Preparation method and application of bacillus siamensis z4 and fermentation liquor thereof

Abstract

This invention provides a Bacillus sicca Z4, Bacillus siamensis The strain Z4 was deposited on March 3, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37830, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The optimal fermentation medium for strain Z4 was 1000 mL of water, with yeast extract at 15 g / L, maltose at 20 g / L, and sodium chloride at 4 g / L. The optimal fermentation conditions were: 1% inoculum in a 250 mL Erlenmeyer flask (75 mL), temperature 35℃, pH 7, rotation speed 289 r / min, and incubation time 18 h. Strain Z4 antagonized the growth and spore germination of *Spodoptera litura*, the causal agent of sorghum smut. The aseptic fermentation filtrate of strain Z4, diluted 10-20 times, could be used to accelerate sorghum seed germination and emergence. The fermentation broth of strain Z4, diluted 10-100 times, could be used for the control of sorghum smut. This invention provides new biocontrol resources for the prevention and control of sorghum smut and other diseases.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to the preparation method and application of Bacillus sicca Z4 and its fermentation broth. Background Technology

[0002] Sorghum smut is caused by Ustilago maydis (Smut fungus) Sporisorium reilianum This is a systemic soil-borne disease caused by infection. The pathogen infects sorghum seeds from germination to emergence. Symptoms are not obvious in the early stages of sorghum growth, but typical symptoms appear only during the heading stage, manifesting as black powder on the entire ear or part of the spikelets. This disease is a very important one in sorghum production, and the degree of damage varies in different years, regions, and varieties. In severe cases, it can lead to complete crop failure. For example, in recent years, in major brewing sorghum producing areas of Shanxi Province, the incidence rate of some varieties has reached over 80%, making it one of the important factors restricting the development of the sorghum industry.

[0003] Integrated green management of plant diseases, aiming at ecological security and sustainable agricultural development, prioritizes environmentally friendly, non-chemical measures such as agriculture and biology for the scientific management of crop diseases and pests, and has become a core concept of modern agricultural plant protection. For a long time, the control of sorghum smut has mainly relied on resistant varieties, chemical control, and agricultural control. However, resistant germplasm resources have a narrow genetic base for resistance, making it difficult to achieve simultaneous resistance to multiple physiological differentiation types of sorghum smut fungus. Furthermore, no sorghum smut-resistant varieties have been found among the main sorghum varieties currently promoted in Shanxi Province. The number of registered chemical agents that can effectively control this disease is limited, and chemical control poses significant environmental pollution and 3R (resources, environment, and ecology) problems. Agricultural control measures that promote sorghum seed germination and emergence can reduce the chance of infection by the fungus, thus mitigating the severity of the disease, but their control effect is limited. Compared with chemical control, biological control has advantages such as no residue, no environmental pollution, less likelihood of pathogens developing drug resistance, simple implementation, and low cost, making it a healthy and effective control method. However, since the target pathogen, *Sorghum smut*, is an obligate parasite, screening biocontrol bacteria is relatively difficult, and currently there are very few biocontrol strains developed and applied. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus sicca that can be used to control sorghum smut, a method for preparing the fermentation broth of the Bacillus sicca, and its application in the control of sorghum smut.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Bacillus sicca Z4B acillus siamensisIt was deposited on March 3, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37830. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] The 16S rDNA sequence of Bacillus sicca Z4 is shown in SEQ ID NO:1.

[0007] A method for preparing Bacillus sicca Z4 fermentation broth, characterized by comprising the following steps: Step 1: Inoculate Bacillus sicca Z4 onto NA agar plates using the streak method. After incubating at 28°C for 24 hours, inoculate onto NB liquid medium and incubate with shaking at 35°C for 12 hours to obtain the seed culture of strain Z4. Step 2: Inoculate the seed culture prepared in Step 1 into the fermentation culture medium, and obtain the Bacillus sicca Z4 fermentation broth through fermentation culture.

[0008] Preferably, the fermentation conditions for the fermentation medium are as follows: maltose 20 g / L, peptone 10 g / L, yeast extract 15 g / L, sodium chloride 4 g / L, inoculum size 1%, liquid volume of 75 mL in a 250 mL Erlenmeyer flask, temperature 35℃, pH value 7, rotation speed 289 r / min, and culture time 18 h.

[0009] Application of Bacillus sicca Z4 and its fermentation broth: antagonizes the growth and spore germination of sorghum smut fungus, and is used for the prevention and control of sorghum smut, and accelerates sorghum seed germination and emergence.

[0010] When using sterile fermentation filtrate of Bacillus sicca Z4 to accelerate germination and emergence of sorghum seeds, it needs to be diluted 10 to 20 times.

[0011] The fermentation broth of Bacillus sicca Z4, diluted 10-100 times, can be used to control sorghum smut. Depending on the field planting density, apply 10 mL per hole with the sorghum sowing.

[0012] The beneficial effects of this invention are as follows: This invention isolates soil bacteria from the rhizosphere soil of sorghum and screens out the biocontrol strain Bacillus sicca Z4, which has both biocontrol and agricultural control effects. It not only antagonizes the growth and spore germination of sorghum smut fungus, but also has the ability to produce amylase, protease, cellulase and IAA, which can accelerate sorghum seed germination and emergence. This provides a new biocontrol resource for the control of sorghum smut and other sorghum diseases, and lays the foundation for further use of this biocontrol strain to control diseases. Attached Figure Description

[0013] Figure 1: Antagonistic effect of antagonistic bacteria against sorghum smut fungus; where CK: sterile PDA treatment control; b: antagonistic effect of strain Z4 against the pathogen.

[0014] Figure 2 The inhibitory effect of antagonistic bacterium Z4 on cultures of *Spodoptera exigua*, the causal agent of sorghum smut; where CK: *Spodoptera exigua*, the causal agent of sorghum smut; a: the inhibitory effect of strain Z4 on the causal agent.

[0015] Figure 3 : Plate culture characteristics and Gram staining of antagonistic strain Z4; where c: plate culture characteristics of strain Z4; d: Gram staining of strain Z4.

[0016] Figure 4 Phylogenetic tree of antagonistic strain Z4 constructed based on 16S rDNA sequence.

[0017] Figure 5 The inhibitory effects of antagonistic bacterium Z4 on different sorghum disease pathogens, including: grain mold (a: Fusarium moniliforme; b: Fusarium oxysporum; c: Curvularia spp.); Fusarium stalk rot (g: Fusarium graminearum; h: Fusarium verticillatum); seedling blight (i: Fusarium moniliforme); black stalk rot (j: Ulva oryzae); top rot (k: Fusarium verticillatum); and panicle rot (l: Fusarium verticillatum).

[0018] Figure 6 The effect of antagonistic bacteria Z4 fermentation broth on sorghum seed germination.

[0019] Figure 7 Detection of IAA production capacity of antagonistic bacteria Z4.

[0020] Figure 8 Detection of extracellular enzymes and siderophores secreted by antagonistic strains; where A is protease; B is amylase; C is cellulase; and D is siderophore production.

[0021] Figure 9 Effects of different culture media on the growth of Bacillus sicca Z4.

[0022] Figure 10 Effects of different carbon sources on the growth of Bacillus sicca Z4.

[0023] Figure 11 Effects of different nitrogen sources on the growth of Bacillus sicca Z4.

[0024] Figure 12 Effects of inorganic salts on the growth of Bacillus sicca Z4.

[0025] Figure 13 Effects of pH and temperature on the growth of Bacillus sicca Z4.

[0026] Figure 14 Effects of rotation speed and sample volume on the growth of Bacillus sicca Z4.

[0027] Figure 15 Effects of different inoculum amounts on the growth of Bacillus sicca Z4.

[0028] Figure 16 Contour plot and response surface plot showing the effects of interactions of various significant influencing factors on Bacillus sicca Z4 OD600. Detailed Implementation

[0029] Bacillus sicca Z4, deposited on March 3, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37830, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0030] The 16S rDNA sequence of Bacillus sicca Z4 is shown in SEQ ID NO:1.

[0031] The preparation method of Bacillus sicca Z4 strain fermentation broth includes the following steps: Step 1: Inoculate the preserved Bacillus sicca Z4 onto the prepared NA plate medium using the streak method. After incubating at 28℃ for 24 hours, inoculate it into NB liquid medium and incubate with shaking at 35℃ for 12 hours at 200 rpm to obtain the seed culture of strain Z4.

[0032] Step 2: Inoculate the seed culture prepared in Step 1 into the fermentation medium, and obtain the Bacillus sicca Z4 fermentation broth through fermentation. The fermentation conditions are: maltose 20 g / L, peptone 10 g / L, yeast extract 15 g / L, sodium chloride 4 g / L, inoculum size 1%, liquid volume of 75 mL in a 250 mL Erlenmeyer flask, temperature 35℃, pH 7, rotation speed 289 r / min, and culture time 18 h.

[0033] Isolation, screening and identification of strain Z4 1.1 Isolation of antagonistic bacteria Beef extract peptone medium (NA): 10g peptone, 3g beef extract, 5g NaCl, 20g agar powder, 1000mL distilled water, pH=7.0-7.2.

[0034] Soil samples were collected from the roots of healthy sorghum plants using a five-point sampling method. An appropriate amount was added to sterile water and incubated for 30 minutes in a shaking incubator at 28℃ and 180 r / min. The samples were then serially diluted to 10⁻⁶ under sterile conditions. -3 g / mL, 10 -4Bacterial suspensions were prepared using a concentration gradient of g / mL, and then bacteria were isolated and purified on NA plates using the plate dilution method. Single colonies were selected for purification and culture based on characteristics such as colony size, protrusion, and transparency.

[0035] 1.2 Screening of antagonistic bacteria PDA medium formula: 200g potato, 10g glucose, 20g agar, 1000mL distilled water.

[0036] The LB liquid culture medium formula is as follows: 5.0g beef extract, 10.0g peptone, 5.0g NaCl, 1000mL distilled water, pH 7.0.

[0037] Antagonistic bacteria capable of inhibiting the growth of *Spodoptera exigua* colonies were screened using the inhibition zone method. After culturing purified bacteria in LB broth with shaking at 180 rpm for 15-17 hours, single-spore colonies of *Spodoptera exigua* were picked from PDA plates and gently dispersed in sterile centrifuge tubes using a pipette tip. 1 mL of sterile water was added, and the suspension was repeatedly pipetted to ensure complete dispersion. 50 μL of the suspension was evenly spread onto PDA plates. Bacterial discs were then punched using a 6 mm sterile punch and inoculated onto plates containing *Spodoptera exigua*. Three bacterial discs were inoculated at equal intervals, 2.5 cm from the center of the plate. A control group inoculated only with PDA agar blocks served. Each treatment was repeated three times. The bacteria were incubated at 28℃ for 4-5 days, and the antibacterial effect was observed.

[0038] Further screening was conducted to identify antagonistic strains that effectively inhibited teliospore germination. Teliospore powder from the pathogen was washed three times with sterile water to prepare a teliospore suspension, with a concentration of 40-50 teliospores per 10×10x microscope field. The antagonistic bacteria were inoculated into liquid culture medium and incubated on a shaker at 28℃ for 3 days. After centrifugation at 12000 rpm for 15 minutes, the supernatant was collected and filtered through a 0.22 μm sterile microporous membrane to obtain a sterile filtrate. The sterile filtrate was diluted 2-fold and 10-fold to prepare culture medium slides. An appropriate amount of spore suspension was evenly spread on the slide and placed in a petri dish lined with moistened filter paper. The humidity for spore germination was maintained at approximately 57% using the small container method, repeated three times. Sterile water was used as a control instead of the sterile filtrate. The mixture was incubated at 28℃ for 24 hours. Whether teliospores germinate or not is determined by the fact that the mycelium exceeds the radius of the teliospore of *Smutella smutella*. The germination status of teliospores is observed and recorded under a microscope, and the germination rate of teliospores is calculated.

[0039] Inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter - 5 mm)] × 100.

[0040] The results showed that strain Z4 had a good inhibitory effect on the colony growth and spore germination of *Spodoptera litura*, the causal agent of sorghum smut. Figure 1 , Figure 2 (Table 1).

[0041] 1.3 Identification of antagonistic bacteria 1.3.1 Morphological characteristics and physiological and biochemical properties of antagonistic bacterial strains Antagonistic strains were streaked onto NA medium and incubated at 28°C for 18-24 hours. Morphological characteristics such as color, size, gloss, elevation, shape, transparency, uniformity, and edge of single colonies were observed, and Gram staining was performed. Morphological identification was based on Bergey's Manual of Bacteriological Identification and the Manual of Systematic Identification of Common Bacteria. Physiological and biochemical characteristics of the strains were identified using gelatin liquefaction, methyl red, VP, and nitrate reduction tests, following the method of Dong Xiuzhu and Cai Miaoying (2001). Strains Z4 were milky white, nearly circular, initially with a smooth, opaque surface, firmly attached to the medium; later, wrinkles appeared on the surface, gradually forming a hill-like shape with smooth edges. Young strains were Gram-positive, and bacilli (…) Figure 3 As shown in Table 2, the physiological and biochemical test results indicate that strain Z4 can decompose gelatin and liquefy it. Its catalase test, VP test, and nitrate reduction test results were positive, while the methyl red, citrate utilization, and hydrogen sulfide tests were negative. The indole reaction test result was positive. The glucose oxidation fermentation test result showed yellowing when the tube was open and green when the tube was closed, indicating the oxidized form. Furthermore, the strain could grow normally on plates containing 1%, 2%, 5%, 7%, and 10% NaCl, indicating its salt tolerance. Based on morphological characteristics and physiological and biochemical test results, strain Z4 was preliminarily identified as belonging to Bacillus (Bacillus). Bacillus sp).

[0042] Note: "+" indicates positive, and "-" indicates negative.

[0043] 1.3.2 Molecular identification of antagonistic bacteria Z4 Strain Z4 was cultured overnight at 28℃ on a shaker at 180 rpm. 1-2 mL of the bacterial culture was used to extract DNA from the antagonistic bacteria Z4 using the BioFlux BSC12S1 bacterial genomic DNA extraction kit. PCR amplification was performed using 16S rDNA gene amplification primers (upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer 1492R: 5'GGTTACCTTGTTACGACTT-3'). The total amplification volume was 25 μL, containing 12.5 μL of 2×Taq PCR Master Mix, 1 μL each of upstream and downstream primers, 1 μL of template DNA, and 9.5 μL of ddH2O. Reaction conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 51.2℃ annealing for 45 s, 72℃ extension for 1 min, for 33 cycles. After amplification, the quality of the amplification products was detected by 1% agarose gel electrophoresis. The purified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared with Blastens in the NCBI database. Strains with high homology were selected, and a phylogenetic tree was constructed using MEGA12 software.

[0044] like Figure 4 The results showed that strain Z4 and Bacillus siamensis The homology of strains KCTC13613, CPU-B1, and ZV051 (with accession numbers NR117274, OR642745, and OR992631, respectively) was 100%, with 99% support and Bacillus siamensis They clustered on the same branch. Therefore, based on morphological characteristics, physiological and biochemical assays, and 16S rDNA sequence developmental analysis, strain Z4 was identified as Bacillus sicca (Bacillus). Bacillus siamensis ).

[0045] Inhibitory effects of Bacillus sicca Z4 on different pathogenic fungi The inhibition spectrum of antagonistic strain Z4 was determined using the plate confrontation method. Twelve pathogens of nine sorghum diseases were activated on PDA plates, and 6 mm diameter mycelial cakes were placed in the center of the PDA plate. Strain Z4 was inoculated 2-3 cm away from the pathogens. The control group was not inoculated with antagonistic bacteria. Each treatment was repeated three times. The plates were incubated at 25℃. When the control group was almost fully covered with mycelium, the colony diameter of the tested pathogens was measured, and the inhibition rate of the antagonistic bacteria against the pathogens of different sorghum diseases was calculated.

[0046] Inhibition rate (%) = [(control colony diameter – treated colony diameter) / (control colony diameter – 5mm)] × 100.

[0047] The results showed that strain Z4 had an inhibitory effect on nine pathogens causing six sorghum diseases, with inhibition rates ranging from 54.01% to 84.38%. The best inhibitory effects were observed against *Ustilago maydis* (rice black stalk rot), *Fusarium graminearum* (causing *Fusarium graminearum* stalk rot in sorghum), and *Fusarium verticillatum* (causing *Fusarium graminearum* top rot in sorghum), with inhibition rates of 84.38%, 83.72%, and 83.39%, respectively. The second best inhibitory effect was against *Curvularia paniculata* (causing *Gnaphalium graminearum* stalk rot in sorghum), with an inhibition rate of 80.60%. The inhibition rate against *Fusarium verticillatum* (causing *Fusarium graminearum* panicle rot in sorghum) also reached 75.49%. The lowest inhibition rate was against *Fusarium verticillatum* (causing *Fusarium graminearum* stalk rot), at 54.01%. Therefore, antagonistic strain Z4 has a good inhibitory effect on the tested pathogens causing various sorghum diseases. Figure 5 ).

[0048] Siamese Bacillus Z4 Growth Promotion Capacity Test 3.1 Effect of antagonistic bacteria Z4 aseptic fermentation filtrate on sorghum seed germination Select plump sorghum seeds, surface disinfect with 2% sodium hypochlorite for 10-15 minutes, rinse 3-4 times with sterile water, place in petri dishes lined with sterile filter paper, and air dry. Inoculate antagonistic bacteria Z4 into LB liquid medium, incubate at 28℃ for 3 days, centrifuge at 12000 rpm for 15 minutes, collect the fermentation supernatant, filter through a 0.22 μm sterile microporous membrane to obtain sterile filtrate, and dilute it successively by 2, 10, and 20 times, pouring 1 mL into the above-mentioned plates containing seeds per plate. A control treatment with only an equal volume of LB medium was used. Each plate contained 20 seeds, and each treatment was repeated 3 times. Incubate at 28℃ for 1 day, observe and record the germination status of the sorghum seeds, and calculate the germination rate. After 3 days, simultaneously measure the length of the radicle and plumule. Figure 6 ).

[0049] Seed germination rate (%) = 100 * number of germinated seeds / total number of seeds The results showed that the sterile fermentation broth of Bacillus sicca Z4 and its 2-fold dilution inhibited the germination of sorghum seeds. The germination rates of the 10-fold and 20-fold dilutions were 48.89% and 53.26%, respectively, which were not significantly different from the control treatment.

[0050] The antagonistic bacteria aseptic fermentation broth, when diluted 2 times, significantly inhibited the elongation of sorghum seed embryos and radicles; dilutions of 10 and 20 times both promoted their elongation, with the 20-fold dilution showing a significantly greater effect on radicle elongation than the 10-fold dilution. This indicates that within a certain dilution range, the ability of the Bacillus sicca Z4 aseptic fermentation broth to promote radicle and embryo elongation increases with increasing dilution ratio (Table 3).

[0051] 3.2 Determination of the growth-promoting ability of antagonistic strain Z4 Growth-promoting culture medium: Ashby's nitrogen-free medium: MgSO4·7H2O 0.2 g, CaCO3 1 g, sucrose 10 g, K2HPO4·3H2O 0.5 g, NaCl 0.12 g, distilled water 1000 mL.

[0052] PKO medium: Ca3(PO4)25 g, glucose 10 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, MgSO4·7H2O 0.3 g, KCl 0.3 g, MnSO4 0.03 g, FeSO4·7H2O 0.03 g, distilled water 1000 mL.

[0053] Silicate culture medium: 10.0 g sucrose, 2.0 g Na2HPO4, 1.0 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 0.1 g NaCl, 0.5 g yeast powder, 10.0 g potassium feldspar powder, 1000 mL distilled water.

[0054] Sodium carboxymethyl cellulose (CMC) medium: (CMC-Na) 15.0 g, NH4NO3 1.0 g, yeast extract 1.0 g, MgSO4·7H2O 0.5 g, KH2PO4 1.0 g, distilled water 1000 mL.

[0055] Protein production medium: Skim milk is added to NA solid medium (NA: milk = 9:1 v / v).

[0056] Iron-producing medium (CAS medium): Chromium azurite S (CAS) 60.5 mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg, ferric chloride hexahydrate FeCl3·6H2O 2.645 mg, peptone 4.5 g, glucose 9 g, beef extract powder 2.7 g, NaCl 4.5 g, agar 20 g, distilled water 1000 mL.

[0057] Methods to promote fertility: Take strain Z4 in LB liquid medium and incubate at 28°C and 180 r·min -1 After culturing for 17 hours, seed culture was obtained.

[0058] (1) The bacterial culture was inoculated onto Ashby nitrogen-free medium, PKO medium, silicate medium, and CAS medium, and cultured at 28°C for 3 to 7 days. The presence or absence of a clear zone around the strain was observed to determine the strain's ability to solubilize phosphorus and potassium. The experiment was repeated 3 times.

[0059] (2) Inoculate the bacterial culture into sodium carboxymethyl cellulose medium and culture at 30 °C for 3 days. Then stain with 1 g / L Congo red for 1 h, discard the staining solution, and then soak in 1 mol / L sodium chloride solution for 1 h. Observe whether there is a clear zone to determine whether the strain produces cellulase. Repeat the test 3 times.

[0060] (3) Inoculate the bacterial culture into the protein-producing medium and incubate at 28°C for 3 days. Observe whether there is a clear zone. Repeat 3 times.

[0061] (4) Take 0.2 mL of bacterial culture and inoculate it into a solution containing 100 mg·L⁻¹ -1 Tryptophan was cultured in King's B liquid medium at 28°C for 4 days in a shaker at 180 rpm, with no inoculation as a control, followed by inoculation at 12000 rpm. -1 Centrifuge for 5 minutes, take 2 mL of the supernatant and add an equal volume of Salkowski colorimetric solution. Mix well and react in the dark for 30 minutes. If the color turns pink, it indicates that the strain has the ability to secrete IAA. The darker the color, the stronger the strain's ability to produce IAA.

[0062] The results showed that *Bacillus sicca* Z4 possesses the ability to produce amylase, protease, cellulase, and IAA, and also exhibits the characteristic of secreting siderophores. No phosphorus-solubilizing, potassium-solubilizing, or nitrogen-fixing activities were detected. Figure 7 , Figure 8 ).

[0063] Optimization of fermentation conditions for strain Z4 4.1 Strain activation Strain Z4 was inoculated onto NA solid medium and cultured at 28°C for 22 h; the activated strain was inoculated onto LB liquid medium and cultured at 28°C with shaking at 180 r / min for 15 h to obtain the fermentation seed liquid.

[0064] 4.2 Single-factor experiments on fermentation medium The prepared seed culture was inoculated at a rate of 1% into 100 mL / 250 mL of YSP, NYBD, PDA, LB, NB, and medium D, respectively. After incubation at 28℃ and 180 rpm for 24 h on a constant temperature shaker, the OD600 nm value of the strains was determined by UV spectrophotometry to screen for the optimal medium. Each treatment was repeated three times. The most suitable medium for the growth of strain Z4 was identified as peptone yeast sucrose medium (YSP). Figure 9 ).

[0065] Yeast extract medium (YSP) (5 g yeast extract, 10 g tryptone, 20 g sucrose, 1000 mL distilled water) was used as the basal fermentation medium. Glucose, mannitol, fructose, lactose, soluble starch, maltose, and sucrose were used as carbon sources, and equal amounts of soybean flour, ammonium sulfate, yeast extract, urea, peptone, ammonium chloride, and sodium nitrate were used as nitrogen sources. Manganese sulfate, ferrous sulfate, calcium carbonate, sodium chloride, zinc sulfate, and magnesium sulfate were used as inorganic salts. The growth rate of the strains was measured, and the optimal carbon and nitrogen sources and the types and amounts of inorganic salts used in the medium were determined to be 20 g / L maltose, 20 g / L yeast extract, and 4 g / L sodium chloride. Figure 10 , Figure 11 , Figure 12 ).

[0066] The culture temperatures were set at 20℃, 25℃, 28℃, 30℃, 35℃, and 40℃, and the pH values ​​were 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively. OD 600 Seed culture volumes of 1%, 2%, 4%, 6%, 8%, 10%, and 12% were added at rotation speeds of 100 r / min, 120 r / min, 150 r / min, 180 r / min, 200 r / min, and 220 r / min. The culture conditions, with 250 mL Erlenmeyer flasks containing 30 mL, 60 mL, 90 mL, 120 mL, 150 mL, and 180 mL of liquid, were followed for 20 h. Strain growth was measured, and the optimal culture conditions were determined to be 35℃, pH = 7, rotation speed 220 r / min, sample volume 90 mL, and 1% added to a 250 mL Erlenmeyer flask. Figure 13 , Figure 14 , Figure 15 ).

[0067] 4.3 Screening of key factors affecting fermentation Based on the results of previous single-factor experiments, the Plackett-Burman experiment was conducted using Design-Expert12 software with N=12 experiments, using OD600nm as the response value (Table 4). Analysis of variance was performed on the data obtained from the PB experiment. The results showed that the most significant factors affecting the fermentation of the strain were shaking speed, yeast extract content, and sample volume. Shaking speed was positively correlated with OD600 value; increasing the shaking speed significantly increased the growth of Bacillus sicca Z4. Yeast extract content and sample volume had negative effects on the experimental results; as these two values ​​increased, they inhibited the growth of Bacillus sicca Z4.

[0068] 4.4 Design of the steepest climb test Using the rotation speed, the most significant factor, as the ramp unit, the steepest ramp experiment was conducted to optimize the rotation speed, yeast extract content, and sample loading to obtain the maximum response region. The results showed that the third experimental design, with a rotation speed of 280 r·min⁻¹, yeast extract content of 15 g, and sample loading of 80 mL, resulted in the largest strain growth and an OD600 of 2.74. Therefore, the third experimental design was used as the center point for the response surface methodology.

[0069] 4.5 Response Surface Analysis of Fermentation Conditions After determining the single-factor condition levels, based on the Box-Behnken central composite principle, and using the bacterial growth OD600 as the response value, a 3-factor, 3-level Box-Behnken experimental design was conducted using Design-Exper.V8.06 software. The experimental results were then fitted with multiple regression, and the significance of the obtained regression model was tested. The final quadratic multinomial regression model for the culture conditions of rotation speed, yeast extract, and sample quantity was: Y = 2.73 - 0.0790A - 0.0172B - 0.0106C - 0.0287AB + 0.0057AC - 0.0118BC - 0.01287A² - 0.356B² - 0.0655C².

[0070] To further investigate the interactions among significant influencing factors, contour plots and 3D response surface plots were generated using Design-Expert software to visualize the interactions between the OD600 values ​​of Bacillus sicca Z4 fermentation broth and the significant influencing factors. Ellipses indicated significant interactions between the two factors, while circles indicated insignificant interactions. The results are as follows: Figure 16 Rotation speed, yeast extract, and sample volume all had interactive effects on *Bacillus sicca* Z4, with the interaction between rotation speed and sample volume being the strongest. The green core region represents the optimal response range for the OD600 value of *Bacillus sicca* Z4. The optimal conditions were determined using a quadratic polynomial regression model: 15 g / L yeast extract, 75 mL sample volume, and a rotation speed of 287 r / min. Further experiments verified the reliability of the optimized fermentation conditions, ultimately determining the optimal fermentation conditions for *Bacillus sicca* Z4 as follows: 20 g / L maltose, 10 g / L peptone, 15 g / L yeast extract, 4 g / L sodium chloride, 1% inoculum size, 75 mL sample volume in a 250 mL Erlenmeyer flask, temperature 35℃, pH 7, and rotation speed 289 r / min.

[0071] The control efficacy of strain Z4 against sorghum smut The pot experiment was used to determine the control efficacy of strain Z4 against sorghum smut. The experiment included four treatments, each with three replicates. Six pots were planted per replicate, with five seeds sown per pot. The fermentation broth of strain Z4 (OD600=1.0) was diluted 10-fold and 100-fold with sterile water before being used to determine its pot control efficacy against sorghum smut. Before sowing, 20 mL of the fermentation broth was poured into the prepared potting soil, and sorghum seeds were sown on the moist soil. The pots were then inoculated with 0.6% teliospores of the sorghum smut fungus using the mulch method. An equal volume of LB broth was used as a positive control instead of the fermentation broth and mulch, while an equal volume of LB broth alone served as a blank control. Sorghum emergence rate was assessed after 15 days, and the disease incidence rate was assessed after heading. The control efficacy was calculated. The results showed that inoculation with the pathogen inhibited sorghum seed emergence, while treatment with the fermented bacterial broth avoided this inhibition (Table 5). This indicates that the Bacillus sicca Z4 fermentation broth significantly reduced the disease incidence in potted sorghum. The control efficacy of the fermented broth diluted 10 times and 100 times was 100% and 69.66%, respectively, and the emergence rate after a 10-fold dilution was significantly higher than the control group without pathogen inoculation (Table 5). This demonstrates that treatment with the antagonistic bacterial fermentation broth not only improved the emergence rate of sorghum seeds but also provided good control of head smut.

[0072]

Claims

1. Bacillus sicca Z4 was deposited on March 3, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37830. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The Bacillus sicca Z4 according to claim 1, characterized in that, The 16S rDNA sequence of the Bacillus sicca Z4 is shown in SEQ ID NO:

1.

3. The method for preparing Bacillus sicca Z4 fermentation broth as described in claim 2, characterized in that, Includes the following steps: Step 1: Inoculate Bacillus sicca Z4 onto NA agar plates using the streak method. After incubating at 28°C for 24 hours, inoculate onto NB liquid medium and incubate with shaking at 35°C for 12 hours to obtain the seed culture of strain Z4. Step 2: Inoculate the seed culture prepared in Step 1 into the fermentation culture medium, and obtain the Bacillus sicca Z4 fermentation broth through fermentation culture.

4. The method for preparing Bacillus sicca Z4 fermentation broth according to claim 3, characterized in that, The fermentation conditions for the fermentation medium were as follows: maltose 20 g / L, peptone 10 g / L, yeast extract 15 g / L, sodium chloride 4 g / L, inoculum size 1%, liquid volume of 75 mL in a 250 mL Erlenmeyer flask, temperature 35℃, pH value 7, rotation speed 289 r / min, and culture time 18 h.

5. The application of Bacillus sicca Z4 as described in claim 1 or 2.

6. The application of Bacillus sicca Z4 according to claim 5, characterized in that, The sterile fermentation filtrate of Bacillus sicca Z4 is used to accelerate the germination and emergence of sorghum seeds. It needs to be diluted 10 to 20 times before use.

7. Application of the Bacillus sicca Z4 fermentation broth prepared by the method described in claim 3 or 4.

8. The application of the Bacillus sicca Z4 fermentation broth according to claim 7, characterized in that, Bacillus sicca Z4 fermentation broth is used for the prevention and control of sorghum smut. It needs to be diluted 10-100 times before use.

9. The application of the Bacillus sicca Z4 fermentation broth according to claim 8, characterized in that, Bacillus sicca Z4 fermentation broth is used for the prevention and control of sorghum smut. When using it in the field, apply 10 mL per hole along with the sorghum sowing and irrigate the planting hole.

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