Bacillus velezensis b143 and application thereof

Abstract

The present application relates to a bacillus velezensis Bv143 and application thereof, the classification and nomenclature of the bacillus velezensis Bv143 is bacillus velezensis Bacillus velezensis , which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.39314, the metabolites thereof have higher levels of antibacterial activity and wider antibacterial range, and have wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to Bacillus belye Bv143 and its applications. Background Technology

[0002] In agricultural production, fungal and bacterial diseases are prevalent in crops. Long-term application of chemical pesticides easily leads to pesticide residues, damage to the soil microecology, and increased drug resistance in pathogens, seriously hindering the development of green agriculture and the quality and safety of agricultural products. *Bacillus belyssus* ( Bacillus velezensis It has strong resistance to adverse conditions, high spore stability, and good environmental compatibility, making it an excellent biocontrol strain with great application prospects.

[0003] However, most existing Bacillus species do not directly inhibit pathogens, primarily relying on nutrient and space competition for control. Their disease control mechanisms are singular, resulting in slow onset of action, fluctuating control efficacy in field environments, and generally narrow inhibitory ranges. This fails to meet the control needs of mixed outbreaks of multiple pathogens and sudden disease epidemics in the field. Therefore, screening for Bacillus species with highly active metabolites as core antibacterial substances, broad-spectrum antibacterial activity, and stable physicochemical properties can effectively improve the effectiveness of green pest control in crops, reduce the use of chemical pesticides, and empower the large-scale and industrialized development of ecological agriculture. Summary of the Invention

[0004] To address the aforementioned technical problems, this application provides a strain of Bacillus belyssus Bv143, which exhibits stronger antibacterial activity and a broader antibacterial spectrum. Furthermore, its antibacterial activity primarily depends on its metabolites. Compared to Bacillus belyssus, which mainly relies on nutrient and space competition, its antibacterial activity is not limited by the colonization environment, is highly efficient, and has superior antibacterial stability. Moreover, it has a better control effect on mixed-infection diseases such as astragalus membranaceus stomatitis.

[0005] According to one aspect of this application, a *Bacillus belyescens* Bv143 is provided, said *Bacillus belyescens* Bv143 being classified and named *Bacillus belyescens*. Bacillus velezensis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 39314.

[0006] According to one aspect of this application, a *Bacillus belyescens* Bv143 is provided, said *Bacillus belyescens* Bv143 being classified and named *Bacillus belyescens*. Bacillus velezensis It satisfies any one or a combination of the following: (1) Contains a molecular marker, the nucleotide sequence of which is shown in SEQ ID NO: 1; (2) Contains a molecular marker, wherein the nucleotide sequence of the molecular marker has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1; (3) The genomic DNA was amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3. The amplification products were analyzed by agarose gel electrophoresis to obtain specific bands. (4) The genomic DNA was amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3. The amplified products were sequenced and analyzed to obtain the nucleotide sequence shown in SEQ ID NO: 1; or The obtained nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0007] According to one aspect of this application, a molecular marker is provided, the nucleotide sequence of which is shown in SEQ ID NO: 1; or The nucleotide sequence of the molecular marker has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0008] According to one aspect of this application, the use of the aforementioned molecular marker in identifying the aforementioned Bacillus belyssus Bv143 is provided.

[0009] According to one aspect of this application, a primer pair is provided, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO:2; The nucleotide sequence of the reverse primer is shown in SEQ ID NO: 3.

[0010] According to one aspect of this application, the use of the above primer pair in identifying the above-mentioned Bacillus belyssus Bv143 is provided.

[0011] According to one aspect of this application, a method for identifying the above-mentioned Bacillus belesiensis Bv143 is provided, comprising the following steps: (1) Sequencing of the Bacillus belyssus Bv143; (2) Obtain a sequence containing a specific molecular marker, wherein the specific molecular marker satisfies any one or a combination of the following: 1) The nucleotide sequence is as shown in SEQ ID NO: 1; and / or 2) The nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0012] Optionally, the sequencing is selected from: (1) Genome sequencing; and / or (2) Sequencing of PCR products.

[0013] Optionally, the PCR product is obtained by amplification using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0014] According to one aspect of this application, the use of the aforementioned Bacillus vesiculosus Bv143 in the preparation of fertilizer products and / or pesticide products is provided.

[0015] According to one aspect of this application, a fertilizer product and / or pesticide product is provided, which contains the aforementioned Bacillus berberis Bv143 and / or its fermentation product.

[0016] Optionally, the fertilizer product and / or pesticide product is selected from any one of fermentation stock solution, fermentation dilution solution, fermentation concentrate, technical material, mother drug, and formulation.

[0017] Optionally, the Bacillus vesiculosus Bv143 content of the fertilizer product and / or pesticide product is 0.1-100 billion CFU / mL.

[0018] Optionally, the dosage form of the formulation is selected from any one of powder, granules, oil, and seed treatment suspension.

[0019] Optionally, the dosage form of the formulation is selected from sustained-release formulations.

[0020] Optionally, the dosage form of the formulation is selected from suspensions.

[0021] Optionally, the formulation is selected from suspension seed coating agents.

[0022] Optionally, the dosage form of the formulation is selected from any one of seed treatment dispersible powder, seed treatment soluble powder, seed treatment liquid, seed treatment emulsion, and seed treatment microcapsule suspension.

[0023] Optionally, the dosage form of the formulation is selected from any one of the following: large granules, fine granules, microparticles, microcapsules, wettable powders, oil-dispersible powders, water-dispersible granules, emulsions, effervescent granules, dispersible tablets, effervescent tablets, sustained-release blocks, sustained-release tubes, sustained-release granules, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsions, latexes, dispersible liquids, pastes, concentrated gels, water emulsions, oil emulsions, microemulsions, liposomes, microcapsule suspensions, oil suspensions, and suspension emulsions.

[0024] According to one aspect of this application, a method for preparing the above-mentioned fertilizer product and / or pesticide product is provided, the method comprising the following steps: The fertilizer and / or pesticide products are obtained by fermentation using Bacillus belye Bv143. The Bacillus belysium Bv143 was classified and named Bacillus belysium. Bacillus velezensis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 39314.

[0025] Optionally, the preparation method includes the following steps: (1) Fermentation using Bacillus vesiculosus Bv143 to obtain the fermentation stock solution, i.e., the fertilizer product and / or pesticide product; or The preparation method further includes the following steps: (2) Further processing of the fermentation stock solution to obtain any one of the following: fermentation dilution, fermentation concentrate, technical material, mother drug, or formulation, which are the fertilizer product and / or pesticide product.

[0026] According to one aspect of this application, the use of the aforementioned Bacillus vesiculosus Bv143, the aforementioned fertilizer product and / or pesticide product, or the fertilizer product and / or pesticide product obtained according to the aforementioned method in promoting plant growth and / or preventing plant diseases is provided.

[0027] According to one aspect of this application, the use of the above-mentioned Bacillus vesiculosus Bv143, the above-mentioned fertilizer product and / or pesticide product, or the fertilizer product and / or pesticide product obtained according to the above method, in the preparation of plant growth promoters and / or plant disease control agents is provided.

[0028] The positive and beneficial effects achieved by this application are as follows: The Bacillus belyssus Bv143 strain provided in this application has stronger antibacterial activity and a broader antibacterial spectrum. Its antibacterial activity mainly depends on its metabolites. Compared with Bacillus belyssus, which mainly depends on nutrient and space competition, its antibacterial activity is not limited by the colonization environment, and it is highly efficient and has better antibacterial stability.

[0029] Preservation Notes: Bacillus belye Bv143 was deposited at the China General Microbiological Culture Collection Center on June 8, 2026, and classified as Bacillus belye. Bacillus velezensis The accession number is CGMCC No. 39314. Attached Figure Description

[0030] Figure 1 Phylogenetic tree of 16S rDNA for Bacillus belyssus Bv143.

[0031] Figure 2 The inhibitory effect of Bacillus belyi Bv135-144 on kiwifruit canker pathogens.

[0032] Figure 3 The inhibitory effect of Bacillus belyssus Bv135-144 on pear fire blight pathogen.

[0033] Figure 4 The inhibitory effect of Bacillus vesiculosus Bv143-148 on Pythium spp., the pathogen of melon and fruit diseases.

[0034] Figure 5 The inhibitory effect of Bacillus belyi Bv143-148 on rice bakanae disease pathogen.

[0035] Figure 6 The inhibitory effect of Bacillus belye Bv143-148 on the damping-off pathogen of pepper.

[0036] Figure 7 The inhibitory effect of Bacillus belyssus Bv143-148 on anthracnose causal agent of pepper anthracnose was investigated.

[0037] Figure 8 The inhibitory effects of Bacillus belyssus Bv143, Bv138 and blank control on apple red heart pathogen were investigated.

[0038] Figure 9 The efficacy of Bacillus vesiculosus Bv135-144 in controlling gray mold in strawberries.

[0039] Figure 10 The control efficacy of Bacillus belyssus Bv143 and Bv138 against powdery mildew in cucumber (in vitro experiment).

[0040] Figure 11 The control effects of Bacillus belyceta var. belyceta Bv143, Bv138 and blank control on cucumber powdery mildew were investigated in a field experiment.

[0041] Figure 12 The image shows the amplification products of the specific primers analyzed by agarose gel electrophoresis. Detailed Implementation

[0042] For the purposes of this invention, unless otherwise stated, the terms used in this application have the following meanings: The term "raw material" refers to the product obtained during the production process, which consists of active ingredients and related impurities, and may include a small amount of additives if necessary.

[0043] The term "mother drug" refers to the product obtained during the production process, which consists of the active ingredient and related impurities, and may contain small amounts of necessary additives and appropriate diluents.

[0044] The term "formulation" refers to a stable product made from pesticide technical (mother drug) and suitable adjuvants, or processed by methods such as bio-fermentation and plant extraction.

[0045] The term "plant disease" refers to the phenomenon where, during the growth and development of a plant, its growth and development are significantly hindered due to infection by other organisms and adverse abiotic factors. This results in pathological changes both internally and externally, in terms of physiology and tissue structure, leading to illness or even death, and consequently, reduced yield and deteriorated quality. In this application, "plant disease" specifically refers to diseases caused by infection of plants by other organisms, including but not limited to fungi and bacteria.

[0046] The reagents and solvents used in the following examples are commercially available or prepared using conventional methods known to those skilled in the art. Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions.

[0047] Preparation of PDA solid culture medium: Weigh 200 g of potato, wash, peel and chop it, add 800 ml of distilled water and boil for half an hour, filter with gauze, then add 20 g of glucose and 20 g of agar, and make up to 1000 ml with distilled water, and sterilize at 121℃ for 30 minutes.

[0048] Preparation of LB solid medium: Weigh 10 g tryptone, 5 g yeast extract, 10 g NaCl and 20 g agar, dissolve in distilled water and bring the volume to 1000 ml, adjust the pH to 7.0, and sterilize at 121℃ for 20 min.

[0049] Preparation of LB liquid medium: Weigh 10 g tryptone, 5 g yeast extract and 10 g NaCl, dissolve in distilled water and bring the volume to 1000 ml, adjust the pH to 7.0, and sterilize at 121℃ for 20 min.

[0050] Genomic DNA was extracted using a bacterial genomic DNA extraction kit from Tiangen Biotech Co., Ltd., following the instructions provided.

[0051] Specific amplification was performed using premixed Taq enzyme Premix Taq™ (TaKaRa Taq™ Version 2.0) from Takara Bio Inc., and the amplification system is as follows:

[0052] The amplification procedure is as follows:

[0053] Example 1: Isolation and Identification of Strains 1. Strains Isolation Ten soil samples were collected from a citrus planting base in Shuangshui Town, Xinhui District, Jiangmen City, Guangdong Province. 10g of each sample was weighed and dissolved in 90mL of sterile water. After being incubated in an 80℃ water bath for 10min, the samples were serially diluted with sterile water to 100 times and 1000 times. An appropriate amount of the 1000-fold dilution was spread on LB agar plates and incubated at 28℃ for 2 days. Several single colonies with different morphologies that grew were further purified and cultured and stored for later use.

[0054] 2. Strain identification The purified strain was identified using the 16S rDNA identification method. The specific identification steps are as follows: (1) Genomic DNA extraction: After each strain was activated and cultured on LB medium plates, genomic DNA was extracted.

[0055] (2) 16S rDNA amplification: Using genomic DNA as a template, 16S rDNA fragments were amplified from genomic DNA using universal 16S rDNA primers (27F sequence as shown in SEQ ID NO: 4 and 1492R sequence as shown in SEQ ID NO: 5). The amplification products were sent to Beijing Liuhe BGI Genomics Co., Ltd. for analysis.

[0056] (3) 16S rDNA sequence analysis: The 16S rDNA sequences obtained from sequencing were compared with the 16S rDNA sequences in GenBank using BLAST. The results showed that 14 strains were Bacillus belye, numbered Bv135-148. A phylogenetic tree of the 16S rDNA of Bv143 was constructed using Mega 5.0 software. This strain is related to... Bacillus velezensis FZB42 clustered on one branch (such as...) Figure 1 (As shown).

[0057] Example 2 Plate Antibacterial Experiment 1. Antibacterial effect Take 200 mL of thawed and cooled LB solid culture medium to approximately 50 °C, and add 1 mL of 1×10⁻⁶ medium. -8A CFU / mL suspension of *Actinidia kiwifruit ulcerans* or *Pyrus pyrifolia* was shaken well, poured into a petri dish, and allowed to solidify to obtain a bacterial layer of *Actinidia kiwifruit ulcerans* or *Pyrus pyrifolia*.

[0058] Take 200 mL of thawed and cooled LB solid culture medium to approximately 50 °C, and add 1 mL of 1×10⁻⁶ medium. -8 The 14 Bacillus belysin suspensions obtained in the CFU / mL step 1 were shaken well, poured into a petri dish, and allowed to solidify to obtain 14 Bacillus belysin layers.

[0059] Mycelial cakes were collected from the 14 *Bacillus belyssus* colonies using a 6mm punch, and inoculated onto the *Actinidia kiwifruit* or *Pyrus pyrifolia* colonies. This process was repeated three times. After incubation at 28℃ for 48 hours, the antibacterial effect was investigated. If a clear transparent inhibition zone existed between *Bacillus belyssus* and the pathogenic bacteria, it indicated that the strain had a direct inhibitory effect on the target pathogen. Its antibacterial effect mainly relied on secreting active substances to inhibit pathogen growth. The average width of the inhibition zone (i.e., the width of the transparent inhibition zone between the edge of the *Bacillus belyssus* colony and the edge of the pathogenic bacterial colony) was measured and calculated. Figure 2-3 (As indicated by the arrow) If Bacillus belye grows normally to the point of contact with the pathogenic bacteria, it indicates that Bacillus belye does not directly inhibit the pathogenic bacteria (e.g., Figure 3 Although the colonies of Bv139 were quite large, there was no clear inhibition zone between them and *Actinidia cuspidatum*, indicating that it had no direct inhibitory effect on *Actinidia cuspidatum*, which was recorded as 0 mm. The results are shown in Table 1 below. The antibacterial effect of strain Bv135-144 against *Actinidia cuspidatum* is shown in the figure below. Figure 2 See the graph showing the antibacterial effect against pear fire blight pathogen. Figure 3 .

[0060] Table 1. Inhibitory effects of various Bacillus species on bacteria (mm)

[0061] 2. Antifungal effect 1) Confrontation Training Method Take 200 mL of thawed and cooled LB solid culture medium to approximately 50 °C, and add 1 mL of 1×10⁻⁶ medium. -8 The 14 Bacillus belysin suspensions obtained in the CFU / mL step 1 were shaken well, poured into a petri dish, and allowed to solidify to obtain 14 Bacillus belysin layers.

[0062] Mycelial cakes were collected from the edges of colonies of *Pythium spp.*, *Bakanaeda bakanae*, and *Dystrophus thuringiensis* using a 6mm punch. Each pathogen was inoculated into the center of a PDA plate. Then, mycelial cakes were collected from the layers of 14 *Bacillus belyssii* strains using a 6mm punch and inoculated around the pathogens. Sterile water was used as a control. The plates were incubated at 28℃ until the control plates were fully covered. The antibacterial effect was then investigated. If a clear antagonistic zone existed between *Bacillus belyssii* and the pathogenic fungus (pathogenic fungal hyphal growth was stagnant), it indicated that the strain had a direct inhibitory effect on the target pathogen. Its antibacterial effect mainly relied on secreting active substances to inhibit the proliferation and growth of the pathogen. The average width of the antagonistic zone (i.e., the width of the antagonistic zone between the edge of the *Bacillus belyssii* colony and the edge of the pathogenic fungus colony, for example...) was measured and calculated. Figure 4 (As indicated by the double arrows) If *Bacillus belyceae* and the pathogenic fungus grow normally to contact, or if the boundary between the two colonies is blurred, it indicates that *Bacillus belyceae* has no direct inhibitory effect on the pathogenic fungus, and is recorded as 0 mm. The investigation results are shown in Table 2 below, where the antibacterial effect of strain Bv143-148 is illustrated in [the image]. Figure 4-7 .

[0063] Table 2. Inhibitory effects of various Bacillus species against fungi (mm)

[0064] 2) Poisoning tablet method The above 14 Bacillus belye strains were inoculated into LB liquid medium and cultured for 48 hours. The bacterial cells were removed by filtration to obtain the fermentation filtrate for later use.

[0065] Take 200 mL of PDA medium that has been melted and cooled to about 50°C, add 1 mL of the above fermentation filtrate, shake well, pour into a petri dish, and let it stand to solidify to obtain 14 Bacillus belye virus plates.

[0066] Use a 6mm punch to puncture apple red heart causal fungus (pathogen: Alternaria). Alternaria alternata (Fr.) Keissl.) colony edges were used to collect mycelial cakes, which were then inoculated into the center of 14 Bacillus belyssin agar plates. A PDA plate without fermentation filtrate was used as a control. The inoculation was repeated three times. The plates were incubated at 28°C until the control plates were nearly fully colonized. The inhibition rate was then investigated and calculated using the following formula: Colony growth inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - mycelial cake diameter) × 100%.

[0067] The results are shown in Table 3 below. The antibacterial effects of Bv143, Bv138, and the blank control (CK) are illustrated in the figure. Figure 8 .Depend on Figure 8It can be seen that the apple red heart pathogen in group Bv143 only grows on the original fungal cake and cannot grow on the virus plate, while the apple red heart pathogen in group Bv138 can basically grow normally, except that the colony diameter is slightly smaller than that of the blank control group.

[0068] Table 3. Antibacterial effects of various Bacillus species against apple red heart causal agent.

[0069] Example 3: In vitro pharmacodynamic test 1. Strawberry gray mold The 14 Bacillus belye strains were inoculated into LB liquid medium and cultured for 48 hours. The bacterial cells were removed by filtration, and the fermentation filtrate was diluted 100 times for later use.

[0070] Diluted filtrates from each Bacillus fermentation were mixed with a suspension of Botrytis cinerea spores (1×10⁻⁶). -5 A 1:1 mixture of CFU / mL and other pathogens was used for needle inoculation of strawberry fruits (10 μL per treatment). Ten strawberries were incubated at 28°C for 6 days, and then the disease incidence and control efficacy were assessed. Disease severity (based on the proportion of the upper surface area of ​​the fruit covered by the pathogen) is as follows: Grade 0: No lesions; Level 1: <5%; Level 2: 6-15%; Level 3: 16-25%; Level 4: 26-50%; Level 5: 51% or higher.

[0071] The calculation formula is as follows: Disease index = 100 × ∑ (number of diseased fruits at each level × disease level) / (total number of fruits surveyed × 5) Prevention and control effect = (Control disease index - Treatment disease index) / Control disease index * 100% The results are shown in Table 4 below, with the antibacterial effect of strain Bv135-144 shown in the figure. Figure 9 .

[0072] Table 4. Control efficacy of various Bacillus species against gray mold in strawberries.

[0073] 2. Cucumber powdery mildew Bacillus belye Bv143 and Bv138 were inoculated into LB liquid medium and cultured for 48 hours. The bacterial cells were removed by filtration, and the fermentation filtrate was diluted 100 times for later use.

[0074] The efficacy of a diluted fermentation filtrate against cucumber powdery mildew under in vitro conditions was tested. The specific test method is as follows: Cucumber leaves with uniform powdery mildew infection were cut in half along the main vein and placed separately in a humidity-controlled container. The petioles were wrapped with moist absorbent cotton to maintain moisture. Each leaf was then sprayed with 2 mL of fermentation filtrate of *Bacillus belycera* Bv143 and Bv138, respectively. Disease symptoms were observed 3 and 6 days after the first treatment, followed by a second treatment. Disease symptoms were re-examined 3 days after the second treatment. Observation results are shown below. Figure 10 The details are as follows: At the time of the first application, the leaf disease was the same in both groups. Three days after the first application, the area of ​​powdery mildew in the Bv138 group expanded, while the powdery mildew in the Bv143 group basically disappeared. Six days after the first application, the area of ​​powdery mildew in the Bv138 group continued to expand, while the Bv143 group showed a recurrence of mild powdery mildew. Three days after the second application, the area of ​​powdery mildew in the Bv138 group worsened, while the powdery mildew in the Bv143 group basically disappeared.

[0075] Example 4 Field Trial 1. Cucumber powdery mildew Bacillus belye Bv143 and Bv138 were inoculated into LB liquid medium and cultured for 48 hours. The bacterial cells were removed by filtration, and the fermentation filtrate was diluted 100 times for later use.

[0076] The efficacy of diluted fermentation filtrate against cucumber powdery mildew under field conditions was tested, with water as a blank control. The specific testing method is as follows: There are 3 treatments, each with 3 replicates, for a total of 9 cells. Each cell is 5m. 2 (1m*5m), each plot was arranged in a randomized block design. The pesticide was applied at the early stage of cucumber powdery mildew, with a dosage of 0.75L per plot. Seven days after application, a disease survey was conducted, recording the total number of leaves, the number of diseased leaves, and the disease severity.

[0077] The classification is based on the percentage of the leaf's lesion area relative to the total leaf area: Grade 0 means no disease spots on the leaves; Grade 1 is defined as lesions covering less than 5% of the total leaf area; Grade 3 is characterized by lesions covering 6% to 15% of the total leaf area; Level 5 is characterized by lesions covering 16% to 25% of the total leaf area; Level 7 indicates that the lesions cover 26% to 50% of the total leaf area; Level 9 indicates that the lesions cover more than 51% of the total leaf area.

[0078] Disease index = [Σ(number of diseased leaves at each level × relative grade value) / (total number of leaves surveyed × highest disease grade value)] × 100 Prevention and control effect (%) = [(Disease index of control group - Disease index of drug-treated group) / Disease index of control group] × 100 The survey results are shown in Table 5 below, and the prevention and control effect diagram is shown in [the image]. Figure 11 .

[0079] Table 5. Control efficacy of Bacillus belyceta var. bacillus Bv143 and Bv138 against cucumber powdery mildew.

[0080] 2. Astragalus membranaceus Bacillus belye Bv143 and Bv138 were inoculated into LB liquid medium and cultured for 48 hours. The bacterial cells were removed by filtration, and the fermentation filtrate was diluted 100 times for later use.

[0081] In selected Astragalus membranaceus planting sites with severe cases of astragalus root rot in previous years, the efficacy of diluted fermentation filtrate against astragalus root rot under field conditions was tested. The specific testing methods are as follows: When transplanting Astragalus membranaceus seedlings, the roots were dipped in the diluted fermentation filtrate, dried, and then transplanted. Each treatment consisted of three plots, each 6.5m in size. 2 (1m*6.5m), with clean water as a control. The control results were investigated at the time of Astragalus harvest that year.

[0082] Disease grading method: Grade 0: No disease spots on the main root Grade 1: The area of ​​diseased spots accounts for less than 5% of the main root area. Grade 3: Lesions cover 6% to 10% of the main root area. Grade 5: The lesion area accounts for 11% to 20% of the main root area. Grade 7: The lesion area accounts for 21% to 50% of the main root area. Grade 9: The lesion area accounts for more than 51% of the main root area. The formulas for calculating the disease index and prevention and control effectiveness are as follows: Disease index = [Σ(Number of diseased plants at each level × Relative level value) / (Total number of plants surveyed × 9)] × 100 Prevention and control effect = [(Disease index in blank control area - Disease index in treatment area) / Disease index in blank control area] × 100% The survey results are shown in Table 6 below.

[0083] Table 6. Control efficacy of various Bacillus species against Astragalus membranaceus stomatitis (%)

[0084] Example 5 SCAR molecular marker analysis Genomic DNA from 14 strains of Bacillus belyssus (Bv135-Bv148) was used as templates. Random amplification was performed using 20 random primers (SEQ ID NO: 6-SEQ ID NO: 25). After agarose gel electrophoresis analysis, a unique band was found in Bv143. The unique band of Bv143 was excised, recovered, and sequenced to obtain the sequence of the unique band.

[0085] Primers were designed based on the obtained unique band sequences (forward primer as shown in SEQ ID NO: 2; reverse primer as shown in SEQ ID NO: 3). Specific amplification was performed using the genomic DNA of the 14 *Bacillus belyssiensis* strains mentioned above as templates. The amplification products were analyzed by agarose gel electrophoresis, and the results are as follows: Figure 12 As shown, lanes 1-14 are Bacillus belyssus Bv135-Bv148, respectively. The figure shows that only Bv143 can amplify the specific band, while the other 13 strains cannot amplify the specific band. This verifies the specificity of the specific band sequence and that the primers (SEQ ID NO: 2 and SEQ ID NO: 3) can specifically identify Bacillus belyssus Bv143.

[0086] The specific bands obtained from the specific amplification of Bacillus belyssus Bv143 by SEQ ID NO: 2 and SEQ ID NO: 3 were recovered and sequenced to obtain the specific band sequence as shown in SEQ ID NO: 1.

[0087] Blast alignment of the specific band sequence shown in SEQ ID NO: 1 in GenBank revealed no identical or similar sequences among all published Bacillus species. This further demonstrates that the specific sequence shown in SEQ ID NO: 1 can be used to identify Bacillus species Bv143.

[0088] The above results indicate that primers SEQ ID NO: 2 and SEQ ID NO: 3 are specific primers for identifying Bacillus belyssus Bv143 strain. Their PCR amplification products, i.e., the sequence shown in SEQ ID NO: 1, are the SCAR molecular markers for Bacillus belyssus Bv143. Primers SEQ ID NO: 2 and SEQ ID NO: 3, as well as the specific band sequence (SEQ ID NO: 1), can rapidly and effectively identify Bacillus belyssus Bv143.

[0089] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Bacillus velezensis Bv143, characterized in that, The Bacillus belye Bv143 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39314.

2. A type of Bacillus belye Bv143, characterized in that, The Bacillus belyssus Bv143 contains a molecular marker, the nucleotide sequence of which is shown in SEQ ID NO:

1.

3. Use of Bacillus belye Bv143 as described in claim 1 in the preparation of fertilizer products and / or pesticide products.

4. A fertilizer product and / or pesticide product, characterized in that, It contains the fermentation stock of Bacillus belyssus Bv143 as described in claim 1.

5. The fertilizer product and / or pesticide product according to claim 4, characterized in that, The fertilizer and / or pesticide products contain 100 billion CFU / mL of Bacillus vesiculosus Bv143.

6. The method for preparing the fertilizer product and / or pesticide product according to claim 4, characterized in that, The preparation method includes the following steps: fermenting Bacillus berberis Bv143 as described in claim 1 to obtain the fermentation stock solution, which is the fertilizer product and / or pesticide product.

7. The preparation method according to claim 6, characterized in that, The preparation method further includes the following steps: further processing the fermentation stock solution to obtain either a fermentation dilution or a fermentation concentrate, which are the fertilizer product and / or pesticide product.

8. The preparation method according to claim 6, characterized in that, The preparation method further includes the following steps: further processing the fermentation broth to obtain a formulation, which is the fertilizer product and / or pesticide product.

9. The preparation method according to claim 8, characterized in that, The dosage form of the preparation is selected from any one of powder, granules, or oil.

10. The preparation method according to claim 8, characterized in that, The dosage form of the formulation is selected from sustained-release formulations.

11. The preparation method according to claim 8, characterized in that, The dosage form of the preparation is selected from suspensions.

12. Use of the Bacillus vesiculosus Bv143 of claim 1, the fertilizer product and / or pesticide product of any one of claims 4-5, or the fertilizer product and / or pesticide product obtained by the method of any one of claims 6-11 in the preparation of plant disease control agents.

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