Bacillus velezensis suitable for non-starch carbon source, fermentation method and application thereof

Abstract

This invention proposes a Bacillus berberis strain suitable for non-grain carbon sources and its fermentation method. This strain has strong stress resistance, nicotinamide metabolism characteristics, and the ability to produce active substances through tryptophan metabolism. It also has a stable preventive effect against tobacco red spot disease. This is of great significance for reducing the use of chemical pesticides, ensuring the quality and safety of tobacco leaves, and promoting the green and sustainable development of the tobacco industry.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a Bacillus berleis, a fermentation method, and its application, which are suitable for use with non-grain carbon sources. Background Technology

[0002] Alternaria ( Alternaria alternata Tobacco red spot disease, caused by , is a fungal disease in tobacco production. It mainly affects mature tobacco leaves, and in severe cases, it causes large areas of leaves to wither and break, which not only leads to a significant reduction in yield, but also damages the chemical composition of tobacco leaves and reduces the grade and commercial value of tobacco leaves.

[0003] Currently, chemical control remains the primary means of preventing and controlling tobacco red spot disease. However, the long-term excessive use of chemical fungicides has led to problems such as increased resistance and decreased efficacy, and it is inconsistent with the green and low-carbon development direction of the tobacco industry. As the disease occurs close to the harvest period, the use of chemical pesticides is limited, and there is an urgent need to develop green, stable, and residue-free alternative technologies.

[0004] Among various biocontrol microorganisms, Bacillus belye ( Bacillus velezensis Due to its broad-spectrum antibacterial activity and growth-promoting properties, this bacterium has become a hot topic in agricultural microbiology research. It can produce a variety of antibacterial substances, inhibiting various plant pathogenic fungi, and also possesses nitrogen-fixing and enzyme-producing functions, promoting crop growth. It shows great promise for green control of tobacco scab and has already been validated in the prevention and control of various crop diseases.

[0005] Some publicly available Bacillus species, *B. belye*, have been reported for use in tobacco disease control, showing some effectiveness against tobacco red spot disease. However, most *B. belye* strains still face application bottlenecks: First, their stress resistance is poor. From a biocontrol mechanism perspective, the colonization and survival ability of strains in acidic, alkaline, or high-salt tobacco field environments directly relates to their niche competition and antagonistic activity against tobacco red spot pathogens. Insufficient stress resistance may lead to a decrease in the colonization rate of strains in complex soils, thereby weakening their field control effect against tobacco red spot disease. Second, existing technologies mostly focus on the antibacterial spectrum or single functions of strains, lacking a systematic analysis of the intrinsic stress-related metabolic characteristics of strains. In particular, there is a lack of in-depth research on nicotinamide metabolic pathways (such as the ability to utilize substrates like NR, NMN, and NADH), and the function of strains in synthesizing various indole derivatives through the tryptophan metabolic pathway has not been verified, making it difficult to meet the needs of stable control in complex field environments.

[0006] Therefore, screening out a strain of Bacillus belyss that possesses strong stress resistance, efficient nicotinamide metabolism (enhancing its own colonization ability), tryptophan metabolism producing indole-like active substances (activating plant disease resistance), and stable control efficacy against tobacco red spot disease is of great significance for reducing the use of chemical pesticides, ensuring the quality and safety of tobacco leaves, and promoting the green and sustainable development of the tobacco industry. Summary of the Invention

[0007] To address the shortcomings of existing Bacillus belyss strains in controlling tobacco red spot disease, such as insufficient stress resistance, weak colonization ability, unstable field control efficacy, and a lack of research on nicotinamide metabolic characteristics and tryptophan metabolic pathways, this invention aims to provide a new Bacillus belyss strain and its application that possesses strong stress resistance, nicotinamide metabolic characteristics, the ability to produce active substances through tryptophan metabolism, and stable control efficacy against tobacco red spot disease.

[0008] To achieve the above-mentioned objectives, this invention provides a strain of Bacillus belye (…). Bacillus velezensis LT3212, this strain was deposited on February 10, 2026 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No. 67847, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0009] Furthermore, the 16S rRNA nucleotide sequence of the Bacillus belyssus LT3212 is shown in SEQ ID NO.1.

[0010] This invention also provides a fermentation method for the above-mentioned Bacillus belye process, specifically including: The first step is to prepare the fermentation medium, adjust the pH, and then sterilize it. The second step involves preparing seed culture using a two-stage liquid culture process, and then transferring the first-stage seed culture to amplify the second-stage seed culture. The third step is to inoculate the secondary seed liquid into the fermentation tank for aerobic fermentation, and control the fermentation temperature and dissolved oxygen. The fourth step involves phased pH control during fermentation: pH is not controlled initially, and an acidic regulator is used to maintain pH stability once the pH naturally rises back to the set value; pH control is stopped when the spore formation rate reaches the preset ratio, and fermentation continues until the spores mature before terminating fermentation.

[0011] In the first step, the fermentation culture medium includes corn steep liquor powder, corn starch, glucose, soybean powder, peptone, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate, and manganese sulfate.

[0012] In the second step, both stages of seed culture were carried out using LB medium, and were cultured at 28°C with shaking to obtain primary and secondary seed solutions.

[0013] In the third step, the fermentation temperature is controlled at 28°C and the dissolved oxygen is maintained at 30%, which is achieved by adjusting the stirring speed and aeration rate.

[0014] In the fourth step, when the pH rises back to 6.55, citric acid is used to control the pH to 6.50; pH control is stopped when the spore formation rate reaches 60%, and fermentation is terminated when the spore rate is ≥90%.

[0015] The invention also provides a microbial agent containing the aforementioned Bacillus belye LT3212, wherein the agent is a liquid agent, a solid agent, or a compound agent, and its viable cell content is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0016] This invention also provides the above-mentioned Bacillus belyssus in the preparation of Alternaria alternata for treating tobacco scab (Alternaria). Alternaria alternata Rice damping-off pathogen Rhizoctonia solani ( Rhizoctonia solani ), Fusarium graminearum, the causal agent of wheat scab ( Fusarium graminearum Fusarium oxysporum, the pathogen of tomato wilt ( Fusarium oxysporum Application of preparations from various plant pathogenic fungi, such as )

[0017] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: Excellent stress resistance: The strain LT3212 of this invention grows vigorously under acidic conditions of pH 5.0, with a 24-hour OD... 600 The value reached 1.03 ± 0.01, which was significantly higher than the control strain. The degradation was slow, showing strong acid resistance and metabolic stability. It can adapt to the complex soil environment of different tobacco-growing areas in my country, laying the foundation for stable colonization in the field and continuous control efficacy.

[0018] Nicotinamide metabolic characteristics: This invention discovered and verified that LT3212 can efficiently utilize nicotinamide precursors such as NR, NMN, and NADH to metabolize and generate nicotinic acid and nicotinamide. This metabolic characteristic provides sufficient precursors for the synthesis and regeneration of intracellular NAD⁺ / NADP⁺ in the strain, thereby enhancing the strain's antioxidant capacity and tolerance to acidic environments, and solving the pain points of existing strains having weak colonization ability and large fluctuations in efficacy in acidic soils.

[0019] Tryptophan metabolic pathway: This invention systematically verified that LT3212 can utilize L-tryptophan to synthesize melatonin, indole-3-acetaldehyde, and other indole derivatives that have been proven to have crop growth-promoting, antioxidant, and stress-resistant functions, providing a core material basis for the strain's growth-promoting, yield-increasing, stress-resistant, and efficiency-enhancing functions in crops such as tobacco and rice.

[0020] Comprehensive growth-promoting functions: LT3212 has multiple growth-promoting functions such as nitrogen fixation, protease production, cellulase production, and iron-loving factor production. It can improve plant nutrient absorption, promote root development, and enhance crop stress resistance from multiple aspects. Its comprehensive function combination has high application value.

[0021] Broad-spectrum pathogen antagonism: LT3212 has significant antagonistic effects against a variety of important plant pathogens, including Alternaria alternata, Rhizoctonia solani, Fusarium graminearum, and Fusarium oxysporum, with inhibition rates of 73.03%, 61.73%, 59.91%, and 54.64%, respectively. It has a broad spectrum of control and has the potential to be developed into a broad-spectrum biocontrol agent.

[0022] Stable efficacy against tobacco red spot disease: Pot experiments have confirmed that LT3212 has a 40% control effect on tobacco red spot disease, effectively reducing the disease index and decreasing leaf scorching and breakage, and has the potential to be developed into a biocontrol agent specifically for tobacco red spot disease.

[0023] Significant crop growth-promoting effects: Pot experiments showed that after root irrigation treatment with LT3212, the chlorophyll content of tobacco increased by 17.3% and the fresh weight of the aboveground parts increased by 20.7%; the plant height of rice increased by 11.7%, the chlorophyll content increased by 26.5%, and the fresh weight of the aboveground parts increased by 26.7%, demonstrating significant growth-promoting effects.

[0024] Excellent compatibility and synergistic growth-promoting effect with organic fertilizer: Pot experiments confirmed that the combined application of strain LT3212 of this invention with bio-organic fertilizer significantly promoted rice growth. The average plant height in the combined treatment group reached 33.00 ± 2.59 cm, an increase of 43.4% compared with the blank control group and 4.2% compared with the bio-organic fertilizer alone group; the average fresh weight of the aboveground parts reached 0.23 ± 0.04 g, an increase of 91.7% compared with the blank control group and 4.5% compared with the bio-organic fertilizer alone group. The growth-promoting effect was significantly better than that of applying bio-organic fertilizer alone or applying the strain liquid alone. This confirms that the two have good compatibility and synergistic growth-promoting effect, greatly expanding the field application scenarios of the strain, reducing the application cost of large-scale promotion, and providing a mature supporting solution for the practical application of the strain in field planting.

[0025] Good biocompatibility: LT3212 is non-hemolytic, safe for humans and animals, has no environmental pollution problems, has simple culture conditions, is easy to preserve, and is suitable for industrial production and large-scale application. Attached Figure Description

[0026] Figure 1 This is a colony morphology diagram of Bacillus belyssus LT3212; Figure 2 The image shows the results of the hemolytic activity assay for Bacillus belyssus LT3212. Figure 3 Figure showing the growth of Bacillus belyssus LT3212 in an electrochemical carbon source medium; Figure 4 A comparison of the growth curves of Bacillus belyssus LT3212 and LL141 at pH 5. Figure 5 HPLC chromatogram for the utilization of nicotinamide substrates by Bacillus belyssus LT3212; Figure 6 Chromatogram for the liquid chromatography detection of indole metabolites of Bacillus belyssus LT3212; Figure 7 Figure showing the cellulase production capacity of Bacillus belyss LT3212; Figure 8 Figure showing the results of casein production capacity of Bacillus belyss LT3212; Figure 9 Figure showing the results of the heptaphilic production capacity of Bacillus belyss LT3212; Figure 10 Figure showing the nitrogen fixation capacity of Bacillus belyssus LT3212; Figure 11 Figure 1 shows the results of the antagonistic ability of Bacillus belyss LT3212 against pathogenic fungi (A: antagonism against Rhizoctonia solani; B: antagonism against Alternaria alternata). Figure 12 Image showing the growth-promoting effect of Bacillus belye LT3212 on potted tobacco; Figure 13 Image showing the results of rice pot cultivation promoted by Bacillus belysin LT3212; Figure 14 The image shows the control efficacy of Bacillus vesicularis LT3212 against tobacco scab in potted plants. Figure 15 Figure showing the results of promoting growth in potted rice plants by applying Bacillus vesiculosus LT3212 in combination with bio-organic fertilizer. Detailed Implementation

[0027] The following embodiments and accompanying drawings are used to describe in detail the implementation of the present invention, so that the process of how the present invention uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0028] Example 1: Isolation and Identification of Bacillus belye LT3212 Fresh fecal samples were collected from Muscovy ducks. 10 g of the sample was added to 90 mL of sterile physiological saline, and thoroughly mixed by shaking. The mixture was then further shaken to prepare 10... -1 The diluent was then used as a basis for a 10-fold serial dilution to obtain 10... -210 -3 10 -4 10 -5 Series of diluents. Take 100 μL of 10 -5 The suspension of the diluted solution was spread onto LB agar plates and incubated at 28°C for 24 h. Based on the differences in colony morphology, single colonies were picked from the plates and streaked for purification. The purification was repeated three times to obtain pure culture strains, which were then numbered and stored for later use.

[0029] The LB solid medium consists of: 10 g tryptone, 5 g yeast extract (Huankai Biotechnology, catalog number: 050090), 10 g sodium chloride, and distilled water to a final volume of 1 L. 15-20 g agar powder is added to the solid medium.

[0030] The cultured bacterial culture was analyzed for 16S rRNA, and the sequence was identified as shown in Seq ID No. 1, confirming it as *Bacillus belyssae*. The single colony morphology of this *Bacillus belyssae* LT3212 on LB agar plates is shown in the image below. Figure 1 The Bacillus belyssus LT3212 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 10, 2026, with accession number GDMCC No. 67847.

[0031] Nucleotide sequence as shown in SEQ ID NO.1: Example 2: Hemolytic activity assay of Bacillus belyssus LT3212 Bacillus belye LT3212 was streaked onto Columbia blood agar plates and incubated at 28°C for 24-48 h. Hemolysis was observed around the colonies. Results are as follows: Figure 2 This indicates that the strain is non-hemolytic, has good biocompatibility, and meets the safety standards for agricultural microbial preparations.

[0032] Example 3: Growth of Bacillus belye LT3212 in an electrochemical carbon source (potassium acetate) Single colonies of *Bacillus belye* LT3212, activated by streak plating, were inoculated into LB broth medium supplemented with an electrochemical carbon source. The electrochemical carbon source replaced 50% of the yeast extract in the LB medium. The specific formulation was: 10 g tryptone, 2.5 g yeast extract (Huankai Biotechnology, catalog number: 050090), 10 g sodium chloride, and 2.5 g potassium acetate (electrochemical carbon source) per 1 L of LB medium. The control group consisted of LB medium. After incubation at 28°C for 16 h, the number of viable bacteria in the medium was determined.

[0033] The results are as follows Figure 3 As shown, after adding 50% yeast extract (Huankai Biotechnology, catalog number: 050090) to the electrochemical carbon source replacement medium, the effective viable count of Bacillus belyss LT3212 was 3.44 × 10⁻⁶. 7 ± 0.23×10 7 CFU / mL, the effective viable count in the control group LB medium was 3.53 × 10⁻⁶. 7 ± 0.28×10 7 The CFU / mL count and the number of viable cells were similar. These results indicate that strain LT3212 can maintain normal growth using an electrochemical carbon source (potassium acetate), and its number of viable cells was essentially the same as the control group. This characteristic reduces the strain's dependence on traditional organic carbon sources such as yeast extract, which is beneficial for reducing fermentation production costs and provides room for process optimization for its industrial-scale cultivation.

[0034] The electrochemical carbon source is potassium acetate powder, which is a non-grain carbon source prepared by catalytic reduction and conversion of industrial source CO2 or directly captured CO2 through an electrochemical reduction device. The industrial source CO2 can come from industrial settings such as thermal power plants and large chemical plants, or it can come from CO2 directly captured from the air or seawater. It is reduced by an electrochemical catalytic device to produce solid low-carbon product potassium acetate, which serves as an alternative carbon source for microbial culture.

[0035] Example 4: Fermentation process of Bacillus belye LT3212 The fermentation medium consisted of the following: 15 g / L corn steep liquor powder, 20 g / L corn starch, 5 g / L glucose, 30 g / L soybean flour, 10 g / L peptone, 0.1 g / L magnesium sulfate heptahydrate, 0.5 g / L sodium chloride, 0.5 g / L potassium dihydrogen phosphate, and 0.05 g / L manganese sulfate. The pH was adjusted to 7.2 with sodium hydroxide, and 0.01% antifoaming agent was added. The medium was sterilized at 121°C for 30 min before use.

[0036] A two-stage seed culture was employed. A single colony of *Bacillus belye* LT3212 was picked from an LB agar plate and inoculated into a 50 mL Erlenmeyer flask containing 10 mL of LB medium. The culture was incubated at 28°C and 220 rpm for 16 h with shaking to obtain the primary seed culture. The primary seed culture was then transferred at a 2% inoculation rate to a 500 mL Erlenmeyer flask containing 100 mL of LB medium and incubated at 28°C and 220 rpm for 12–16 h with shaking to obtain the secondary seed culture.

[0037] A 5 L fermenter was used, with a liquid volume of 2.5 L. The secondary seed culture was inoculated into the fermenter at a 4% inoculum rate. The fermentation temperature was controlled at 28℃, and the dissolved oxygen (DO) was set at 30%, maintained by adjusting the stirring speed (200~800 rpm) and aeration rate (0.5~2 vvm). No pressure control was performed on the glass fermenter. The initial pH after sterilization was approximately 6.8. pH was not controlled during the early stages of fermentation. After the pH naturally decreased and then rose back to 6.55, 500 g / L citric acid was used to control the pH at 6.50 to prevent a rapid pH rise that could reduce the viable cell count. When the number of spores reached 60% under microscopic examination, pH adjustment was stopped, allowing the pH to rise naturally. Spore formation was then examined every hour, and fermentation was terminated when the spore count exceeded 90%. The total fermentation time was approximately 22~24 hours.

[0038] The viable cell count in the fermentation broth can reach 76 × 10⁶. 8 CFU / mL (i.e., 7.6 billion CFU / mL), spore count can reach 50 × 10⁻⁶. 8 CFU / mL (i.e., 5 billion CFU / mL).

[0039] Example 5: Determination of stress resistance of Bacillus belyssus LT3212 The OD of the Bacillus berberis LT3212 fermentation broth prepared in Example 4 was adjusted. 600The pH was adjusted to 1.0 ± 0.05 as the test bacterial culture. Inoculation was performed at a rate of 2% (v / v) into the fermentation medium (adjusted to 0.1 mol / L HCl) at pH 5.0 as in Example 4, with three biological replicates per treatment. The culture was incubated at 28°C with shaking. Samples were taken continuously at 54-hour intervals after inoculation, and the absorbance (OD) of the bacterial culture at 600 nm was measured using a UV spectrophotometer. 600 (Value), and plot the growth curve of the strain.

[0040] like Figure 4 As shown, strain LT3212 showed an OD value of 24 hours. 600 The value reached 1.03 ± 0.01, indicating a relatively rapid growth rate; by 54 hours, OD... 600 The value remained at 0.88 ± 0.07, showing a slow decline. Throughout the observation period, the strain's OD... 600 The pH value remained consistently high, and the growth curve was flat and stable, demonstrating strong acid resistance and metabolic stability. This indicates that the strain can effectively survive and metabolize in acidic environments and has good growth capacity under pH 5.0 conditions.

[0041] Comparative Example 1: Determination of stress resistance of Bacillus belysin in commercially available products The same method as in Example 5 was used, except that *Bacillus lanceolata*, a commercially available bacterium with good resistance to tobacco red spot disease pathogens, was used to test its stress resistance. For example... Figure 4 As shown, the OD of the control strain at 24 hours 600 The value was only 0.66 ± 0.01, and although it increased somewhat afterward, it was still lower than that of Bacillus belyssus LT3212; by 54 hours, OD 600 The value decreased to 0.73 ± 0.01, indicating a significantly accelerated rate of decay. Throughout the observation period, the OD value of the control strain... 600 The pH value is relatively low and decays quickly, and its growth ability is relatively weak under acidic conditions of pH 5.0.

[0042] As demonstrated in Example 5 and Comparative Example 1 of this application, not all Bacillus belyes exhibit resistance to acidic environments. However, Bacillus belyes LT3212 demonstrates stronger resistance, more stable metabolic activity, and better survival adaptability in acidic environments.

[0043] Example 6: Utilization of Nicotinamide Precursors and Detection of Metabolites by Bacillus belyssus LT3212 The *Bacillus belyssioides* LT3212 fermentation broth prepared in Example 4 was transferred at a 1% inoculum to the fermentation medium of Example 4 supplemented with 0.5 mg / mL nicotinamide ribomatate (NR), 0.5 mg / mL β-nicotinamide mononucleotide (NMN), and 0.5 mg / mL reduced coenzyme I (NADH). A blank control without inoculation was also included. Each group was repeated three times. After incubation at 28°C and 220 rpm for 48 h with shaking, the fermentation broth was centrifuged at 4°C and 12000 rpm for 15 min. The supernatant was filtered through a 0.22 μm organic phase filter and analyzed by liquid chromatography. The chromatographic conditions were: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase of 15% acetonitrile-water solution containing 0.1% phosphoric acid and 5 mM sodium decanesulfonate, isocratic elution, detection wavelength of 260 nm, flow rate of 1.0 mL / min, column temperature of 30°C, and injection volume of 10 μL. Figure 5 As shown, LT3212 can utilize NR, NMN, and NADH in the culture medium, and the characteristic metabolites of nicotinic acid and nicotinamide can be stably detected in the fermentation broth. No obvious target product peaks were detected in the blank control. These results indicate that LT3212 can efficiently utilize nicotinamide precursors such as NR, NMN, and NADH to metabolize and produce nicotinic acid and nicotinamide, confirming its possession of the nicotinamide metabolic pathway. This metabolic pathway provides the strain with highly efficient NAD+. + / NADP + The synthesis and regeneration capabilities significantly enhance the survival rate of the strain under oxidative stress. Since the infection of tobacco scab pathogen induces the production of large amounts of reactive oxygen species in host leaves, creating a high-oxidative-stress microenvironment, the strain of this invention, with its aforementioned nicotinamide metabolic characteristics, can maintain a high viable bacterial count and metabolic activity at the site of disease occurrence, thereby continuously antagonizing the pathogen and maintaining a stable control effect. Therefore, this metabolic capacity is a crucial intrinsic basis for the excellent and stable control efficacy of the strain of this invention in the complex environment of tobacco fields.

[0044] Example 7 HPLC Detection and Analysis of Indole-like Growth-Promoting Metabolites from Bacillus belyssus LT3212 To verify the tryptophan metabolic pathway activity and the ability to synthesize growth-promoting functional metabolites in *Bacillus belyssiensis* LT3212 of this invention, this embodiment employs high-performance liquid chromatography (HPLC) to simultaneously qualitatively and quantitatively detect five indole derivatives in the strain's fermentation broth: L-tryptophan (Trp), indole-3-propionic acid (IPA), indole-3-acetic acid (IAA), indole-3-acetaldehyde (IAAld), and melatonin. The *Bacillus belyssiensis* LT3212 fermentation broth prepared in Example 4 was used, and the OD was adjusted... 600=1.00 ± 0.05 as seed culture, transferred at an inoculum rate of 1% (v / v) to the fermentation medium of Example 4 supplemented with 0.5 g / L L-tryptophan. An uninoculated culture medium of the same type was also used as a blank control. Each treatment was performed in triplicate. The culture was carried out at 28°C and 220 rpm in the dark with shaking. After 48 h of culture, samples were collected. The bacterial culture was centrifuged at 4°C and 12000 rpm for 15 min to collect the supernatant, which was then filtered through a 0.22 μm organic phase filter membrane before analysis. HPLC detection conditions were set as follows: mobile phase A was acetonitrile, and mobile phase B was aqueous solution. The gradient elution program was as follows: 0–1 min elution with 10% A + 90% B isocratic elution; 1–8 min linear gradient to 95% A + 5% B; 8–9.5 min elution with 95% A + 5% B isocratic elution; 9.6–12 min linear gradient recovery to 10% A + 90% B; 12–15 min elution with 10% A + 90% B. A 90%B balanced column was used, with a flow rate of 1.0 mL / min, a column temperature of 40℃, an injection volume of 10 μL, and a detection wavelength of 280 nm.

[0045] like Figure 6 As shown, the *Bacillus belyssus* LT3212 strain of this invention can efficiently utilize L-tryptophan in the culture medium to synthesize various indole-based growth-promoting and stress-resistance metabolites. This strain possesses the tryptophan metabolic pathway and can efficiently synthesize melatonin, indole-3-acetaldehyde, and other indole derivatives. Melatonin activates the antioxidant enzyme system in tobacco leaves, reducing oxidative damage caused by *Alternaria alternata* infection, thereby inhibiting the expansion of tobacco scab lesions. Indole-3-acetaldehyde, as a precursor for IAA synthesis, also participates in plant immune regulation, indirectly enhancing the plant's disease resistance. Therefore, effective control of tobacco scab is achieved from two levels: enhancing the plant's own disease resistance and reducing oxidative damage.

[0046] Example 8: Experiment on the casein production capacity of Bacillus belyssus LT3212 Take 10 μL of the *Bacillus belye* LT3212 fermentation broth prepared in Example 4 and spot it onto a protease detection medium plate. Use 10 μL of LB liquid as a control. Each treatment is repeated three times. After drying, incubate upside down at 28°C for 2 days and observe for the appearance of a clear zone. Figure 7 As shown, the control group plate showed no change, while the detection plate inoculated with strain LT3212 culture medium exhibited a clear hydrolysis zone around the inoculated colonies, indicating that the proteins in the plate were decomposed. This demonstrates that strain LT3212 has the ability to produce casein. This ability enables the strain to decompose protein-based organic nitrogen sources in the soil, releasing amino acids and small peptides that can be absorbed and utilized by plants, thereby promoting crop root development and nutrient absorption. This is one of the important mechanisms by which the strain of this invention achieves its growth-promoting function.

[0047] Casein assay medium: 10 g casein, 3 g beef extract powder, 2 g disodium hydrogen phosphate, 5 g sodium chloride, 0.05 g bromothymol blue, 15 g agar, pH adjusted to 7.4 ± 0.2, and distilled water brought to a final volume of 1000 mL.

[0048] Example 9: Cellulase Production Capacity Experiment of Bacillus belyssus LT3212 Take 10 μL of the *Bacillus belye* LT3212 fermentation broth prepared in Example 4 and inoculate it onto a Congo red carboxymethyl cellulose sodium agar plate. Use 10 μL of LB liquid as a control. Each treatment is repeated three times. After drying, incubate upside down at 28°C for 4 days, then observe for the presence or absence of a clear zone. Figure 8 As shown, the control group plate showed no change, while the detection plate inoculated with strain LT3212 culture medium exhibited a clear zone around the inoculated colonies, indicating that the cellulose in the plate was decomposed. This demonstrates that strain LT3212 possesses the ability to produce cellulase. This ability enables the strain to decompose cellulose-based organic matter in the soil, improve the rhizosphere microenvironment, and simultaneously provide a carbon source for its own growth, enhancing its colonization competitiveness in the soil. This is an important auxiliary mechanism for the strain of this invention to achieve rhizosphere colonization and long-lasting control efficacy.

[0049] Congo red sodium carboxymethyl cellulose medium: 20 g sodium carboxymethyl cellulose, 2.5 g disodium hydrogen phosphate, 1.5 g potassium dihydrogen phosphate, 2.5 g peptone, 0.5 g yeast extract, 20 g agar, pH adjusted to 7.0-7.2, and distilled water brought to a final volume of 1000 mL.

[0050] Example 10: Experiment on the heparin-producing capacity of Bacillus belyssus LT3212 Take 10 μL of the *Bacillus belyssioides* LT3212 fermentation broth prepared in Example 4, and spot it onto a CAS blue qualitative detection medium plate. Incubate at 28°C with the plate upside down for 3 days, and observe whether a yellow halo appears around the colony. Because the heparin competes with EDTA for iron ions in the medium, causing the medium to change from blue to yellow, the appearance of a yellow halo around the colony indicates the production of heparin. Figure 9 As shown, the yellow halo around the LT3212 colony demonstrates that strain LT3212 possesses the ability to produce heparin. Heparin can chelate iron ions in the environment, inhibiting the growth of iron-dependent pathogens (including Alternaria) on the one hand, and providing available iron to plants on the other, promoting chlorophyll synthesis. This ability gives the strain of this invention a dual function of nutrient competition and pathogen inhibition, which is one of the important mechanisms for the synergistic control of tobacco red spot disease.

[0051] CAS blue qualitative detection medium: acid-hydrolyzed casein 3 g, chromaine S 0.0605 g, cetyltrimethylammonium bromide 0.0729 g, ferric chloride hexahydrate 0.002645 g, disodium hydrogen phosphate 3.58 g, ammonium chloride 1 g, potassium dihydrogen phosphate 3 g, sodium chloride 0.5 g, magnesium sulfate 0.05 g, calcium chloride 0.01 g, agar 15 g, and distilled water to a final volume of 1000 mL.

[0052] Example 11: Experiment on nitrogen fixation capacity of Bacillus belyssus LT3212 Take 10 μL of the *Bacillus belyssioides* LT3212 fermentation broth prepared in Example 4, and inoculate it onto an Ashby medium plate. Incubate at 28°C inverted for 5-7 days, observing colony growth. A plate without inoculation serves as a negative control. Figure 10 As shown, LT3212 grows well on nitrogen-free medium, forming distinct colonies, indicating its nitrogen-fixing ability. This ability enables the strain to convert atmospheric molecular nitrogen into ammonia nitrogen that can be used by plants, providing natural nitrogen nutrition for crops such as tobacco, promoting robust plant growth, and thus indirectly enhancing tobacco's tolerance to red spot disease infection.

[0053] Ashube nitrogen-free medium: 0.2 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 0.2 g sodium chloride, 5 g calcium carbonate, 10 g mannitol, 0.1 g calcium sulfate, pH adjusted to 7.0 ± 0.1, and distilled water brought to a final volume of 1000 mL.

[0054] Example 12 Determination of the antibacterial spectrum of Bacillus belyss LT3212 The antibacterial activity of strain LT3212 was determined using the plate confrontation method: Using a 0.7 cm diameter punch, freshly cultured pathogenic fungal pellets on a PDA plate at 25°C were collected. The pellets were picked up with an inoculation needle and inoculated onto the center of the fresh PDA plate with the hyphae facing down. Then, 10 μL of *Bacillus belyssioides* fermentation broth prepared in Example 4 was inoculated approximately 2.5 cm away from the pellet. After drying, the plates were incubated upside down at 25°C. Plates inoculated only with the pathogen served as controls. Each treatment was replicated three times. The width of the inhibition band was observed and measured after 5-7 days of incubation. The inhibition rate was calculated using the formula: Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%.

[0055] like Figure 11As shown, strain LT3212 exhibited good inhibitory effects against all tested plant pathogenic fungi, with inhibition rates of 73.03% against Alternaria alternata, 61.73% against Rhizoctonia solani, 59.91% against Fusarium graminearum, and 54.64% against Fusarium oxysporum, indicating that strain LT3212 possesses broad-spectrum anti-plant pathogenicity capabilities.

[0056] Example 13 Pot experiment on the promotion of tobacco growth by Bacillus belyceta LT3212 Tobacco seeds were sown in seedling trays and cultured in a light incubator (light / dark duration 16 / 8 h, temperature 28℃). After 4 weeks of culture, tobacco seedlings with uniform growth were selected for growth promotion experiments.

[0057] Growth promotion experiment: The Bacillus berberis LT3212 fermentation broth prepared in Example 4 was diluted with distilled water to a concentration of 1×10⁻⁶. 8 CFU·mL -1 The bacterial suspension was applied as a root drench of tobacco plants at 20 mL for two consecutive weeks, for a total of two drenchments. Each treatment consisted of four plants, replicated three times, with distilled water used as a control. Growth was observed and recorded daily. On day 30 after root drenching, plant height, stem diameter, chlorophyll content, leaf area, and above-ground weight were measured. Results are as follows: Figure 12 As shown, tobacco plants treated with LT3212 bacterial suspension showed vigorous growth, with an average chlorophyll content (SPAD) of 23.54 ± 1.12, significantly higher than the control group's chlorophyll content of 20.06 ± 1.25; and an average aboveground part weight of 4.573 ± 0.52 g, significantly higher than the control group's aboveground part weight of 3.79 ± 0.24 g.

[0058] Example 14: Pot experiment on the promotion of rice growth by Bacillus belyceta LT3212 After surface disinfection and germination, rice seeds were soaked in distilled water for 24 hours and then sown in seedling trays. When the seedlings reached the three-leaf stage, strong seedlings with uniform growth were selected and transplanted into pots for growth promotion experiments.

[0059] Growth promotion experiment: The Bacillus berberis LT3212 fermentation broth prepared in Example 4 was diluted with distilled water to a concentration of 1×10⁻⁶. 8 CFU·mL -1 The bacterial suspension was applied as a root drench of rice at 20 mL for two consecutive weeks, for a total of two drenchments. Each treatment had nine biological replicates, three times in total, with distilled water used as a control. Growth was observed and recorded daily. On day 21 after root drench, plant height, chlorophyll content, and above-ground weight of the rice were measured. Results are as follows: Figure 13As shown, rice treated with LT3212 bacterial suspension for root irrigation exhibited vigorous growth, with an average chlorophyll content (SPAD) of 21.34 ± 1.94, significantly higher than the control group's chlorophyll content of 16.87 ± 2.34; an average plant height of 32.68 ± 2.41 g, significantly higher than the control group's plant height of 29.25 ± 2.66 g; and an average aboveground fresh weight of 0.484 ± 0.04 g, significantly higher than the control group's aboveground fresh weight of 0.382 ± 0.06 g.

[0060] Example 15: Pot experiment on the control of tobacco red spot disease by Bacillus belyceta LT3212 Healthy tobacco plants with uniform growth were selected, and three treatments were set up: a control group (sprayed with an equal volume of sterile water and then inoculated with pathogens), a treatment group (sprayed with 20 mL of the preparation in Example 4 and diluted to 1×10⁻⁶), and a treatment group (sprayed with 20 mL of the preparation prepared in Example 4 and diluted to 1×10⁻⁶). 8 Two weeks after foliar spraying with the bacterial suspension, Alternaria alternata spore suspension (1×10⁻⁶ CFU / mL) was inoculated with pathogens, and a blank control group (no bacterial suspension or pathogen inoculation) was also included. Three tobacco plants were included in each group. 6 Spores / mL), after inoculation, keep in a moist environment (temperature 25-28℃, relative humidity above 85%), inoculate each tobacco plant with 4 pathogen sites, investigate the disease index and calculate the control efficacy after 7 days.

[0061] Disease severity grading criteria: Grade 0, no lesions; Grade 1, lesion size 0-0.5 mm; Grade 2, lesion size 0.5-2.0 mm; Grade 3, lesion size 2.0-5.0 mm; Grade 4, lesion size 5.0 mm or larger. Disease index = Σ(number of diseased leaves at each grade × representative value for each grade) / (total number of leaves surveyed × 4) × 100. Control efficacy (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%. Results are as follows: Figure 14 As shown, the disease index of the control group was 40.10, while that of the treatment group was 23.96, with a control efficacy of 40%. This indicates that strain 3212 has a good control effect on tobacco red spot disease.

[0062] Example 16: Pot experiment on the promotion of rice growth by the combined application of Bacillus belye LT3212 and bio-organic fertilizer. After surface disinfection and germination, rice seeds were soaked in distilled water for 24 hours and then sown in seedling trays. When the seedlings reached the three-leaf stage, strong seedlings with uniform growth were selected and transplanted into pots for growth promotion experiments.

[0063] Growth-promoting experiment: A blank control group, a bacterial strain treatment group (root irrigation with LT3212 bacterial solution only), a bio-organic fertilizer treatment group (mixed with bio-organic fertilizer only), and a combined application treatment group (mixed with bio-organic fertilizer and root irrigation with LT3212 bacterial solution) were set up. Each treatment had 9 biological replicates, repeated 3 times. The bio-organic fertilizer was mixed with the soil according to the product instructions and then potted as a base fertilizer. The Bacillus belyes LT3212 fermentation broth prepared in Example 4 was diluted with distilled water to a concentration of 1×10⁻⁶. 8 CFU·mL -1 The bacterial suspension was used. 20 mL was applied to the roots of rice seedlings for two consecutive weeks after transplanting, for a total of two applications. The control group was treated with an equal volume of distilled water. Growth was observed and recorded daily. On the 21st day after root irrigation, the plant height and above-ground fresh weight of the rice were measured.

[0064] like Figure 15 As shown, rice plants treated with a combination of Bacillus vesiculosus LT3212 and bio-organic fertilizer exhibited excellent growth. The average plant height in the combined treatment group was 33.00 ± 2.59 cm, significantly higher than the control group's 23.01 ± 1.31 cm and the bio-organic fertilizer treatment group's 31.68 ± 2.54 cm. The average aboveground fresh weight reached 0.23 ± 0.04 g, significantly higher than the control group's 0.12 ± 0.02 g and the bio-organic fertilizer treatment group's 0.22 ± 0.03 g. These results indicate that strain LT3212 and bio-organic fertilizer have good compatibility. Their combined application can further improve rice growth indicators while maintaining the growth-promoting effect of bio-organic fertilizer, demonstrating synergistic potential.

[0065] All of the foregoing primary implementations of this intellectual property right do not limit other forms of implementation of this new product and / or new method. Those skilled in the art will utilize this important information to modify the foregoing to achieve similar implementations. However, all modifications or alterations based on this new product invention are reserved rights.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A Bacillus belesii strain suitable for non-grain bio-based carbon sources, characterized in that: The Bacillus berberis Bacillus velezensis It is named LT3212 and its accession number is GDMCC No. 67847.

2. The *Bacillus belye* strain with a non-grain bio-based carbon source as described in claim 1, characterized in that: The nucleotide sequence of the Bacillus belyssus is shown in SEQ ID NO.

1.

3. The fermentation method of Bacillus belye using a non-grain bio-based carbon source as described in claim 1 or 2, characterized in that, include: The first step is to prepare the fermentation medium, adjust the pH, and then sterilize it. The second step involves preparing seed culture using a two-stage liquid culture process, and then transferring the first-stage seed culture to amplify the second-stage seed culture. The third step is to inoculate the secondary seed liquid into the fermentation tank for aerobic fermentation, and control the fermentation temperature and dissolved oxygen. The fourth step involves phased pH control during the fermentation process: pH is not controlled in the early stage, and an acidic regulator is used to maintain pH stability after the pH naturally rises back to the set value. When the spore formation rate reaches the preset ratio, pH adjustment is stopped, and fermentation continues until the spores mature before stopping fermentation.

4. The fermentation method of Bacillus belye using non-grain bio-based carbon sources as described in claim 3, characterized in that: In the first step, the fermentation medium contains corn steep liquor powder, corn starch, glucose, soybean powder, peptone, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate, and manganese sulfate.

5. The fermentation method of Bacillus belye using a non-grain bio-based carbon source as described in claim 3 or 4, characterized in that: In the second step, both stages of seed culture were carried out using LB medium, and were cultured at 28°C with shaking to obtain primary and secondary seed solutions.

6. The fermentation method of Bacillus belye using a non-grain bio-based carbon source as described in claim 3 or 4, characterized in that: In the third step, the fermentation temperature is controlled at 28°C and the dissolved oxygen is maintained at 30%, which is achieved by adjusting the stirring speed and aeration rate.

7. The fermentation method of Bacillus belye using a non-grain bio-based carbon source as described in claim 3 or 4, characterized in that: In the fourth step, when the pH rises back to 6.55, citric acid is used to control the pH to 6.50; pH control is stopped when the spore formation rate reaches 60%, and fermentation is terminated when the spore rate is ≥90%.

8. A microbial inoculum containing *Bacillus belye* as described in claim 1 or 2, suitable for use with non-grain bio-based carbon sources, characterized in that: The microbial agent is a liquid microbial agent, a solid microbial agent, or a compound microbial agent, and its viable bacteria content is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

9. The *Bacillus belye* strain of claim 1 or 2, which is a suitable non-grain bio-based carbon source, is used in the preparation of a strain of *Alternaria* for treating tobacco scab. Alternaria alternata Rice damping-off pathogen Rhizoctonia solani Rhizoctonia solani Fusarium graminearum, the pathogen of wheat scab Fusarium graminearum Fusarium oxysporum, the pathogen that causes tomato wilt Fusarium oxysporum Application of preparations from plant pathogenic fungi.

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