Bacillus velezensis and application thereof in prevention and treatment of sweet potato black spot

Abstract

The invention discloses bacillus velezensis and application thereof in prevention and treatment of sweet potato black spot, and belongs to the field of microorganisms, 15 bacteria are successfully obtained through systematic separation of sweet potato rhizosphere soil and healthy plant tissues, screening is performed through a plate confrontation culture method, and the bacillus velezensis is obtained. The bacillus velezensis N-J-5 with a remarkable inhibiting effect on the sweet potato alternaria alternate is obtained. The antibacterial radius of the strain reaches 7.5 mm, and the antibacterial rate reaches up to 51.61%. And technologies such as morphological observation, physiological and biochemical characteristic determination, 16S rRNA gene sequence analysis and the like are comprehensively utilized to finally identify the bacillus velezensis as bacillus velezensis. The discovery of the strain provides a high-quality strain resource for research and development of a sweet potato black spot biological control preparation, and has important theoretical significance and practical value for promoting green and sustainable development of the sweet potato industry.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Bacillus berberis NJ-5 and its application in the prevention and control of sweet potato black spot disease. Background Technology

[0002] Sweet potato black rot is a devastating disease caused by fungi of the genus *Ascomycota*, severely impacting the global sweet potato industry. This disease is infectious throughout the entire growth cycle. In seedlings, dark brown spindle-shaped lesions appear at the stem base, developing into black sunken spots as the plant grows. The diseased tissue softens and becomes easily broken. Tubers initially appear water-soaked, later forming and expanding black necrotic spots, which turn brown and produce toxins, rendering the tubers inedible and unusable for processing. Its spores are remarkably resilient, surviving for years in soil and diseased plant debris, and are widely spread by wind, rain, agricultural machinery, and seed transport. It thrives in high temperatures and humidity, with rampant infection occurring at temperatures between 25 and 30°C and relative humidity above 80%.

[0003] Traditional chemical control of sweet potato black rot faces challenges such as a limited range of pesticides and increasing pathogen resistance. For example, the antifungal effect of benzimidazole fungicides like carbendazim has significantly decreased with long-term use; physical control methods (such as high-temperature treatment) can easily damage tuber viability. Biological control has become a research focus due to its environmental and sustainability advantages, with Bacillus and Klebsiella showing significant potential in inhibiting pathogens and enhancing host resistance.

[0004] Based on this, developing a Bacillus species that can mitigate the damage of sweet potato black spot disease to sweet potato crops has great potential as a biological control agent and can provide data support for the subsequent preparation of microbial agents. Summary of the Invention

[0005] The purpose of this invention is to provide a Bacillus berberis NJ-5 and its application in the prevention and control of sweet potato black spot disease.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A new strain of Bacillus velezensis, NJ-5, has been deposited on July 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35279. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] Preferably, a bacterial agent containing Bacillus belye NJ-5 is also provided.

[0009] Preferably, a biopesticide, biofertilizer, or plant growth promoter prepared from Bacillus vesalis NJ-5 or an agent containing Bacillus vesalis NJ-5 is also provided.

[0010] Preferably, the application of Bacillus belyssus NJ-5 in the preparation of biocontrol agents is also provided.

[0011] Preferably, the application of Bacillus belyssus NJ-5 in inhibiting pathogens and plant diseases caused by pathogens is also provided.

[0012] Preferably, the application of Bacillus berberis NJ-5 in the prevention and control of sweet potato black spot disease is also provided.

[0013] Preferably, the pathogen is *Saccharomyces nigra*.

[0014] Preferably, the plant disease is sweet potato black spot disease.

[0015] Preferably, the fungus *Saccharomyces cerevisiae*, which causes sweet potato black spot disease, exhibits a significant antagonistic effect, with an inhibition radius of 7.5 mm and an inhibition rate as high as 51.61%.

[0016] The present invention has the following advantages:

[0017] The *Bacillus belyceae* NJ-5 strain isolated from sweet potato rhizosphere soil in this invention is harmless to humans and animals and environmentally friendly, meeting the requirements of green plant protection and sustainable agricultural development. In practical applications, it was found to have an inhibition rate of up to 51.61% against sweet potato black rot fungus, demonstrating a significant control effect. This strain provides a new and effective bacterial resource for the biological control of sweet potato black rot, and its antagonistic characteristics indicate its potential for development into a microbial agent, providing a theoretical basis and data support for subsequent agent preparation process development. Attached Figure Description

[0018] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0019] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0020] Figure 1 This is a Gram result diagram of Bacillus belyssus NJ-5 of the present invention;

[0021] Figure 2 This is a diagram showing the spore staining results of Bacillus belyss NJ-5 in this invention;

[0022] Figure 3 This is a diagram showing the glycosylation results of Bacillus belyss NJ-5 in this invention;

[0023] Figure 4 This is a phylogenetic tree of the 16S rRNA gene sequence of Bacillus belyssus NJ-5 of the present invention;

[0024] Figure 5 This diagram illustrates the antagonistic effect of Bacillus vesiculosus NJ-5 on sweet potato black spot pathogens in a plate confrontation experiment according to the present invention. Detailed Implementation

[0025] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] To address the shortcomings of existing technologies, this invention provides a Bacillus velezensis NJ-5, classified as Bacillus velezensis, which was deposited on July 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35279, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0027] Preferably, a bacterial agent containing Bacillus belye NJ-5 is also provided.

[0028] Preferably, a biopesticide, biofertilizer, or plant growth promoter prepared from Bacillus vesalis NJ-5 or an agent containing Bacillus vesalis NJ-5 is also provided.

[0029] Preferably, the application of Bacillus belyssus NJ-5 in the preparation of biocontrol agents is also provided.

[0030] Preferably, the application of Bacillus belyssus NJ-5 in inhibiting pathogens and plant diseases caused by pathogens is also provided.

[0031] Preferably, the application of Bacillus berberis NJ-5 in the prevention and control of sweet potato black spot disease is also provided.

[0032] Preferably, the pathogen is *Saccharomyces nigra*.

[0033] Preferably, the plant disease is sweet potato black spot disease.

[0034] Preferably, the fungus *Saccharomyces cerevisiae*, which causes sweet potato black spot disease, exhibits a significant antagonistic effect, with an inhibition radius of 7.5 mm and an inhibition rate as high as 51.61%.

[0035] To better illustrate the technical effects of this application, the usage process of this invention is as follows:

[0036] Example 1

[0037] This embodiment provides a method for isolating, purifying, and screening strain NJ-5 obtained from sweet potato rhizosphere soil.

[0038] 1. Materials and Methods

[0039] 1.1 Target pathogens

[0040] One strain of *Ceratocystis fimbriata* Ellis et Halsted, a pathogen causing sweet potato black rot, was provided by our laboratory (Microbiology Laboratory of Xingtai University).

[0041] 1.2 Test Culture Medium

[0042] Beef extract peptone (NA) medium: Weigh 3.0g beef extract, 10.0g peptone, 5.0g NaCl, and 20g agar, place them in a clean beaker, add an appropriate amount of distilled water (about 800mL), stir until all components are completely dissolved, continue to add distilled water to make up to 1000mL, and adjust the pH value to the range of 7.4 to 7.6.

[0043] Potato glucose agar (PDA) medium: Weigh 200g of peeled potatoes, 20g of agar, and 20g of glucose, put them in a pot and cook until the potatoes are soft. Filter through eight layers of gauze, and take the filtrate and dilute it to 1000mL with distilled water.

[0044] GM1 medium: Weigh 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g sodium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, and 20g agar. Place them in a clean beaker, add an appropriate amount of distilled water (about 800mL), heat and stir until all components are completely dissolved. Continue to add distilled water to bring the volume to 1000mL and adjust the pH to 7.4-7.6.

[0045] The prepared culture medium was autoclaved at 121°C for 15–30 minutes.

[0046] 1.3 Isolation of test strains

[0047] 1.3.1 Isolation of rhizosphere strains

[0048] Weigh 10g of sweet potato rhizosphere soil and place it in a 90mL Erlenmeyer flask containing sterile water and glass beads. Place the flask on a shaker at 37℃ and 180r / min for 30min. Measure the bacterial suspension and dilute it to 10⁻¹⁰ using a 10-fold serial dilution method. -6 10 -7 10 -8 Concentration. Using a pipette, 100 μL of each concentration dilution was applied for isolation: bacteria were isolated using beef extract peptone medium; fungi were isolated using PDA medium; and actinomycetes were isolated using GM1 medium. Each concentration was tested in triplicate. All media were incubated at 25°C for 24 hours. Different strains were then purified. Using a pipette, 100 μL of each concentration dilution was applied for isolation: bacteria were isolated using beef extract peptone medium; fungi were isolated using PDA medium; and actinomycetes were isolated using GM1 medium. Each concentration was tested in triplicate. All media were incubated at 25°C for 24 hours. Single colonies of each strain were purified. The purified strains were transferred to test tube slant agar, numbered, and stored at 4°C for later use.

[0049] 1.3.2 Isolation of sweet potato tuber strains

[0050] Healthy sweet potato tubers were selected and crushed and ground under sterile conditions. The microorganisms were then subjected to gradient dilution, separation, purification, and preservation, using the same method as in 1.3.1.

[0051] 1.4 Screening of antagonistic strains

[0052] The antibacterial activity of the selected strains was determined using the plate confrontation method, and the steps are as follows:

[0053] Pathogen activation: Transfer the stored sweet potato black rot pathogen to the center of a PDA medium plate and incubate at 25°C for 5 days until the colonies spread evenly and basically fill the entire plate.

[0054] Mycelial cake preparation: Using a sterile punch, circular mycelial cakes of the same age and 6 mm in diameter were cut from the edge of the activated pathogen colony. Mycelial cakes from the same pathogen plate were taken for each experimental batch as a negative control.

[0055] Experimental grouping and inoculation: Prepared mycelial discs were placed in the center of PDA medium plates, and three isolated test bacteria were inoculated at equal intervals 2 cm from the edge, with one inoculation point for each bacterial species. A control group was also set up, inoculating only the same pathogenic fungal mycelial discs in the center of the plate, without bacterial inoculation. Three replicates were prepared for each group. All plates were then placed in a 25°C incubator for static incubation. After 3 days, colony growth was observed, and the radius of pathogenic bacterial colonies in each plate of both the control and treatment groups was measured and recorded in detail.

[0056] 2 Results and Analysis

[0057] 2.1 Isolation and screening of antagonistic strains

[0058] Ten strains with potential antagonistic activity against *Hydrocotyle vulgaris*, the causal agent of sweet potato black spot disease, were isolated and purified from rhizosphere soil and sweet potato tubers. The inhibition rates of these 10 antagonistic strains against *Hydrocotyle vulgaris* were determined using the plate confrontation method, and the specific calculation formula is as follows:

[0059]

[0060] The table below shows the inhibition rates of different strains against the pathogen causing sweet potato black rot.

[0061]

[0062] As can be seen from the table above, among the 10 antagonistic strains screened, strain NJ-5 had the strongest inhibitory effect on sweet potato black spot fungus, reaching 51.61%.

[0063] Example 2

[0064] Identification of strain NJ-5

[0065] 1.1 Morphological identification of strain NJ-5

[0066] The selected strain NJ-5 was identified using a combination of colony morphology, Gram staining, and molecular biology methods.

[0067] On GM1 agar plates, the colonies of strain NJ-5 are irregular, brown and opaque, with wavy edges, viscous texture, and easy to pick up.

[0068] 1.2 Physiological and biochemical identification of strain NJ-5

[0069] Gram staining of strain NJ-5 showed the following results (e.g.) Figure 1 The bacteria is purple in color, identified as a Gram-positive bacterium, and has a rod-shaped morphology.

[0070] Spore staining of this strain showed (e.g.) Figure 2 It has the ability to form spores.

[0071] The strain was stained with sugars, and the results showed (e.g.) Figure 3 The blue areas indicate the presence of bacterial glycocalyx structures. Glycocalyx staining typically employs special methods, such as negative staining, to highlight these structures. The blue areas represent the location of the glycocalyx, indicating its presence in the sample. Glycocalyx is a loose, transparent, mucous or gelatinous polysaccharide secreted by bacteria onto the cell wall surface under specific nutritional conditions. It can be divided into capsules, microcapsules, mucus layers, and bacterial flocs.

[0072] 1.3 Molecular biological identification of strain NJ-5

[0073] To clarify the taxonomic position of strain NJ-5, 16S rRNA gene sequencing analysis was performed using colony PCR.

[0074] The PCR amplification primers used are universal primers for the bacterial 16S rRNA gene, as follows:

[0075] Forward primer 27F (SEQ ID NO:2): 5'-AGAGTTTGATCMTGGCTCAG-3';

[0076] Reverse primer 1492R (SEQ ID NO:3): 5'-TACGGYTACCTTGTTACGACTT-3'.

[0077] The PCR reaction system and amplification procedure are as follows:

[0078] The 25 μL reaction system contained 2.5 μL of 10×PCR Buffer, 2 μL of dNTP Mixture (2.5 mmol / L each), 1 μL each of forward and reverse primers (10 μmol / L), 1 μL of template DNA, 0.2 μL of Taq DNA polymerase (5 U / μL), and 17.3 μL of ddH2O.

[0079] The amplification program was as follows: pre-denaturation at 94℃ for 5 min, followed by denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, for 35 cycles, and finally extension at 72℃ for 10 min, and storage at 4℃.

[0080] The 16S rRNA gene sequence (SEQ ID NO:1) of strain NJ-5 obtained by the above method was subjected to NCBI BLAST homology alignment analysis. The results showed that its gene sequence coverage with Bacillus velezensis CR-502 (NCBI accession number: MZ573380) was as high as 100%. To further verify its taxonomic position, a phylogenetic tree was constructed using MEGA 7.0 based on the 16S rRNA gene sequence using the nearest neighbor (NJ) method. The results showed that (as shown in the figure)... Figure 4 Strain NJ-5 clustered with several strains of the Bacillus genus, such as Bacillus velezensis CR-502 and Bacillus velezensis DSM 7 (NCBI accession number: NR118950), into the same branch, and was the closest in genetic distance to Bacillus velezensis CR-502.

[0081] Therefore, based on morphological, physiological and biochemical characteristics and molecular biological results, strain NJ-5 was finally identified as Bacillus belyssus.

[0082] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A type of Bacillus belye NJ-5, characterized in that, The specimen, classified as Bacillus velezensis, was deposited on July 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35279. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. An inoculum containing the Bacillus belyssus NJ-5 as described in claim 1.

3. A biopesticide, biofertilizer, or plant growth promoter prepared from Bacillus berreatus NJ-5 as described in claim 1 or the microbial agent as described in claim 2.

4. The use of Bacillus belyssus NJ-5 as described in claim 1 in the preparation of biocontrol agents.

5. The use of Bacillus belye NJ-5 as described in claim 1 in inhibiting pathogens and plant diseases caused by pathogens.

6. The application as described in claim 5, characterized in that, The pathogen is *Saccharomyces nigra*.

7. The application as described in claim 5, characterized in that, The plant disease mentioned is sweet potato black spot disease.

8. The application as described in claim 5, characterized in that, The fungus *Saccharomyces cerevisiae*, which causes black spot disease in sweet potatoes, showed a significant antagonistic effect, with an inhibition radius of 7.5 mm and an inhibition rate as high as 51.61%.

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