5 This is a diagram illustrating the effect of compound microbial agents BAA, single bacteria CJ-796, and CJ-365 on the growth of tobacco in greenhouse potted plants, according to one embodiment of the present invention. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0047] It should be noted that, unless otherwise specified, the embodiments and features described in 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 this is not intended to limit the scope of the invention.

[0048] Other manufacturers not specifically indicated in the embodiments of this invention can be obtained through commercial purchases.

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

[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 samples were collected from healthy tobacco plants in severely diseased tobacco habitats in Chengjiang County, Yuxi City, Yunnan Province, China. During sampling, loose soil and leaf debris around the roots were removed, and the tobacco plant was carefully removed along with its roots using a shovel, ensuring a sufficiently wide and deep shovel area while avoiding damage. Large clumps of soil were shaken off, and the 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 the collected samples. Weigh 5g of rhizosphere soil and pour it into an Erlenmeyer flask containing glass beads. Add 45mL of sterile water to the flask and incubate on a shaker at 28℃ and 150rpm for 30min. After thorough shaking, let it stand for 5min. Perform serial dilutions with sterile water, and select 10-4, 10-5, and 10-6 times dilutions for plate spreading. Take 100μL of each of these three gradient suspensions and place them on pre-pouring NA plates, spreading them using a disposable spreader. Incubate the plates at 28℃ for 48h. Select colonies with different morphologies and colors from the plates for purification and store them in an ultra-low temperature freezer with glycerol.

[0056] 3. Filtering

[0057] Antagonistic bacteria were screened using plate antagonism. Single colonies of the purified strain were collected using a sterile toothpick and placed in LB liquid medium. The culture was incubated at 28°C and 200 rpm for 16 hours to obtain the bacterial suspension. The plate confrontation method was used: *Tobacco Black Shank* was inoculated onto the center of an OA plate using a 5 mm perforator, and streaked 2.5 cm from both ends of the pathogen. The plate was then incubated at 28°C for 5 days. A blank control was included. Each isolated strain was tested three times. Strains with obvious inhibition zones and stable inhibitory effects were selected for purification. The purified strains were then subjected to confrontation culture again to verify the antagonistic effect. The diameter of the inhibition zone of the antagonistic bacterial group was measured, and its inhibition rate was calculated.

[0058] Inhibition rate (%) = (Coronary diameter of control plate - Coronary diameter of treatment plate) / Coronary diameter of control plate × 100.

[0059] Based on the relative inhibition rate results, two strains with the best inhibitory effect on the pathogen of tobacco black shank disease were identified: *Bacillus amyloliquefaciens* CJ-796 and *Bacillus glaber* CJ-365. The results are as follows: Figure 1 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 moist surface, irregular edges, raised wrinkles, and opaque; the colonies of strain CJ-796 are milky white, round, with a smooth and moist surface and regular edges; Bacillus amyloliquefaciens CJ-796 and Bacillus hygroscopicus CJ-365 have antagonistic effects against tobacco black shank pathogen.

[0061] Table 1. Inhibitory effect of biocontrol strains on Phytophthora indicum

[0062]

[0063] 4. Identification

[0064] Cell preparation: Pick a single colony with a pipette tip and place it in LB liquid medium. Incubate overnight at 28°C (200 rpm) for 12–16 h. Add the bacterial solution to a 2 mL centrifuge tube and centrifuge at 10,000 rpm for 1 min. Discard the supernatant.

[0065] Genomic DNA extraction: Genomic DNA of the strain was extracted using the TIANGEN plasmid miniprep kit (centrifuge column type, Beijing Tiangen). The specific operation steps were performed according to the instructions. The DNA product was stored at -20℃.

[0066] 16S rDNA PCR amplification: PCR amplification was performed using universal 16S rDNA amplification primers, and the expansion 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 subjected to 1% agarose gel electrophoresis to detect the bands. The obtained PCR product was sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing.

[0070] The sequencing results were aligned with the NCBI website. The results showed that CJ-796 is *Bacillus amyloliquefaciens*, and CJ-365 is *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 sequences were deposited, and the deposit information is as follows:

[0071] Bacillus amyloliquefaciens, named Bacillus amyloliquefaciens CJ-796, was deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34788; deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0072] Bacillus altitudinis, named Bacillus altitudinis CJ-365, was deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.34787. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

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

[0074]

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

[0076]

[0077] Example 2 - Enzyme activity assay of strains

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

[0079] 1. Protease activity assay

[0080] Using a toothpick, pick up a bacterial strain in its vigorous growth phase and spot it onto protein culture medium (A: 8g skim milk powder dissolved in 300mL water, sterilized at 121℃ for 10min; B: 8g agar, diluted to 300mL, sterilized at 121℃ for 20min; A and B are sterilized separately and then mixed). After inoculation, incubate at 30℃ for 3 days and observe the presence or absence of a clear zone. Record the inner and outer diameters of the clear zone. The results are as follows: Figure 2 As shown in Table 3.

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

[0082] After inoculation on dextran plates (β-1,3-glucan 0.1g, TSB 0.4g, agar 1.6g, Congo red 1mL, 4g / L, diluted to 100mL), the culture was incubated at 30℃ for 48h. The inner and outer diameters of the clear zone were observed and measured. The results are as follows: Figure 2 As shown in Table 3.

[0083] 3. Cellulase activity assay

[0084] The prepared bacterial strain was inoculated onto a cellulase activity assay plate (10g peptone, 10g yeast extract, 10g sodium carboxymethyl cellulose, 5g sodium chloride, 1g potassium dihydrogen phosphate, 18g agar, diluted to 1000mL, pH=7.0), and incubated at 30℃ for 48h. After staining with 1g / L Congo red for 1h, the stain was discarded, and the plate was soaked in 1M NaCl for 1h. Then, the presence and inner and outer diameters of the transparent zone were measured. The results are as follows: Figure 2 As shown in Table 3.

[0085] 4. Assay for ferrophilic activity

[0086] Preparation of ferroptosis detection culture medium:

[0087] Solution A: 60.5 mg of Chromium Azurite S dissolved in 50 mL of ultrapure water; 10 mL of ferric solution (1 mM FeCl3·6H2O, 10 mM hydrochloric acid as solvent); 72.9 mg of HDTMA dissolved in 40 mL of ultrapure water. Mix the above three solutions thoroughly and bring the volume 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 30.24g Pipes in 900mL WA medium, adjust the pH to 6.8 with 1mol / L NaOH, and sterilize at 121℃ for 20min.

[0089] When using, mix solutions A and B thoroughly, pour the mixture onto a plate, inoculate with the bacteria, and incubate at 28°C for 3 days. Observe and record the size of the transparent zone. The results are as follows: Figure 2 As shown in Table 3.

[0090] Table 3 Results of enzyme activity assays by 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 them, strain CJ-796 has weaker iron-producing activity, and strain CJ-365 does not have iron-producing enzyme activity.

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

[0095] Example 3 - Preparation of Compound Microbial Agent BAA

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

[0097] 1. Strain compatibility testing

[0098] Bacillus amyloliquefaciens CJ-796 and Bacillus glomeratus CJ-365 were inoculated into 5 mL of NB medium and cultured overnight in a constant-temperature shaking incubator at 28 °C and 150 rpm / min. The bacterial concentration was measured using a microplate reader (OD600 = 1.0–1.2), and then diluted with ultrapure water to maintain the concentration within the range of OD600 = 0.1 ± 0.02. Using CJ-796 as the test strain, 200 μL of a single bacterial culture was evenly spread on the surface of an NA plate. After drying, 5 μL of the test strain CJ-365 was inoculated onto a sterile dry filter paper disc, which was then placed in the center of the NA plate inoculated with CJ-796. Conversely, CJ-365 was used as the test strain, spread on NA plates, while CJ-796 was used as the test strain, inoculated onto a filter paper disc and placed in the center of the NA plate. Each treatment was performed in triplicate. The treated NA medium was incubated in a 28℃ incubator for 2 days, and the results were observed.

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

[0100] 2. Preparation of compound microbial agent BAA

[0101] Strains CJ-796 and CJ-365 were streaked onto LB agar plates and incubated upside down in a 28°C biochemical incubator for 14–16 h. After single colonies appeared, they were inoculated into test tubes containing 5 mL of liquid LB agar and cultured on a shaker at 28°C and 200 rpm / min to prepare seed culture. Seed culture was then inoculated at a 1% inoculation rate into Erlenmeyer flasks containing 200 mL of LB agar and cultured on a shaker at 28°C and 200 rpm / min for 14–16 h. Both bacterial cultures were centrifuged separately at 8000 rpm for 5 min, resuspended in sterile distilled water, and the concentration adjusted to 1 × 10⁻⁶. 6 ~1×10 9 After mixing CFU / mL, the mixture is prepared at a volume ratio of 1:1 and then set aside for use. This is the prepared biocontrol compound bacterial agent BAA.

[0102] Example 4 - Verification of the efficacy of compound microbial agent BAA and single strain against tobacco black shank in greenhouse potted plants.

[0103] This embodiment uses the compound microbial agent BAA prepared in Example 3 to verify its disease prevention effect on tobacco black shank in greenhouse potted plants.

[0104] 1. Preparation of Blackleg Fungus Suspension

[0105] Pour sterilized 10% V8 culture medium into sterile petri dishes. Place 15-20 mL of activated *Phytophthora inoculum* mycelium in each dish, and add 8-10 mycelium dishes to each dish. Incubate in the dark at 26°C for 2-3 days. Once mycelial colonies have formed, discard the supernatant and resuspend the mycelium in 15-20 mL of sterile water. Change the sterile water every 12-24 hours, changing it 3-4 times. After each cycle, pick mycelium to observe for the production of numerous sporangia. If numerous sporangia are produced, place the petri dish in a 4°C refrigerator for 30 minutes of low-temperature induction. Then, remove it and allow it to stand at room temperature for 30 minutes, at which point a large number of zoospores will be released. Add 1% glucose solution to adjust the zoospore concentration to 1×10⁻⁶. 8 Prepare a suspension of black shank bacteria by collecting samples per mL.

[0106] 2. Experimental group setup and treatment

[0107] Select healthy and uniform tobacco seedlings that have fully sprouted 4-5 leaves, and transplant them into small flower pots containing 125g of substrate, one seedling per pot. Place 10 pots for each treatment in a bread box, adding 3g of tobacco seedling fertilizer and 3L of tap water to the box.

[0108] Preparation of single-strain inoculum: Strawberries CJ-796 and CJ-365 were streaked onto LB solid medium and incubated upside down in a 28°C biochemical incubator for 14–16 h. After single colonies grew, they were inoculated into test tubes containing 5 mL of liquid LB medium and cultured on a shaker at 28°C and 200 rpm / min to prepare seed culture: 1% of the seed culture was inoculated into Erlenmeyer flasks containing 500 mL of LB medium and cultured on a shaker at 28°C and 200 rpm / min for 14–16 h. Both bacterial cultures were centrifuged at 8000 rpm for 5 min, resuspended in sterile distilled water, and the concentration was adjusted to 1 × 10⁻⁶. 8 CFU / mL available for use.

[0109] Compound microbial agent: The above concentration is 1×10 8 Two single-strain inoculants with CFU / mL were mixed in a 1:1 volume ratio to prepare a biocontrol compound inoculant BAA for later use.

[0110] On the day of transplanting, roots were drenched with 50 mL of fungal agent per plant, while the control group was treated with plain water. 24 hours after transplanting, 20 mL of black shank spore suspension per plant was applied, repeated 3 times. Seven days after inoculation with tobacco black shank spores, the incidence of black shank in each treatment was recorded, and the disease index was calculated per plant.

[0111] The grading of tobacco black shank disease is based on the People's Republic of China Tobacco Industry Standard—Grading and Survey Methods for Tobacco Diseases and Pests GB / T23222-2008. The grading standards are as follows:

[0112] Grade 0, the entire plant is disease-free;

[0113] Grade 1: Stem lesions do not exceed 1 / 3 of the stem circumference, or the leaves wither.

[0114] Grade 3: Stem lesions surround 1 / 3 to 1 / 2 of the stem circumference or 1 / 3 to 1 / 2 of the leaves, causing slight wilting, or lesions appear on a few lower leaves;

[0115] Grade 5: Stem lesions extend beyond 1 / 2 of the stem circumference, but do not completely encircle the stem circumference or cause 1 / 2 to 2 / 3 of the leaves to wither;

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

[0117] Level 9, the diseased plants are basically dead.

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

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

[0120] Table 4. Effects of compound microbial agents and single microorganisms on the prevention and control of tobacco black shank disease.

[0121]

[0122] The above greenhouse pot experiment showed that when tobacco is at the 4-5 leaf stage, a concentration of 1×10⁻⁶ is effective. 8 A root drench application of 50 mL of the compound microbial agent BAA (CFU / mL) per plant effectively controlled black shank disease. Compared with the control group, the control effect reached 75.71%. The control efficacy of single-strain CJ-796 and CJ-365 was 56.52% and 65.22%, respectively. Therefore, the compound microbial agent BAA has a better control effect on tobacco black shank disease than single-strain agents.

[0123] Example 5 - The promoting effect of compound microbial agent BAA and single strain on tobacco growth

[0124] This embodiment uses the compound microbial 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 fully emerged leaves and transplant them into small pots containing 125g of substrate, one seedling per pot, with 10 pots per treatment placed in a bread box containing 3g of tobacco seedling fertilizer and 3L of tap water. On the day of transplanting, perform a root drench treatment, applying 50mL of bacterial agent per seedling; the control group was treated with plain water. Seven days after transplanting, perform a second root drench, for a total of two drench treatments. Seven days after the second root drench, observe and record the seedling growth, including plant height, stem circumference, number of effective leaves, maximum leaf length, maximum leaf width, leaf fresh weight, and dry weight.

[0127] 2. Experimental Results

[0128] The growth records of seedlings in different experimental groups are shown in Table 5.

[0129] Table 5. Growth-promoting effects of compound microbial agent BAA and single strains 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 compound microbial agent BAA were significantly higher than those of the control and single-agent treatment groups. Among them, compound microbial agent BAA promoted the growth of leaf dry weight and leaf area of ​​tobacco plants by 50.68% and 34.32%, respectively.

[0136] In summary, the compound microbial agent BAA prepared in this invention not only has a preventive effect against tobacco black shank disease, but also significantly promotes the growth of tobacco plants. Specifically, after root irrigation treatment of tobacco plants with compound microbial agent BAA, the biocontrol effect against tobacco black shank disease reached 75.71%, and the effects on leaf dry weight and leaf area of ​​tobacco plants increased by 50.68% and 34.32%, respectively. It has significant advantages of being environmentally friendly, highly safe, and sustainable, and can significantly promote the healthy growth of crops, meeting the requirements for safe pesticide use in tobacco production. It has important application significance and value in the biological control of tobacco black shank disease and the promotion of tobacco plant growth.

[0137] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of compound microbial agents in the prevention and control of tobacco black shank disease and the promotion of tobacco growth, characterized in that, The effective component of the compound microbial agent is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Bacillus amyloliquefaciens ) and Highland Bacillus ( Bacillus altitudinis The compound consists of: *Bacillus amyloliquefaciens* named *Bacillus amyloliquefaciens* CJ-796, deposited on June 6, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 34788; and *Bacillus glacialis* named *Bacillus glacialis* CJ-365, deposited on June 6, 2025 at the same center with accession number CGMCC NO. 34787. The viable concentration of *Bacillus amyloliquefaciens* CJ-796 in the compound is 1 × 10⁻⁶. 6 ~1×10 9 The viable bacterial concentration of the *Bacillus hygroscopicus* CJ-365 was 1 × 10⁻⁶ CFU / mL. 6 ~1×10 9 CFU / mL, the *Bacillus amyloliquefaciens* CJ-796 exhibits protease activity, β-1,3-glucanase activity, cellulase activity, and heparin-producing activity; the *Bacillus glaber* CJ-365 exhibits protease activity, β-1,3-glucanase activity, and cellulase activity; wherein... The promotion of tobacco growth includes promoting leaf area growth, increasing leaf dry weight, and increasing the number of leaves; the application method is to drench the plants with the compound microbial agent when the tobacco plants have 4-5 leaves.

2. The method for preparing the compound microbial agent as described in claim 1, characterized in that, Includes the following steps: S1. Inoculate Bacillus amyloliquefaciens CJ-796 and Bacillus hygroscopicus CJ-365 separately onto LB solid medium and culture in a biochemical incubator until single colonies grow. Then, inoculate them into liquid LB medium and culture on a shaker to prepare seed culture. S2, inoculate the seed liquid into LB culture solution with 1% inoculation amount, shake bed culture for 14-16 h, centrifuge, resuspend and adjust the viable bacterial concentration of the bacterial liquid to 1 x 10 6 ~1 x 10 9 CFU / mL to obtain bacillus amyloliquefaciens bacterial liquid and bacillus altitudinis bacterial liquid; S3. Mix the Bacillus amyloliquefaciens bacterial solution and the Bacillus hygroscopicus bacterial solution in a volume ratio of (1~2):(1~2) to obtain the compound bacterial agent.

3. The method according to claim 2, characterized in that, In step S1, the biochemical incubator culture is performed by inverting the incubator at 26-30℃ for 14-16 hours; and / or In step S1, the shaking culture is performed by incubating on a shaking table at 26-30℃ and 150-300 rpm / min for 14-16 h; and / or In step S2, the shaking culture is performed at 26-30℃ and 150-300 rpm / min for 14-16 h; and / or In step S2, the centrifugation is performed at 7000-9000 rpm for 4-6 minutes; and / or In step S2, the resuspension is performed using sterile distilled water.

4. A biocontrol agent, characterized in that, The biocontrol agent is prepared using the method described in any one of claims 2 to 3. The plant diseases controlled by the biocontrol agent include tobacco black shank disease. The biocontrol agent has a promoting effect on tobacco growth, including promoting leaf area growth, increasing leaf dry weight, and increasing the number of leaves.

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