Bacillus sicca and its applications

Abstract

This invention relates to Bacillus sicca and its applications. This invention discovers Bacillus sicca (… Bacillus siamensis Strain Bs-1117 has antagonistic and inhibitory effects on Fusarium solani, Fusarium oxysporum, Fusarium pseudograss, Fusarium verticillatum, Fusarium graminearum, or Botrytis cinerea, and therefore can be used to control root rot, wilt, stem base rot, ear rot, Fusarium head blight, or gray mold.

Description

Technical Field

[0001] This invention relates to the field of microorganisms, and particularly to biocontrol microorganisms. Background Technology

[0002] In recent years, the damage caused by tobacco root rot to tobacco has been increasing. This disease is caused by various Fusarium species, among which *Fusarium solani* (also known as bark rot fungus) is a major contributing factor. Fusarium solani Fusarium solani is the main pathogen. It can infect tobacco alone or in combination with Phytophthora indicum, root-knot nematodes, etc., leading to the death of tobacco plants, resulting in significant reduction in tobacco yield or even crop failure. It has become a serious root and stem disease in tobacco production.

[0003] Currently, the control of tobacco root rot mainly relies on disease-resistant variety breeding, chemical control, and biological control, with chemical control remaining the mainstream approach. However, long-term application of chemical pesticides can easily induce pesticide resistance in pathogens, reducing control effectiveness and potentially causing ecological risks such as environmental pollution. In contrast, biological control has advantages such as being environmentally friendly, safe for humans and animals, having a long-lasting effect, and being easy to coordinate with other plant protection measures, providing a feasible path to reduce the use of chemical pesticides. Summary of the Invention

[0004] One of the inventions provides Bacillus sicca ( Bacillus siamensis The strain Bs-1117 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33939.

[0005] The second invention provides a composition comprising Bacillus sicca strain Bs-1117 as described in the first invention, and decomposed vetch bran.

[0006] In one specific embodiment, the ratio of the *Bacillus sicca* strain Bs-1117 CFU to the mass of well-rotted vetch bran with a moisture content of 50 wt% is 1 × 10⁻⁶. 11 CFU: (0.1 to 10) kg.

[0007] In one specific embodiment, the ratio of the *Bacillus sicca* strain Bs-1117 CFU to the mass of well-rotted vetch bran with a moisture content of 50 wt% is 1 × 10⁻⁶. 11 CFU: 1kg.

[0008] In one specific embodiment, the preparation process of the decomposed vetch bran is as follows: collect the above-ground parts of vetch plants from the full bloom stage to the early pod stage, air-dry them naturally until the moisture content is below 3wt%, and then crush them to within 2 cm to obtain dry material; add water at (100 to 200) L / 100 kg of dry material and stir evenly; adjust the pH value to 6 to 8, and then carry out aerobic composting for 35 to 45 days, turning and ventilating during the period to obtain the decomposed vetch bran.

[0009] The third invention provides the *Bacillus sicca* strain Bs-1117 according to the first invention or the composition according to the second invention for inhibiting *Fusarium* (…). Fusarium ) and / or Staphylococcus aureus ( Botrytis Applications in ).

[0010] In one specific embodiment, the Fusarium is *Fusarium solani* (…). Fusarium solani Fusarium oxysporum ( Fusarium oxysporum Fusarium pseudograss () Fusarium pseudograminearum Fusarium pseudoverticum ( Fusarium verticillioides ) and Fusarium graminearum ( Fusarium graminearum At least one of the following.

[0011] In one specific embodiment, the *Botrytis cinerea* is *Botrytis griseus* (…). Botrytis cinerea ).

[0012] The fourth invention provides the use of the Bacillus sicca strain Bs-1117 according to the first invention or the composition according to the second invention for the control of at least one of root rot (caused by Fusarium solani, especially tobacco root rot), wilt (caused by Fusarium oxysporum, especially banana wilt), stem base rot (caused by Fusarium pseudogracilis, especially wheat stem base rot), ear rot (caused by Fusarium verticillatum, especially maize ear rot), Fusarium head blight (caused by Fusarium gracilis, especially wheat Fusarium head blight), and gray mold (caused by Botrytis cinerea).

[0013] Beneficial effects of the present invention: The present invention discovered Bacillus sicca ( Bacillus siamensis strain Bs-1117 is effective against Fusarium ( ) Fusarium ) or Staphylococcus aureus ( Botrytis ) has antagonistic and inhibitory effects. Among them, Fusarium can be Fusarium solani ( Fusarium solani Fusarium oxysporum ( Fusarium oxysporum Fusarium pseudograss () Fusarium pseudograminearum Fusarium pseudoverticum ( Fusarium verticillioides ) and Fusarium graminearum ( Fusarium graminearum ); Staphylococcus can be Staphylococcus griseus ( Botrytis cinereaTherefore, Bacillus sicca strain Bs-1117 can be used to control root rot, wilt, stem base rot, ear rot, Fusarium head blight, or gray mold. Furthermore, when Bacillus sicca strain Bs-1117 is used in combination with well-rotted vetch bran to control Fusarium solani, the two can have a synergistic effect.

[0014] Strain Preservation: The microorganism Bs-1117 strain screened in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 33939, deposited on March 24, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is Bacillus sicca. Bacillus siamensis . Attached Figure Description

[0015] Figure 1 Photographs showing the plate confrontation inhibition of strain Bs-1117 against Fusarium solani.

[0016] Figure 2 The colony morphology of Bs-1117 on LB plates is shown.

[0017] Figure 3 The phylogenetic tree of strain Bs-1117 is shown.

[0018] Figure 4 A circular map of the Bs-1117 strain genome is shown.

[0019] Figure 5 The phylogenetic tree of the core genes of the Bs-1117 genome is shown.

[0020] Figure 6 The images show plate confrontation inhibition of strain Bs-1117 against different pathogens.

[0021] Figure 7 The effects of crude extracts of secondary metabolites from strain Bs-1117 on Fusarium solani mycelia were shown.

[0022] Figure 8 The results of phosphorus solubilization by strain Bs-1117 are shown.

[0023] Figure 9 The absorbance ratio (λ / λ0) of strain Bs-1117 and the negative control at different color development times is shown.

[0024] Figure 10 The results of the assay for indoleacetic acid production by strain Bs-1117 are shown.

[0025] Figure 11This shows the content of rhizosphere pathogens in flue-cured tobacco in a pot experiment, with different letters indicating significant differences between treatments. P <0.05). Detailed Implementation

[0026] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0027] Unless otherwise specified, the strains and reagents used in the embodiments of this invention can be purchased commercially.

[0028] Fusarium solani ( Fusarium solani For details, see the pathogenic biological characteristics of Fusarium root rot in tobacco and the metabolic phenotypic characteristics of its dominant species [J]; Jiangsu Agricultural Sciences; Authors: Huang Yufeng, Li Fei, Wang Hancheng, et al.; 2024, 52(07):124-132.

[0029] Fusarium oxysporum ( Fusarium oxysporum f.sp. cubense ) For details, see A smooth vetch (Viciavillosa var.) strain endogenous to the broad-spectrum antagonist Bacillussiamensis JSZ06 alleviates banana wilt disease[J]; Frontiers in Plant Science; Author: Ruan Y, Nong C, Jintrawet A et al; 2024, 15: 1410197.

[0030] Fusarium graminearum ( Fusarium pseudograminearum See the susceptibility of Fusarium graminearum in Henan Province to flutriafol [J]; Acta Phytopathologica Sinica; Authors: Hou Ying, Xin Hewen, Zhang Xin et al.; 2023, 53(2): 307-316.

[0031] Fusarium pseudoverticum ( Fusarium verticillioides For details, see the staining method and fluorescent strain construction of Fusarium oxysporum [J]; Journal of North China Agricultural University; Authors: Kong Ming, Zhu Jinjie, Qi Xiantao et al.; 2024, 39(S1): 252-258.

[0032] Fusarium graminearum ( Fusarium graminearum For details, see the Isolation and Identification of Pathogen Populations of Wheat Stem Base Rot and Fusarium Head Blight in Henan Province in 2022 [J]; Journal of Triticeae Crops; Authors: Yan Shuwei, Bai Nima, Pan Xin et al.; 2024, 44(5): 667-674.

[0033] Staphylococcus grayi ( Botrytis. cinerea See Resistance of Petunia Phenotypesto[J];HORTSCIENCE;Authors: HI, Krahl, Randle et al;2019. 34(4): 690-692.

[0034] R2A solid culture medium: yeast extract 0.5g, peptone 0.5g, casein hydrolysate 0.5g, glucose 0.5g, soluble starch 0.5g, dipotassium hydrogen phosphate 0.3g, magnesium sulfate 0.024g, sodium pyruvate 0.3g, agar 15g, distilled water 1000ml, pH 7.2±0.2. Sterilize at 121°C for 20 min.

[0035] LB liquid medium: 10g peptone, 5g yeast extract, 10g sodium chloride, 1000ml distilled water, natural pH. Sterilize at 121°C for 20 min.

[0036] LB solid medium: 10g peptone, 5g yeast extract, 10g sodium chloride, 15g agar, 1000ml distilled water, natural pH. Sterilize at 121°C for 20 min.

[0037] PDA medium: 200g peeled potatoes, 20g glucose, 15g agar, 1000ml distilled water, natural pH. Sterilize at 121°C for 20 min.

[0038] PDB medium: 200g peeled potatoes, 20g glucose, 1000ml distilled water, natural pH. Sterilize at 121°C for 20 min.

[0039] Pikovskaya liquid culture medium: glucose 10.0g, calcium phosphate 5.0g, ammonium sulfate 0.5g, sodium chloride 0.3g, magnesium sulfate 0.3g, potassium chloride 0.3g, yeast extract 0.1g, deionized water 1000mL, adjust pH to 7.0. Sterilize at 121 degrees Celsius for 20min.

[0040] Siderogenic capacity determination (CAS plate assay) Solid culture medium: composed of solution A (CAS-iron-CTAB mixture) and solution B (basal culture medium): Solution A: Weigh 0.06g Chrome Azurol S and dissolve it in 50 mL of deionized water, add 10 mL of 1mmol / L FeCl3·6H2O (dissolved in 10mmol / L HCl), stir well, and slowly add 40 mL of 2mmol / L hexadecyltrimethylammonium bromide (CTAB) aqueous solution, and mix well; Solution B: Weigh 2.0g sucrose, 0.8g casein amino acids, 0.5g potassium dihydrogen phosphate, 0.1g sodium chloride, 0.1g magnesium sulfate, and 15g agar, and make up to 900mL with deionized water, adjust the pH to 7.0, and sterilize at 121°C for 20min. Once solution B has cooled to 50 to 60 degrees Celsius, under aseptic conditions, slowly pour 100 mL of solution A into 900 mL of solution B, shake well, and pour into petri dishes, 20 mL per dish.

[0041] Example 1: Isolation of strains

[0042] Rhizosphere soil from healthy flue-cured tobacco plants was collected from diseased plots in an experimental tobacco field in Longjie Town, Chengjiang City, Yunnan Province. The soil was sealed in sterile self-sealing bags and brought back to the laboratory, where it was temporarily stored at 4 degrees Celsius for the purpose of isolating antagonistic bacteria.

[0043] Take 10 grams of soil and place it in a 200 mL Erlenmeyer flask. Add 90 mL of water and shake at 180 rpm for 30 minutes to prepare a suspension. Let the suspension stand for 10 minutes, then use the supernatant as the stock solution for serial dilution, with the concentration gradient set at 10%. -3 10 -4 10 -5 and 10 -6 .

[0044] Take 100 μL of each of the four different dilutions and spread them onto R2A solid medium. Then, incubate the R2A plates at 30°C for 72 hours. Select single bacterial colonies with inconsistent morphology and color, pick them with an inoculation loop, and inoculate them onto LB solid medium. Repeat this process 2 to 3 times to achieve purification. Transfer the culture to LB slant for storage.

[0045] Example 2: Biological activity analysis of isolated strains

[0046] The plate confrontation method was used to determine the activity of isolated bacterial strains against Fusarium solani (Fusarium solani). F. solani The antagonistic effect of ).

[0047] Preparation of bacterial suspension for test strains: Each test strain preserved on LB slant was inoculated onto an LB agar plate and incubated at 30°C for 1 day. A certain amount of bacterial cells was picked up using a sterile inoculation loop and transferred to 200 mL of LB liquid medium under sterile conditions. The medium was incubated at 30°C and 180 r / min with shaking for 3 days. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in sterile water and diluted to 1×10⁻⁶ with sterile water. 8 CFU / mL was used to obtain bacterial suspensions of each test strain.

[0048] Fusarium solani preserved on PDA slant F. solani The strain was inoculated onto PDA plates and activated by incubation at 30°C for 72 hours. After sufficient mycelium growth, *Fusarium solani* was prepared using a 5 mm diameter punch. The *Fusarium solani* mycelial cake was inoculated into the center of a new PDA plate. 10 μL of the test strain suspension was then spotted 2.5 cm from the edge of the mycelial cake. Four spots were evenly inoculated on each plate. The plates were then incubated in the dark at 30°C for 7 days, and the presence or absence of an inhibition zone was observed. A control group was also set up, inoculated only with *Fusarium solani*.

[0049] For the test strains with antibacterial activity, the same screening procedure was performed, and the width of the inhibition band was measured after 7 days of incubation. Each treatment was repeated in triplicate. The results showed that 33 bacteria with antibacterial activity were isolated and purified from the rhizosphere soil of flue-cured tobacco, including 4 antagonistic strains with inhibition band widths greater than 5 mm. Among them, Bs-1117 had an inhibition band width greater than 20 mm. Figure 1 .

[0050] While measuring the above-mentioned inhibition zone, the colony radius of the pathogen treated with Bs-1117 strain was measured, and the inhibition rate was calculated based on formula (1).

[0051] Antibacterial rate = [(colony radius of control group - colony radius of treatment group) / radius of control group] × 100% Formula (1).

[0052] The Bs-1117 strain showed an inhibition rate of 92.5% against Fusarium rotundifolium as determined by the plate confrontation method.

[0053] Example 3: Classification and identification of strain Bs-1117

[0054] The Bs-1117 strain was identified using a combination of colony morphology and molecular biology methods.

[0055] 3.1 Colony morphology of strain Bs-1117: The colonies were incubated upside down on LB solid medium at 30°C for 4 days. Colony morphology was observed and recorded daily. Initially, Bs-1117 colonies were milky white and translucent with regular edges. Over time, the color gradually changed to milky white and opaque, with slightly wrinkled edges. Finally, the colonies exhibited a morphology with multiple notches at the edges, irregular growth, a rough appearance, and a wrinkled surface. (See attached image). Figure 2 This can serve as a basis for the identification of Bacillus spp.

[0056] 3.2 16S rRNA gene sequence analysis of strain Bs-1117: Genomic DNA was extracted from strain Bs-1117 using the TSINGKE Plant DNA Extraction Kit (universal type). PCR amplification was performed using the extracted genomic DNA as a template, with primers for the universal primers for bacterial identification at 27F (as shown in SEQ ID No. 1) and 1492R (as shown in SEQ ID No. 2). The amplified PCR products were detected by gel electrophoresis and then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA gene sequence of strain Bs-1117 is shown in SEQ ID No. 3. The sequence results were compared and analyzed on the Ezbiocloud website, showing that strain Bs-1117 is similar to the type strain. Bacillus siamensis KCTC-13613 is the most closely related. A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 6.0 software, with all parameters kept at default settings. The resulting phylogenetic tree is shown below. Figure 3 .

[0057] 3.3 Whole genome sequencing of strain Bs-1117

[0058] Whole-genome sequencing of strain Bs-1117 was performed by Shanghai Paisenuo Biotechnology Co., Ltd. using the Novaseq PE150 & PacBio Revio sequencing platform. Filtered data were assembled using Canu (v1.6), and high-quality sequencing reads were spliced ​​into a complete bacterial genome. Genome coverage depth was analyzed using pbalign (BLASR v0.4.1). A circular map of the JLU-1 strain genome was constructed using Circos (v0.64). Whole-genome annotation was performed using Glimmer (v3.02), and alignment analyses were conducted using GO, KEGG, COG, SwissProt, and the non-redundant protein database (NR).

[0059] Whole-genome sequencing of strain Bs-1117 revealed a genome size of 3,929,414 bp, with a GC content of 46.5%, containing 27 rRNA genes, 86 tRNA genes, and 3,868 protein-coding genes (average length 899 bp). Functional annotation of 3,858 (99.7%) coding genes was successfully achieved through alignment with NR, GO, KEGG, eggNOG, Pfam, SwissProt, and TrEMBL databases. Genome analysis predicted 25 genomic islands, ranging in length from 4,113 bp to 15,345 bp. Figure 4 .

[0060] Mean nucleotide identity (ANI) analysis was performed between Bs-1117 and 20 other Bacillus strains. Bacillus siamensis KCTC-13613 is the most closely related (98.1%) to Bacillus thaliana. A core gene phylogenetic tree was constructed, which further confirmed its relationship with Bacillus thaliana. B. siamensis Highest similarity, see Figure 5 .

[0061] In summary, based on colony morphology, 16S rRNA gene sequence, ANI analysis, and core gene tree analysis, Bs-1117 was identified as Bacillus sicca. Bacillus siamensi .

[0062] The strain Bs-1117 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33939, on March 24, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is Bacillus sicca. Bacillus siamensis .

[0063] Example 4: Determination of the antibacterial activity of strain Bs-1117

[0064] 4.1 The antibacterial spectrum of strain Bs-1117 was determined using the plate confrontation method.

[0065] Preparation of bacterial suspension of strain Bs-1117: Bs-1117 strain preserved on LB slant was inoculated onto LB agar plates and incubated at 30°C for 24 hours. A certain amount of bacterial cells was picked up using a sterile inoculation loop and transferred to 200 mL of LB liquid medium under sterile conditions. The medium was incubated at 30°C and 180 r / min with shaking for 3 days. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in sterile water and diluted to 1×10⁻⁶ with sterile water. 8 CFU / mL was used to obtain a Bs-1117 bacterial suspension.

[0066] The preparation of the pathogenic fungal cake was the same as in Example 2, both involving incubation at 30 degrees Celsius for 72 hours. The selected pathogenic fungus was *Fusarium oxysporum* (…). Fusarium oxysporum f.sp. cubense ), Fusarium graminearum ( Fusarium. pseudograminearum Fusarium pseudoverticum ( Fusarium verticillioides Fusarium graminearum ( ), Fusarium graminearum ), Staphylococcus aureus ( Botrytis cinerea ).

[0067] The plate confrontation operation was the same as in Example 2, with three replicates for each treatment. Among them, after 7 days of dark incubation in a 30°C biochemical incubator, the colony radius was measured, and the inhibition rate was calculated based on formula (1).

[0068] Photos of plate confrontation inhibition of Bs-1117 strain against different pathogens are shown below. Figure 6 As shown.

[0069] The Bs-1117 strain showed a plate inhibition rate of 90.2% against Fusarium oxysporum; 88.8% against Fusarium pseudograss; 86.3% against Fusarium verticillatum; 84.7% against Fusarium grass; and 92.1% against Botrytis cinerea.

[0070] The above results indicate that strain Bs-1117 not only has a strong inhibitory effect on Fusarium solani, but also on Fusarium oxysporum, Fusarium pseudograss, Fusarium verticillatum, Fusarium grass, and Botrytis cinerea.

[0071] 4.2 Inhibitory effect of crude extract of strain Bs-1117 on Fusarium solani

[0072] The extraction process of secondary metabolites from strain Bs-1117 is as follows: First, the Bs-1117 seed culture obtained in LB liquid medium was inoculated into 200 mL of LB liquid medium and cultured with shaking at 32°C and 200 rpm for 24 hours. Then, 5 mL of macroporous resin (Amberlite™ XAD-4, 50 nm) pretreated with methanol was added, and the culture was continued under the same conditions for another 24 hours to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 15 minutes at ambient temperature, the supernatant was discarded, and the precipitate was resuspended in 35 mL of ethyl acetate. The precipitate was extracted with shaking at 37°C and 200 rpm for 4 hours, and centrifuged again at 8000 rpm for 15 minutes. The supernatant was collected, and the ethyl acetate was removed from the supernatant by rotary evaporation. The obtained solid crude extract was dissolved in 2 mL of methanol to obtain the crude extract of secondary metabolites of strain Bs-1117, which was stored at -20°C for use.

[0073] The antibacterial activity of crude extract of secondary metabolites of strain Bs-1117 was determined by plate diffusion method. The specific steps were as follows: the pathogenic fungal cake was inoculated in the center of a PDA plate, four holes were punched evenly around the cake using a sterile punch, 50 μL of crude extract of secondary metabolites of strain Bs-1117 was added to the holes, methanol was used as a negative control, and a blank control was set up in which no substance was added to the four holes. Each treatment was repeated three times. After culturing in the dark in a biochemical incubator at 30 degrees Celsius for 7 days, the hyphal morphology of Fusarium solani was observed using an inverted fluorescence microscope (Lecia DMT4000B, Germany), the colony radius was measured, and the antibacterial rate was calculated based on formula (1).

[0074] The crude extract of secondary metabolites of strain Bs-1117 showed antifungal activity against Fusarium solani on agar plates, as shown in the following images: [Image of Fusarium solani mycelium morphology]. Figure 7 As shown.

[0075] The experimental results showed that the crude extract of secondary metabolites of strain Bs-1117 could inhibit the mycelial growth of *Fusarium solani*, with an inhibition rate of 68%. Further microscopic observation revealed that the *Fusarium solani* mycelia treated with the crude extract of secondary metabolites of strain Bs-1117 exhibited significant curling and formed swollen structures, indicating that strain Bs-1117 has the ability to inhibit the mycelial growth of *Fusarium solani*.

[0076] Example 5: Determination of the phosphorus solubilization, siderophore production, and indoleacetic acid production capabilities of strain Bs-1117

[0077] 5.1 Quantitative determination of inorganic phosphorus solubility: First, a standard curve for phosphorus was prepared: Accurately weigh analytical grade potassium dihydrogen phosphate that had been dried to constant weight at 105°C. Dissolve the potassium dihydrogen phosphate in Pikovskaya liquid medium (10.0 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g magnesium sulfate, 0.3 g potassium chloride, 0.1 g yeast extract, 1000 mL deionized water, pH adjusted to 7.0, sterilized at 121°C for 20 min) to prepare standard working solutions with concentrations of 0, 1.0, 2.0, 5.0, 10.0, 20.0, and 30.0 mg / L as concentration points (X values) for the standard curve. Then, the molybdenum blue colorimetric method was used, i.e., molybdenum antimony anti-colorimetric agent was added to each working solution, and after the colorimetric reaction, the absorbance value (Y value) was measured at a wavelength of 882 nm. The standard curve was then plotted and the linear equation was obtained.

[0078] The Bs-1117 strain was inoculated at a 1% inoculum into Pikovskaya liquid medium rich in calcium phosphate and cultured at 30°C. Samples were taken at 6, 12, 24, 48, and 72 hours, and the supernatant was obtained by centrifugation at 8000 rpm for 10 minutes at 4°C. The absorbance of the supernatant at 882 nm was measured using the molybdenum blue colorimetric method at each time point. The phosphorus content was calculated by substituting the values ​​into the standard curve equation, thereby quantitatively evaluating the strain's ability to dissolve inorganic phosphorus at different culture times. Results are shown below. Figure 8 The results showed that strain Bs-1117 can dissolve inorganic phosphorus, with the highest solubility of mineral phosphate reaching 261.8 mg / L, demonstrating outstanding phosphorus-dissolving ability.

[0079] 5.2 Quantitative Determination of Siderophore Production: Following the method for quantitative determination of siderophores, strain Bs-1117 was cultured in PDB medium for 48 hours. The supernatant was collected by centrifugation at 8000 rpm for 10 minutes at 4°C. The supernatant was mixed with an equal volume of CAS detection solution and reacted in the dark at 30°C for 72 hours for color development. Subsequently, the absorbance was measured at 630 nm using a spectrophotometer after 6, 12, 24, 48, and 72 hours of color development. A negative control was prepared by mixing strain-free PDB medium with an equal volume of CAS detection solution; the absorbance was also measured at 630 nm using a spectrophotometer after 6, 12, 24, 48, and 72 hours of color development. The absorbance ratio of the bacterial strain sample to the negative control sample during the same color development time was calculated (λ / λ0: "λ" represents the absorbance of the bacterial strain sample, and "λ0" represents the absorbance of the negative control). The lower the absorbance ratio, the stronger the siderophore-producing ability. See [link to relevant documentation]. Figure 9 The experimental results show that strain Bs-1117 can produce siderophores and belongs to the category of high siderophore-producing bacteria.

[0080] 5.3 Quantitative Determination of Indoleacetic Acid (IAA) Yield: After activation, strain Bs-1117 was inoculated into LB liquid medium supplemented with 500 mg / L tryptophan (as a precursor) and cultured with shaking at 30°C. Fermentation broth samples were collected at 6, 12, 24, 48, and 72 hours of culture, with uninoculated medium serving as a negative control. The fermentation broth was centrifuged at 8000 rpm at ambient temperature, and the supernatant was collected. The absorbance at 450 nm was measured using an indoleacetic acid (IAA) ELISA kit (double-antibody one-step sandwich method, Beijing Solarbio Science & Technology Co., Ltd.). The concentration of IAA in the fermentation broth at each time point was calculated. The results are shown in [Figure number missing]. Figure 10The results showed that strain Bs-1117 could effectively synthesize IAA, and its yield showed a trend of first increasing and then stabilizing: IAA (26.3 mg / L) could be detected after 6 hours of cultivation, and the yield continued to accumulate with the extension of cultivation time, reaching a peak of 64.7 mg / L at 48 hours, showing that this strain has a significant ability to synthesize indoleacetic acid.

[0081] Example 6: Pot experiment on the control of root rot in flue-cured tobacco by strain Bs-1117

[0082] The test soil was the topsoil from the 0-20cm layer of flue-cured tobacco experimental field at the Songming Experimental Station of the Yunnan Academy of Agricultural Sciences. The soil type was red limestone soil. Before the experiment, the soil pH was 6.7, and the total organic matter, total nitrogen, total phosphorus, and total potassium were 22.1, 0.9, 1.6, and 9.8 g / kg, respectively. The available nitrogen, phosphorus, and potassium in the soil were 76.1, 30.7, and 144.5 mg / kg, respectively.

[0083] The flue-cured tobacco variety tested was K326, a major variety cultivated in Yunnan, provided by the Institute of Agricultural Environment and Resources, Yunnan Academy of Agricultural Sciences.

[0084] The method for preparing the bacterial suspension of strain Bs-1117 was the same as in Example 4, and the concentration of the obtained bacterial suspension was 1×10⁻⁶. 8 CFU / mL.

[0085] Preparation of *Fusarium solani* conidia suspension: *Fusarium solani* preserved on PDA slant agar plates were inoculated onto PDA plates and cultured at 30°C for 3 days. 8 mm *Fusarium solani* mycelial cakes were prepared using a sterile punch and transferred to 200 mL PDB medium under sterile conditions. The mixture was cultured at 30°C and 180 r / min with shaking for 5 days. Mycelia were removed by filtration. The filtrate was centrifuged, and the supernatant was discarded to obtain conidia precipitate. The conidia precipitate was resuspended in sterile water and diluted to 1×10⁻⁶ with sterile water. 7 CFU / mL was used to obtain a suspension of Fusarium solani conidia.

[0086] The pot experiment was conducted at the Songming Experimental Station of the Yunnan Academy of Agricultural Sciences (25°21′11.21″N, 103°6′47.24″E) from February 20 to April 11, 2025.

[0087] Pot experiment: 10 kg of sterile soil was placed in each pot, and healthy flue-cured tobacco seedlings with 5 to 6 true leaves were transplanted into the pots, 1 seedling per pot. Four treatments were set up: 1) F+Bs group: Immediately after transplanting, 150 mL of strain Bs-1117 bacterial suspension was applied to the tobacco seedlings, and one week later, 150 mL of Fusarium solani conidia suspension was applied to the roots; 2) Fs group: One week after transplanting, 100 mL of Fusarium solani conidia suspension was applied to the roots; 3) Bs group: Immediately after transplanting, 150 mL of strain Bs-1117 bacterial suspension was applied to the tobacco seedlings; 4) Control group (CK): Immediately after transplanting, 150 mL of sterile water was applied. Each treatment was replicated 10 times (1 pot per replicate). The growth of the tobacco seedlings was measured every 15 days for a total of 45 days after application of Fusarium solani. Water each pot with sterile water in the morning and evening to keep the soil moist. Calculate the incidence rate, disease index, and relative efficacy according to formulas (2), (3), and (4). The results are shown in Table 1.

[0088] Incidence rate = (ni / N) × 100% (2).

[0089] Disease index = ∑ni×vi×100% / N×4 (3).

[0090] Relative efficacy (%) = (F−T)×100% / F (4).

[0091] In the formula, ni refers to the number of diseased plants at each disease level, vi = disease level (0, 1, 2, 3, 4), N refers to the total number of flue-cured tobacco seedlings used in each treatment, F refers to the disease index of the control group, and T refers to the disease index of each treatment group. Disease severity grading (graded by plant): Level 0, no disease in the entire plant; Level 1, 0 to 25% of leaves wilted; Level 2, 26% to 50% of leaves wilted; Level 3, 51% to 75% of leaves wilted; Level 4, 76% to 100% of leaves wilted, diseased plants are basically dead.

[0092] Table 1

[0093]

[0094] The results in Table 1 show that 45 days after inoculation, the disease incidence rate of Fs-treated tobacco seedlings reached 80%, and the disease incidence rate of Fs+Bs-treated seedlings was reduced by 40.0% compared with Fs-treated seedlings. The relative control efficacy of Fs+Bs-treated seedlings reached 64%.

[0095] Forty-five days later, soil microbial DNA was extracted from the rhizosphere of flue-cured tobacco using a soil FastDNA Spin kit (MP Bio, Santa Ana, CA, USA) to quantify the amount of Fusarium solani in the rhizosphere using qPCR. Using FFs (as shown in SEQ ID No. 4) and RFs (as shown in SEQ ID No. 5) as primers and the soil microbial DNA from the rhizosphere of flue-cured tobacco as a template, qPCR quantification was performed. Serial 10-fold dilutions of plasmids containing the target gene were used as qPCR standards, with amplification efficiencies ranging from 90% to 110%, and the standard curve showed good linearity (R0). 2 >0.99). The quality of qPCR amplification was verified by melting curve analysis; non-specific amplification was negligible. Results are shown below. Figure 11 .

[0096] Figure 11 The quantitative results of pathogen detection showed that the abundance of pathogens in the Fs+Bs treatment was significantly reduced by 95% compared with the Fs treatment. These results indicate that Bs-1117 has good indoor control efficacy against tobacco root rot.

[0097] Example 7: Field trial of Bs-1117 strain combined with vetch husk for the control of root rot in flue-cured tobacco

[0098] The experiment was conducted at the Songming Experimental Station of the Yunnan Academy of Agricultural Sciences (25°21′11.21″N, 103°6′47.24″E) from April 10 to September 20, 2025.

[0099] The method for preparing the bacterial suspension of strain Bs-1117 was the same as in Example 4, and the concentration of the obtained bacterial suspension was 1×10⁻⁶. 10 CFU / mL.

[0100] The method for preparing the Fusarium solani conidia suspension is the same as in Example 6, and the concentration of the obtained suspension is 1×10⁻⁶. 7 CFU / mL.

[0101] Fermentation treatment of *Vigna glabra* husk: Collect the above-ground parts of *Vigna glabra* plants from full bloom to early pod stage, air-dry them naturally until the moisture content is below 3 wt%, then crush them to within 2 cm to obtain dry material. Add water at a ratio of 142.5 L / 100 kg of dry material, stir well. If the pH value is below 6 (too acidic), adjust it using light calcium carbonate by gradually sprinkling it in and thoroughly mixing to neutralize the acidity. If the pH value is above 8 (too alkaline), spray a citric acid solution evenly and stir thoroughly to neutralize the alkalinity. Adjustment should follow the principle of "small amounts, multiple times," and after each addition, mix thoroughly and monitor the pH value in real time until the pH value stabilizes within the ideal range of 6 to 8. In this example, the pH value is 7.0. Then, perform aerobic composting for 40 days, turning and ventilating during this period to obtain fully decomposed *Vigna glabra* husk. The fully decomposed *Vigna glabra* husk has a uniform appearance, no layering, a loose flocculent or powdery structure, is brown or black, and has no pungent odor.

[0102] The moisture content of the decomposed vetch leaves was adjusted to 50 wt% to obtain decomposed vetch leaves with a moisture content of 50 wt%.

[0103] 1 L of concentration is 1×10 10 A Bs-1117 bacterial suspension at CFU / mL was diluted with water and mixed with well-rotted vetch bran to a moisture content of 50 wt%, yielding a Bs-vetch bran composition. The mass ratio of Bs-1117 CFU to well-rotted vetch bran with a moisture content of 50 wt% was 1 × 10⁻⁶. 11 CFU: 1kg.

[0104] Each plot has an area of ​​50 square meters, and 75 tobacco plants are planted in each plot. The experiment is set up with 4 treatments, 3 replicates per treatment, and 1 plot per replicate.

[0105] Treatment 1: At the time of tobacco transplanting, inoculate the roots of the tobacco plants with a Bs-1117 bacterial suspension at a rate of 1×10⁻⁶ per plant. 10 CFU, which is an application rate of 1×10 13 CFU / acre.

[0106] Treatment 2: When transplanting flue-cured tobacco, first apply 100 g of well-rotted vetch husk with a moisture content of 50 wt% to the transplanting hole, that is, the application rate is 100 kg / mu.

[0107] Treatment 3: When transplanting flue-cured tobacco, first apply Bs-sunflower vetch bran composition to the transplanting hole, with an application rate of 1×10⁻⁶ Bs-1117. 13 CFU / mu, and the application rate of well-rotted vetch bran is 100 kg / mu.

[0108] Control group: When transplanting flue-cured tobacco, water was poured onto the roots of the tobacco plants.

[0109] Fifteen days after transplanting, 200 mL of Fusarium solani conidia suspension was inoculated onto the roots of each of the four treatment groups. All other procedures followed standard management practices, with consistent management conditions across all four treatments.

[0110] Disease incidence was investigated during the tobacco clump stage and maturity stage, and the incidence rate was calculated based on formula (2). The results are shown in Table 2.

[0111] Table 2

[0112]

[0113] The results in Table 2 show that during the tobacco clump stage, the incidence rate of disease in Treatment 1 was reduced by 35% compared to the control group, in Treatment 2 by 15%, and in Treatment 3 by 60.0%. The reduction in incidence rate in Treatment 3 (60%) was significantly higher than the combined reduction of Treatments 1 and 2 (35% + 15% = 50%). During the tobacco maturity stage, the incidence rate of disease in Treatment 1 was reduced by 48.4% compared to the control group, in Treatment 2 by 26.7%, and in Treatment 3 by 83.4%. The reduction in incidence rate in Treatment 3 (83.4%) was significantly higher than the combined reduction of Treatments 1 and 2 (48.4% + 26.7% = 75.1%). Therefore, the synergistic application of Bs-1117 bacteria and well-rotted vetch bran has a synergistic effect.

Claims

1. Bacillus sicca ( Bacillus siamensis The strain Bs-1117 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33939.

2. A composition comprising the Bacillus sicca strain Bs-1117 as described in claim 1, and decomposed vetch bran, wherein the decomposed vetch bran is prepared as follows: the above-ground parts of vetch plants from full bloom to early pod stage are collected, naturally air-dried to a moisture content of less than 3 wt%, and then pulverized to a thickness of less than 2 cm to obtain dry material; water is added at a ratio of (100 to 200) L / 100 kg of dry material, and stirred evenly; the pH is adjusted to 6 to 8, and then aerobic composting is carried out for 35 to 45 days, during which time the compost is turned and ventilated to obtain the decomposed vetch bran.

3. The composition according to claim 2, characterized in that, The ratio of the *Bacillus sicca* strain Bs-1117 CFU to the mass of well-rotted vetch bran with a moisture content of 50 wt% was 1 × 10⁻⁶. 11 CFU: (0.1 to 10) kg.

4. The composition according to claim 2, characterized in that, The ratio of the *Bacillus sicca* strain Bs-1117 CFU to the mass of well-rotted vetch bran with a moisture content of 50 wt% was 1 × 10⁻⁶. 11 CFU: 1kg.

5. The *Bacillus sicca* strain Bs-1117 according to claim 1 or the composition according to any one of claims 2 to 4 for inhibiting *Fusarium* (… Fusarium ) and / or Staphylococcus aureus ( Botrytis Applications in ).

6. The application according to claim 5, characterized in that, The Fusarium species is *Fusarium solani* ( ). Fusarium solani Fusarium oxysporum ( Fusarium oxysporum Fusarium pseudograss () Fusarium. pseudograminearum Fusarium pseudoverticum ( Fusarium verticillioides ) and Fusarium graminearum ( Fusarium graminearum At least one of the following.

7. The application according to claim 5, characterized in that, The staphylococcus is *Botrytis cinerea* (…). Botrytis cinerea ).

8. The use of the Bacillus sicca strain Bs-1117 according to claim 1 or the composition according to any one of claims 2 to 4 in the prevention and control of at least one of root rot, wilt, stem base rot, ear rot, Fusarium head blight and gray mold.

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