Bacillus velezensis for biocontrol and growth promotion as well as preparation method and application of bacillus velezensis

Abstract

The invention belongs to the technical field of application of agricultural microorganisms, and particularly relates to bacillus velezensis as well as a preparation method and application thereof. The bacillus velezensis disclosed by the invention has good stress resistance, can resist salt, alkali and ultraviolet radiation with certain intensity, has the function of antagonizing pathogenic bacteria, has the highest inhibition rate of rhizoctonia solani up to 80.88%, and also has relatively high phosphate solubilizing capacity and IAA (Indoleacetic Acid) producing capacity. The bacillus velezensis disclosed by the invention can obviously promote the growth of rice, wheat, pepper, tomato, Chinese cabbage and the like and improve the biomass, shows a good growth promoting effect, and is widely applied as an agricultural soil inoculant. The bacillus velezensis disclosed by the invention has relatively good phosphorus dissolving, alkali resistance, salt resistance and high-temperature resistance, and can be used for reducing dependence of chemical fertilizers, improving salinized soil and promoting plant growth.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of application of agricultural microorganisms, and particularly relates to a bacillus velezensis with strong stress resistance and functions of promoting growth and biological control, and a preparation method and application thereof. BACKGROUND

[0002] The increase of grain yield has long relied on a large amount of chemical fertilizers and pesticides, but excessive application leads to soil compaction, secondary salinization, excessive nitrate in groundwater and the risk of pesticide residues in agricultural products, which seriously threatens the ecological environment and food safety. In particular, chemical fertilizers represented by phosphorus fertilizer are prone to combine with Ca 2+ , Al 3+ , Fe 3+ in the soil to form insoluble phosphates, which makes it difficult for plants to efficiently utilize phosphorus fertilizer and causes soil compaction and salinization. Therefore, it is necessary to develop environmentally friendly, long-lasting and effective agricultural products that can replace or reduce the use of chemical fertilizers and pesticides.

[0003] It has been explored that the use of growth-promoting microbial inoculants can serve as a practical alternative method. Plant growth-promoting rhizobacteria (PGPR) can colonize on or in the roots of crops, improve nutrient utilization and enhance plant stress resistance through multiple mechanisms such as nitrogen fixation, phosphorus solubilization, secretion of plant hormones (IAA) and induction of systemic resistance (ISR). Bacillus is widely distributed in nature and inside plants, shows effective colonization in the rhizosphere of plants, and Bacillus spp. is considered as one of the most promising PGPR groups due to its strong stress resistance, rich secondary metabolism and ease of industrial fermentation.

[0004] As an important member of PGPR, Bacillus has been reported in crops such as rice, tomato, pepper and corn, but under the influence of human and natural conditions, the harsh environment (such as soil salinization, strong ultraviolet pressure and temperature pressure) causes the adaptability of PGPR to decline, which in turn reduces its promotion effect on crop growth, and cannot fully exert the potential of Bacillus as a growth-promoting strain. Therefore, it is of great significance to develop a stress-resistant Bacillus, and to improve its survival rate in soil under adverse conditions, for reducing the use of chemical fertilizers and increasing yield in agriculture. SUMMARY

[0005] The present application is to overcome the defects of the prior art that Bacillus has poor adaptability and weak stress resistance in harsh environments, and provides a Bacillus velezensis for biological control and growth promotion, and a preparation method thereof, and applies it to reduce the dependence on chemical fertilizers, improve salinized soil and promote plant growth.

[0006] To achieve the above-mentioned application purposes, the present application realizes the following technical solutions: A bacillus velezensis for biocontrol and growth promotion, the preservation number of the bacillus velezensis for biocontrol and growth promotion is CCTCC NO: M20252291, and the bacillus velezensis for biocontrol and growth promotion is preserved in China Center for Type Culture Collection.

[0007] The application provides an anti-stress bacillus, which is a rhizosphere growth-promoting microorganism bacillus velezensis ZJB-2025241 (Bacillus velezensis) preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M20252291 and a preservation date of October 22, 2025.

[0008] The bacillus velezensis for biocontrol and growth promotion has excellent comprehensive anti-stress ability, can maintain a good survival and growth state under high-salt, wide-pH-range, strong ultraviolet and high-temperature conditions, and good environmental adaptability is a prerequisite for successful colonization and function in actual farmland, effectively overcoming the problem that the growth-promoting strain is unstable under stress. In addition, the bacillus velezensis for biocontrol and growth promotion can not only efficiently dissolve inorganic phosphorus fixed by soil and convert the inorganic phosphorus into an effective form that can be absorbed by plants, thereby improving the utilization rate of phosphorus fertilizer, but also can secrete IAA to promote plant growth. The two mechanisms jointly stimulate plant growth and development from two aspects of nutrient supply and internal physiological regulation. In addition, the bacillus velezensis for biocontrol and growth promotion also has a broad-spectrum antagonistic pathogenic fungus ability, and has obvious inhibitory effect on a plurality of pathogenic fungi causing soil-borne diseases, thereby endowing the bacillus velezensis for biocontrol and growth promotion with a biological control function.

[0009] A microbial agent, comprising the bacillus velezensis for biocontrol and growth promotion.

[0010] As preferred, the microbial agent is a liquid agent or a solid agent.

[0011] As preferred, the microbial agent comprises any one or a combination of a plurality of fermentation liquor, metabolite, culture, exosome, lysate and extract of the bacillus velezensis for biocontrol and growth promotion.

[0012] Application of the bacillus velezensis for biocontrol and growth promotion or the microbial agent in agricultural production.

[0013] As preferred, the application is to promote plant growth.

[0014] As preferred, the plant is a food crop, a vegetable or an economic crop.

[0015] As further preferred, the plant is at least one selected from rice, wheat, pepper, tomato and Chinese cabbage.

[0016] Preferably, the application is to increase the plant height, stem diameter, root length, fresh weight, dry weight, chlorophyll content, and soluble sugar content.

[0017] Preferably, the application is for the prevention and control of plant diseases.

[0018] Preferably, the disease is a soil-borne disease.

[0019] Preferably, the disease is caused by at least one of Rhizoctonia solani, Botrytis cinerea, and Fusarium graminearum.

[0020] Preferably, the application includes improving unfavorable conditions in the plant growth environment.

[0021] Preferably, the adverse conditions include at least one of high salt, high alkali, high acid, high temperature, and ultraviolet irradiation.

[0022] Preferably, the application is the production of plant hormones.

[0023] Preferably, the application is for dissolving insoluble phosphates.

[0024] Preferably, the insoluble phosphate is any one or a combination of Ca3(PO4)2, Fe3(PO4)2, and AlPO4.

[0025] A method for promoting plant growth includes root irrigation, direct spraying or fertigation of a bacterial solution of Bacillus vesicles ZJB-2025241 or its fermentation product for biocontrol and growth promotion as described above, onto plants, leaves, or soil.

[0026] Preferably, the application concentration is 1×10⁻⁶. 9 ~9×10 9 CFU / mL.

[0027] A method for preparing Bacillus belye for biocontrol and growth promotion as described above includes the step of culturing Bacillus belye for biocontrol and growth promotion.

[0028] Preferably, a method for preparing Bacillus belye for biocontrol and growth promotion as described above includes the following steps: The *Bacillus belye* strain used for biocontrol and growth promotion was streaked onto LB agar plates and incubated at 30 °C for 12 h. Single colonies were then picked and inoculated onto LB liquid medium, and subsequently cultured at 30 °C with shaking at 180 r / min until OD reached [value missing]. 600 The concentration was 0.6~0.8 to complete the activation. The activated Bacillus berberis used for biocontrol and growth promotion was inoculated into the culture medium for culture to obtain the culture.

[0029] Preferably, the culture medium used for cultivation contains peptone, yeast extract, sodium chloride, glucose, and magnesium sulfate.

[0030] Preferably, the culture medium used for cultivation contains 10.0 g / L peptone, 5.0 g / L yeast extract, 5.0 g / L sodium chloride, 2.0 g / L glucose, and 0.3 g / L MgSO4·7H2O.

[0031] As a preferred method, the cultivation conditions are: a cultivation temperature of 30-35 ℃, a shaking speed of 150-200 rpm, and a cultivation time of 18-24 h.

[0032] Therefore, the present invention has the following beneficial effects: (1) The Bacillus berleis, used for biocontrol and growth promotion in this invention, has good stress resistance, can resist salt, alkali and a certain intensity of ultraviolet radiation, has the function of antagonizing pathogens, and the inhibition rate against Rhizoctonia solani can reach up to 80.88%. It also has high phosphorus solubility and IAA production capacity. (2) The Bacillus beryl of the present invention for biocontrol and growth promotion can significantly promote the growth of rice, wheat, pepper, tomato, cabbage and other crops, increase biomass and show good growth promotion effect. It has a wide range of applications as an agricultural soil inoculant. (3) The Bacillus berreatus of the present invention for biocontrol and growth promotion has good phosphorus solubility, alkali resistance, salt resistance and high temperature resistance, and can be used to reduce dependence on chemical fertilizers, improve saline soil and promote plant growth. Attached Figure Description

[0033] Figure 1 This image shows the growth of Bacillus belyssus ZJB-2025241 on an inorganic phosphorus plate.

[0034] Figure 2 This is the IAA standard curve.

[0035] Figure 3 This is a standard curve for phosphorus.

[0036] Figure 4 Phylogenetic tree of Bacillus belyssus ZJB-2025241 based on 16S rDNA sequence.

[0037] Figure 5 Plate image showing the antagonistic effect of Bacillus belyssus ZJB-2025241 against Rhizoctonia solani and Botrytis cinerea.

[0038] Figure 6 The image shows the cell morphology of the prepared Bacillus belyssus ZJB-2025241 liquid inoculum.

[0039] Figure 7 This is a comparison chart of the growth of potted Chinese cabbage inoculated with Bacillus vesicles ZJB-2025241, the control group, and the commercial growth-promoting bacterial agent group.

[0040] Figure 8 This is a comparison chart of growth indicators between the group inoculated with Bacillus vesicles ZJB-2025241 in a potted cabbage experiment, the control group, and the commercially available growth-promoting bacterial agent group.

[0041] Figure 9 The graph shows the chlorophyll content of the group inoculated with Bacillus vesicles ZJB-2025241 in a potted cabbage experiment, the control group, and the commercial growth-promoting bacterial agent group.

[0042] Figure 10 The graph shows the difference in soluble sugar content between the group inoculated with Bacillus vesicles ZJB-2025241 in a potted cabbage experiment, the control group, and the commercial growth-promoting bacterial agent group.

[0043] Figure 11 The graph shows the control effect of treatment group 2, the control group, and treatment group 1 on tomato wilt disease. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0045] Example 1: Isolation and Screening of Rhizosphere Growth-Promoting Microorganisms 1. Sample collection Plant rhizosphere soil was collected from near the Biology Building on the Moganshan Campus of Zhejiang University of Technology, the riverbank in the off-campus industrial park, and near the West Annex Building on the Zhaohui Campus. At each sampling point, tall and healthy plants were taken, the above-ground parts of the plants were removed, and the roots and root soil were placed in sterile bags and placed in foam boxes with ice packs. The samples were processed within 24 hours.

[0046] 2. Strains Isolation and Screening Bring the plant rhizosphere soil back to the laboratory, gently shake off the soil adhering to the roots, and weigh 10 g of the root system with a small amount of soil attached into a 90 mL sterile Erlenmeyer flask containing physiological saline. Add 5 sterile glass beads to the flask, shake on a shaker (180 r / min) for 30 min, and let it stand for 10 min. Next, prepare a sterile test tube and add 9 mL of physiological saline, then add 1 mL of the soil suspension to it to prepare a 10 mL solution.-2 Prepare a soil gradient dilution solution; then, take 1 mL of the suspension from the previous test tube and add it to another identical 9 mL sterile test tube. Repeat this process to prepare 10 different solutions. -3 10 -4 and 10 -5 Soil gradient dilution solution.

[0047] The prepared soil gradient dilutions were streaked three times onto LB solid medium and incubated at 37 °C for 24 h. Single colonies with significant morphological differences were selected. These single colonies were inoculated into LB liquid medium containing L-tryptophan, and strains producing plant hormones (IAA) were screened using the Salkowski colorimetric method. The obtained single colonies were then inoculated onto Monkina inorganic phosphate medium (PVK medium), and the phosphorus-solubilizing function of the strains was qualitatively characterized based on the clear zones on the PVK plates. The growth of *Bacillus belyssiensis* ZJB-2025241 on inorganic phosphate plates is shown in the figure below. Figure 1 As shown.

[0048] from Figure 1 Analysis showed that Bacillus belye ZJB-2025241 could produce a clear zone after being inoculated on PVK solid medium and cultured statically for 5 days, indicating that the strain has phosphate solubilization function.

[0049] Preparation of LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, agar powder 15 g / L, adjust pH to 7.

[0050] Phosphorus solubilization capacity test medium (PVK medium, with no change in dosage when changing phosphorus source): glucose 10 g / L, (NH4)2SO4 0.5 g / L, MgSO4·7H2O 0.3 g / L, FeSO4·7H2O 0.03 g / L, MnSO4·H2O 0.03 g / L, Ca3(PO4)2 5 g / L, NaCl 0.3 g / L, KCl 0.3 g / L; adjust pH to 7, and add 15 g / L agar powder to the solid medium.

[0051] (1) Initial screening of IAA-producing strains: Screening of IAA-producing bacteria: The isolated and purified bacteria were inoculated into LB liquid medium containing L-tryptophan (100 mg / L) and cultured in the dark with shaking at 30 ℃ and 180 r / min for 24 h. Then, 300 μL of the bacterial suspension was transferred to a sterile 1.5 mL Eppendorf tube, along with 300 μL of Salkowski colorimetric solution (50 mL 35% HClO4 + 1 mL 0.5 mol / L FeCl3). An equal volume of uninoculated liquid medium was added as a control. The thoroughly mixed Eppendorf tubes were placed at room temperature in the dark, and the results were observed after 30 min. A red color indicated that the bacteria were capable of producing IAA.

[0052] (2) Secondary screening of IAA-producing bacteria: Quantitative determination of IAA-producing strains was performed under the same culture conditions as above. OD values ​​were measured immediately after color development. 530 The value was zeroed using CK with added colorimetric solution, and the standard curve was prepared using analytical grade IAA through serial dilutions. The IAA standard curve is shown below. Figure 2 As shown.

[0053] (3) Determination of phosphorus solubility activity of strains - molybdenum antimony colorimetric method: (a) Plotting the phosphorus standard curve Phosphorus standard solution: Weigh 0.4394 g KH2PO4, dry for 2 h, cool and dissolve in 200 mL deionized water, add 5 mL concentrated sulfuric acid, transfer to a 1 L volumetric flask and make up to volume to obtain a 100 mg / L phosphorus standard solution.

[0054] Accurately pipette 0, 1, 2, 4, 6, 8, and 10 mL of phosphorus standard solution into separate 50 mL stoppered colorimetric tubes. Add 5 mL of molybdenum-antimony anti-chromic reagent, then add water to the mark and mix well. After 30 min, using tube 0 as a blank, measure the absorbance at 700 nm. Plot a phosphorus standard curve with the measured absorbance as the ordinate and the soluble phosphorus content in the solution as the abscissa. The prepared phosphorus standard curve is shown below. Figure 3 As shown.

[0055] (b) Determination of phosphorus solubilization effect The OD of the seed culture was adjusted to 0.8, and 2% of the culture was inoculated into Erlenmeyer flasks containing PVK medium. 24 h after inoculation, 5 mL of the inoculum was added to a 50 mL colorimetric tube, diluted with water to 3 / 5 of the total volume, and 5 mL of molybdenum antimony anti-chromic solution was added. The mixture was shaken well, brought to a final volume, and the absorbance was measured at 700 nm after 30 min. The phosphorus solubilization effect of the strain was calculated based on the phosphorus standard curve. The strain screening results are shown in Table 1 below.

[0056] Table 1: Comparison of functions of rhizosphere growth-promoting strains Serial number Strain IAA production capacity (mg / L) Dissolved Ca3(PO4)2activity (mg / L) Dissolved Fe3(PO4)2activity (mg / L) Dissolved AlPO4activity (mg / L) 1 MGS-5 — 27.47 4.28 15.65 2 MGS-8 7.95 8.90 — — 3 SWL-12 5.51 9.23 5.13 7.10 4 ZH-8 — 4.81 — 1.61 5 ZJB-2025241 28.31 167.42 27.30 67.42 6 GJ-6 11.99 30.50 13.53 18.20 7 GJ-15 12.45 — — — 8 GJ-18 3.85 25.30 10.58 15.19 .

[0057] Analysis of the data in Table 1 shows that the Bacillus belye strain ZJB-2025241 of this invention has significantly higher IAA production and phosphate solubilization capabilities than other strains.

[0058] Example 2: Molecular identification of Bacillus belyssus strain ZJB-2025241 The genus and species of the isolated and screened bacterial strain ZJB-2025241 were identified using 16S rDNA gene sequence analysis. DNA extraction: Strain DNA was extracted using the FastDNA® SPIN Kit bacterial genomic DNA extraction kit. Using the obtained bacterial DNA as a template, the universal primer pair 27F / 1492R was used. 27F (5'-AGAGTTTGATCCTGGCTCAG- 3'); 1492R (5'-TACGGTTACCTTGTTACGACTT-3'); The 16S rDNA sequence was amplified and detected by electrophoresis. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for 16S rDNA gene sequencing. The successfully sequenced sequences were assembled into a single sequence, and txt and FASTA format files were created for Blast homology comparison. The highest similarity (99.79%) was found with Bacillus velezensis strain CBMB205, and strain ZJB-2025241 was identified as Bacillus velezensis. A phylogenetic tree was then constructed using the Neighbor-Joining method in MEGA (10.2.4) software. The phylogenetic tree of Bacillus velezensis ZJB-2025241 based on the 16S rDNA sequence is shown below. Figure 4 As shown in the image. This strain was deposited on October 22, 2025, at the China Center for Type Culture Collection (address: Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China), with accession number CCTCC NO: M20252291. The 16S rDNA sequence of Bacillus belyssus ZJB-2025241 is shown in SEQ ID NO.1.

[0059] from Figure 4 Analysis shows that by performing nucleic acid sequence homology comparison (Blastn) in the GenBank database on NCBI, the preliminary identification results show that the strain belongs to the genus Bacillus.sp. The strain ZJB-2025241 has the highest homology with strain Bacillus velezensis strain CBMB205, reaching 99.79%.

[0060] Example 3: Evaluation of salt (NaCl) tolerance of Bacillus belyssus strain ZJB-2025241 Using LB as the basal medium, the concentration of sodium chloride in the medium was adjusted to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, and 9%, respectively. The medium was then inoculated with 1% OD200. 600 ZJB-2025241 bacterial suspension at 0.8 μL was cultured at 37 °C and 180 rpm, with uninoculated culture media at various concentrations as controls. OD values ​​were measured at 3 h, 6 h, 12 h, and 24 h after inoculation. 600 The results are shown in Table 2 below.

[0061] Table 2: Effects of different concentrations of NaCl in the culture medium on the growth of the strains

[0062] Analysis of the data in Table 2 shows that Bacillus belye strain ZJB-2025241 still grows at a 9% NaCl concentration, indicating that it can tolerate high-salt adverse environments.

[0063] Example 4: Study on the acid and alkali resistance of Bacillus belyssus ZJB-2025241 strain The pH gradient of LB medium was adjusted to 4, 5, 6, 7, 8, and 9, and then inoculated with 1% OD. 600 0.8% ZJB-2025241 bacterial culture was incubated in LB medium at 37 ℃ and 180 rpm. OD values ​​were measured at 3 h, 6 h, 12 h, and 24 h after inoculation. 600 The results are shown in Table 3 below.

[0064] Table 3: Effects of different pH values ​​on bacterial growth .

[0065] Analysis of the data in Table 3 shows that the Bacillus vesiculus strain ZJB-2025241 can grow at pH 4 to 9, indicating that the Bacillus vesiculus strain ZJB-2025241 of this invention has a certain acid and alkali resistance.

[0066] Example 5: Study on UV resistance of Bacillus belyssus strain ZJB-2025241 Bacillus belye strain ZJB-2025241 was activated and its bacterial suspension was irradiated under a UV lamp for 0, 1, 2, 3, 4 and 5 min, respectively. The suspension was then inoculated onto LB agar plates using the dilution plating method and incubated at 37 ℃ for 24 h. A control group without UV irradiation was used. Viable bacterial counts were determined using the plate count method.

[0067] Table 4: Effects of Different Ultraviolet Irradiation Times on the Growth of Strains Irradiation time (min) Survival rate (%) 0 100 1 86.93 2 68.88 3 45.52 4 34.65 5 25.13 。

[0068] From the data analysis in Table 4, it can be seen that the Bacillus velezensis ZJB-2025241 strain remains active under ultraviolet irradiation, indicating that it has strong tolerance and can well adapt to adverse environments.

[0069] Example 6: Study on the High Temperature Tolerance Characteristics of Bacillus velezensis ZJB-2025241 Strain The bacterial liquid prepared by activating the Bacillus velezensis ZJB-2025241 strain was treated at 40, 50, 60, 70, and 80 °C for 5 min respectively, then inoculated on the LA medium plate by the dilution coating method, placed in a 37 °C incubator for 24 h, and the non-temperature treatment after culturing at 35 °C was used as a control. The viable bacteria count was determined by the plate counting method.

[0070] Table 5: Effects of Different Temperature Treatments on the Growth of Strains Temperature (℃) Survival rate (%) 35 100 40 97.33 50 90.5 60 86.6 70 78.21 80 70.4 。

[0071] From the data analysis in Table 5, it can be seen that the Bacillus velezensis ZJB-2025241 strain still maintains high activity after being treated at 80 °C for 5 min, indicating that it has strong tolerance and can well adapt to high temperature environments.

[0072] Example 7: Antibacterial Ability Test of Bacillus velezensis ZJB-2025241 Strain Select a single colony of Bacillus velezensis ZJB-2025241, inoculate and culture it, take a fresh mycelium block at the edge of the fungal colony, transfer it to the center position of a clean PDA medium plate, and spot the ZJB-2025241 sample around the pathogen. Among them, the PDA medium formula is 6 g / L potato extract powder, 20 g / L glucose, and 15 g / L agar powder. At the same time, inoculating sterile water is used as a blank control. These plates are placed in an incubator at 28 °C for culturing. When culturing for 7 days, measure the colony diameter. Each group of experiments is repeated 3 times, and the inhibition rate is calculated. The results are shown in Table ⑥ below. The plate diagrams of Bacillus velezensis ZJB-2025241 antagonizing Rhizoctonia solani and Botrytis cinerea are as Figure 5 shown. Among them, [[ID=​​​​​ .

[0074] From the data in Table 6 and Figure 5 Analysis shows that in the sterile water group cultured for 7 days, all pathogens grew well and covered the surface of the culture medium. The inhibitory effect of Bacillus berberis ZJB-2025241 of this invention was 48.2% against Colletotrichum gloeosporioides, while the inhibition rate of the other pathogens was above 57%, confirming that ZJB-2025241 has a good broad-spectrum antibacterial effect.

[0075] Example 8: Preparation of Bacillus belyssus ZJB-2025241 bacterial suspension (1) After overnight activation, Bacillus belye strain ZJB-2025241 was picked and cultured in LB medium at 30 ℃ and 200 rpm for 10-12 h as seed inoculum. The seed inoculum was then transferred at 2% (v / v) to the following medium for culture: peptone: 10.0 g / L, yeast extract 5.0 g / L, NaCl 5.0 g / L, glucose 2.0 g / L, MgSO4·7H2O 0.3 g / L. The culture conditions were: 35 ℃, 180 rpm, culture for 24 h.

[0076] (2) Collect bacterial cells by centrifugation at 8000 rpm for 10 min. Wash the bacterial cells 1-2 times with sterile 0.9% sodium chloride solution, and then resuspend the bacterial cells with sterile 0.9% sodium chloride solution to adjust the concentration to 10. 9 The concentration of CFU / mL was then used as a liquid microbial inoculum (i.e., Bacillus belyssus ZJB-2025241 bacterial suspension inoculation preparation) and observed under a microscope. The bacterial morphology of the prepared Bacillus belyssus ZJB-2025241 liquid inoculum is shown in the figure below. Figure 6 As shown.

[0077] from Figure 6 Analysis revealed that, compared with the short rod-shaped morphology observed under an optical microscope, the morphological characteristics of the Bacillus vesiculosus ZJB-2025241 bacterial inoculation preparation prepared in this invention are uniformly straight or slightly curved short rods, indicating that the prepared Bacillus vesiculosus ZJB-2025241 bacterial inoculation preparation was not contaminated by other bacteria.

[0078] Example 9: Experiment on promoting growth of potted Chinese cabbage 1. Seed treatment Select plump, uniformly sized Chinese cabbage seeds, disinfect them with 6% sodium hypochlorite for 5 minutes, rinse them three times with distilled water, and germinate them in sterile vermiculite at room temperature. After 7 days of germination, select seedlings with uniform growth and place them in small pots (7 cm in diameter and 10 cm in height) containing 100 g of sterile substrate (vermiculite and soil 1:1, v / v), with one seedling per pot. A total of 3 groups were set up: 1 blank group (i.e., control group) and 2 experimental groups, with each group being tested in parallel 5 times. Spraying with sterile water served as a blank control. The experimental setup is shown in Table 7 below.

[0079] Table 7: Potted Plant Experiment Group Settings Serial number Treatment Bacterial liquid concentration CK Sterile water — Promoting growth of bacteria agent commodity Promoting growth of bacteria agent commodity 10 9 CFU / mL ZJB-2025241 ZJB-2025241 bacterial liquid 10 9 CFU / mL .

[0080] 2. Preparation of Bacillus vesiculosus ZJB-2025241 bacterial inoculation formulation: The preparation method of the Bacillus vesiculosus ZJB-2025241 bacterial inoculum preparation is the same as that in Example 8.

[0081] Inoculum application: The inoculum was applied by root drenching, with 10 mL per pot, every 7 days, applied to the roots of the plants, for a total of 2 applications. For the control treatment, 10 mL of sterile aqueous solution was applied to the roots of the plants. The cabbages were harvested 25 days after sowing.

[0082] 3. Results of the growth-promoting effect Plant height reflects plant growth vitality, measured from the ground to the top using a ruler. Higher plant height generally indicates better vertical growth, reflecting more active cell elongation and division. Furthermore, there is a positive correlation between plant height and yield in cabbage. Higher plant height usually means more above-ground biomass accumulation, resulting in higher yields at harvest. Similarly, taller plants have thicker stems, measured using calipers. The growth of the cabbage was recorded after 20 days of growth. The growth comparison of the group inoculated with Bacillus thuringiensis ZJB-2025241 in potted cabbage, the control group, and the group treated with a commercial growth-promoting agent is shown in the figure below. Figure 7 As shown.

[0083] Figure 7 The results showed that the growth of the experimental groups (the commercial growth promoter group and the ZJB-2025241 group) was significantly better than that of the control group (CK group). The plant height of both experimental groups treated with the growth promoter was higher than that of the CK group (control group), with the ZJB-2025241 treatment showing the highest height. The plant height after inoculation with the Bacillus belyssus ZJB-2025241 was the highest, significantly increasing by 14.84% compared to the commercial growth promoter.

[0084] Root length is a key indicator of Chinese cabbage's adaptation to soil conditions. The growth of its root system directly affects the growth and yield of the above-ground parts. Longer roots mean a more developed root system, enabling better absorption of water and nutrients from the soil, thus providing sufficient material for the growth of the stems and leaves. Therefore, research on the root length of Chinese cabbage plants has a significant impact on yield. The following is a comparison of growth indicators between the group inoculated with Bacillus thuringiensis ZJB-2025241 bacterial solution, the control group, and the group treated with a commercial growth-promoting agent in a potted Chinese cabbage experiment. Figure 8 As shown.

[0085] from Figure 8 Analysis showed that inoculation with different rhizosphere growth-promoting bacteria increased the root length of Chinese cabbage. The root length of Chinese cabbage treated with Bacillus belycera var. spp. ZJB-2025241 was significantly longer than that of the control (CK), increasing by 174.4%, and significantly higher than that of the commercial growth-promoting bacteria agent by 57.93%.

[0086] The dry weight per plant of Chinese cabbage directly reflects the total amount of organic matter accumulated by the plant during its growth process and is a key indicator for measuring the degree of nutrient accumulation. A higher dry weight per plant indicates that the Chinese cabbage has synthesized and stored a large amount of organic matter such as carbohydrates, proteins, and fats through photosynthesis. The fresh weight and dry weight of Chinese cabbage plants after application of rhizosphere growth-promoting microorganisms are shown in the figure. Figure 8 .

[0087] Figure 8 The results showed that the fresh weight and dry weight of plants treated with growth-promoting bacteria were higher than those of the control group. The fresh weight of a single plant treated with Bacillus belyssus ZJB-2025241 bacterial inoculation preparation reached 1.549g, which was 6.38% higher than that of the commercial growth-promoting bacteria group.

[0088] 4. Quality determination of Chinese cabbage Chlorophyll content is related to photosynthesis and provides energy for plant metabolism; therefore, this study investigated whether the strain could increase chlorophyll content. Soluble sugars are not only a source of energy but also participate in various metabolic processes within plants. High sugar content usually means higher nutritional and commercial value. Active accumulation of soluble sugars by plants can also enhance stress resistance. This sugar accumulation not only helps plant survival but may also indirectly affect the sugar content at fruit ripening, thus influencing the final quality. 0.2 g of fresh young leaves were weighed and placed in a mortar. A small amount of quartz sand and calcium carbonate powder were added, followed by 2-3 mL of 95% ethanol. The sample was ground into a homogenate, and then 10 mL of ethanol was added and ground until the tissue turned white. Chlorophyll a (C) content was then measured at 663 nm and 645 nm using a UV-Vis spectrophotometer. a ), chlorophyll b (C b The chlorophyll content of the group inoculated with Bacillus berberis ZJB-2025241 bacterial solution, the control group, and the group treated with commercial growth-promoting bacteria in the potted cabbage experiment is shown in the figure below. Figure 9As shown. The formula for calculating chlorophyll content is as follows: C a (mg / g) = (12.7A663 − 2.69A645)×(V / m×1000); C b (mg / g) =(22.9A645 − 4.68A663)×(V / m×1000); C (a + b) (mg / g) = C a + C b ; V represents the volume of the extraction solution used (mL), and m represents the fresh weight of the sample (g).

[0089] Soluble sugar content was determined using the anthrone colorimetric method. The sample was chopped, homogenized with a small amount of ddH2O, and then centrifuged at 4℃ and 8000 rpm for 10 min. The supernatant was collected and repeatedly extracted with ddH2O. The combined extraction solution was adjusted to a final volume of 10 mL, and 5 mL of a 10 g / L anthrone-sulfuric acid reaction solution was added to 1 mL of the extract. A calibration curve was constructed using glucose standard solutions ranging from 0 to 100 mg / L. The absorbance was measured at 620 nm using a UV-Vis spectrophotometer, and the soluble sugar content was calculated. The difference in soluble sugar content between the *Bacillus belycera* ZJB-2025241 inoculated group, the blank group, and the commercially available growth-promoting agent group in the potted Chinese cabbage experiment is shown in the figure below. Figure 10 As shown.

[0090] from Figure 9~10 Analysis showed that the chlorophyll and soluble sugar content of the bacterial agent treatment group (i.e., the experimental group) were higher than those of the blank group (i.e., the control group). The effects of the bacterial strain on improving the quality of Chinese cabbage can be reflected by the two indicators of chlorophyll content and soluble sugar content.

[0091] Example 10: Study on the control efficacy of Bacillus belyceta var. salina ZJB-2025241 against Fusarium wilt in tomato Select plump, uniformly sized tomato seeds, disinfect them with 6% sodium hypochlorite for 5 minutes, rinse them three times with distilled water, and germinate them in sterile vermiculite at room temperature. After 7 days of germination, select seedlings of uniform growth and place them in small pots (7 cm in diameter, 10 cm in height) containing 100 g of sterile substrate (vermiculite and soil 1:1, v / v), one seedling per pot. A total of 3 groups were set up: 1 control group and 2 experimental groups, with each group tested in parallel 5 times. Spraying with sterile water served as the control (CK group), spraying with *Fusarium oxysporum* was treatment 1, and drenching the roots with *Bacillus vesiculosus* ZJB-2025241 bacterial suspension 7 days after spraying with *Fusarium oxysporum* was treatment 2. After 30 days, the disease incidence and growth of tomatoes were recorded, and the specific data are shown in Table 8 below. The control effects of treatment 2, the control group, and treatment 1 on tomato wilt disease are shown in the figure below. Figure 11 As shown.

[0092] Disease severity classification is as follows: Grade 0: Disease-free leaves; Grade 1: Diseased leaves ≤ 25% of total leaves; Grade 2: Diseased leaves account for 25% to 50% of the total number of leaves; Grade 3: Diseased leaves account for 50% to 75% of the total leaves; Level 4: Diseased panicles account for 75% to 100% of the total number of leaves.

[0093] Table 8: Growth of Tomatoes in Pot Experiments Serial number Disease level Plant height (cm) Stem diameter (mm) Plant dry weight (g) CK 0 7.43±0.87 2.73±0.38 1.074±0.03 Treatment 1 4 5.35±0.66 1.43±0.29 0.722±0.05 Treatment 2 2 8.88±0.80 2.35±0.90 1.212±0.04 .

[0094] From Table 8 and Figure 11 Data analysis shows that applying Bacillus vesiculosus ZJB-2025241 inoculum can control Fusarium oxysporum during tomato growth, reducing the disease grade of tomatoes from level 4 to level 2. Simultaneously, Bacillus vesiculosus ZJB-2025241 inoculum also promotes tomato growth. In terms of plant height, treatment 2 increased by 19.51% compared to the control (CK) and by 65.98% compared to treatment 1. In terms of plant dry weight, treatment 2 increased by 12.85% compared to the control (CK) and by 67.88% compared to treatment 1.

[0095] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A Bacillus belye strain for biocontrol and growth promotion, characterized in that, The Bacillus belye used for biocontrol and growth promotion has the accession number CCTCC NO:M20252291 and is deposited at the China Center for Type Culture Collection.

2. A microbial inoculant, characterized in that, It includes Bacillus belye, as described in claim 1, for biocontrol and growth promotion.

3. The application of Bacillus berberis, as described in claim 1, for biocontrol and growth promotion, or the microbial agent as described in claim 2, in agricultural production.

4. The application according to claim 3, characterized in that, The application is to promote plant growth.

5. The application according to claim 3, characterized in that, The application is for the prevention and control of plant diseases.

6. The application according to claim 3, characterized in that, The applications include improving unfavorable conditions in the plant growth environment.

7. The application according to claim 3, characterized in that, The application is for the production of plant hormones.

8. The application according to claim 3, characterized in that, The application is for dissolving insoluble phosphates.

9. A method for promoting plant growth, characterized in that, This includes applying the bacterial solution or fermentation product of Bacillus vesicles of claim 1 for biocontrol and growth promotion by root irrigation, direct spraying or fertigation onto plants, leaves, or soil.

10. A method for preparing *Bacillus belye* for biocontrol and growth promotion as described in claim 1, characterized in that, This includes the steps of culturing Bacillus belye for biocontrol and growth promotion.

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