Bacillus velezensis strain nj157 and application thereof

Abstract

The application provides a bacillus velezensis NJ157 and application thereof, and relates to the technical field of biological pesticides.The bacillus velezensis NJ157 provided by the application can resist salt and alkali, has good inhibitory effects on ralstonia solanacearum, rhizoctonia solani, fusarium oxysporum and phytophthora capsici, and can effectively prevent and treat tomato bacterial wilt, root rot and fruit rot.The bacillus velezensis NJ157 provided by the application also has strong abilities of producing protease, cellulase and IAA, and has good growth promotion effects on tomatoes.The biocontrol agent prepared by using the biocontrol agent does not cause pollution to the ecological environment and has no public hazards in the use process.The use of the biocontrol agent can reduce the use amount of other chemical pesticides, reduce the prevention and control cost of tomato diseases, promote the growth of tomatoes, and has good popularization value.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, and in particular to a strain of Bacillus belye NJ157 and its applications. Background Technology

[0002] The greenhouse vegetable production model, characterized by low input, low energy consumption, intensification, and year-round cultivation, has led to increasingly severe continuous cropping obstacles and high incidence of diseases, becoming a bottleneck restricting the sustainable utilization of greenhouse vegetables in my country. Examples include tomato gray mold caused by *Fusarium oxysporum* infection and tomato late blight caused by *Phytophthora*. Currently, the control of pathogenic microorganisms mainly includes physical, chemical, and biological control. However, most methods still rely on traditional chemical control, such as the use of organophosphate pesticides, organoarsenic compounds, and benzene pesticides. While these chemicals are fast-acting, long-term use can lead to increased resistance in pathogens, harm human health, and pollute the environment, contradicting the concept of sustainable development. Biological control is a newly emerging method in recent years. Because it produces no environmental pollution, leaves no pesticide residues, and meets the requirements for pollution-free vegetable production, research in this area is increasing, and biocontrol bacteria are gaining popularity.

[0003] Currently, most newly identified unknown microorganisms are isolated from ordinary habitats, and the number of new species is gradually decreasing. Due to the unique geological and climatic characteristics of Antarctica, which creates a natural environment of low temperature, high salinity, and strong radiation, polar microorganisms possess unique molecular biological mechanisms and physiological and biochemical characteristics in terms of gene composition, enzymatic properties, and metabolic regulation. Antarctica is the origin of novel and uniquely functional chemical substances worldwide. Polar microorganisms have great potential in the application of biopesticides. Therefore, discovering different strains of polar microorganisms for the biological control of plant diseases is of great significance for the development and utilization of polar microbial resources. However, no salt-tolerant strains with biocontrol functions isolated from Antarctica have been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus belyssus NJ157 and its applications. The Bacillus belyssus NJ157 was isolated from the soil of a penguin colony near the Great Wall Station earthquake station in Antarctica. It is resistant to low temperatures and salinity, and can inhibit various pathogens and tomato diseases, thus solving the problem of limited functionality of strains isolated from ordinary habitats in existing technologies.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a strain of Bacillus belye NJ157, characterized in that it is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31984, deposit date of September 14, 2024, and deposit address of No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides a method for preparing the bacterial culture of Bacillus vesiculus NJ157, wherein Bacillus vesiculus NJ157 is inoculated into a fermentation medium for fermentation culture to obtain the bacterial culture.

[0008] Preferably, the fermentation temperature is 28–32°C, the fermentation speed is 180–220 rpm, and the fermentation time is 22–26 h.

[0009] The present invention also provides the use of the Bacillus belyssus NJ157 in all or part of the following (1) to (6):

[0010] (1) Application in inhibiting pathogenic oomycetes, pathogenic bacteria and pathogenic fungi;

[0011] (2) Application in suppressing tomato diseases;

[0012] (3) Application in promoting tomato growth in severely saline-alkali soil;

[0013] (4) Applications in the breakdown of proteins or cellulose;

[0014] (5) Application in the preparation of tomato growth promoters in severely saline-alkali soil;

[0015] (6) Application in the preparation of microbial agents that decompose proteins or cellulose.

[0016] Preferably, the pathogenic oomycete includes *Phytophthora capsici*; the pathogenic bacteria include *Pseudomonas syringae* and *Ralstonia solanacearum*; and the pathogenic fungi include *Botrytis cinerea*, *Fusarium moniliforme*, *Fusarium solanum*, and *Rhizoctonia solani*.

[0017] Preferably, the tomato diseases include bacterial wilt, root rot, and fruit rot.

[0018] The present invention also provides a microbial agent comprising a bacterial suspension of Bacillus belye NJ157 as described in claim 1.

[0019] Preferably, the spore concentration in the bacterial suspension is 1–9 × 10⁻⁶. 7 CFU / mL.

[0020] The present invention also provides a method for promoting tomato growth in severely saline-alkali soil, wherein the tomato plants are treated with root irrigation using the aforementioned microbial agent.

[0021] By adopting the above technical solution, the present invention has the following beneficial effects: The *Bacillus vesiculus* NJ157 of the present invention is tolerant to saline-alkali conditions and exhibits good inhibitory effects against *Ralstonia solanacearum*, *Rhizoctonia solani*, *Fusarium moniliforme*, and *Phytophthora capsici*, effectively controlling bacterial wilt, root rot, and fruit rot in tomatoes. The *Bacillus vesiculus* NJ157 of the present invention also possesses a strong ability to produce protease, cellulase, and IAA, and has a good growth-promoting effect on tomatoes. The inoculant prepared using this biocontrol bacterium will not pollute the ecological environment during use and is harmless. Using the biocontrol bacterium of the present invention can reduce the amount of other chemical pesticides used, lower the cost of tomato disease control, and promote tomato growth, thus having good promotional value. Attached Figure Description

[0022] Figure 1 Phylogenetic tree of Bacillus belyss NJ157.

[0023] Figure 2 The diagram shows the inhibitory effect of Bacillus belyssus NJ157 on pathogens, where A is Ralstonia solanacearum, B is Pseudomonas syringae, C is Botrytis cinerea, D is Phytophthora capsici, E is Fusarium moniliforme Sheld, and F is Rhizoctonia solani.

[0024] Figure 3 The image shows the results of the salt stress tolerance test of Bacillus belyss NJ157. A and B are the phenotypic results of salt tolerance (11%) and salt tolerance (13%) plate of Bacillus belyss NJ157, FZB42, 168, and DMW1, respectively.

[0025] Figure 4 The image shows the results of the alkaline stress resistance test of Bacillus belyss NJ157. A and C are the alkali resistance (pH=8, 9, 10) plate phenotype results of Bacillus belyss NJ157, FZB42, 168, and DMW1, respectively.

[0026] Figure 5 The HPLC-MS identification results of secondary metabolites of Bacillus belyssus NJ157 are shown in the figure.

[0027] Figure 6The image shows the antibacterial effect of crude extracts of secondary metabolites of Bacillus belyssus NJ157; where A represents Ralstonia solanacearum, B represents Pseudomonas syringae, C represents Phytophthora capsici, D represents Rhizoctonia solani, and E represents Botrytis cinerea.

[0028] Figure 7 The image shows the detection results of protease, cellulase, amylase, and indoleacetic acid (IAA) produced by Bacillus belyssus NJ157. In the image, A represents protease, B represents cellulase, C represents amylase, and D represents indoleacetic acid.

[0029] Figure 8 This image shows the disease control effect of Bacillus vesicularis NJ157 on bacterial wilt infection in tomato plants.

[0030] Figure 9 This image shows the disease control effect of Bacillus vesicles NJ157 against P. capsici infection on tomato plants.

[0031] Figure 10 This image shows the disease control effect of Bacillus belye NJ157 against P. capsici infection on tomato fruits.

[0032] Figure 11 This image shows the disease control effect of Bacillus belye NJ157 against Fusarium solani infection in tomato fruit.

[0033] Figure 12 The graph shows the effect of Bacillus belyss NJ157 on the germination of tomato seeds. A represents the effect of Bacillus belyss NJ157 on the germination of tomato seeds; BC represents the effect of NJ157 on the root length and shoot length of germinating tomato seeds.

[0034] Figure 13 The graph shows the effect of Bacillus vesiculosus NJ157 on the growth of tomato plants. A is the effect of Bacillus vesiculosus on tomato plant height and root length; B and C are statistical graphs showing that Bacillus vesiculosus can increase tomato plant height, root length, chlorophyll content, stem diameter, above-ground fresh weight, above-ground dry weight, root fresh weight, and root dry weight.

[0035] Figure 14The graph shows the effect of Bacillus vesiculosus NJ157 on the growth of tomato plants under salt stress. A is the effect of Bacillus vesiculosus NJ157 on tomato plant height and root length under salt stress; B1 and B2 are statistical graphs showing that Bacillus vesiculosus NJ157 can increase tomato plant height, root length, chlorophyll content, stem diameter, aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight under salt stress.

[0036] Biological Preservation Instructions

[0037] The Bacillus velezensis NJ157 provided by this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31984, deposit date of September 14, 2024, and address of No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1. Taxonomic analysis of Bacillus belyssus NJ157 based on its core genome.

[0040] Bacillus belye NJ157 strain was inoculated into liquid LB medium and incubated at 37°C for 12 hours at 200 rpm. The bacterial cells were collected by centrifugation, and genomic DNA was extracted using a bacterial extraction kit (Beijing Zhuangmeng International Biotechnology Co., Ltd.). The 16S rDNA gene was then amplified by PCR using forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 3) and reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 4). The PCR conditions were: 95°C for 5 min; 95°C for 15 s; 56°C for 1 min; 72°C for 1 min; 32 cycles; 72°C for 10 min; and stored at 4°C. The amplification of the 16S rRNA fragment was observed by gel electrophoresis, and the PCR stock solution was sent for sequencing. The 16S rRNA gene sequence is shown in SEQ ID No. 1.

[0041] 16S rRNA gene sequence:

[0042]

[0043] PCR amplification and sequencing of the gyrB gene

[0044] Forward primer gyrB-F:

[0045] 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3'

[0046] (SEQ ID No. 5);

[0047] Reverse primer gyrB-R:

[0048] 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'

[0049] (SEQ ID No. 6), the PCR reaction conditions for the gyrB gene are the same as above. The partial gene sequence of gyrB is approximately 1500 bp in length, and the result is shown in SEQ ID No. 2 of the sequence listing.

[0050] gyrB gene sequence:

[0051]

[0052] 16S rDNA and gyrB identification results showed that strain NJ157 belongs to *Bacillus velezensis*. Secondly, based on the genome sequence of NJ157 and 31 housekeeping genes from all samples, 19 strains most closely related at the species level were selected, and a phylogenetic tree was constructed using MEGA 6.0 software selection. The results showed that strain NJ157 is most closely related to *Bacillus velezensis* in evolutionary distance. These results confirm that strain NJ157 belongs to *Bacillus velezensis*.

[0053] Example 2. Determination of the antibacterial activity of Bacillus belyssus NJ157

[0054] Preliminary assays were performed to determine the antibacterial activity of Bacillus belyi NJ157. Several representative pathogenic bacteria, fungi, and oomycetes were selected. The pathogenic bacteria included Ralstonia solanacearum and Pseudomonas syringae; the pathogenic fungi included Botrytis cinerea, Fusarium moniliforme Sheld, and Rhizoctonia solani; and the oomycetes included Phytophthora capsici.

[0055] Determination of the antibacterial activity of Bacillus belysium NJ157 against pathogenic bacteria: First, the pathogenic bacteria were activated. A single colony of the pathogenic bacteria was picked and added to 20 mL of the corresponding culture medium, and incubated overnight at 30°C and 200 rpm. LB or NA solid medium was heated and cooled to body temperature. The bacterial suspension was then added to the medium at a concentration of 1%, and after thorough mixing, the culture was poured into a plate containing the bacteria. 5 μL of Bacillus belysium bacterial suspension (OD500) was dropped onto the solidified culture plate at a position 2.5 cm from the center. 600 =2.0), LB culture medium was used as a control. After being fully dried, the culture medium was placed upside down in a 30℃ constant temperature incubator. The antibacterial effect was observed after 24 hours, and the diameter of the transparent inhibition zone was measured.

[0056] Determination of the antifungal activity of Bacillus belyss NJ157 against pathogenic fungi: A certain number of mycelial cakes were punched from the outermost edge of the original pathogenic fungal culture plate using a sterile punch with a diameter of approximately 0.5 cm. The mycelial cakes (with the mycelial side) were then transferred to the center of a new PDA culture plate and incubated at a suitable temperature for 10-18 hours until new mycelia grew around the mycelial cakes. Afterward, sterile filter paper was placed at the same distance from the mycelial cakes, and 5 μL of Bacillus belyss NJ157 bacterial suspension (OD) was added to the center of the filter paper.600 =2.0), LB culture medium was used as a blank control. After drying, the culture was returned to the 25℃ incubator. The antibacterial effect was observed after 48 hours. The results are as follows: Figure 2 As shown in the figure. The results indicate that *Bacillus belye* NJ157 has good antagonistic effects against various plant pathogenic fungi and bacteria, with the best inhibitory effects against *Ralstonia solanacearum*, *Rhizoctonia solani*, and *Fusarium moniliforme*. These results suggest that *Bacillus belye* NJ157 has good biocontrol efficacy and can be used as a biocontrol strain for further research.

[0057] Example 3. Determination of the stress resistance of Bacillus belyssus NJ157

[0058] Antarctica is a permafrost environment. Bacillus species that survive and can grow and reproduce normally in this environment may have a strong ability to resist abiotic stress. Therefore, the abiotic tolerance ability of Bacillus belesiensis NJ157 was determined.

[0059] 1. Determination of salt stress tolerance of Bacillus belyss NJ157

[0060] The salt stress resistance of Antarctic strain NJ157, model strain 168, FZB42, and DMW1 was determined by simulating different salt stresses (salt gradients: 11% and 13% NaCl concentrations). Single colonies of the tested strains were picked and cultured in LB liquid medium at 37°C and 200 rpm overnight for 24 h until the bacterial concentration reached OD200. 600 When the concentration was 2.0, 5 μL of bacterial culture was transferred to LB solid medium with different salt gradients (11%, 13%). The medium was then placed in a 37°C incubator. After 5 days, the colony growth of each strain under different salt gradients was observed. The biological replicate was performed three times. The results are as follows: Figure 3 As shown in the figure. The results indicate that the Antarctic strain NJ157 can grow under salt stress of 13%, while the model strain FZB42 can only tolerate salt stress of 11% NaCl. The salt stress tolerance of the Antarctic strain NJ157 is higher than that of the model strain FZB42.

[0061] 2. Determination of the alkaline stress tolerance of Bacillus belyssus NJ157

[0062] Different alkaline stresses (pH gradients: 8, 9, 10) were simulated to determine the resistance of *Bacillus belyssiensis* NJ157, model strain 168, FZB42, and DMW1 to alkaline stress. Single colonies of the tested strains were picked and placed in LB broth, incubated overnight at 37°C and 200 rpm for 24 h on a shaker, until the bacterial concentration reached OD500. 600When the pH was 2.0, 5 μL of bacterial culture was transferred to LB solid medium with different alkalinity gradients (pH 8, 9, and 10). The medium was then placed in a 37°C incubator. After 5 days, the colony growth of each strain under different salt gradients was observed. The biological replicate was performed three times. The results are as follows: Figure 4 As shown in the figure. The results indicate that Bacillus belye NJ157 can grow under strong alkaline stress conditions at pH 10, demonstrating a strong ability to withstand alkaline stress.

[0063] Example 4. Extraction, identification, and antibacterial activity determination of secondary metabolites of Bacillus belyssus NJ157

[0064] 1. Extraction and identification of secondary metabolites of Bacillus belyssus NJ157

[0065] Activate Bacillus belye NJ157 on LB solid medium and incubate at 37°C until single colonies grow; pick a single NJ157 colony and add it to 20 mL. In LB broth, the culture was incubated overnight at 37°C with shaking at 200 rpm. The next day, 1% of the *Bacillus belye* NJ157 bacterial suspension was transferred to 200 mL of LB broth and incubated at 37°C with shaking at 200 rpm for 24 h. 5 mL of resin (XAD-16N) was added to each NJ157 bacterial suspension, and the culture was continued with shaking for another 24 h. The next day, the bacterial suspension was centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected. The bacterial cells were dissolved in 30 mL of methanol, and the bacterial suspension was then incubated at 37°C with shaking at 200 rpm for 4 h. The bacterial suspension was centrifuged at 8000 rpm for 10 min, the supernatant was collected and filtered through filter paper. The supernatant was concentrated to 2 mL using a rotary evaporator, and the crude extract was filtered through a 0.22 μm organic filter. The production of lipopeptides was identified and detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The results are as follows: Figure 5 As shown in the figure, high performance liquid chromatography-mass spectrometry identification results showed that Bacillus belye NJ157 can produce iturin, fengycin, and surfactant secondary metabolites.

[0066] 2. Determination of the antibacterial effect of secondary metabolites of Bacillus belyssus NJ157

[0067] From the original pathogenic fungal culture plate, a certain number of mycelial cakes were punched at the outermost edge of the colony using a sterile punch with a diameter of approximately 0.5 cm. The mycelial cakes (with the mycelial side) were then transferred to the center of a new PDA culture plate and incubated in a suitable temperature incubator for 10-18 hours until new mycelia grew around the mycelial cakes. Afterwards, holes were punched at the same distance from the mycelial cakes using a sterile punch with a diameter of 0.5 cm. 50 μL of secondary metabolites of *Bacillus belyssae* strain was added to the center of each hole, with methanol solution used as a control. After drying, the plates were returned to the incubator, and the antibacterial effect was observed after 48 hours. The results are as follows. Figure 6 As shown in the figure. The plate confrontation experiment results showed that the secondary metabolites of Bacillus belye NJ157 had good antagonistic effects on a variety of plant pathogenic fungi and bacteria, among which the inhibitory effect on Ralstonia solanacearum, Rhizoctonia solani and Botrytis cinerea was the best.

[0068] 3. Determination of the ability of Bacillus belyssus NJ157 to produce extracellular hydrolases

[0069] Qualitative detection of protease production by Bacillus belyssus NJ157 was performed on skim milk agar plates: The ability of Bacillus belyssus to produce protease was determined in agar medium containing skim milk powder (20g skim milk powder, 20g agar, pH 7.0, sterilized at 115℃ for 10min). Single colonies of Bacillus belyssus were picked and incubated in LB broth at 37℃ and 200 rpm overnight for 12 h; 5 μL (OD) of protease was added to the broth. 600 A bacterial suspension of 1.0 g (e.g., 1.0 g) was incubated in a 37°C incubator. After 24-96 hours, the presence of a clear zone around the colonies was observed. The results were as follows: Figure 7 As shown in A in the diagram.

[0070] The cellulase production capacity of *Bacillus belyssiensis* NJ157 was determined using CMC-Na medium: Single colonies of *Bacillus belyssiensis* were picked and incubated overnight at 37°C and 200 rpm for 12 h. Sterile filter paper discs with a diameter of 4 mm were placed in a cross shape on CMC-Na plates; 5 μL of bacterial suspension (OD) was then drawn. 600 =1.0), inoculate the plate in the center of a filter paper disc, seal the plate, and incubate it in a 37℃ incubator; after 2 days, remove the petri dish, pour Gram's iodine stain solution to submerge the surface of the plate, let it stand for 4 minutes, then pour off the stain solution, and observe whether a clear zone forms around the colony. If a clear hydrolysis zone appears around the bacterial cell, it indicates that the strain can produce the corresponding enzyme; if no clear hydrolysis zone appears, it indicates that the strain cannot produce this type of enzyme. The results are as follows. Figure 7 As shown in B in the diagram.

[0071] Detection of amylase activity in Bacillus belyssus NJ157 on starch agar medium: Colonies of Bacillus belyssus were picked and incubated in LB liquid medium at 37°C and 200 rpm overnight for 12 h. 5 μL of bacterial suspension (OD200) was then added dropwise. 600 =1.0) to starch-containing culture medium, and incubate at 37℃; after 2 days, remove the petri dish, pour in Gram's iodine stain to submerge the surface of the plate, let it stand for 4 minutes, then pour off the stain, observe and record the experimental results, as shown in the figure. Figure 7 As shown in C.

[0072] The results showed that strain NJ157 could produce large transparent hydrolysis zones in skim milk powder medium and CMC-Na medium, indicating that it can produce proteases and cellulases.

[0073] 4. Determination of IAA production capacity of Bacillus belyssus NJ157

[0074] Preparation of Salkowski colorimetric solution: Weigh 0.81 g of FeCl3 and dissolve it in 10 mL of ddH2O. Ensure complete dissolution and mix thoroughly to obtain a 0.5 mol / L stock solution. Prepare a 35% perchloric acid solution, then add the 0.5 mol / L FeCl3 stock solution at a ratio of 1:50 and mix thoroughly.

[0075] First, the bacteria were cultured in LB medium at 37°C and 180 rpm to obtain a stock solution. Then, the stock solution was transferred at a concentration of 1% to LB liquid medium containing 100 mg / L tryptophan. After incubation at 37°C and 180 rpm for 24 hours, 1 mL of the bacterial culture was centrifuged at 12000 rpm to collect the supernatant. An equal volume of Salkowski colorimetric solution was added to the supernatant, mixed thoroughly, and reacted in the dark for 30 minutes. If the solution turned red, it indicated that the bacteria could produce IAA. An equal volume of LB liquid medium containing 100 mg / L tryptophan was used as a blank control. The results are as follows. Figure 7 As shown in D in the figure. The results indicate that Bacillus belye NJ157 can produce IAA, and the IAA production capacity of strain NJ157 is similar to that of the model strain FZB42, indicating that it has great potential in promoting plant growth and development and has the potential to be developed into a biocontrol agent.

[0076] Example 5. Determination of the control efficacy of Bacillus vesiculosus NJ157 against tomato diseases.

[0077] 1. Determination of the control efficacy of Bacillus belyceta var. nigra NJ157 against bacterial wilt of tomato.

[0078] Sterilized seedling substrate was placed into 50-cell seedling trays, with one tomato seed of similar growth stage placed in each cell. The seed was planted root-down in the soil using tweezers and covered with a thin layer of soil. After two weeks of cultivation at 28℃, one uniformly grown tomato seedling was retained in each cell of the tray and transplanted using 350mL plastic cups. After one month of cultivation in a 28℃ greenhouse, tomato seedlings of similar growth stage were selected for disease prevention experiments. *Bacillus belye* NJ157 and the model bacterium FZB42 were activated on LB solid medium. Single colonies were picked with a sterile toothpick and placed in 20mL of LB liquid medium, incubated overnight at 37℃ and 200rpm with shaking. The next day, a 1% inoculum was transferred, and after 4-5 hours of cultivation, the OD of the bacterial solution was measured. 600 Adjust to 0.5(10) 7 (CFU / mL) was prepared for use. Tomato seedlings were treated with root drenching, with 12 seedlings per treatment. Each seedling was drenched with 10 mL of Bacillus belye bacterial solution. Sterile water was used as a control. The seedlings were placed in a 30℃ greenhouse. After 7 days, the same root drenching treatment was repeated, while simultaneously treating the roots with root damage by drenching each seedling with 10 mL of OD (CFU / mL) solution. 600 A 0.1% solution of Ralstonia solanacearum was used. Five days later, the disease incidence on the tomato plants was observed and recorded, and the incidence rate was calculated. The results are as follows: Figure 8 As shown in Table 1.

[0079] Incidence rate (%) = (Number of infected plants / Total number of plants) × 100%

[0080] Disease grading survey standards:

[0081] 0: No wilting;

[0082] 1:1%-25% of leaf area wilting;

[0083] 2:26%-50% of leaf area wilted;

[0084] 3:51%-75% of leaf area wilted;

[0085] 4:76%-100% of leaf area wilts or dies.

[0086] Disease index (%) = [∑(disease grade value × number of plants at that disease grade) / (highest grade number × number of plants surveyed)] × 100.

[0087] Prevention and control effect = (disease index of blank control - disease index of treatment) / disease index of blank control × 100%.

[0088] Table 1. Control efficacy of Bacillus belyceta var. nigra NJ157 against bacterial wilt of tomato.

[0089]

[0090] Note: Columns with the same letter indicate no significant difference (P>0.05), while columns with different letters indicate significant differences (P<0.05).

[0091] The experimental results showed that Bacillus vesicles NJ157 treatment of tomato plants reduced the incidence of bacterial wilt in tomatoes, with an incidence rate of 61.11%, which was 22.22% lower than the blank control group. In addition, the disease index of tomatoes treated with Bacillus vesicles NJ157 was significantly reduced, and the control effect on bacterial wilt in tomatoes reached 56.18%. NJ157 has a very good control effect on bacterial wilt.

[0092] 2. Determination of the control efficacy of Bacillus belyceta var. nigra NJ157 against tomato root rot

[0093] Sterilized seedling substrate was placed into 50-cell seedling trays, with one tomato seed of similar growth stage placed in each cell. The seed was planted root-down in the soil using tweezers and covered with a thin layer of soil. After two weeks of cultivation at 25℃, one uniformly grown tomato seedling was retained in each cell of the tray and transplanted using 350mL plastic cups. After one month of cultivation in a 25℃ greenhouse, tomato seedlings of similar growth stage were selected for disease prevention experiments. *Bacillus belye* NJ157 and the model bacterium FZB42 were activated on LB solid medium. Single colonies were picked with a sterile toothpick and placed in 20mL of LB liquid medium, incubated overnight at 37℃ and 200rpm with shaking. The next day, a 1% inoculum was transferred, and after 4-5 hours of cultivation, the OD of the bacterial solution was measured. 600 Adjust to 0.5(10) 7 (CFU / mL) was prepared for use. Tomato seedlings were treated with root drenching, with 12 seedlings per treatment. Each seedling was drenched with 10 mL of *Bacillus belye* solution. Sterile water was used as a control. After 7 days, the same root drenching treatment was repeated, and root damage was also performed. Four *Phytophthora capsici* fungal discs were inoculated at the root zone of each seedling for infection. After 5 days, the disease incidence was observed and recorded, and the disease rate was calculated. The results are as follows: Figure 9 As shown in Table 2.

[0094] Disease grading survey standards:

[0095] 0: No wilting;

[0096] 1:1%-25% of leaf area wilting;

[0097] 2:26%-50% of leaf area wilted;

[0098] 3:51%-75% of leaf area wilted;

[0099] 4:76%-100% of leaf area wilts or dies.

[0100] Disease index (%) = [∑(disease grade value × number of plants at that disease grade) / (highest grade number × number of plants surveyed)] × 100.

[0101] Prevention and control effect = (disease index of blank control - disease index of treatment) / disease index of blank control × 100%.

[0102] Table 2. Control efficacy of Bacillus belyceta var. nigra NJ157 against tomato root rot.

[0103]

[0104] Note: Columns with the same letter indicate no significant difference (P>0.05), while columns with different letters indicate significant differences (P<0.05).

[0105] The experimental results showed that treatment of tomato plants with Bacillus vesicles NJ157 reduced the incidence of tomato root rot. The disease incidence was lowest in the Bacillus vesicles NJ157-treated plants (55.55%), a 25% reduction compared to the control group. The Bacillus vesicles NJ157 treatment significantly reduced the root rot disease index, demonstrating the best control effect on tomato root rot, reaching 54.33%.

[0106] 3. Determination of the disease control ability of Bacillus vesalis NJ157 against Phytophthora capsici on tomato fruit.

[0107] Bacillus belye NJ157 and the model strain FZB42 were activated on LB solid medium. Single colonies were picked with a sterile toothpick and placed in 20 mL of LB liquid medium, incubated overnight at 37°C and 200 rpm. The next day, 1% was transferred, and after 4-5 hours of incubation, the OD of the bacterial culture was measured. 600 Adjust to 0.5 (5×10) 7 (CFU / mL) for later use. Activate the preserved *Phytophthora capsici* on a PDA plate, and obtain mycelial discs using a pipette tip. Disinfect the surface of tomato fruits of roughly the same size using 75% anhydrous ethanol. After disinfection, invert the tomato fruits and immerse them in different treatment solutions for 30 minutes. Remove them and place them upright in a perforated tray (with a layer of sterile water at the bottom). Allow the tomato surface to air dry naturally. Using a sterile syringe, gently poke a hole in the top of the tomato fruit. Place the obtained *P. capsici* mycelial discs tightly against the hole and wrap them with absorbent cotton to retain moisture. Wrap with plastic wrap and place in a 25℃ greenhouse. After 4 days, observe the disease incidence on the tomato fruits and conduct a disease severity survey. The disease severity classification criteria are shown in Table 3. The survey results are as follows: Figure 10 As shown in Table 4.

[0108] Table 3 Disease Grading Survey Standards

[0109]

[0110]

[0111] Disease index (%) = [∑(disease grade value × number of plants at that disease grade) / (highest grade number × number of plants surveyed)] × 100.

[0112] Prevention and control effect = (disease index of blank control - disease index of treatment) / disease index of blank control × 100%.

[0113] Table 4. Control efficacy of Bacillus belyceta var. NJ15 against Phytophthora capsici on tomato fruits.

[0114]

[0115] Note: Columns with the same letter indicate no significant difference (P>0.05), while columns with different letters indicate significant differences (P<0.05).

[0116] Experimental results showed that Bacillus belye NJ157 treatment of tomato plants inhibited the disease of P. capsici on tomato fruits. The disease index of tomato fruits treated with strain NJ157 was significantly reduced to 29.17%, and the control effect on tomato bacterial wilt was the best, reaching 58.82%.

[0117] 4. Determination of the resistance of Bacillus vesiculosus NJ157 to Fusarium solani (fruit rot) on tomato fruit.

[0118] Bacillus belye NJ157 and the model strain FZB42 were activated on LB solid medium. Single colonies were picked with a sterile toothpick and placed in 20 mL of LB liquid medium, incubated overnight at 37°C and 200 rpm. The next day, 1% was transferred, and after 4-5 hours of incubation, the OD of the bacterial culture was measured. 600 Adjust to 0.5 (5×10) 7(CFU / mL) was prepared for later use. The preserved *Fusarium solani* was activated on a PDA plate, and the mycelium was collected using a pipette tip. Tomato fruits of similar size were surface-sterilized with 75% anhydrous ethanol. After sterilization, the tomatoes were inverted and immersed in different treatment solutions for 30 minutes, then removed and placed upright in a perforated tray (with a layer of sterile water at the bottom). The different treatment solutions were allowed to air dry naturally. A small hole was gently made at the top of the tomato fruit using a sterile syringe. The collected *F. solani* mycelium was then placed tightly against the hole, and the mycelium was wrapped with absorbent cotton to retain moisture. The fruit was then wrapped with plastic wrap and placed in a 25°C greenhouse to observe subsequent disease development. The diseased area of ​​*F. solani* on the tomato fruit was calculated, and the results are as follows: Figure 11 As shown in Table 5.

[0119] Table 5. Control efficacy of Bacillus belyceta var. nigra NJ157 against fruit rot on tomato fruits.

[0120]

[0121] Note: Columns with the same letter indicate no significant difference (P>0.05), while columns with different letters indicate significant differences (P<0.05).

[0122] The experimental results showed that Bacillus vesicles NJ157 treatment of tomato plants inhibited the disease of F. solani on tomato fruits. The disease index of tomato fruits treated with Bacillus vesicles NJ157 was significantly lower than that of tomatoes treated with CK. Bacillus vesicles had the best control effect on tomato fruit rot, reaching 81.75%.

[0123] Example 6. Investigation on the growth-promoting effect of Bacillus belye NJ157 on tomato

[0124] 1. Effects of Bacillus belye NJ157 bacterial suspension on tomato seed germination

[0125] Tomato seed treatment: Select healthy, plump, and uniformly sized tomato seeds. Soak them in 30% sodium hypochlorite solution for 2 minutes, then soak them in 75% alcohol solution for 30 seconds. Rinse them 5 times with sterile water. Add 50 μL of the rinsing solution to PDA solid medium and LB solid medium, spread it evenly with a sterile spreader, and incubate at 25℃ for 24 hours. If no colonies appear, the seed treatment is complete.

[0126] The *Bacillus belye* bacterial culture was washed twice with water to remove secondary metabolites produced by the strain, and then diluted to obtain a final concentration of 10. 7CFU / mL of Bacillus belysin suspension was used. Sterilized tomato seeds were placed in seed germination bags, and 25 mL of Bacillus belysin suspension was injected into the bags. The seed germination bags were placed in a 25℃ greenhouse. After 7 days, samples were collected, and the root and shoot lengths of the tomato seedlings were measured. The results are as follows: Figure 12 As shown.

[0127] The results showed that Bacillus belye NJ157 had a significant growth-promoting effect on tomato seeds. Strain NJ157 had the best effect on promoting tomato root length, with a growth-promoting rate as high as 52.64%. Strain FZB42 had a growth-promoting rate of 20.21% on tomato shoots, while strain NJ157 had a growth-promoting rate of 35.19%. The growth-promoting rate of Bacillus belye NJ157 on tomato growth was higher than that of the model strain FZB42, indicating that this strain has a good growth-promoting effect.

[0128] 2. Determination of the growth-promoting effect of Bacillus belyssus NJ157 on tomato plants

[0129] Tomato seed disinfection and germination: Select intact and plump seeds, soak them in 30% sodium hypochlorite solution for 120 seconds, then soak them in 75% ethanol for 30 seconds, and then rinse them repeatedly with ddH2O 5 times until there is no irritating odor remaining on the surface of the tomato seeds; place sterile filter paper in a 9cm sterilized glass petri dish, add an appropriate amount of sterile water with a pipette so that the filter paper in the dish is completely wetted with water; use sterile tweezers to pick up the tomato seeds and place them on the filter paper, making sure that there is a certain distance between the seeds so that they can germinate; after sealing the glass dish, place it in a 25℃ greenhouse and cultivate it in the dark for 3-5 days. Once the radicle has grown, it can be transplanted to 1 / 2MS.

[0130] Planting tomato seedlings: After sifting the black soil, mix it evenly with vermiculite in a 1:1 ratio. Sterilize twice at 121℃ for 20 minutes under high temperature and pressure. Use a hole punch with a diameter of about 2-3cm to make a hole in the center of a disposable plastic cup. Then add 80% of the mixture of black soil and vermiculite. Use tweezers to pick up the tomato seeds that have grown radicles and plant them in the soil with the roots facing down. Cover the top with another layer of soil. Place the disposable plastic cup in a tray with an appropriate amount of water and place it in a greenhouse at 28℃ for about 20 days.

[0131] Root irrigation treatment with Bacillus vesicles NJ157 fermentation broth: After the tomato seedlings have emerged from the soil and unfolded their leaves, select plants with uniform growth and dilute the seed broth of Bacillus vesicles NJ157 and the model strain FZB42 with sterile water to a concentration of 10. 7 CFU / mL, inject 50mL into each tomato root to a final concentration of 10. 7 A bacterial suspension of CFU / mL was cultured with sterile water as a blank control. Growth promotion data were collected after 20 days of incubation. Results are as follows: Figure 13 As shown.

[0132] The results showed that NJ157 had a good promoting effect on the growth of tomato plants. Among them, strain NJ157 had the best promoting effect on the dry weight of the aboveground parts, as well as the dry weight and fresh weight of the underground parts, with promotion rates as high as 43.75%, 81.25%, and 172.73%, respectively. Strain NJ157 also had a good promoting effect on the production of chlorophyll and stem diameter in tomatoes, with promotion rates of 42.43% and 36.00%, respectively. The growth promotion rate of this strain on tomato growth was higher than that of the model strain FZB42, indicating that Bacillus belyeis NJ157 has a good growth-promoting effect and certain development potential.

[0133] Example 7. Investigation on the growth-promoting effect of Bacillus belye NJ157 on tomato plants

[0134] Tomato seed disinfection and germination: Select whole and plump seeds, disinfect them with 30% sodium hypochlorite solution for 3 minutes, disinfect them with 75% ethanol for 45 seconds, and then rinse them 4 times with sterile water until there is no irritating odor remaining on the surface of the tomato seeds; place sterile filter paper in a 9cm sterilized glass petri dish, add an appropriate amount of sterile water with a pipette so that the filter paper in the dish is completely wetted with water; use sterile tweezers to pick up the tomato seeds and place them on the filter paper, with a certain distance between the seeds to facilitate germination; after sealing the glass dish, place it in a 25℃ greenhouse and cultivate it in the dark for 3-5 days. Once the radicle has grown, it can be transplanted into a disposable plastic cup.

[0135] Planting tomato seedlings: After sifting black soil, mix it evenly with vermiculite in a 1:1 ratio, and sterilize it twice at 121℃ for 20 minutes under high temperature and pressure. Use a hole punch with a diameter of about 2-3cm to make a hole in the center of a disposable plastic cup, then add the sterilized mixture of black soil and vermiculite. Use tweezers to pick up the tomato seeds that have grown radicles, plant them in the soil with the roots facing down, cover them with another layer of soil, place the disposable plastic cup in a tray with an appropriate amount of water, and place it in a greenhouse at 25℃ for about 20 days.

[0136] Root irrigation treatment with Bacillus vesicles NJ157 fermentation broth: After the tomato seedlings have emerged from the soil and unfolded their leaves, select plants with uniform growth and dilute the seed broth of Bacillus vesicles NJ157 and the model strain FZB42 with sterile water to a concentration of 10. 7 CFU / mL, inject 50mL into each tomato root to a final concentration of 10. 7 CFU / mL bacterial suspension was used, with sterile water as a blank control. Salt stress treatment began 2 days after inoculation. A 200mM NaCl solution was added to each treatment tray, and the salt solution was applied every three days. Growth promotion data were collected after 30 days of incubation. Results are shown below. Figure 14 As shown.

[0137] Experimental results showed that *Bacillus belyssus* NJ1507 had a good promoting effect on tomato growth under salt stress. Among them, *Bacillus belyssus* NJ157 had the best promoting effect on tomato plant height, chlorophyll content, above-ground fresh weight, underground fresh weight, and dry weight, with growth promotion rates as high as 71.21%, 63.63%, 246.62%, 468.75%, and 328.57%, respectively. Furthermore, the growth promotion rate of strain NJ157 on tomato growth was no lower than that of the model strain FZB42, indicating that *Bacillus belyssus* NJ157 still has a good growth-promoting effect under salt stress and has high development potential.

[0138] As can be seen from the above embodiments, the present invention provides a strain of Bacillus vesiculus NJ1507 and its applications. The Bacillus vesiculus NJ1507 of the present invention has salt and alkali tolerance and can effectively control bacterial wilt, root rot, and fruit rot in tomatoes. It also has a good growth-promoting effect on tomatoes and has great application potential in the field of biological pesticides.

[0139] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis NJ157, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31984, deposited on September 14, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The method for preparing the bacterial suspension of Bacillus belyssus NJ157 as described in claim 1, characterized in that, The Bacillus berberis NJ157 was inoculated into a fermentation medium for fermentation culture to obtain the bacterial solution.

3. The preparation method according to claim 2, characterized in that, The fermentation temperature is 28–32°C, the fermentation speed is 180–220 rpm, and the fermentation time is 22–26 h.

4. The use of Bacillus belyssus NJ157 according to claim 1 in all or part of the following (1) to (6): (1) Application in inhibiting pathogenic oomycetes, pathogenic bacteria and pathogenic fungi; The pathogenic oomycete is *Phytophthora capsici*; the pathogenic bacteria are *Pseudomonas syringae* and *Ralstonia solanacearum*; the pathogenic fungi are *Botrytis cinerea*, *Fusarium moniliforme*, *Fusarium solanum*, and *Rhizoctonia solani*. (2) Application in suppressing tomato diseases; The tomato diseases mentioned are bacterial wilt, root rot, and fruit rot; (3) Application in promoting tomato growth under salt stress; (4) Applications in the breakdown of proteins or cellulose; (5) Application in the preparation of tomato growth promoters under salt stress; (6) Application in the preparation of microbial agents that decompose proteins or cellulose.

5. A microbial inoculant, characterized in that, The microbial agent comprises a bacterial suspension of Bacillus berleis NJ157 as described in claim 1.

6. The microbial agent according to claim 5, characterized in that, The spore concentration in the bacterial suspension is 1–9 × 10⁻⁶. 7 CFU / mL.

7. A method for promoting tomato growth under salt stress, characterized in that, The tomato plants were treated with the microbial agent described in claim 5 or 6 by root irrigation.

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