Bacillus velezensis bac8 and application thereof in preventing and treating black spot of cabbage

Abstract

The application belongs to the field of agricultural microorganism technology, and discloses bacillus velezensis Bac8 and application thereof in prevention and treatment of black spot disease of cabbage. The strain is isolated from soil of a cabbage planting field, and has been preserved in China Center for Type Culture Collection on February 28, 2026, with a preservation number of CCTCC NO: M 2026354. The strain has significant antagonistic effect on alternaria brassicae, and can be used for development of a biocontrol product, and has a good application prospect in prevention and treatment of black spot disease of cabbage caused by alternaria brassicae.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology and relates to Bacillus belye strain Bac8 and its application in the prevention and control of black spot disease in cabbage. Background Technology

[0002] Cabbage ( Brassica oleracea var capitata Cabbage (L.) is an important cruciferous leafy vegetable in my country, with an annual cultivation area exceeding 13 million mu (approximately 800,000 hectares), playing a vital role in my country's year-round vegetable production and export earnings. With the promotion of intensive cultivation methods and the continuous expansion of planting area, cabbage production faces increasingly severe disease threats, among which black spot disease has become a significant factor restricting the healthy development of the industry.

[0003] Black spot disease of cruciferous plants is a widespread fungal disease occurring globally. Its pathogens are complex, primarily including *Alternaria brassicae* (…). Alternaria brassicicola Alternaria brassicae ( Alternaria brassicae ) and Alternaria radicatorius ( Alternaria raphani In my country's main cabbage-producing areas, *Alternaria brassicae* is the dominant pathogen causing the prevalence of this disease. This pathogen can infect multiple parts of cabbage, including leaves, heads, stems, and seed pods, forming typical dark brown to black necrotic lesions with concentric rings on the surface of infected tissues. These lesions not only severely impair the photosynthetic capacity of the leaves, leading to weakened plant growth and reduced yield, but more importantly, their presence directly reduces the appearance quality and storage life of cabbage, causing significant economic losses to growers.

[0004] Faced with the increasingly frequent and severe situation of black spot disease in cabbage, and due to the current lack of commercially available cabbage varieties with high-level and long-lasting resistance, chemical control remains the primary means of agricultural production. Although chemical control can control the occurrence of the disease to some extent, long-term reliance on chemical pesticides can easily lead to environmental pollution, fungicide resistance, and excessive pesticide residues. Therefore, seeking an environmentally friendly, safe, efficient, and sustainable green control strategy for black spot disease in cabbage has become an urgent need to ensure the healthy development of my country's cabbage industry.

[0005] Biological control is a method of controlling harmful organisms by using beneficial organisms or their metabolites to inhibit, reduce, or eliminate them. Biocontrol bacteria, as the core of biological control, possess advantages such as high environmental compatibility, diverse mechanisms of action, and low resistance rates, making them an ideal alternative to traditional chemical pesticides. However, despite the widespread recognition of the concept and potential of biological control, its research and application in different agricultural scenarios exhibit significant imbalances. Existing research and commercial applications are mostly concentrated on the control of field crop diseases, while the development of biocontrol products for diseases in vegetable crops such as cabbage lags behind. Therefore, screening and identifying biocontrol strains capable of stably inhibiting *Alternaria brassicae* is of significant application value for the green control of black spot disease in cabbage. Summary of the Invention

[0006] To address the lack of existing biological control products for cabbage black spot disease, this invention provides a Bacillus belyssus strain and its application in the control of cabbage black spot disease.

[0007] A type of Bacillus belesi ( Bacillus velezensis Bac8, the strain in question, was deposited at the China Center for Type Culture Collection on February 28, 2026, with accession number CCTCC NO: M 2026354.

[0008] A biocontrol agent, the active ingredient of which is the aforementioned Bacillus berleis Bac8.

[0009] Preferably, the concentration of Bacillus berberis Bac8 in the biocontrol agent is OD. 600 =1.0 (approximately 10) 8 (cfu / mL).

[0010] The preparation method of the above-mentioned biocontrol agent includes the following steps: (1) The Bacillus belye was inoculated into LB liquid medium to obtain seed culture; (2) The obtained seed culture was inoculated into LB liquid medium at an inoculation rate of 1% (volume fraction) for expansion culture; (3) Collect bacterial cells by centrifugation, resuspend in 10 mM phosphate buffer, and adjust OD. 600 =1.0 (approximately 10) 8 The biocontrol agent was obtained by (cfu / mL).

[0011] A biocontrol agent, the active ingredient of which is the sterile fermentation broth of Bacillus berberis Bac8.

[0012] Preferably, the sterile fermentation broth is obtained by inoculating Bacillus berberis Bac8 into LB liquid medium, culturing at 28°C and 180 rpm for 72 h, and then centrifuging and filtering.

[0013] The application of the Bac8 strain of Bacillus belyssus, the biocontrol agent, or the biocontrol preparation in the prevention and control of black spot disease in cabbage.

[0014] Preferably, the pathogen causing black spot disease in cabbage is *Alternaria brassicae* (…). Alternaria brassicicola ).

[0015] Preferably, the application includes spraying the biocontrol agent onto cabbage plants.

[0016] Beneficial effects:

[0017] This invention isolated and screened a strain of Bac. belesiensis, Bac8, from soil in cabbage-growing fields. This strain exhibits significant antagonistic activity against *Alternaria brassicae*, the pathogen causing black spot disease in cabbage. Screening using the plate confrontation method confirmed that Bac8 can form a clear inhibition zone, significantly inhibiting the mycelial growth of the pathogen. More notably, the aseptic fermentation broth of Bac8 exhibits excellent antibacterial activity; at a volume fraction of 8%, it shows an average inhibition rate of up to 96.59% against *Alternaria brassicae* mycelial growth, demonstrating strong antibacterial ability of its metabolites. This characteristic allows this invention to be developed into both live bacterial preparations and preparations containing metabolites without live bacteria, offering flexible application options to meet the needs of different scenarios. Regarding field control efficacy, OD... 600 =1.0 (approximately 10) 8 Spraying cabbage plants with a Bac8 bacterial suspension (20 mL / plant) at a concentration of CFU / mL followed by inoculation with Alternaria brassicae spore suspension 12 hours later, the results after 7 days showed that the average disease index of the treatment group was 11.85, while that of the control group was as high as 54.32, with an average control effect of 77.78%, which significantly reduced the incidence of black spot disease in cabbage and effectively protected the photosynthetic capacity and appearance quality of the leaves.

[0018] The preparation process of the biocontrol product of this invention is simple, requiring only the inoculation of Bac8 into LB liquid medium, followed by shaking culture at 28°C and 180 rpm, centrifugation to collect the cells, or filtration to obtain the fermentation broth. The raw materials are readily available, and the cost is controllable, making it suitable for large-scale production and widespread application. Compared with existing chemical control methods, this invention has significant advantages such as high environmental compatibility, low likelihood of developing resistance, and no pesticide residues. It aligns with the direction of green agricultural development and provides a safe and efficient biological control alternative for the sustainable control of cabbage black spot disease, possessing broad application prospects and significant industrial value. Attached Figure Description

[0019] Figure 1. Inhibitory effect of Bac8 on Alternaria brassicae. Figure 2. Multi-gene consortium tree construction analysis of Bac8 strain Figure 3. Colony morphology of Bac8 (left) and transmission electron microscopy observation (right). Figure 4. Inhibitory effect of Bac8 aseptic fermentation broth on the growth of Alternaria brassicae hyphae. Figure 5. Control effect of Bac8 bacterial suspension on black spot disease in cabbage. Information on the preservation of biological materials

[0020] Bac8, categorized and named Bacillus velezensis Bac8 was deposited on February 28, 2026, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026354, located at Wuhan University, Wuhan, China. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0022] The culture media and culture solutions involved in the following examples include: PDA medium: 200 g / L potato, 20 g / L glucose, 10 g / L agar powder, diluted with deionized water, sterilized at 121℃ for 20 min; PDB medium: 200 g / L potato, 20 g / L glucose, diluted with deionized water, sterilized at 121℃ for 20 min; LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 10 g / L, diluted with deionized water, sterilized at 121℃ for 20 min; LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, diluted to volume with deionized water, sterilized at 121℃ for 20 min.

[0023] Example 1

[0024] (1) Isolation and purification of soil bacteria Take 10 g of fresh soil sample and add it to 90 mL of sterile water. Place it in a constant temperature shaker at 180 rpm and mix for 30 min. Then repeat the process according to the following steps: 10¹, 10², 10³, 10 4Serial dilutions were performed; 100 μL of each dilution was evenly spread onto LB agar plates, with three replicates for each concentration gradient. The plates were incubated at 28°C for 3 days. After colony growth, single colonies were selected based on morphology, color, size, and elevation. These colonies were then streaked onto LB agar plates for purification. A small sample (about the size of a toothpick tip) of the purified strain was cultured overnight in LB liquid medium to obtain the corresponding seed culture. 500 μL of the seed culture was diluted 1:1 with 50% glycerol and stored at -80°C. Finally, the samples were classified and numbered according to their collection location.

[0025] (2) Preliminary screening of antagonistic strains The plate confrontation method was used for initial screening. A cross was drawn on a PDA medium plate, and a *Alternaria brassicae* mycelial cake was inoculated at the center of the cross. Purified soil bacterial strains were inoculated at the four corners of the plate, 3 cm from the center. Sterile water was drawn at the four corners of the control group. Each treatment was repeated three times. The plates were placed in a 25°C incubator for 7 days. The inhibition of *Alternaria brassicae* mycelial growth by the bacteria was then observed and recorded, and antagonistic strains that could form clear inhibition zones were screened.

[0026] (3) Rescreening of antagonistic strains Conidia of *Alternaria brassicae* were inoculated into 50 mL of PDB medium and incubated at 25°C and 180 rpm for 36 h. Mycelia were collected by centrifugation and disrupted by shaking. 100 μL of the mycelial suspension was evenly spread onto a PDA plate. Bacteria were collected by centrifugation, resuspended in 0.75% sodium chloride solution, and OD was adjusted. 600 =1.0; Place a sterile filter paper disc in the center of the plate, add 10 μL of bacterial suspension, and add an equal volume of 0.75% sodium chloride solution to the control group. After incubation at 25℃ for 7 days, measure the diameter of the inhibition zone using the cross-sectional method. Figure 1 As shown, strain Bac8 with strong antagonistic effect was obtained through screening.

[0027] Example 2

[0028] (1) PCR amplification and sequencing Using extracted Bac8 genomic DNA as a template, 16S, gyrA , rpoB , purH , polC , groEL PCR amplification was performed, and the primer sequences are shown in Table 1. The PCR products were sent to General Biotechnology (Anhui) Co., Ltd. for bidirectional sequencing. The 16S sequence of Bac8 is shown in SEQ ID NO.1, consisting of five genes ( gyrA , rpoB , purH , polC ,groEL The assembled sequence is shown in SEQ ID NO.2.

[0029] (2) Cluster analysis Sequencing and BLAST alignment of the 16S sequence of Bac8 revealed that it belongs to the genus Bacillus, but its differentiation from several other bacteria in the same genus is not significant. Further amplification of five conserved genes of Bac8 (…) gyrA , rpoB , purH , polc and groEL After sequencing, multi-gene phylogenetic tree analysis was performed. Using MEGA12 software, the maximum likelihood method was employed to construct a phylogenetic tree with 1000 bootstrap values. Results are as follows: Figure 2 As shown, strain Bac8 clustered with Bacillus belyssus BD-569, BD-568, B-23190, and B-23189 into a separate branch.

[0030] Table 1 Primer Information Table

[0031] (3) Morphological identification like Figure 3 As shown, Bac8 colonies are milky white, with a smooth, slightly convex surface and a viscous texture. Under a transmission electron microscope, Bac8 cells are rod-shaped and have obvious peritrichous flagella.

[0032] Based on the combined molecular and morphological identification results, strain Bac8 was identified as Bacillus belye, which was deposited at the China Center for Type Culture Collection on February 28, 2026, with accession number CCTCC NO: M 2026354.

[0033] Example 3

[0034] (1) Preparation of sterile fermentation broth Pick a single colony of Bac8 and inoculate it into 10 mL of LB liquid medium. Incubate at 28°C and 180 rpm for 24 h with shaking to obtain seed culture. Take 500 μL of seed culture and inoculate it into 500 mL of LB liquid medium. Incubate at 28°C and 180 rpm for 72 h with shaking to obtain fermentation broth. Collect the fermentation broth into a 50 mL centrifuge tube and centrifuge at 7500 rpm and 4°C for 10 min. Take the supernatant and filter it through a 0.22 μm sterile filter membrane to obtain sterile fermentation broth of Bac8.

[0035] (2) Determination of antibacterial activity of aseptic fermentation broth After the PDA medium cooled to approximately 50°C, sterile fermentation broth was added at volume fractions of 1%, 2%, 4%, and 8%, respectively. After thorough mixing, the mixture was poured onto plates. An equal volume of sterile water was added to the control group. Each treatment was replicated three times. A 6mm diameter *Alternaria brassicae* mycelial cake was inoculated in the center of the plate and incubated at 25°C for 7 days. Subsequently, the diameter of the pathogenic mycelium was measured, and the inhibition rate was calculated as follows: Inhibition rate (%) = (Control group mycelial diameter - Experimental group mycelial diameter) / Control group mycelial diameter × 100%. Results are shown below. Figure 4 As shown, the sterile fermentation broth has a significant inhibitory effect on the growth of Alternaria brassicae mycelia, with an average inhibition rate of 96.59% at a volume fraction of 8%.

[0036] Example 4

[0037] (1) Preparation of biocontrol bacteria suspension Single colonies of Bac8 were picked and inoculated into LB liquid medium. The culture was incubated at 28°C and 180 rpm for 24 h with shaking to obtain a seed culture. The seed culture was then inoculated into LB liquid medium at a 1% (v / v) inoculation rate for further culture. The culture was incubated at 28°C and 200 rpm for 24 h with shaking. The bacterial cells were collected by centrifugation at 7500 rpm for 10 min and resuspended in 10 mM phosphate buffer (0.530 g sodium dihydrogen phosphate dihydrate and 2.364 g disodium hydrogen phosphate dodecahydrate, dissolved in deionized water and brought to a final volume of 1 L). The OD of the bacterial suspension was adjusted. 600 =1.0 (approximately 10) 8 (cfu / mL).

[0038] (2) Spraying of biocontrol bacteria suspension and inoculation with Alternaria brassicae Take the above bacterial solution and spray it evenly onto 35-day-old cabbage seedlings, 20 mL per seedling. A phosphate buffer solution was sprayed as a control. Each treatment was repeated in triplicate, with each replicate containing 15 cabbage seedlings. After 12 hours, each cabbage seedling was sprayed with 2 mL of a 1×10⁻⁶ solution. 5 The plants were treated with a suspension of Alternaria brassicae spores per mL and then placed in a humidified environment at 25°C for 7 days with a photoperiod of 16 h light / 8 h dark.

[0039] (3) Calculation of disease index Based on the percentage of diseased area on the third true leaf of cabbage seedlings, the severity of the disease was classified into six levels: Level 0: no obvious diseased spots; Level 1: diseased area ≤ 5% of leaf area; Level 3: 5% < diseased area ≤ 25% of leaf area; Level 5: 25% < diseased area ≤ 50% of leaf area; Level 7: 50% < diseased area ≤ 75% of leaf area; Level 9: 75% < diseased area ≤ 100% of leaf area. Based on these statistical results, the disease index (DI) for each treatment was calculated using the formula: DI = Σ (representative value for each level × number of plants affected at that disease level) / (highest representative value × total number of plants surveyed) × 100.

[0040] (4) Prevention and control effect like Figure 5 As shown in Table 2, spraying with Bac8 bacterial suspension reduced the severity of black spot disease in cabbage compared to the control group. The average disease index of cabbage in the control group was 54.32, while the average disease index of cabbage sprayed with Bac8 bacterial suspension was 11.85. Compared to the control, the average control efficacy after spraying with Bac8 bacterial suspension was 77.78% (Table 2). This result indicates that Bac8 has a good effect on controlling black spot disease in cabbage.

[0041] Table 2 Evaluation of the efficacy of Bac8 bacterial suspension in controlling black spot disease of cabbage

[0042] SEQ ID NO. 1:

[0043]

Claims

1. A type of Bacillus belesii ( Bacillus velezensis Bac8, characterized in that, The strain was deposited at the China Center for Type Culture Collection on February 28, 2026, with accession number CCTCC NO: M 2026354.

2. A biocontrol agent, characterized in that, The active ingredient of the biocontrol agent is Bac8 of Belize as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that, The concentration of Bacillus vesiculus Bac8 in the biocontrol agent is OD. 600 =1.

0.

4. A method for preparing the biocontrol agent according to any one of claims 2 to 3, characterized in that, Includes the following steps: (1) The Bac8 of claim 1 is inoculated into LB liquid medium and cultured to obtain seed culture; (2) Inoculate the seed culture obtained in step (1) into LB liquid medium at an inoculation rate of 1% by volume for expansion culture; (3) Collect the bacterial cells by centrifugation, resuspend them in 10 mM phosphate buffer, and adjust the OD. 600 =1.0, to obtain the biocontrol agent.

5. A biocontrol agent, characterized in that, The biocontrol agent is the sterile fermentation broth of Bacillus berberis Bac8 as described in claim 1.

6. The biocontrol agent according to claim 5, characterized in that, The sterile fermentation broth is obtained by inoculating the Bacillus berberis Bac8 of claim 1 into LB liquid medium, culturing at 28°C and 180 rpm for 72 h, and then centrifuging and filtering.

7. The use of the Bac8 strain of Bacillus berberis as described in claim 1, the biocontrol agent as described in any one of claims 2 to 3, or the biocontrol preparation as described in claim 5 in the prevention and control of black spot disease in cabbage.

8. The application according to claim 7, characterized in that, The pathogen causing black spot disease in cabbage is Alternaria brassicae (Alternaria brassicae). Alternaria brassicicola ).

9. The application according to claim 8, characterized in that, The application includes spraying the biocontrol agent onto cabbage plants.

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