Air bacillus, microbial agent and application thereof

Abstract

The present application belongs to the field of microbiology and its agricultural application technology, and particularly relates to a bacillus aerius, a microbial inoculum and application thereof. The present application screens a strain with good antagonistic effect on tobacco black shank and fusarium root rot from a tomato root, and identifies the strain as bacillus aerius. The strain can inhibit the growth of pathogenic fungi by secreting chitinase, cellulase and protease, dissolving the cell wall structure of pathogenic fungi, and losing the invasion ability. The strain has an inhibitory effect on more than 10 kinds of plant pathogenic fungi, and the inhibitory rate is greater than 80%. Especially, the inhibitory rates of the strain on tobacco phytophthora and fusarium oxysporum are greater than 90%. The pot experiment and field experiment verify that the strain has a better effect on the prevention and treatment of tobacco black shank and fusarium root rot than the commercially available biocontrol inoculum and chemical agents. Meanwhile, the strain can promote tobacco seed germination, promote the growth and development of tobacco plants in the field, has good stability in subculture, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and its agricultural applications, specifically relating to an airborne Bacillus, a microbial agent, and its applications. Background Technology

[0002] Tobacco black shank and Fusarium root rot are global soil-borne fungal diseases that severely restrict tobacco production, causing serious economic losses in tobacco-producing countries such as the United States, Australia, India, South Africa, South Korea, Japan, and Canada. They have also seriously damaged tobacco yield and quality in my country. These two diseases often occur together, and due to their strong infectivity, short disease cycle, and rapid spread, they often become devastating regional diseases. Currently, field production mainly relies on the application of chemical pesticides, the breeding of disease-resistant varieties, and improved cultivation practices for control. However, long-term and excessive use of chemical agents can easily lead to the development of resistance in pathogens, reducing control efficacy and harming the environment and human health. Highly resistant tobacco varieties to these two diseases are rare in production, and in tobacco fields with limited arable land and an ever-increasing multiple cropping index, relying solely on cultivation practices for disease control is very limited in effectiveness. Biological control, due to its high efficiency and environmental friendliness, has become an important means of integrated pest management in tobacco both domestically and internationally, playing an increasingly important role in the control of soil-borne root and stem diseases in tobacco.

[0003] Continuously exploring new antagonistic bacterial resources and elucidating their mechanisms of action is the primary task of current tobacco biological control. Wang Yayue et al. isolated a strain of *Streptomyces violascens* from marine sponges, achieving a field control efficacy of 83.5% against tobacco black shank disease. This strain can inhibit the pathogen by secreting proteases and cellulases to disrupt the cell wall structure of the pathogen. Liu Tianbo et al. screened *Acinetobacter pietroides*... Acinetobater pittiiThe control efficacy against tobacco black shank reached 55.37%, and it significantly affected the root growth and development of tobacco plants. Qiu Rui et al. screened *Pseudomonas aeruginosa* YZ66 and *P. chlororaphis* YX33 from tobacco rhizosphere soil, which effectively inhibited *Fusarium oxysporum* and *Fusarium oxysporum*. Previous studies have mostly focused on screening antagonistic strains targeting only tobacco black shank or *Fusarium oxysporum* root rot, and there are no reports of simultaneous control of both diseases. Qian Huimin et al. screened *Pseudomonas aeruginosa* and *Pseudomonas granadensis*, which showed good control effects against both tobacco black shank and root rot; Song Yujuan et al. found that *Trichoderma echinococcus* T-6 had good control effects against both tobacco black shank and root rot. This indicates that screening biocontrol bacteria that can simultaneously control multiple diseases has become a new strategy for biological control. Tobacco black shank and Fusarium root rot often co-infect tobacco plants, and the symptoms are difficult to distinguish. Therefore, it is urgent to screen out biocontrol bacteria that can control both diseases at the same time. Summary of the Invention

[0004] To address the problems existing in the prior art, one of the objectives of this invention is to provide an airborne Bacillus that has an inhibitory effect on a variety of pathogens that can cause plant diseases, especially showing good control efficacy against tobacco black shank and Fusarium root rot. At the same time, it can promote tobacco seed germination and field growth, thus having both disease prevention and growth promotion effects.

[0005] The second objective of this invention is to provide a microbial agent containing Bacillus aerogenes provided by this invention, which can be used for the dual prevention and control of tobacco black shank disease and Fusarium root rot, and promote tobacco seed germination and field growth.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An airborne Bacillus species, with accession number CGMCC NO.21278.

[0007] Microbial agents containing the aforementioned Bacillus aeruginosa.

[0008] The aforementioned microbial agent is a lyophilized powder or a liquid preparation; specifically, the liquid preparation may be the aforementioned *Bacillus aerobicus* fermentation broth. In a specific embodiment of the present invention, the fermentation culture temperature of the *Bacillus aerobicus* fermentation broth is 30°C. Specifically, the concentration of *Bacillus aerobicus* in the liquid preparation is 1.0 × 10⁻⁶. 7 ~1.0×10 11 cfu / mL; preferably, the concentration of *Bacillus aeruginosa* in the liquid formulation is 1.0 × 10⁻⁶. 8 ~1.0×10 10cfu / mL.

[0009] The aforementioned Bacillus aerosus and microbial agents have antibacterial effects against a variety of plant pathogenic fungi and can be used to prevent and control plant diseases caused by plant pathogenic fungi, including Anthracnose sicca, Fusarium oxysporum, Trichoderma fulvidraco, Alternaria solani, Fusarium wiltii, Fusarium solani, Fusarium wiltii, Fusarium oxysporum, Fusarium oxysporum, Fusarium oxysporum, Fusarium oxysporum, Fusarium oxysporum, Botrytis cinerea, Phytophthora indicum, and Fusarium oxysporum.

[0010] The aforementioned *Bacillus aerobicans* and microbial agents can be used to control tobacco black stem disease and Fusarium root rot. Preferably, the concentration of *Bacillus aerobicans* in the microbial agent for controlling tobacco black stem disease and Fusarium root rot is 1.0 × 10⁻⁶. 8 CFU / mL; the above-mentioned Bacillus aerobicans and microbial agents can also be used to improve the disease resistance of tobacco plants, improve the germination rate of tobacco seeds, and / or the field growth of plants. Preferably, the concentration of Bacillus aerobicans in the microbial agents for improving the disease resistance of tobacco plants, improving the germination rate of tobacco seeds, and / or the field growth of plants is 1.0 × 10⁻⁶. 9 cfu / mL.

[0011] Beneficial effects of this invention: (1) The Bacillus aerius of this invention was isolated from the rhizomes of tomato and identified as Bacillus aerius based on its morphological characteristics, physiological and biochemical properties and 16S rRNA homology analysis. It has a high-efficiency and broad-spectrum antibacterial activity against plant pathogens. The antibacterial rate against 17 kinds of plant pathogenic fungi is >80%, and the antibacterial rate against Phytophthora tobaccoii, Fusarium oxysporum, Anthracnose sicca, Fusarium graminearum, and Trichoderma pulveratum is >90%. (2) The antimicrobial proteins (chitinase, cellulase and protease) derived from Bacillus aerosus of the present invention can cause mycelial malformation, cell wall dissolution, cytoplasmic leakage and loss of infectivity of Phytophthora tobacco and Fusarium oxysporum; pot experiment showed that the relative control efficacy against tobacco black shank disease and Fusarium root rot was 63.6% and 82.8% respectively, and the relative control efficacy in field experiment reached 72.4% and 75.1% respectively, which is significantly better than commercially available biocontrol agents and chemical agents; (3) The airborne Bacillus of the present invention can simultaneously induce the activity of defense enzymes (POD, PAL, PPO) in tobacco plants to increase in a short period of time (3d to 6d), thereby improving the disease resistance of tobacco plants; (4) The airborne Bacillus of the present invention can promote the germination of tobacco seeds and promote the growth and development of tobacco plants in the field; (5) The Bacillus aerobicus of the present invention has good stability in subculture. After 50 subcultures, it still maintains high antibacterial activity (>87%) against the pathogens of tobacco black shank and Fusarium root rot. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0013] Figure 1 The plate confrontation effect of strain C-1 against Phytophthora tobaccoii and Fusarium oxysporum; Figure 2 Colony morphology (A and B) and microscopic morphology (C) of strain C-1; Figure 3 Electrophoresis diagram of 16S rDNA amplification products of strain C-1; Figure 4 Phylogenetic diagram of the 16S rDNA sequence of strain C-1 Figure 5 The following are images of the effects of strain C-1 on pathogens under scanning electron microscopy: a. normal Fusarium oxysporum hyphae; b. Fusarium oxysporum hyphae treated with fermentation broth of strain C-1; c. normal Phytophthora tobacco hyphae; d. Phytophthora tobacco hyphae treated with fermentation broth of strain C-1. Figure 6 The effects of different concentrations of fermentation broth from strain C-1 on the activities of POD, PAL, and PPO enzymes in tobacco leaves were investigated. Figure 7 The effect of different concentrations of C-1 strain fermentation broth on tobacco seed germination; Figure 8 The effects of different concentrations of C-1 strain fermentation broth on soluble protein content and total amylase activity during tobacco seed germination were investigated. Figure 9 The inhibitory effect of strain C-1 on 12 pathogenic bacteria. Detailed Implementation

[0014] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0015] The tobacco variety used in the following examples and test cases is "Zhongyan 100"; the pathogen of tobacco black shank (Phytophthora parasitica var. nicotianae) and the pathogen of tobacco Fusarium root rot (Fusarium oxysporum) were isolated by the inventor's research group from tobacco plants infected with tobacco black shank and Fusarium root rot in the tobacco fields of Pingdingshan.

[0016] Example 1: Screening and Identification of Target Antagonistic Bacteria 1.1. Strain isolation and screening: Endophytic bacteria in tomato roots were isolated and purified using the tissue homogenization method. Twenty strains were isolated from five tomato root samples. The purified single colonies were preserved for later use using the slant method and the glycerol method, respectively. 1.2. The antimicrobial spectrum of antagonistic bacteria was detected using the plate confrontation method: *Phytophthora tobaccoii* and *Fusarium oxysporum* were respectively inoculated into 1 cm mycelial discs in the center of PDA medium. The fermentation broth of the isolated strains (cell concentration 1.0 × 10⁻⁶) was then added. 8 (cfu / mL) Inoculate the pathogen in a triangular pattern at equal intervals 2 cm from the center of the plate. Use a plate inoculated only with the pathogen as a control. Incubate at 30℃ for 5 days. Repeat each treatment three times. When the control colonies reach near the edge of the plate, measure the diameter of the pathogen colony and the diameter of the inhibition zone. Calculation formula: Inhibition diameter = Pathogen colony diameter in control group - Pathogen colony diameter in treatment group Inhibition rate = [(Coronary diameter of control group - Colony diameter of treatment group) / (Coronary diameter of control group - Diameter of mycelial cake)] × 100% One strain, designated C-1, was selected based on its antagonistic effect against *Phytophthora tobaccoii* and *Fusarium oxysporum*, as shown in Table 1. Figure 1 As shown, the inhibition diameter against both Phytophthora tobaccois and Fusarium oxysporum reached 72 mm, and the inhibition rate was greater than 90%, indicating a good antibacterial effect in plate confrontation.

[0017]

[0018] 1.3. Strain identification: 1.3.1 Observation of colony morphology and microscopic morphology Using NA solid medium, C-1 colonies were streaked and incubated at 30℃ and humidity for 2-3 days to form single colonies. Their colony morphology was then observed. Figure 2 As shown, the C-1 strain colonies are generally beige with a pale milky-white edge. They are flat and irregular in shape, with scattered petal-like edges. The colony surface is moist with a central concave protrusion and wrinkles. The surface is rough and opaque, containing spores that are centrally or eccentrically located, elliptical in shape, with slightly enlarged sporangia. Gram staining is positive, and microscopic examination reveals that the bacterium is rod-shaped.

[0019] 1.3.2 Physiological and biochemical characteristics were measured, and the results are shown in Table 2:

[0020] The results shown in Table 2 indicate that strain C-1 can liquefy gelatin, hydrolyze casein, starch, urea, and cellulose, reduce nitrate, and is positive for oxidase, methyl red, and VP tests. However, it is negative for lipase, tryptophan degradation, and catalase tests. It can utilize carbon sources such as glucose, citric acid, and sucrose.

[0021] Based on comparison with Bergey's Manual of Bacteriology and combined with the morphological characteristics of C-1 colonies, it was preliminarily identified as Bacillus spp. Bacillus ).

[0022] 1.3.3 Molecular biological identification of the strain: Genomic DNA was extracted from strain C-1 using a bacterial genome extraction kit. PCR was performed using universal primers 27F and 1492R for bacterial 16S rDNA. The amplified products were then subjected to agarose gel electrophoresis. Figure 3 As shown, the 16S rDNA gene sequence of strain C-1 is 1434 bp in length. The 16S rDNA gene sequence of strain C-1 was sent to Tianjin Genewise Biotech Co., Ltd. for sequencing. The obtained base pair sequences were compared for similarity using BLAST software. The eight sequences with the highest homology to strain C-1 were selected, and a phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 7.0. Figure 4 As shown, after 16S rDNA sequence alignment using Blast, strain C-1 was found to be similar to *Bacillus aeruginosa* (…). Bacillus aerius They belong to the same branch and have the highest homology.

[0023] Therefore, based on the morphological characteristics, physiological and biochemical properties, and 16S rDNA homology analysis of strain C-1, it was determined to be *Bacillus aeruginosa* (…). Bacillus aerius It was deposited on December 2, 2020 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.21278.

[0024] Strain C-1 was cultured on a large scale to obtain the fermentation broth for subsequent experiments. The specific conditions for the large-scale culture were as follows: seed culture was inoculated at a ratio of 1:100 into a 500 mL shake flask (200 mL liquid NA / flask), and cultured at 30 ℃ and 200 r / min for 3-5 days.

[0025] Example 2: Identification of the biocontrol effect and antibacterial mechanism of strain C-1 2.1 Biocontrol potential against tobacco black shank and Fusarium root rot: The efficacy of C-1 bacterial solution in potted plants and field plants was determined by root irrigation treatment of tobacco seedlings. The pot experiment included four treatments: (1) CK (blank control): only pathogens were inoculated; (2) C-1 treatment: the fermentation broth of the expanded C-1 strain was diluted to prepare a fermentation solution (cell concentration approximately 1.0 × 10⁻⁶). 8 (3) Wangtaibao treatment: Wangtaibao (Dajing Biotechnology, effective strain: Trichoderma harzianum, commercially available) (biocontrol agent control) solution concentration was 1%, 65 mL per tobacco plant was irrigated; (4) Metalaxyl-mancozeb treatment: 58% metalaxyl-mancozeb wettable powder (Jiangsu Baoling Chemical Co., Ltd., metalaxyl content: 10%, mancozeb content: 48%, chemical agent control, commercially available) was diluted 600 times, 20 mL per plant was irrigated. Seven days after the first application of fungicide or pesticide, each treatment was inoculated with 20 mL of fermentation liquid of Fusarium oxysporum or Phytophthora indicum. After that, each treatment was irrigated with fungicide or pesticide once every 7 days, for a total of 3 times. 8 plants per treatment, 3 replicates, and the disease incidence was investigated 14 days after inoculation.

[0026] The field trial was conducted in Bailongmiao Village, Pingdingshan City. Except for the control (CK), which involved natural disease development, the other treatments were identical to those used in potted plants. Each treatment consisted of 50 plants, replicated three times. The tobacco plants were transplanted on May 1, 2022. After transplanting, each treatment was treated with the same dosage as the potted plants, applied via root drenching every 15 days for a total of three applications. Disease incidence was assessed 15 days after the final root drenching. Other measures were implemented according to the standards for high-quality tobacco production.

[0027] The disease index grading standards for tobacco black shank and Fusarium root rot are based on the "Tobacco Disease Grading and Investigation Methods" in the "Tobacco Industry Standards of the People's Republic of China" (GB / T23222-2008, GB / T23224-2008).

[0028] Calculation formula: Incidence rate = (Number of infected plants / Total number of plants surveyed) × 100% Disease index = ∑(Number of diseased plants at each level × Disease level value) / (Total number of plants surveyed × Highest level value) × 100 Relative efficacy = (Control disease index - Treatment disease index) / Control disease index × 100% The results of pot and field efficacy tests are shown in Table 3:

[0029] Pot experiment results showed that the control group (CK) inoculated only with pathogens had a tobacco black shank incidence rate of 91.7% and a Fusarium root rot incidence rate of 100%, indicating successful pathogen inoculation. The corresponding disease indices were 57.3 and 89.3, respectively. Under the treatment of the commercially available biocontrol agent, Wantaibao, the incidence rates of tobacco black shank and Fusarium root rot were 75.0% and 100%, respectively, with disease indices of 44.8 and 78.6, and relative control efficacies of 21.7% and 11.9%. The chemical agent, metalaxyl-mancozeb, showed incidence rates of 54.2% and 66.7%, with disease indices of 33.1 and 42.9, and relative control efficacies of 42.1% and 51.9%, respectively. The C-1 treatment showed incidence rates of 29.2% and 20.8%, with disease indices of 20.8 and 15.3, and relative control efficacies of 63.6% and 82.8%, respectively. The incidence and disease index of the two diseases under the three treatments of commercially available biocontrol agents, chemical agents, and C-1 were all lower than the control, and all had a certain relative control effect. The differences between the treatments were significant. The two diseases treated with strain C-1 had the lowest incidence and disease index, and the relative control effect was also significantly higher than the other treatments.

[0030] Field trials showed that the incidence rates of tobacco black shank and Fusarium root rot in the naturally occurring control (CK) treatment were 12.0% and 32.7%, respectively; the incidence rates of the two diseases treated with the commercially available biocontrol agent Wantaibao were 7.3% and 16.7%, with disease indices of 6.2 and 11.0, and relative control efficacy of 38.4% and 31.6%, respectively; the incidence rates of the disease treated with the chemical agent metalaxyl-mancozeb were 6.7% and 10.0%, with disease indices of 6.0 and 7.3, and relative control efficacy of 38.4% and 53.1%, respectively; and the incidence rates of the disease treated with the C-1 treatment were 4.0% and 5.3%, with disease indices of 2.8 and 4.0, and relative control efficacy of 72.4% and 75.1%, respectively. Compared with the control, the commercially available biocontrol agents, chemical agents and C-1 treatment in the field trials all significantly reduced the incidence and disease index of tobacco black shank and Fusarium root rot, and all had certain relative control efficacy, especially C-1, which had the best control efficacy. The relative control efficacy against the two diseases in the field trials was higher than 70%.

[0031] 2.2 Determination of the antibacterial mechanism of antagonistic strains 2.2.1 Assay of cell wall degrading enzyme activity in antagonistic strains Supernatant filtrate of fermentation broth from strain C-1 (whole bacterial culture centrifuged at 12000 rpm for 5 min, then filtered) and cell lysate of fermentation broth were collected separately (100 mL of whole bacterial culture was collected, centrifuged at 6500 rpm for 10 min at 4℃, then 5 mL of 50 mmol / L pH 7.5 KH2PO4-K2HPO4 buffer solution was added, and the culture was sonicated). The activities of antimicrobial proteins (protease, chitinase, and cellulase) in the supernatant filtrate and cell lysate of strain C-1 fermentation broth were determined separately: cellulase content was determined using a cellulase activity assay kit (Shanghai Yuanxin Biotechnology, YX-SH-TQ22025), chitinase content was determined using a chitinase activity assay kit (Shanghai Yuanxin Biotechnology, YX-W-B917), and protease content was determined using a protease activity assay kit (Wuhan Merck Biotechnology Co., Ltd., KT59429).

[0032] The results are shown in Table 4: Protease, chitinase and cellulase activities were detected in both the supernatant filtrate of the fermentation broth and the cell lysate of strain C-1, and there was no significant difference in the enzyme activities of the three cell wall degrading enzymes between the two treatments.

[0033]

[0034] 2.2.2 Scanning electron microscopy observation of inhibited pathogen hyphae Scanning electron microscopy was used to observe *Fusarium oxysporum*, *Phytophthora nicotineae*, and pathogens treated with C-1 fermentation broth, respectively. Figure 5 As shown: from Figure 5 Figure a shows that the normal hyphae of *Fusarium oxysporum* are tubular, with a smooth, velvety surface and no branching. From... Figure 5 As shown in Figure b, treatment with C-1 bacterial solution significantly inhibited the growth and sporulation of the pathogen. The inhibited pathogen exhibited increased hyphal branching, hyphae entanglement, and swollen tips. C-1 parasitizes the interior of Fusarium oxysporum hyphae, secreting substances that inhibit the pathogen's growth, causing it to shrivel and eventually die.

[0035] from Figure 5 As shown in Figure c, the hyphae of the control *Phytophthora indicum* grow normally, with full morphology and smooth cell walls. From... Figure 5 As shown in Figure d, after antagonistic treatment with the fermentation broth of strain C-1, the growth and sporulation of Phytophthora were significantly inhibited. The surface of the inhibited pathogenic hyphae shrank, became sunken and shriveled, twisted and deformed, and some hyphal walls collapsed, eventually losing their infectivity.

[0036] 2.2.3 Effects of strain C-1 on the activity of defensive enzymes in tobacco seedlings A pot experiment was conducted. Thirty days after transplanting, the cells were inoculated with the original C-1 fermentation broth (cell concentration approximately 1.0 × 10⁻⁶).8 (cfu / mL), 10-fold (cell concentration 1.0×10⁻⁶) 9 (cfu / mL), 100-fold (cell concentration 1.0×10⁻⁶) 10 10 ml of fermentation broth (cfu / mL) was used, with water treatment as the control (CK). Each treatment was replicated 10 times. The activity of the defense enzyme was measured on the third leaf at 0, 3, 6, and 9 days after inoculation.

[0037] Peroxidase (POD) was determined using the guaiacol method; polyphenol oxidase (PPO) was determined using the catechol method; and phenylalanine ammonia-lyase (PAL) was determined using the L-phenylalanine method.

[0038] The results are as follows Figure 6 As shown in Figure a, after inoculation with fermentation broth of strain C-1 at different concentrations, the activity of POD (peroxidase) in tobacco plants showed a trend of slightly decreasing on day 3 after inoculation, significantly increasing on day 6, and then decreasing again; Figures b and c show that the activities of PAL (phenylalanine ammonia-lyase) and PPO (polyphenol oxidase) showed a trend of steadily increasing from day 0 to day 6 after inoculation, and then significantly decreasing.

[0039] Compared with the control, after inoculation with different concentrations of fermentation broth from strain C-1, the POD activity increased with increasing C-1 fermentation broth concentration, especially on days 6 and 9 post-inoculation, showing significant differences. The PAL activity generally showed an increasing trend with increasing fermentation broth concentration, but on day 9, the inoculated broth was significantly lower than the control (CK). The PPO activity, however, was significantly higher than the control after inoculation with the original C-1 broth and a 10-fold concentration of fermentation broth, but decreased after inoculation with a 100-fold concentration. This indicates that inoculation with C-1 fermentation broth induces a short-term (3-6 days) increase in the defensive enzymes (POD, PAL, PPO) of tobacco plants, with the increase increasing at cell concentrations up to 1.0 × 10⁻⁶. 9 A cfu / mL ratio is preferable.

[0040] Example 3: Feasibility evaluation of strain C-1 in promoting tobacco seed germination and field tobacco plant growth and development. 3.1 Effects on tobacco seed germination Tobacco seeds were soaked in sterile petri dishes using water (CK), fermentation broth stock solution of strain C-1, and fermentation broth at concentrations of 10x and 100x, respectively. The dishes were then incubated in a constant temperature incubator at 25°C. Starting from day 3, the germination rate was recorded every 12 hours (with the germination standard being root length equal to seed length or shoot length equal to 1 / 2 seed length), and the results were recorded until day 6.

[0041] The results are as follows Figure 7As shown, the germination rates of tobacco seeds treated with water (CK), C-1 stock solution, 10-fold dilution, and 100-fold dilution of fermentation broth on day 4 were 52.67%, 60.00%, 61.33%, and 59.00%, respectively. Compared with the water control, different concentrations of C-1 strain fermentation broth significantly promoted tobacco seed germination. From day 4 to day 6 after soaking, the germination rate was significantly higher with a 10-fold dilution of fermentation broth (cell concentration 1.0 × 10⁻⁶). 9 The highest germination rate was observed in the fermentation broth (cfu / mL), followed by the stock fermentation broth (1.0 × 10⁻⁶ cells / mL). 8 (cfu / mL).

[0042] 3.2 Effects on soluble protein content and total amylase activity during tobacco seed germination In the above seed germination experiment, 0.5 g of germinated tobacco seeds were taken on the 9th day after germination, and 5 mL of phosphate buffer (pH 6.9) was added. The mixture was thoroughly homogenized and allowed to stand for 30 min. The homogenate was then transferred to a centrifuge tube and centrifuged at 4 ℃ and 12000 r / min for 10 min. All supernatants were collected for later use. The soluble protein content was determined using the Coomassie Brilliant Blue method, and the amylase activity was determined using the 3,5-dinitrosalicylic acid method.

[0043] The results are as follows Figure 8 As shown, after soaking tobacco seeds in water (CK), C-1 stock solution, 10-fold and 100-fold fermentation solution, the soluble protein content in the seeds was 7.32, 9.25, 10.70 and 8.01 mg / g, respectively. Figure 8 The soluble protein content of all C-1 strain fermentation broth treatments at different concentrations was higher than that of the control (CK), with a significant difference between the 10-fold and original broth treatments. After seed soaking, the amylase activity in tobacco seeds increased with increasing fermentation broth concentration, and significant differences were observed among different concentrations of fermentation broth treatments.

[0044] 3.3 Effects on tobacco agronomic traits At maturity, three tobacco plants were randomly selected from each treatment group in Example 2, and their plant height, stem circumference, internode distance, number of effective leaves, maximum leaf length, leaf width, leaf area, fresh leaf weight, root weight, and root length were measured. The results are shown in Table 5.

[0045] Compared with the control, the C-1 treatment showed significant increases in plant height, stem circumference, internode distance, leaf length of the largest leaf, leaf width, leaf area, number of effective leaves, fresh leaf weight, root length, and root weight. Except for stem circumference, all agronomic traits in the C-1 treatment were also increased compared with those treated with the microbial inoculant Wantaibao and the chemical agent metalaxyl-mancozeb.

[0046] Example 4: Determination of the stability of strain C-1 in subculture Strain C-1 was subcultured on NA medium, with subculture every 48 h for 50 consecutive subcultures. The antifungal effects of the primary and subcultured strains against *Phytophthora tobaccoii* and *Fusarium oxysporum* were determined using a plate confrontation test. The influence of subculturing on the antifungal effect of the antagonistic bacteria was analyzed based on the changes in the inhibition band width. The results are shown in Table 6.

[0047] As shown in Table 6, after 1, 5, 10, and 50 generations of subculturing, the inhibition rates of strain C-1 against *Phytophthora indicum* were 90.55%, 87.97%, 89.45%, and 89.07%, respectively; and the inhibition rates against *Fusarium oxysporum* were 90.15%, 89.67%, 90.13%, and 89.24%, respectively. The inhibition rates of strain C-1 against the two pathogens showed good stability after different subculturings, without significant differences.

[0048] Example 5: Determination of the antibacterial spectrum of strain C-1 The antifungal effect of strain C-1 against other plant pathogenic fungi was detected using the plate confrontation method. Five plates were used for each treatment, and the experiment was repeated three times. The results are as follows: Figure 9 And as shown in Table 7 below:

[0049] The results showed that strain C-1 had a significant inhibitory effect on the growth of 12 pathogenic fungi. Figure 9 The inhibition rates against *Anthrax sicca*, *Fusarium fusiforme*, *Trichoderma pinkis*, *Alternaria tomato*, *Fusarium rotundifolium*, *Fusarium solanum*, *Fusarium fusiforme*, *Fusarium moniliforme*, *Fusarium solani*, *Fusarium moniliforme*, *Fusarium solani*, *Fusarium moniliforme*, *Anthrax moniliforme*, and *Botrytis cinerea* reached 93.99%, 93.69%, 90.39%, 89.50%, 87.69%, 86.18%, 85.59%, 84.98%, 84.08%, 82.89%, 82.28%, and 81.68% respectively (Table 7), indicating a significant inhibitory effect. This indicates that strain C-1, screened in this invention, is a highly efficient and broad-spectrum antagonistic bacterium with significant potential application value in the control of plant diseases. Besides its dual protective effects against tobacco black shank and Fusarium root rot, it also shows potential application value in the control of strawberry stem base rot, anthracnose; strawberry root rot; corn ear rot and stem rot; rice bakanae disease; sugarcane top rot; corn ear rot; wheat scab; melon powdery mildew / white mold; tomato powdery mildew; cucumber powdery mildew; mushroom powdery mildew; apple heart rot; mango fruit rot; tomato early blight; corn ear rot and stem rot; tomato fruit rot; tobacco Fusarium root rot; tomato Fusarium fruit rot; zucchini Fusarium fruit rot; sweet potato brown spot; strawberry root rot; eggplant flower rot; cucumber Fusarium wilt; sweet potato dry rot; mango anthracnose; and gray mold in various crops.

[0050] The characterization results of the above embodiments show that the *Bacillus aeruginosa* C-1 isolated from tomato roots in this invention, with its derived antimicrobial proteins (chitinase, cellulase, and protease), can cause mycelial malformation, cell wall dissolution, cytoplasmic leakage, and loss of infectivity in *Phytophthora tobaccois* and *Fusarium oxysporum*. It exhibits good control efficacy against both tobacco black shank and Fusarium root rot, with field control efficacy exceeding 70%. It can increase the activity of defense enzymes such as PPO, POD, and PAL in tobacco plants, inducing disease resistance, and simultaneously promotes tobacco seed germination and field growth, thus possessing both disease prevention and growth-promoting effects. Furthermore, this strain can effectively inhibit multiple crop diseases, exhibiting good, high-efficiency, broad-spectrum antibacterial activity. It is a highly promising disease-preventing and growth-promoting bacterium, and its good stability in subculturing makes it suitable for widespread application in biocontrol formulations.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An airborne Bacillus, characterized in that, The Bacillus aerosporium is Bacillus aerosporium C-1, with accession number CGMCC NO.21278, and is deposited at the China General Microbiological Culture Collection Center.

2. A microbial agent containing Bacillus aeruginosa as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The microbial agent is a freeze-dried powder.

4. The microbial agent as described in claim 2, characterized in that, The microbial agent is a liquid preparation.

5. The microbial agent as described in claim 4, characterized in that, The microbial agent is the fermentation broth of the *Bacillus aeruginosa* strain as described in claim 1.

6. The microbial agent as described in claim 5, characterized in that, The concentration of *Bacillus aeruginosa* in the microbial agent is 1.0 × 10⁻⁶. 7 ~1.0×10 11 cfu / mL.

7. The microbial agent as described in claim 6, characterized in that, The concentration of *Bacillus aeruginosa* in the microbial agent is 1.0 × 10⁻⁶. 8 ~1.0×10 10 cfu / mL.

8. The application of *Bacillus aeruginosa* as described in claim 1 or the microbial agent as described in any one of claims 2 to 7 in the prevention and control of tobacco diseases caused by pathogenic fungi in tobacco, characterized in that, The tobacco diseases mentioned are tobacco black stem disease and Fusarium root rot.

9. The application of the *Bacillus aeruginosa* as described in claim 1 or the microbial agent as described in any one of claims 2 and 4 to 7 in improving the disease resistance of tobacco plants, increasing the germination rate of tobacco seeds, and / or the growth of tobacco plants; The concentration of *Bacillus aeruginosa* in the microbial agent is 1.0 × 10⁻⁶. 9 cfu / mL.

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