Bacillus siamensis F019, microbial inoculant, and preparation method and application thereof

Abstract

This invention relates to the field of microbial technology. This invention provides a strain of Bacillus sicca (…). Bacillus siamensis This invention provides strain F019, which was deposited on July 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 35129. The invention also provides a microbial inoculant. Furthermore, the invention provides the application of Bacillus sicca strain F019 or the microbial inoculant. The Bacillus sicca strain F019 provided by this invention exhibits highly effective antibacterial activity against various plant diseases, including black shank, and significantly promotes plant disease resistance, plant growth, and seed germination. Strain F019 and AM fungi mutually promote each other's colonization in the tobacco rhizosphere; the combined application of strain F019 and AM fungi can significantly promote tobacco growth and enhance tobacco resistance to black shank.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus sicca F019, a microbial agent, its preparation method, and its application. Background Technology

[0002] With the continuous planting of crops and the development of protected cultivation, soil-borne diseases have become increasingly serious year by year, and various diseases are frequently occurring throughout the country.

[0003] Biological control, an environmentally friendly control technology, has gained attention. Biological control utilizes antagonistic microorganisms and their secondary metabolites to reduce the reproduction and survival rate of pathogens through mechanisms such as competition, antibiosis, lysis, and induction, thereby inhibiting plant diseases in a complex process. Typical biocontrol bacteria, such as Bacillus, have advantages such as direct acquisition, rapid colonization, strong adaptability, stable effects, and ease of operation. Meanwhile, beneficial fungi (AM fungi) can enhance host resistance by expanding root absorption area and activating systemic resistance in plants. However, existing research often focuses on single species, and using a single strain for black shank control often reveals shortcomings such as a limited mechanism of action and insufficient environmental adaptability.

[0004] Therefore, there is an urgent need to discover a Bacillus strain that combines AM fungal interaction capabilities with highly efficient disease resistance. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a strain of Bacillus sicca F019, a microbial inoculant, its preparation method, and its applications. This invention provides a strain of Bacillus sicca F019 that can effectively control tobacco black shank disease and promote the efficient colonization of AM fungi. Furthermore, the combined application of these two strains has a certain growth-promoting effect on tobacco. The microbial inoculant prepared using this strain can be applied to tobacco disease control and growth promotion.

[0006] To address the aforementioned problems in the prior art, the present invention provides the following technical solution: In a first aspect, the present invention provides a strain of Bacillus sicca ( Bacillus siamensis Strain F019 was deposited on July 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35129. The morphological characteristics of this strain are: irregularly round growth on LB medium; rough surface with protrusions, wrinkled edges, and a pale yellowish-white color; Gram staining identification shows it to be Gram-positive; spore staining identification shows that F019 can produce spores. Based on the comprehensive results of various physiological and biochemical experiments and molecular biological analysis, this bacterium is identified as *Bacillus sicca*.

[0007] Secondly, the present invention provides a microbial agent containing Bacillus sicca strain F019.

[0008] Based on the highly efficient antagonistic activity of Bacillus sicca F019, it can be used as an active ingredient to prepare a microbial agent.

[0009] In addition, to further optimize and improve the bactericidal or growth-promoting effects of the microbial agent, the microbial agent also includes other bactericidal active ingredients (such as other biocontrol bacteria and antibacterial active substances with complementary bactericidal spectrum) or growth-promoting active ingredients.

[0010] The microbial agent described above contains, as a whole, or as a spore, or fermentation supernatant of Bacillus sicca strain F019. Alternatively, the bacterial solution, bacterial cells (e.g., dry powder), and spores can be used in combination.

[0011] The microbial agent described above, wherein the total viable bacterial concentration of Bacillus sicca strain F019 in the microbial agent is 1×10⁻⁶. 9 ~2×10 9 CFU / mL.

[0012] Thirdly, the present invention provides a method for preparing a microbial inoculant, the specific steps of which include: inoculating a loop of Bacillus sicca strain F019 into LB liquid medium, and culturing it in a shake flask at a temperature of 30-36°C and a rotation speed of 180-220 r / min for a certain period of time to obtain the microbial inoculant. Preferably, in the inoculation step, the inoculation amount is 1-5%, and the bottling amount is 10-30%; preferably, the liquid medium includes the following components: maltodextrin content of 5-9 g / L, beef extract content of 14-18 g / L, and magnesium chloride content of 10-14 g / L. Preferably, the culturing time is 15-17 h. Most preferably, the culturing temperature is 34°C, the rotation speed is 209-210 r / min, the pH is 8.0, the inoculation amount is 3%, and the bottling amount is 15-16.65%; the liquid medium includes the following components: maltodextrin content of 7 g / L, beef extract content of 16 g / L, and magnesium chloride content of 12 g / L. Under these conditions, the growth of strain F019 can be better promoted.

[0013] Fourthly, the present invention provides an application of the above-mentioned Bacillus sicca strain F019 or the above-mentioned microbial agent in any one of the following (a1)-(a10): (a1) controlling plant diseases; (a2) preparing products for controlling plant diseases; (a3) ​​promoting AM fungal infection of plant roots; (a4) preparing products that promote AM fungal infection of plant roots; (a5) synergistically promoting colonization in the rhizosphere with AM fungi; (a6) synergistically promoting plant growth with AM fungi; (a7) synergistically enhancing plant disease resistance with AM fungi; (a8) promoting plant seed germination; (a9) promoting the improvement of plant leaf quality; (a10) improving the physical and chemical properties of plant soil.

[0014] In the applications described above, the plant is tobacco, tomato, grape, watermelon, or peanut. Preferably, the plant is tobacco.

[0015] In the applications described above, in (a1), (a2) and (a7), the plant diseases are tobacco black shank, tomato gray mold, grape anthracnose, grape white rot, watermelon wilt, or peanut root rot.

[0016] As described above, in (a8), the plant seeds include: tobacco 'Zhongchuan 208', tobacco 'Yunyan 301', tobacco 'Honghua Dajinyuan', tomato 'Zhongshu No. 4', tomato '72-69', and zucchini 'Jingyingcui'.

[0017] As described above, in (a9), the plant is tobacco. Specifically, the leaf quality refers to the total sugar, reducing sugar content, total nitrogen, potassium content, nicotine content, and sugar-to-alkali ratio of tobacco. Experiments have shown that the total sugar, reducing sugar content, total nitrogen, potassium content, nicotine content, and sugar-to-alkali ratio of field tobacco treated with F019 fermentation liquid are significantly higher than those of the control group of field tobacco without F019 fermentation liquid.

[0018] As described above, in (a10), the plant is tobacco. Specifically, the soil physicochemical properties refer to soil pH, available phosphorus, hydrolyzable nitrogen, and available potassium. Experiments have shown that applying F019 fermentation broth can regulate soil pH, adjusting slightly alkaline soil to a slightly acidic soil more suitable for tobacco growth. Compared with the control group without F019 fermentation broth, it significantly increased the content of available phosphorus, hydrolyzable nitrogen, and available potassium in the soil.

[0019] Fifthly, the present invention provides a method for controlling plant diseases, wherein the method utilizes the *Bacillus sicca* strain F019 as described above or a microbial inoculant as described above. Preferably, the plant is tobacco.

[0020] Sixthly, the present invention provides a method for promoting plant seed germination, wherein the method utilizes the *Bacillus sicca* strain F019 as described above or a microbial inoculant as described above to soak plant seeds. Preferably, the plant is tobacco.

[0021] Seventhly, the present invention provides a method for improving the growth and colonization of Bacillus sicca in the rhizosphere of tobacco. Tobacco is selected as the experimental subject, and a seedling substrate is prepared by mixing peat moss and sand in a ratio of 1:9. After the seedling substrate is thoroughly stirred, it is placed in flowerpots that have been sterilized with alcohol. Tobacco seedlings with good growth are selected for transplanting, and after one week of recovery, the fermentation liquid of Bacillus sicca strain F019 as described above is used for root irrigation.

[0022] Compared with existing technologies, the effects and advantages of this invention are: (1) This invention provides a strain of Bacillus sicca F019 that has a high-efficiency antibacterial effect against a variety of plant diseases, including black shank disease, and has a significant promoting effect on plant disease resistance, plant growth and plant seed germination.

[0023] (2) The Bacillus sicca F019 and AM fungi of the present invention have a mutual promoting effect on each other's colonization in the tobacco rhizosphere. The combined application of F019 and AM fungi can significantly promote the growth of tobacco and enhance the tobacco's resistance to black shank disease.

[0024] (3) The present invention provides the optimal culture conditions for Bacillus sicca F019, further optimizes and improves the yield of the strain, saves the fermentation cost of the strain, and improves the production efficiency of the inoculant. Attached Figure Description

[0025] Figure 1 Morphological identification of strain F019; Figure 2 Phylogenetic analysis of 16S rDNA for strain F019; Figure 3 For strain F019 Gyrb Molecular phylogenetic analysis; Figure 4 Plate confrontation experiment between strain F019 and different pathogens; Figure 5 Tobacco phenotype was treated with fermentation broth of strain F019; Figure 6 The effect of fermentation broth of strain F019 on tobacco growth; Figure 7 This refers to the infection of tobacco roots by AM fungi. Figure 8 The growth status of tobacco rhizosphere microorganisms in media with different concentrations of rifampicin; Figure 9 F019 grown on LB resistant medium containing 200 μg / mL R Single colony; Figure 10 strains F019 and F019 R Growth curve; Figure 11 The effect of AM fungi on the colonization of strain F019 in the tobacco rhizosphere; Figure 12 Tobacco phenotypes were determined by treatment with strain F019 and AM fungi; Figure 13 The effects of applying strain F019 and AM fungi on tobacco growth; Figure 14 The growth curve of strain F019; Figure 15 Screening for the optimal carbon source, nitrogen source, and inorganic salts for the culture medium of strain F019; Figure 16 Screening for the optimal carbon source, nitrogen source, and inorganic salt content in the culture medium for strain F019; Figure 17 Orthogonal optimization experiment for fermentation conditions of strain F019; Figure 18 Optimize fermentation conditions for strain F019; Figure 19 Pareto Chart of culture conditions for strain F019; Figure 20 The effect of different fermentation conditions on the fermentation biomass of strain F019; Figure 21 Comparison of the cell growth curve of strain F019 before and after fermentation optimization; Figure 22 Comparison of spore growth curves of strain F019 before and after fermentation optimization. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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.

[0027] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] In this invention, Bacillus sicca F019 may be simply referred to as "strain F019" or "F019".

[0031] Example 1: Isolation and screening of strain F019 Strain F019 was isolated from the rhizosphere soil of peanut plants in Daxinjia Village, Liugezhuang Town, Haiyang City, Shandong Province. The specific procedure was as follows: 10g of soil sample was added to a 250mL Erlenmeyer flask containing 90mL of sterile water and mixed by shaking at 28℃ and 180rpm for 60min. Then, serial dilutions were performed with sterile water, and 100μL of the diluted solution was evenly spread onto LB agar plates. After incubation at 28℃ for 24 hours, single colonies of varying morphology and size were selected, purified by streak plating, and transferred to fresh LB agar plates until single colonies grew for further research.

[0032] Example 2 Identification of strain F019 2.1 Morphological identification like Figure 1 As shown in a and 1b, observation of single F019 colonies isolated by streaking on LB agar plates revealed that F019 colonies are generally irregularly shaped; the surface is rough with protrusions, the edges are wrinkled, and the colonies are pale yellowish-white, with a diameter of approximately 2–3 mm. Gram staining results are as follows: Figure 1 As shown in c, the Gram staining result of strain F019 differs from the red color of the Gram-negative bacterium *Escherichia coli*, but is consistent with the purple color of the Gram-positive bacterium *Bacillus subtilis*. Therefore, strain F019 is identified as a Gram-positive bacterium; the spore staining results are as follows... Figure 1 As shown in d, it can be observed that the green spores are attached around the red bacterial cells of F019, indicating that strain F019 can produce spores.

[0033] 2.2 Physiological and Biochemical Identification Referring to the "Handbook for Systematic Identification of Common Bacteria", strain F019 was preliminarily identified by physiological and biochemical assays. The identification results are shown in Table 1.

[0034] Table 1. Physiological and biochemical identification of strain F019

[0035] 2.3 Molecular biological identification (1) Molecular identification of 16S rDNA: DNA was extracted from strain F019 and used as a template for 16S rDNA amplification. The universal upstream primer for 16S rDNA was 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 3 in the sequence listing), and the downstream primer was 1492R: 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO: 4 in the sequence listing). The amplified fragment was directly sequenced. The PCR reaction conditions for 16S rDNA are shown in Table 2.

[0036] Table 2 Reaction conditions for 16S rDNA PCR system

[0037] (2) Molecular identification of the gyrB gene: DNA was extracted from strain F019 as an amplification template. The gyrB nucleotide fragment of F019 was amplified using the universal gyrB upstream primer gyrB UP-1S:5'-GAAGTCATCATGACCGTTCTGCA-3' (see SEQ ID NO:5 in the sequence listing) and the downstream primer gyrB UP-2Sr:5'-AGCAGGGTACGGATGTGCGAGCC-3' (see SEQ ID NO:6 in the sequence listing). The amplified fragment was then directly sequenced. The PCR reaction conditions for gyrB are shown in Table 3.

[0038] Table 3. Reaction conditions for the gyrB PCR system

[0039] (3) Experimental results and analysis: The 16S rDNA of strain F019 (see SEQ ID NO:1 in the sequence listing) and the gyrB nucleotide sequence (see SEQ ID NO:2 in the sequence listing) showed the highest homology to Bacillus Siamensis in the GenBank gene bank; in addition, such as Figure 2 and Figure 3As shown, genetic evolutionary analysis of sequences 1 and 2 was performed using DNAMAN 6.0 and MEGA 11, and a phylogenetic tree was constructed. The results also showed that the 16S rDNA and gyrB of the antagonistic bacterium F019 had the highest homology with Bacillus Siamensis. Therefore, this bacterium F019 was identified as Bacillus siamensis of the genus Bacillus. Bacillus siamensis ).

[0040] The above-mentioned Bacillus simonii ( Bacillus siamensis Strain F019 was deposited on July 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35129. The requesting institution for deposit was Qingdao Agricultural University.

[0041] Example 3: Identification of the antibacterial function of strain F019 3.1 Experimental Methods: (1) Preparation of F019 fermentation broth: One loop of strain F019 was inoculated into a 100 mL Erlenmeyer flask containing 20 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride). The flask was then cultured with shaking at 30 °C, 180 r / min, and pH 7.00 until the OD value reached 0.6–0.8.

[0042] (2) Plate confrontation experiment between strain F019 and pathogenic fungus: Invert sterilized PDA medium plates and allow them to solidify. Make 5mm diameter holes on both sides of the plate, 3cm from the center. Inoculate the pathogenic fungus in the center of the plate, and add 2μL of F019 fermentation broth to the two side holes. No bacterial culture is added to the control plate. After incubation at 28℃ for 7 days, measure the diameter of the inhibition zone and calculate the inhibition rate of the selected strain against the pathogenic fungus using the following formula.

[0043] Antibacterial ability assessment criteria: Measure and calculate the antibacterial rate.

[0044] The formula for calculating the inhibition rate is: Inhibition rate = [(Control colony diameter - Mycelium cake diameter) - (Treatment colony diameter - Mycelium cake diameter)] / (Control colony diameter - Mycelium cake diameter) × 100%.

[0045] 3.2 Experimental Results and Analysis: like Figure 4As shown in Table 4, strain F019 exhibits excellent broad-spectrum antibacterial activity, demonstrating good antagonistic effects against various pathogens, including tobacco black shank, tomato gray mold, grape anthracnose, grape white rot, watermelon wilt, and peanut root rot. Among these, strain F019 showed the most significant antibacterial effect against tobacco black shank, with an estimated inhibition rate of approximately 92.50%.

[0046] Table 4. Inhibition rate of strain F019 against different pathogenic fungi

[0047] Example 4: Effects of strain F019 on the germination of different plant seeds 4.1 Experimental Methods: (1) Preparation of F019 fermentation broth: One loopful of strain F019 was inoculated into 20 mL of LB liquid medium and cultured with shaking at 30 °C, 180 rpm, and pH 7.00 for 12 h. The culture was then diluted with sterile water to a concentration of 5 × 10⁻⁶. 7 CFU / mL.

[0048] (2) Treatment of plant seeds: Mature, plump, and uniformly sized healthy seeds of tobacco varieties 'Zhongchuan 208', 'Yunyan 301', 'Honghua Dajinyuan', tomato varieties 'Zhongshu No. 4', '72-69', and 'Jingyingcui' were selected. Using sterile water as a control (CK), the seeds of different crops were soaked in F019 fermentation liquid for 5 hours. After soaking, the seeds were repeatedly rinsed with sterile water until the surface bacterial solution was washed away. The seeds were then sown and cultured. The number of germinations was recorded daily during the seed germination test until the number of germinating seeds no longer changed.

[0049] Germination rate / % = (Total number of germinated seeds / Number of seeds tested) × 100%; Germination potential / % = [Number of normally germinated seeds when germination reaches its peak (specified number of days) / Number of tested seeds] × 100%.

[0050] 4.2 Experimental Results and Analysis: As shown in Table 5, seeds treated with F019 fermentation broth exhibited better germination performance compared to the control group. The germination potential and germination rate of tobacco, tomato, and zucchini seeds treated with F019 fermentation broth were all improved. This result reveals that the application of F019 strain fermentation broth can effectively promote the germination process of plant seeds.

[0051] Table 5. Effects of strain F019 on seed germination of different plants.

[0052] Example 5: Detection of the effect of strain F019 on the growth of potted tobacco and its control effect on black shank disease. 5.1 Experimental Methods: Select the tobacco variety 'Nakagawa 208', and transplant tobacco plants of uniform size that have grown to the four-leaf stage. Three weeks later, subject them to five different treatments: (1) CK1: Water treatment - negative control group; (2) CK2: Inoculated pathogen-positive control group; (3) CT1: Root irrigation F019 fermentation broth treatment group; (4) CT2: Treatment group inoculated with pathogens and treated with root irrigation with F019 fermentation broth; (5) CT3: Group inoculated with pathogens and treated with root drenching with the chemical agent metalaxyl-mancozeb; A cross-shaped cut of approximately 5 cm was made at the base of the tobacco plant using a sterile needle, and a 5 mm diameter mycelial cake of the tobacco black shank pathogen was inoculated. Control groups were established, including one without pathogen inoculation and the other with pathogen inoculation but without any treatment. The application rates of F019 fermentation broth and water were both 50 mL. When significant differences in tobacco disease were observed under different treatment conditions, photographs were taken and the disease index was calculated to determine the control effect.

[0053] Evaluation criteria for control efficacy: Disease index is statistically analyzed. The evaluation and grading of the disease index mainly observes the number of lesions on branches, and the criteria are as follows: Level 0: No symptoms; Grade 1: Stem lesions do not exceed 1 / 3 of the stem, or less than 1 / 3 of the leaves wither; Grade 3: Stem lesions surround 1 / 3 to 1 / 2 of the stem, or 1 / 3 to 1 / 2 of the leaves are slightly wilted, or a few leaves at the bottom have lesions; Grade 5: Stem lesions extend beyond 1 / 2 of the stem but do not completely encircle the stem, or 1 / 2 to 2 / 3 of the leaves wither; Level 7: Stem lesions completely surround the stem, or more than 2 / 3 of the leaves wither; Level 9: The diseased plants are basically dead.

[0054] Disease index = ∑(Number of diseased plants at each level × Number of disease levels) / (Total number of plants × Highest disease level) × 100% 5.2 Experimental Results and Analysis: Experimental results are as follows Figure 5As shown in the figure, compared with the control, the overall growth of black-shank-infected tobacco without F019 fermentation liquid treatment showed obvious signs of disease, with blackening of the stem base, yellowing and wilting of leaves, indicating severe disease. However, the symptoms of black-shank-infected tobacco treated with F019 fermentation liquid and the chemical agent metalaxyl-mancozeb through root drenching were significantly alleviated, and the disease index was significantly reduced. The control efficacy test results are shown in Table 6. The control effect of F019 fermentation liquid on black-shank was 78.79%, while that of metalaxyl-mancozeb was 75.76%. The control effect of F019 fermentation liquid was slightly higher than that of metalaxyl-mancozeb, indicating that F019 has a significant antagonistic function against black-shank.

[0055] Further testing of growth indicators was conducted on the tobacco treated as described above, and the results are as follows: Figure 6 As shown, compared to the control group, the tobacco plants treated with F019 fermentation broth through root irrigation exhibited significantly increased plant height, leaf number, and leaf area. In potted tobacco plants infected with black shank, those treated with F019 fermentation broth through root irrigation showed better growth traits. The treatment group CT2 showed significantly higher stem diameter, leaf area, leaf number, plant height, and chlorophyll content compared to the control group CK2. These results indicate that root irrigation with F019 fermentation broth can promote tobacco growth and improve the growth traits of tobacco plants infected with black shank.

[0056] Table 6. Effects of F019 fermentation broth on the control of blackleg disease in potted plants.

[0057] Example 6: Effect of strain F019 on colonization of AM fungi in tobacco roots 6.1 Experimental Methods: The AM fungal strain was *Glomus mosierifolium* (Gs), provided by the laboratory. It was a rhizosphere soil containing root segments of *Wheatgrass*, corresponding mycorrhizal fungal spores, and extra-root mycelium, propagated from the original species using *Wheatgrass* as the host. This experiment used the tobacco variety 'Nakagawa 208' as the experimental subject, employing a seedling substrate of peat moss and sand mixed in a 1:9 ratio. After thorough mixing, the substrate was placed in pots sterilized with alcohol. High-quality tobacco seedlings were transplanted and treated one week after transplanting. The effects of F019 fermentation broth on AM fungal colonization in tobacco roots were investigated through different treatments. The experiment included a control group (cultured by root irrigation with water) and two treatment groups: Treatment group one was inoculated with AM fungi only, while Treatment group two was treated with F019 fermentation broth for root irrigation seven days after AM fungal inoculation. Twenty days after inoculation with AM fungi, tobacco roots were cut into 1 cm segments and stained with acetic blue ink to observe the structure of the AM fungal symbiosis (including vesicles, arbuscular structures, and spores) and to determine the infection and colonization rates of AM fungi in the tobacco rhizosphere.

[0058] AM fungal infection rate detection: Using tweezers and a needle, uniformly sized root segments from stained tobacco rhizosphere samples were precisely laid flat on a glass slide. Two to five root segments were evenly arranged on each slide and covered with a coverslip. The samples were then observed under a Leica DM750 digital microscope to record the formation of structures such as vesicles, arbuscular mycelia, and spores. Forty root segment samples were observed for each treatment group. The AM fungal infection rate was defined as the percentage of infected root segments out of the total number of observed root segments.

[0059] AM fungal colonization rate detection: The infection status and mycelial morphology of each root segment were observed and recorded under a microscope. The grades were determined according to the percentage of the length of each infected root segment that formed mycorrhizae to the total length of the root segments: Grade 1 (0% < infection rate < 20%), Grade 2 (20% ≤ infection rate < 40%), Grade 3 (40% ≤ infection rate < 60%), Grade 4 (60% ≤ infection rate < 80%), and Grade 5 (infection rate ≥ 80%).

[0060] Colonization rate / % = ∑(Number of root segments at each level × Colonization level) / (Total number of root segments observed × Highest colonization level) × 100% 6.2 Experimental Results and Analysis: The effects of AM fungal infection on tobacco roots, such as Figure 7 As shown, compared with uninfected root systems ( Figure 7 Compared to a), the structures of AM fungi stained with acetic acid ink were clearly visible in all samples. Figure 7 bf), where AM fungal hyphae inside and outside the roots can be observed ( Figure 7 bc) and vesicles ( Figure 7 Table 7 shows the statistical results of AM fungal rhizosphere infection under different treatments. In the control group, the AM fungal infection rate was 0%. When AM fungi infected tobacco alone, the infection rate was 63.33% and the colonization rate was 41.67%. However, when AM fungi were applied together with F019, the infection rate of AM fungi in tobacco was 73.89% and the colonization rate was 47.00%, both of which were significantly increased. This indicates that strain F019 can enhance the infection of tobacco by AM fungi.

[0061] Table 7. Effects of F019 on infection and colonization rates of AM fungi.

[0062] Example 7 Screening and stability analysis of antibiotic-labeled strain F019 7.1 Experimental Methods: The wild-type strain F019 was induced to resist rifampicin by gradually increasing the concentration of rifampicin in the culture medium until the selected strain could grow stably on a medium containing 200 μg / mL rifampicin and maintain the same colony morphology as the original strain. Thus, the rifampicin-resistant labeled strain F019 was successfully obtained. R Meanwhile, the labeled strain and the original strain were inoculated into LB medium using a sterile inoculation loop and cultured with shaking at 34℃ and 210 r / min. Uninoculated LB medium served as a control group. Samples were taken at different time points, and the absorbance at OD600 was measured using a spectrophotometer to analyze whether there were significant differences in the growth curves of the labeled strain and the original strain.

[0063] 7.2 Experimental Results and Analysis: To investigate the resistance of tobacco rhizosphere microorganisms to rifampin after root irrigation with F019 fermentation broth, rhizosphere soil samples were collected from tobacco plants treated with F019 fermentation broth. The samples were serially diluted and evenly spread on LB solid medium without rifampin, as well as on LB solid medium containing 200 μg / mL, 300 μg / mL, and 400 μg / mL rifampin, respectively. The results are as follows: Figure 8 As shown, after 3 days of cultivation and observation, colony growth was observed only on LB solid medium without rifampin, while no colonies were observed on media containing different concentrations of rifampin. This result indicates that the tobacco rhizosphere microorganisms treated with F019 fermentation broth showed no resistance to the three concentrations of rifampin mentioned above. Based on this finding, future research could use the F019 strain induced and screened to develop resistance to 200 μg / mL rifampin, to detect the colonization status of F019 in tobacco roots and name this resistant strain F019. R ,like Figure 9 As shown.

[0064] Meanwhile, to evaluate strain F019 R Whether the growth of wild-type strain F019 and its marker strain F019 is potentially affected, this study investigated. R The growth rate was measured. The results are as follows: Figure 10 The display shows F019 R The growth curves of F019 and F019 are highly similar, indicating that F019... R The growth was not significantly affected.

[0065] Example 8 Colonization analysis of strain F019 in tobacco rhizosphere 8.1 Experimental Methods: The resistant marker strain F019 screened in Example 7 R To determine whether AM fungi affect the growth of F019, F019 was...R 50 mL of bacterial solution was applied to the roots of both normally growing tobacco plants and potted tobacco plants inoculated with AM fungus for 7 days. One week after root irrigation, samples of the tobacco rhizosphere soil were taken, diluted, and spread onto LB medium containing 200 μg / mL rifampicin. The F019 levels under different treatments were observed and statistically analyzed. R The number of bacterial colonies. 8.2 Experimental Results and Analysis: Statistical results are as follows Figure 11 The results showed that F019 could colonize the tobacco rhizosphere, and the colonization rate increased significantly in the presence of AM fungi. Compared with the control, AM fungi application increased the F019 colonization rate in the tobacco rhizosphere by 59.23%. These results demonstrate that AM fungi can promote the colonization of F019 in the tobacco rhizosphere. Combined, these results indicate that F019 and AM fungi mutually promote each other's colonization in the tobacco rhizosphere.

[0066] Example 9: Effects of co-application of strain F019 and AM fungi on tobacco growth and black shank control efficacy. 9.1 Experimental Methods: The tobacco variety 'Nakagawa 208' was selected. Three weeks after transplanting tobacco plants of uniform size that had reached the four-leaf stage, eight different treatments were administered: (1) CK1: Water treatment - negative control group; (2) CK2: Inoculated pathogen-positive control group; (3) CT1: Root irrigation F019 fermentation broth treatment group; (4) CT2: AM fungal inoculation treatment group; (5) CT3: Treatment group inoculated with AM fungi and treated with root irrigation F019 fermentation broth; (6) CT4: Treatment group inoculated with pathogens and treated with root irrigation F019 fermentation broth; (7) CT5: Inoculated with pathogens and AM fungi; (8) CT6: Treatment group inoculated with pathogens, AM fungi and root irrigation F019 fermentation broth.

[0067] When tobacco disease showed significant differences, photos were taken to record the data, and the disease index, control efficacy, and morphological parameters of each treatment plant were statistically analyzed, including stem diameter, leaf area, number of leaves, chlorophyll content, and plant height.

[0068] 9.2 Experimental Results and Analysis: The results are as follows Figure 12As shown in Table 8, the control efficacy of F019 fermentation broth against black shank disease in potted tobacco was 76.47%, while that of AM fungi was 52.94%. The combined application of both resulted in a control efficacy of 82.35% against black shank disease, an increase of 7.65% compared to F019 alone. These results indicate that the combined application of AM fungi and F019 can enhance tobacco's resistance to black shank disease. Various growth indicators of tobacco under different treatments were tested, and the results are as follows... Figure 13 As shown, compared with the control, the combined application of AM fungi and F019 can significantly improve various growth indicators of tobacco, such as stem diameter, leaf area, number of leaves, plant height, and chlorophyll content. Moreover, the improvement effect is better than that of F019 or AM fungi alone. In summary, the results indicate that the combined application of AM fungi and F019 has a significant effect on improving the growth of tobacco and its resistance to black shank disease.

[0069] Table 8. Control effects of F019 and AM fungi on black shank disease in potted tobacco.

[0070] Example 10 Fermentation optimization of strain F019 I. Growth curve determination of strain F019 10.1.1 Experimental Methods: Using the conventional shake-flask fermentation method, the seed culture was inoculated at a rate of 2% into an Erlenmeyer flask containing 200 mL of LB liquid medium and cultured with shaking at 28 °C, 160 rpm, and pH 7. During the early stages of fermentation, samples were taken at regular intervals to measure the absorbance of the cells at 600 nm, and growth curves were plotted based on the results.

[0071] 10.1.2 Experimental Results and Analysis: The growth curve of strain F019 was measured to determine the fermentation time of the seed culture and the detection time points for subsequent fermentation optimization experiments. Observation Figure 14 Data showed that strain F019 was in the logarithmic growth phase before 12 hours of fermentation, during which the biomass growth rate was the fastest. The OD value of the strain reached its peak at 17 hours, after which the growth rate gradually slowed down. Based on this, 17 hours was selected as the detection time point for subsequent fermentation optimization experiments.

[0072] II. Screening of optimal culture medium components and concentrations for strain F019 10.2.1 Experimental Methods: Carbon source optimization: Yeast extract, lactose, mannitol, citric acid, maltodextrin, soluble starch, corn flour, glucose, maltose, sucrose, and chitin were selected as carbon sources to replace the yeast extract in LB medium in equal amounts (ensuring the carbon source concentration remained consistent with that in LB medium). Other components were fixed, and different experimental groups were constructed, with three replicate experiments conducted. Subsequent experimental steps were the same as described above.

[0073] Nitrogen source optimization: Nitrogen sources such as tryptone, ammonium molybdate, beef extract, soybean flour, ammonium sulfate, urea, peptone, ammonium bicarbonate, and yeast extract were used to replace the tryptone in LB medium in equal amounts (ensuring the nitrogen source concentration remained consistent with LB medium). Other components were fixed, and different experimental groups were constructed, with three replicates performed. Seed culture was inoculated into each treatment group at a 1% inoculum and cultured at 28℃ and 160 r / min for 17 hours with shaking. The absorbance of F019 in each treatment group at 600 nm was then measured.

[0074] Inorganic salt optimization: Sodium chloride, magnesium chloride, sodium dihydrogen phosphate, calcium carbonate, zinc sulfate heptahydrate, calcium chloride, dipotassium hydrogen phosphate, copper sulfate, and ferrous sulfate were used to replace sodium chloride in LB medium in equal amounts (ensuring the inorganic salt concentration remained consistent with that in LB medium). Other components were fixed, and different experimental groups were constructed, with three replicate experiments performed. Subsequent experimental procedures were the same as described above.

[0075] Concentration optimization: After determining the optimal culture medium composition, a single-variable method was used for concentration optimization. This involved setting five different concentration gradients for the third component while keeping the concentrations of two components constant, with each concentration tested in triplicate. Subsequent experimental procedures were the same as described above.

[0076] 10.2.2 Experimental Results and Analysis: like Figure 15 As shown, through shake-flask fermentation, 17 hours was selected as the fermentation optimization test time point. Finally, maltodextrin was selected as the most suitable carbon source for F019 fermentation, beef extract as the most suitable nitrogen source, and magnesium chloride as the most suitable inorganic salt.

[0077] like Figure 16 As shown, the OD value of F019 was highest when the maltodextrin content was 7 g / L, the beef extract content was 16 g / L, and the magnesium chloride content was 12 g / L. Therefore, the preliminary conclusion is that the optimal concentrations of the above three nutrients required for F019 fermentation are 7 g / L, 16 g / L, and 12 g / L, respectively.

[0078] III. Orthogonal Design Experiment for Strains F019 10.3.1 Experimental Methods: An orthogonal design was conducted, and three factors with significant influence were selected to adopt a three-factor, three-level orthogonal design. The experimental design details are shown in Tables 9 and 10. The optimal culture medium formulation was then screened.

[0079] Table 9. Orthogonal Design Factor Levels

[0080] Table 10 Orthogonal Design Experimental Design

[0081] 10.3.2 Experimental Results and Analysis: The synergistic effect of various nutrients in the culture medium can better promote the growth of strain F019. Orthogonal experiments were used to optimize the nutrient composition. The results are as follows... Figure 17 As shown, the optimal combination of conditions is A2B2C2. Therefore, the optimal nutrient components and concentrations of the culture medium for strain F019 are 7 g / L maltodextrin, 16 g / L beef extract, and 12 g / L magnesium chloride.

[0082] IV. Screening for optimal fermentation conditions for strain F019 10.4.1 Experimental Methods: Based on previous orthogonal experimental results, this study determined the optimal culture medium formulation for strain F019, specifically comprising 7 g / L maltodextrin, 16 g / L beef extract, and 12 g / L magnesium chloride. Using this formulation, the culture conditions for strain F019 (including pH, temperature, stirring speed, culture medium volume, and inoculum size) were further screened through single-factor experiments. The specific experimental conditions were set as follows: Temperature screening: The temperature gradients were set to 30℃, 32℃, 34℃, 36℃ and 38℃, and each temperature gradient was repeated three times.

[0083] Speed ​​selection: Set speed gradients of 180 r / min, 190 r / min, 200 r / min, 210 r / min and 220 r / min, and conduct three repeated experiments for each speed gradient.

[0084] pH screening: pH gradients of 5, 6, 7, 8 and 9 were set, and each pH gradient was repeated three times.

[0085] Inoculation rate screening: Inoculation rate gradients of 1%, 2%, 3%, 4% and 5% were set, and corresponding experiments were conducted.

[0086] Bottling amount screening: Set bottling amount gradients of 10%, 15%, 20%, 25% and 30% and conduct corresponding experiments.

[0087] 10.4.2 Experimental Results and Analysis: Depend on Figure 18 It can be seen that the optimal fermentation temperature for strain F019 is 34℃, the optimal bottling volume is 15%, the optimal rotation speed is 210 r / min, the optimal pH is 8, and the optimal inoculum size is 3%.

[0088] V. Experiment with strain F019 PB 10.5.1 Experimental Methods: Based on the single-factor experiment, temperature (A), pH (B), rotation speed (C), inoculum size (D), bottling size (E), and time (F) were selected as independent variables. Low-level (-1) and high-level (1) experiments were designed to conduct Plackett-Burman (PB) experiments to quickly screen the components that have the greatest impact on the F019 cell mass. The design factors and levels of the PB experiment are shown in Table 11.

[0089] Table 11. Factor Levels in the Plackett-Burman Experiment

[0090] 10.5.2 Experimental Results and Analysis: Based on the effect analysis of the Plackett-Burman Design and considering the required variables of the experimental factors, the optimal factors affecting the growth of strain F019 were screened, and the results are as follows: Figure 19 As shown, the factors that have the most significant impact on the growth of F019 are, in descending order, temperature, bottling volume, and rotation speed.

[0091] VI. Optimization of F019 Response Surface 10.6.1 Experimental Methods: Three key factors significantly influencing F019 biomass were identified through PB experiments: temperature, bottling volume, and rotation speed. To further investigate the interactions among these three factors and their specific effects on F019 biomass, this study employed a three-factor, three-level response surface methodology (see Tables 12 and 13). The results were analyzed using Design-Expert 13 software for multiple regression fitting, and response surface plots were constructed to fit regression equations between each factor level and F019 biomass.

[0092] Table 12 Factors and Levels of the Response Surface

[0093] Table 13 Response Surface Experimental Design and Results

[0094] Table 14 Analysis of Variance for Response Surface Model

[0095] 10.6.2 Experimental Results and Analysis: Through single-factor optimization experiments of fermentation conditions for strain F019, the three factors with the greatest impact on F019 fermentation biomass were identified as temperature, rotation speed, and bottling volume. To investigate the interaction among these three factors and their influence on F019 fermentation biomass, a three-factor, three-level response surface methodology was employed. Based on the biomass test results, data analysis was performed using Design-Expert 13 software, yielding the regression equation: 0.9746 + 0.0281A - 0.0139B - 0.0155C - 0.0363AB - 0.041AC - 0.02BC - 0.0569A² - 0.0389B² - ​​0.0572C². This regression equation represents the relationship between temperature, rotation speed, and bottling volume.

[0096] According to the significance analysis results of the response surface model shown in Table 14, the P-value is 0.0098 (less than 0.05), indicating that the model results are significant; the P-value for the model lack of fit is 0.2698 (greater than 0.05), indicating that the model fits well and there is no significant bias. The response surface obtained through analysis ( Figure 20 The influence of the three factors and their interactions on the response value in the above model equation can be visually observed, and the figure shows that the response value has a maximum value. Further analysis using Design-Expert 13 software revealed that the conditions corresponding to the maximum cell content were a temperature of 34.70℃, a bottling volume of 16.65%, and a rotation speed of 209.21 r / min. Based on the above analysis, the optimal fermentation conditions for strain F019 were determined using response surface methodology: rotation speed 209.21 r / min, inoculum size 3%, temperature 34.70℃, pH 8.00, fermentation time 17 hours, and bottling volume 16.65%.

[0097] VII. Comparison of F019 fermentation before and after optimization 10.7.1 Experimental Methods: Based on the optimized fermentation medium composition and concentration, the F019 seed culture was inoculated at a ratio of 3% into a 100mL Erlenmeyer flask containing 16.65mL of optimized medium (7g / L maltodextrin, 16g / L beef extract, and 12g / L magnesium chloride). The culture was then incubated with shaking at 210 rpm, 34.70℃, and pH 8.00. Samples were taken at specific time intervals to determine the cell concentration and spore formation.

[0098] Following traditional fermentation conditions, the F019 seed culture was inoculated at a ratio of 2% into 100ml Erlenmeyer flasks containing 20mL LB liquid medium, and cultured with shaking at 30℃, 160r / min, and pH 7.00. Samples were taken at specific time intervals to determine cell concentration and spore formation. Based on the results, growth curves of cells and spores before and after fermentation optimization were plotted, and the changes in cell concentration and spore formation before and after optimization were compared and analyzed.

[0099] 10.7.2 Experimental Results and Analysis: As shown in Tables 15 and 16, Figure 21 , 22 As shown, after fermentation optimization, the cell count of strain F019 increased from 112.33 × 10⁻⁶. 7 CFU / mL increased to 232.33 × 10⁻⁶ 7 CFU / mL increased by 106.83%; spore count increased from 13.07 × 10⁻⁶. 7 CFU / mL increased to 28.63×10 7 The CFU / mL level increased by 119.05%.

[0100] Table 15 Comparison of cell mass before and after fermentation optimization

[0101] Table 16 Comparison of spore count before and after fermentation optimization

[0102] Example 11: Effects of F019 fermentation broth on the quality of field tobacco leaves 11.1 Experimental Methods: After transplanting 'Nakagawa 208' tobacco seedlings to the field and allowing them to recover for one week, the optimized culture broth of F019 was diluted to 5×10⁻⁶. 7 CFU / mL, 100mL per tobacco seedling was applied to the roots. Field tobacco without F019 fermentation liquid was used as a control. After the tobacco reached maturity, tobacco leaves from different parts of the tobacco plant were collected and sent to Yunnan Sanbiao Technology Co., Ltd. to test the quality of the tobacco leaf samples.

[0103] 11.2 Experimental Results and Analysis: Total sugar and reducing sugar are closely related to the sweetness and aroma characteristics of tobacco leaves; total nitrogen affects the taste of tobacco leaves; nicotine determines the irritation of tobacco leaves; potassium is related to the combustibility of tobacco leaves; and the sugar-to-nicotine ratio is a key indicator for evaluating the balance between the "sweetness" and "irritation" of tobacco leaves. The results of the above indicators are shown in Table 17. F019 fermentation broth has a multifaceted effect on improving the quality of field-grown tobacco leaves. The results show that after F019 treatment, the total sugar, reducing sugar, total nitrogen, potassium, nicotine, and sugar-to-nicotine ratio of the upper and lower leaves were significantly higher than those of the control group (CK). In conclusion, F019 fermentation broth can significantly promote the improvement of the quality of field-grown tobacco leaves. Table 17 Effects of F019 fermentation broth on the quality of field-grown tobacco leaves

[0104] Example 12 Effects of F019 fermentation broth on the physicochemical properties of tobacco field soil 12.1 Experimental Methods: The processing method is the same as in 11.1. Field soil samples were collected using the five-point sampling method and sent to Qingdao Yiming Testing Service Co., Ltd. to test the relevant physical and chemical properties of the soil.

[0105] 12.2 Experimental Results and Analysis: The results of soil physicochemical property analysis are detailed in Table 18. Compared with the control treatment, the application of F019 fermentation broth can regulate the soil pH, adjusting the slightly alkaline soil to a slightly acidic soil more suitable for tobacco growth. Simultaneously, this treatment can also promote the enrichment of nutrients in the soil. Compared with the control, it significantly increased the content of available phosphorus, hydrolyzable nitrogen, and available potassium in the soil. Table 18 Effects of F019 fermentation broth on the physical and chemical properties of field soil

[0106] It should be noted that the specific embodiments are merely representative examples of the present invention, and the technical solution of the present invention is obviously not limited to the above embodiments, and there can be many variations. Those skilled in the art who obtain the present invention based on its explicit disclosure or without objection from the written description should consider it to be within the scope of protection of this patent.

Claims

1. A strain of Bacillus sicca ( Bacillus siamensis strain F019, characterized in that, This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 8, 2025, with accession number CGMCC No. 35129.

2. A microbial inoculant, characterized in that, The microbial agent contains Bacillus sicca strain F019.

3. The microbial agent according to claim 2, characterized in that, The effective active ingredient of the microbial agent is the bacterial cell or fermentation broth of Bacillus sicca strain F019.

4. The microbial agent according to claim 2, characterized in that, The total viable bacterial concentration of Bacillus sicca strain F019 in the microbial agent was 1×10⁻⁶. 9 ~2×10 9 CFU / mL.

5. The method for preparing the microbial inoculant according to claim 2, characterized in that, The specific steps include: inoculating Bacillus sicca strain F019 into LB liquid medium and culturing it in a shaker at 30–36°C and 180–220 r / min for a certain period of time to obtain the microbial agent.

6. The application of the Bacillus sicca strain F019 according to claim 1 or the microbial agent according to claim 2 in any one of the following (a1)-(a6), characterized in that, (a1) Promote AM fungal infection of tobacco roots; (a2) Prepare a product that promotes AM fungal infection of tobacco roots; (a3) ​​Apply AM fungi to tobacco to promote the colonization of strain F019 in the tobacco rhizosphere; (a4) Synergistically promote tobacco growth with AM fungi; (a5) Synergistically enhance resistance to tobacco black shank with AM fungi; (a6) Improve the physical and chemical properties of tobacco field soil, wherein the soil physical and chemical properties are pH, organic matter, available phosphorus, hydrolyzable nitrogen and available potassium, and the AM fungi is *Glomus mosie* (…). Glomus mosseae ).

7. A method for preventing and controlling tobacco black shank disease, characterized in that, The method utilizes the Bacillus sicca strain F019 of claim 1 or the microbial agent of claim 2 for treatment.

8. A method for the colonization of Bacillus sicca in the rhizosphere of tobacco, characterized in that, A seedling substrate is prepared by mixing peat moss and sand in a 1:9 ratio. After thorough mixing, the seedling substrate is placed in flowerpots that have been sterilized with alcohol. Tobacco seedlings with good growth are selected for transplanting. After one week of recovery, the fermentation liquid of Bacillus sicca strain F019 as described in claim 1 is used for root irrigation.

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