Bacillus velezensis and application thereof in prevention and treatment of bacterial wilt of solanaceae crops

Abstract

The invention belongs to the technical field of biocontrol bacteria, and discloses bacillus velezensis and application thereof in preventing and treating bacterial wilt of solanaceae crops. The bacillus velezensis is named as bacillus velezensis PR-7, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address of the preservation unit is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation time is November 13, 2023, and the preservation number is CGMCC No.28979. The bacillus velezensis has the advantages that the bacillus velezensis is named as bacillus velezensis PR-7; the bacillus velezensis can effectively inhibit the growth of a bacterial wilt pathogen, i.e., the evolutionary I type (main evolutionary type of bacterial wilt pathogen in South China) of bacterial wilt pathogen, improves the bacterial wilt resistance of solanaceae crops, and is of great significance to the prevention and treatment of bacterial wilt of solanaceae crops in South China.

Description

Technical Field

[0001] This invention belongs to the field of biocontrol technology, specifically relating to a Bacillus berberis and its application in controlling bacterial wilt in Solanaceae crops. Background Technology

[0002] Rhesus solani ( Ralstonia solanacearum Bacterial wilt, caused by bacterial infection, is known as "plant cancer." It is a devastating soil-borne bacterial disease that often causes severe yield reduction or even crop failure in solanaceous crops. Currently, bacterial wilt has become a major disease affecting the production of solanaceous crops such as tomatoes and eggplants in southern my country.

[0003] Foreign studies have shown that rhizosphere microorganisms can participate in the process of bacterial wilt resistance in tomatoes, and beneficial bacteria present in the roots of disease-resistant tomato plants can help tomatoes resist bacterial wilt fungus. For example, adding specific Flavobacterium isolated from the rhizosphere of tomatoes can enhance resistance to bacterial wilt. The improved resistance of tomatoes to bacterial wilt is mainly due to the enrichment of Flavobacterium in the disease-resistant variety Hawaii 7996. Other researchers have obtained a series of rhizosphere bacteria with good biocontrol effects against bacterial wilt in laboratory and greenhouse environments, such as Pseudomonas (…). Pseudomonas spp. ), Bacillus spp. ( Bacillus spp. Flavobacterium ( Flavobacterium johnonia ) and Agrimonia pilosa ( Chryseobacterium daecheongense )wait.

[0004] Due to my country's vast territory and significant regional climate differences, South China exhibits highly specific climatic conditions (high temperature and high humidity). From April to July each year, temperatures exceed 33℃ for dozens of consecutive days, with relative humidity often exceeding 80%. This makes it difficult for some existing biological control agents to effectively control bacterial wilt in solanaceous crops. Therefore, further research and development of biocontrol bacterial strains for controlling bacterial wilt in solanaceous crops in South China are needed. Summary of the Invention

[0005] This invention aims to at least solve one of the problems existing in the prior art. To this end, this invention proposes a *Bacillus belyssae* strain and its application in the control of bacterial wilt in solanaceous crops. This *Bacillus belyssae* strain can effectively inhibit the growth of *Ralstonia solanacearum* evolution type I (the main evolutionary form of bacterial wilt pathogen in South China), thereby improving the resistance of solanaceous crops to bacterial wilt, which is of great significance for the control of bacterial wilt in solanaceous crops in South China.

[0006] This invention provides a Bacillus belye, named Bacillus velezensisPR-7, preserved in China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Beichen West Road, Chaoyang District, Beijing, preservation time: November 13, 2023, and preservation number: CGMCC No. 28979.

[0007] The strain is isolated and screened from the rhizosphere soil of a Solanaceae field with serious bacterial wilt in South China, and the strain is identified as Bacillus velezensis, named Bacillus velezensis PR-7. The strain can antagonize the pathogenic bacteria of bacterial wilt Ralstonia solanacearum , and can effectively improve the resistance of Solanaceae plants to bacterial wilt, and has good application value and prospect as a biocontrol agent.

[0008] The application also provides application of the above-mentioned Bacillus velezensis in preventing and treating bacterial wilt of Solanaceae crops.

[0009] Preferably, the Solanaceae crop is tomato or eggplant.

[0010] Preferably, the pathogenic bacteria of the Solanaceae crop bacterial wilt is Ralstonia solanacearum Ralstonia solanacearum .

[0011] The application also provides a bacterial wilt biological control agent comprising the above-mentioned Bacillus velezensis.

[0012] The application also provides a method for preventing and treating bacterial wilt of Solanaceae crops, which uses the above-mentioned Bacillus velezensis or bacterial wilt biological control agent to treat Solanaceae crops.

[0013] Compared with the prior art, the application has the following beneficial effects: The application is isolated and screened from the rhizosphere soil of Solanaceae crops, and an antagonistic bacterium with obvious inhibitory effect on the evolution type I of the dominant pathogenic bacteria of bacterial wilt in South China Ralstonia solanacearum is obtained, and is named Bacillus velezensis Bacillus velezensis PR-7. The strain can better adapt to the high-temperature and high-humidity environment in South China, can stimulate the immune resistance of tomato, eggplant and other Solanaceae plants, and has a significant and stable effect on preventing and treating bacterial wilt under laboratory and greenhouse conditions, which has important significance for preventing and treating bacterial wilt of Solanaceae crops in South China. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The Bacillus velezensis PR-7 and the pathogenic bacteria Ralstonia solanacearum GM1000 plate confrontation culture results.

[0015] Figure 2 The Bacillus velezensis PR-7 strain evolution analysis results.

[0016] Figure 3 For Bacillus velezensis PR-7 control effect diagram of tomato bacterial wilt.

[0017] Figure 4 For Bacillus velezensis PR-7 control effect diagram of eggplant bacterial wilt.

[0018] Figure 5 For Bacillus velezensis Effect of PR-7 on the incidence of bacterial wilt; wherein A is a tomato plant, and B is an eggplant plant.

[0019] Figure 6 For Bacillus velezensis Effect of PR-7 on the protective enzyme activity in tomato; wherein A is catalase (CAT), B is polyphenol oxidase (PPO), and C is phenylalanine ammonialyase (PAL).

[0020] Figure 7 For Bacillus velezensis Effect of PR-7 on the protective enzyme activity in eggplant; wherein A is catalase (CAT), B is polyphenol oxidase (PPO), and C is phenylalanine ammonialyase (PAL).

[0021] Figure 8 Second messenger content in tomato Figure 9 Second messenger content in eggplant Figure 10 Real-time fluorescent quantitative PCR detection Bacillus velezensis PR-7 metC Gene copy number results; wherein "PR-7" and "R" represent Bacillus velezensis PR-7 and Ralstonia solanacearum Ralstonia solanacearum Evolution I type GM1000.

[0022] Biological material preservation instructions The Beijerinckia present in the application Bacillus velezensis PR-7 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation unit address is No. 3, Beichen West Road, Chaoyang District, Beijing, the preservation time is November 13, 2023, and the preservation number is CGMCC No. 28979. DETAILED DESCRIPTION

[0023] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0024] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0025] Example 1: Bacillus velezensis PR-7 screening and identification 1. Preparation of rhizosphere soil suspension from healthy eggplant Soil sample pretreatment: From an eggplant field in South China with an age ≥8 years and severe bacterial wilt disease, rhizosphere soil samples were collected from 3-5 healthy eggplant plants (sampling depth 10-20cm, soil close to the roots, removing stones and root debris). 50g of soil was collected from each plant, mixed, and placed in a sterile sampling bag. The samples were stored at 4℃ and processed within 24 hours. 10g of the mixed rhizosphere soil was then placed in a 50mL centrifuge tube containing 90mL of sterile physiological saline. 3-5 sterile glass beads (3mm in diameter) were added, and the tube was vortexed for 30 minutes (2000rpm) to fully disperse the soil particles, yielding 10... -1 Soil suspension of a certain concentration; Serial dilution: Let the soil suspension stand for 5 minutes (to allow large soil particles to settle), then use a sterile pipette to extract 1 mL of the supernatant and inject it into a centrifuge tube containing 9 mL of sterile physiological saline. Vortex to mix, obtaining 10... -2 Concentration dilution; repeat this operation to prepare 10 solutions sequentially. -3 10 -4 10 -5 A soil dilution solution of a certain concentration.

[0026] 2. Isolation and purification of candidate antagonistic bacterial communities Coating separation: Take 10 -3 10 -4 10 -5 Add 0.1 mL of each of the three concentrations of the diluted solution to the center of an LB agar plate, and spread the bacterial solution evenly over the entire surface of the plate using a sterile spreader (3 replicates for each concentration). Culture and selection: Invert the LB plate and place it in a 28℃ constant temperature incubator for 24-48 hours. After single colonies appear on the plate, select single colonies of different morphologies (color, size, edge, elevation) according to their morphology (color, size, edge, elevation) (select 3-5 colonies of each morphology) and inoculate them into the corresponding fresh LB slant medium. After incubating at 28℃ for 24-48 hours, store at 4℃ for later use. This is the "pure culture of candidate antagonistic bacterial community" (if it is a mixed community, it needs to be purified to single colonies through multiple streaking to ensure the purity of the strain).

[0027] 3. Activation and suspension preparation of bacterial wilt pathogen (evolution type I) in South China Pathogen activation: A small number of single colonies of bacterial wilt pathogens were picked from the preserved NA slant agar medium. Ralstonia solanacearum Evolutionary type I GM1000 was inoculated into fresh NA liquid medium and cultured at 28°C and 180 rpm for 24 h to obtain an activated suspension of the pathogen. Bacterial concentration adjustment: Take a small amount of activated bacterial suspension, dilute it with sterile water, and then measure the OD using a UV spectrophotometer. 600 Adjust the bacterial suspension concentration to OD value. 600 =0.5 (corresponding to a pathogen concentration of approximately 10) 8 CFU / mL, this concentration is the optimal initial concentration for the confrontation experiment to ensure a stable growth rate of the pathogen.

[0028] 4. Plate confrontation culture experiment Pathogen plating: Take 0.1 mL of the adjusted concentration of pathogen suspension and drop it into the center of the NA solid medium plate. Use a sterile spreader to spread it evenly over the entire plate (the amount of plating on each plate should be the same to ensure uniform pathogen density). Let it stand at room temperature for 10 min to allow the bacterial suspension to be completely absorbed. Antagonistic bacteria inoculation: Use a sterile toothpick to pick a single colony (approximately 2 mm in diameter) of a pure culture of the candidate antagonistic bacteria and spot-inoculate it on a NA plate already coated with pathogens: Using the center of the plate as the center, evenly spot-inoculate 4 antagonistic colonies on a circle with a radius of 2.5 cm (one type of antagonistic bacteria is spot-inoculated on each plate to avoid cross-interference between different strains). At the same time, set up a "blank control" (spot-inoculated with sterile water, without antagonistic bacteria) and a "pathogen control" (only pathogens are coated, without spot-inoculation of antagonistic bacteria), with 3 colonies for each treatment, and label the antagonistic bacteria number, control type, and date; 3. Standoff culture: Invert all standoff plates and place them in a 28℃ constant temperature incubator for static culture for 48-72 hours (avoid shaking to prevent uneven diffusion of antibacterial substances).

[0029] like Figure 1 As shown, this antagonistic bacterium can effectively antagonize... Ralstonia solanacearumGrowth of Evolutionary Type I GM1000 strain. Phylogenetic tree sequencing and average nucleotide abundance (ANI) tests were performed on the entire genome of this antagonistic bacterium using snippy, and the sequences were compared with reference strain sequences in the NCBI GenBank database to obtain a phylogenetic tree (e.g., Figure 2 (As shown). Whole-genome sequence analysis (the gold standard for strain differentiation) showed that it is most similar to the currently known strains. Bacillus velezensis strain The average nucleotide identity (ANI) between FZB42 and the antagonistic bacterium was 98.0475%. This indicates that the antagonistic bacterium belongs to *Bacillus belyssae*. Bacillus velezensis They named it PR-7.

[0030] Example 2: Greenhouse pot experiment evaluation Bacillus velezensis PR-7's resistance to bacterial wilt Under greenhouse conditions (40℃ during the day, 38℃ at night, and 90% humidity), tomato and eggplant plants were inoculated with Ralstonia solanacearum and / or... Bacillus velezensis Forty-eight hours after PR-7 administration, the incidence of bacterial wilt in tomato and eggplant plants was investigated, and the levels of protective enzymes and second messengers in the plants were measured to determine whether PR-7 could prevent the occurrence of bacterial wilt in tomatoes and eggplants and whether it could induce bacterial wilt resistance in plants.

[0031] like Figures 3-5 As shown, Bacillus velezensis PR-7 can effectively alleviate the symptoms of bacterial wilt in tomatoes and eggplants after inoculation with Ralstonia solanacearum. It can reduce the incidence of bacterial wilt in tomatoes from 91.67% to 38.33% and in eggplants from 88.33% to 33.33%, indicating that PR-7 can significantly improve the resistance of tomatoes and eggplants to bacterial wilt.

[0032] Since defensive enzymes play an important role in host plants' resistance to pathogen invasion, this invention also compared the activities of phenylalanine ammonialyase (PAL), catalase (CAT), polyphenol oxidase (PPO), and superoxide (O2) to evaluate the physiological effects and disease resistance of PR-7 against bacterial wilt in tomatoes and eggplants. − Changes in the content of nitric oxide (NO) and hydrogen peroxide (H2O2).

[0033] like Figures 6-7As shown, compared with the control group, after being treated by Ralstonia solanacearum for 48 hours, the PAL and PPO activities of tomato and eggplant in which PR-7 was applied were significantly improved, while the CAT activity was significantly inhibited. It is shown that PR-7 can inhibit the CAT activity of tomato and eggplant, enhance the PPO and PAL activities, and thus enhance the protection of Solanaceae plants such as tomato and eggplant.

[0034] In addition, signal molecules such as H2O2 and NO also participate in the regulation of various metabolic and signal transduction pathways and the regulation of plant defense responses. Figures 8-9 It can be known that PR-7 can change the content of second messengers in tomato and eggplant, regulate the plant signal transduction pathway, and thus activate the immune response and enhance the resistance of Solanaceae plants (such as tomato, eggplant, etc.) to Ralstonia solanacearum.

[0035] In order to further evaluate Bacillus velezensis whether PR-7 can enter the plant body and work, the present application detects the Ralstonia solanacearum infection and Bacillus velezensis After PR-7 treatment, the copies of PR-7 target gene (metC) in the roots, stems and leaves of plants (copies / μL DNA) are detected. Bacillus velezensis As shown, Figure 10 Bacillus velezensis PR-7 can enter the plant body and work.

[0036] The above embodiments of the present application are described in detail in combination with the drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application.​

Claims

1. A type of Bacillus belye, characterized in that, Named Bacillus velezensis PR-7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28979.

2. The application of Bacillus berberis as described in claim 1 in the control of bacterial wilt in Solanaceae crops.

3. The application according to claim 2, characterized in that, The Solanaceae crop mentioned is tomato or eggplant.

4. The application according to claim 2, characterized in that, The pathogen causing bacterial wilt in solanaceous crops is Rhesus solani.

5. A biological control agent for bacterial wilt, characterized in that, It includes Bacillus berberis as described in claim 1.

6. A method for controlling bacterial wilt in Solanaceae crops, characterized in that, Solanaceae crops are treated with Bacillus berberis as described in claim 1 or the bacterial wilt biological control agent as described in claim 5.

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