Bacillus Velez and its application

Abstract

The invention relates to the technical field of bioengineering, and in particular to a Bacillus velezensis NHKJ0001 and an application thereof; the Bacillus velezensis NHKJ0001 has a deposit number of CGMCC No.27608; the application discloses a Bacillus velezensis NHKJ0001 and an application thereof; the Bacillus velezensis NHKJ0001 disclosed in the application has a good antibacterial effect on the pathogenic fungus Fusarium oxysporum of broccoli wilt disease; the Bacillus velezensis NHKJ0001 can not only promote the growth of broccoli, but also has a preventive and control effect on broccoli wilt disease.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to Bacillus velez NHKJ0001 and application thereof. Background Art

[0002] Bacillus spp. is an important biocontrol microbial resource in agricultural production, which has antagonistic effects on a variety of plant pathogens. In recent years, microbial pesticides and fertilizers made from Bacillus spp. have played an important role in plant disease prevention and control.

[0003] Bacillus velezensis is a new type of biocontrol bacillus. Its wide and effective antibacterial activity against many plant pathogenic fungi and even some bacteria is one of the important reasons why people have paid attention to it in recent years. Bacillus velezensis is a type of Gram-positive bacteria, rod-shaped, 0.5× (1.5-3.5μm) in size. Under adverse conditions, it can produce spores in the middle or tail of the bacteria. The spores are oval in shape. The bacteria move by flagella and grow in a temperature range of 15-45℃. Bacillus velezensis is widely distributed in nature and can be isolated from habitats such as marine and river sediments, soil, plant rhizosphere, plant tissues and animal intestines. It is harmless to humans and animals and does not pollute the environment.

[0004] Bacillus Velez currently plays a certain role in the prevention and control of plant diseases, but the relevant technology does not disclose its role in preventing and controlling broccoli wilt. Therefore, screening and obtaining Bacillus Velez that has a good antibacterial effect on the pathogen of broccoli wilt Fusarium oxysporum, promotes the growth of broccoli, and has a preventive and control effect on broccoli wilt has become an urgent problem to be solved. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present application provides a Bacillus velezensis NHKJ0001 and applications thereof.

[0006] In a first aspect, the present application provides a Bacillus velezensis NHKJ0001, which adopts the following technical solution:

[0007] A Bacillus velezensis, wherein the Bacillus velezensis is Bacillus velezensis (Bacillus velezensis) NHKJ0001, the preservation number is CGMCC No.27608, the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation date is June 12, 2023.

[0008] Bacillus velezensis is a Gram-positive bacterium. This experiment found that a strain of Bacillus velezensis had a good antibacterial effect on Fusarium oxysporum, the pathogen of broccoli wilt, and had a certain effect on promoting the growth of broccoli and preventing and controlling broccoli wilt.

[0009] The Bacillus Velez was isolated from the rhizosphere soil of a broccoli plant at the broccoli planting base of Lingyang Farm in Rudong, Nantong. The soil at the base is slightly salinized sandy soil.

[0010] Preferably, the Bacillus velezensis NHKJ0001 is a salt-tolerant strain.

[0011] In a second aspect, the present application provides a fermentation liquid, using the following technical solution:

[0012] A fermentation broth is obtained by fermenting Bacillus velezensis NHKJ0001.

[0013] In a third aspect, the present application provides a biocontrol agent, which adopts the following technical solution:

[0014] A biocontrol agent comprises Bacillus velezensis NHKJ0001 or a fermentation liquid of Bacillus velezensis NHKJ0001.

[0015] In a fourth aspect, the present application provides a use of Bacillus velezensis NHKJ0001 or its fermentation liquid, or its biocontrol agent in preventing and controlling plant diseases caused by plant pathogens and / or promoting plant growth, using the following technical solution:

[0016] The invention discloses an application of Bacillus velezensis NHKJ0001 or its fermentation liquid or its biocontrol agent in preventing and controlling plant diseases caused by plant pathogens and / or promoting plant growth.

[0017] Preferably, the plant pathogen is Fusarium oxysporum.

[0018] Preferably, the plant disease is broccoli wilt.

[0019] When the Bacillus velezensis NHKJ0001 of the present application is antagonistic to the pathogen causing broccoli wilt on a plate, the inhibition rate reaches 58% or more, indicating that Bacillus velezensis NHKJ0001 has a good antibacterial effect on Fusarium oxysporum, the pathogen of broccoli wilt.

[0020] The Bacillus velezensis NHKJ0001 of the present application can treat a colony of the pathogen Fusarium oxysporum with a diameter of 3.5 cm, indicating that it has a preventive and control effect on broccoli wilt disease.

[0021] The Bacillus velezensis NHKJ0001 isolated in the present application has a promoting effect on the growth of broccoli. The stem diameter of the strain treated with root irrigation increases by 30%-50% compared with the water control treatment, and the leaf color is greener.

[0022] In a fifth aspect, the present application provides a use of Bacillus velezensis NHKJ0001 or its fermentation liquid or its biocontrol agent in the preparation of a drug for preventing and treating broccoli wilt caused by Fusarium oxysporum and / or promoting plant growth, using the following technical solution:

[0023] The invention discloses an application of Bacillus velezensis NHKJ0001 or its fermentation liquid or its biocontrol agent in preparing a medicine for preventing and treating broccoli wilt disease caused by Fusarium oxysporum and / or promoting plant growth.

[0024] In a sixth aspect, the present application provides a drug, which adopts the following technical solution:

[0025] A medicine comprises Bacillus velezensis NHKJ0001 or its fermentation liquid or its biocontrol agent.

[0026] In a seventh aspect, the present application provides a use of Bacillus velezensis NHKJ0001 or its fermentation liquid or its biocontrol agent or medicine in the preparation of pesticides, antibacterial agents, growth promoters or fertilizers, using the following technical solution:

[0027] The invention discloses an application of Bacillus velezensis NHKJ0001 or its fermentation liquid or its biocontrol agent or its medicine in the preparation of pesticides, bacteriostatic agents, growth promoters or fertilizers.

[0028] In a specific embodiment, a growth promoter includes Bacillus velezensis NHKJ0001 or its fermentation broth or its biocontrol agent or its medicine.

[0029] In a specific embodiment, a fertilizer includes Bacillus velezensis NHKJ0001 or its fermentation liquid or its biocontrol agent or its medicine.

[0030] In a specific embodiment, a pesticide includes Bacillus velezensis NHKJ0001 or its fermentation broth or its biocontrol agent or its medicine.

[0031] In a specific embodiment, a bacteriostatic agent includes Bacillus velezensis NHKJ0001 or its fermentation broth or its biocontrol agent or its medicine.

[0032] In summary, the present application includes at least one of the following beneficial technical effects:

[0033] The present application discloses a Bacillus velezensis NHKJ0001 and an application thereof; the Bacillus velezensis NHKJ0001 disclosed in the present application has a good antibacterial effect on Fusarium oxysporum, the pathogen of broccoli wilt disease; it can not only promote the growth of broccoli, but also prevent and control broccoli wilt disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a comparison chart of the plate growth antagonism test between Fusarium oxysporum and Bacillus velez;

[0035] Figure 2 After potted broccoli was inoculated with Fusarium oxysporum, the protective effect of the control treatment (water) and the root irrigation treatment with Bacillus Velez on broccoli;

[0036] Figure 3 A diagram showing the effect of Bacillus Velez on promoting broccoli growth. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further illustrated by specific embodiments below. The specific embodiments do not limit the protection scope of the present invention; some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0038] The raw materials involved in this application are all commercially available products, and the application is further described in detail below in conjunction with the embodiments and comparative examples.

[0039] Embodiment 1:

[0040] Screening, Isolation and Identification of Bacillus velezensis NHKJ0001:

[0041] 1. Soil sample separation

[0042] The soil sample was isolated from the rhizosphere soil of a broccoli plant at the broccoli planting base of Lingyang Farm in Rudong, Nantong. The soil at the base was slightly salinized sandy soil.

[0043] Prepare 0.85% saline solution in a mass-volume ratio, select 250mL flasks, fill each bottle with 50mL, sterilize at 121℃ for 20min, cool and set aside. Weigh 5g of the mixed root soil sample and add it to the flask containing 0.85% saline solution in a mass-volume ratio, shake on a shaker at 28℃ and 180rpm for 30min, let it stand for 5min, and take the supernatant to obtain 10 -1 dilution of soil (i.e. diluted 10 times); dilute with sterile water to 10 -7 ,spare.

[0044] 2. Initial screening

[0045] The above 10 -1 -10 -7 The soil gradient dilution solution was subjected to high temperature treatment at 85°C for 15 min and then allowed to cool naturally.

[0046] Prepare LB plates containing 15% NaCl (high salinity environment) in advance, and pipette 10 -3 -10 -7 50 μL of the dilution solution was coated on the plate, and each concentration was repeated 3 times. The coated LB plate was placed in a 37°C incubator, and the growth of the colonies on the plate was observed every day.

[0047] 3. Rescreening

[0048] The colonies grown on the LB plate in step (2) were picked and streaked on a high-salt LB medium (containing 15% NaCl) for pure culture, and the growth of Bacillus on the LB medium was observed.

[0049] 4. Identification

[0050] 4.1 Morphological identification

[0051] Activate the dominant strain on the plate and culture it in a constant temperature box for 48-72 hours. Observe and record the colony color, growth rate, shape, edge, degree of protrusion, surface condition, etc. Note: If the colony grows slowly, the culture time can be extended.

[0052] The culture morphology of Bacillus Velezii NHKJ0001: On LB solid culture medium, it appears as white opaque colonies with a rough surface, wrinkled edges, and a slight depression in the middle.

[0053] 4.2 Molecular identification

[0054] The screened strains were identified by molecular biology using 16S rDNA universal primers 27F and 1492R.

[0055] The sequence of primer 27F is: 5′-AGAGTTTGATCATGGCTCAG-3′, SEQ ID NO: 1;

[0056] The sequence of primer 1492R is: 5'-TACGGYTACCTTGTTACGACT T-3', SEQ ID NO: 2; PCR amplification reaction system (25 μL): 2×TaqMix 12.5 μL, primer 27F, primer 1492R 1 μL each, template DNA 1 μL, ddH2O 9.5 μL.

[0057] PCR amplification reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 54°C for 30 s; extension at 72°C for 1 min, 34 cycles; extension at 72°C for 10 min.

[0058] The PCR product was detected by 1% agarose gel electrophoresis at 130V, and the product was sent to Sangon Biotech for sequencing. The obtained sequence was uploaded to NCBI (https: / / www.ncbi.nlm.nih.gov) for BLAST sequence homology comparison, and the strain with the highest comparison score was used as the identification species. The sequence is shown in SEQ ID NO: 3.

[0059] The screened strain was named Bacillus velezensis NHKJ0001 and deposited in the General Microbiology Center of China Culture Collection Administration. The deposit date was June 12, 2023, and the deposit number was CGMCC No.27608. The Bacillus velezensis NHKJ0001 has salt tolerance.

[0060] 5. Save

[0061] The strain was cultured in LB liquid culture medium at 37°C and 180 rpm shaking on a shaker until the OD value was above 1.0, then an appropriate amount of the culture medium was mixed with an equal volume of glycerol solution (volume concentration of 30%) and then frozen at -80°C.

[0062] Embodiment 2:

[0063] 1. Preparation of biocontrol bacteria of Bacillus Velezii NHKJ0001:

[0064] Use an inoculation needle to streak the frozen Bacillus on the LB plate and incubate at 37°C for 1-2 days. After a visible single colony grows, pick a single colony and transfer it to liquid LB medium for incubation at 37°C with a shaker at 180 rpm until 1×10 9 cfu / ml.

[0065] 2. Antagonistic experiment of biocontrol bacteria against Bacillus velez NHKJ0001. The specific steps are as follows:

[0066] (1) First, Bacillus Velez NHKJ0001 biocontrol bacteria and Bacillus (control bacteria) were inoculated on PDA plates. Each plate was inoculated with Bacillus Velez NHKJ0001 biocontrol bacteria and Bacillus (control bacteria) at the same time. The same Bacillus were inoculated oppositely and placed in a 28°C incubator for culture.

[0067] (2) Observe the growth of Bacillus and inoculate Fusarium oxysporum after about one day of growth. Take a freshly grown Fusarium oxysporum plate, take a 5 mm diameter bacterial disc from the periphery of the colony, with the mycelium facing down, and inoculate it in the center of the PDA plate; in addition to inoculating the plate antagonistic to Bacillus, inoculate a Fusarium oxysporum blank control and place them in a 28°C incubator for culture.

[0068] (3) 5-7 days after the inoculation of Fusarium oxysporum, observe the antagonistic effect on the plate. The plate is considered complete when the Fusarium oxysporum blank control fills the plate.

[0069] The inhibition rate was calculated according to the inhibition rate (%) = (control pathogen colony diameter - treated pathogen colony diameter) / control diameter * 100%.

[0070] When Bacillus Velezii NHKJ0001 was antagonized against the pathogen causing broccoli wilt on a plate, the inhibition rate reached 58% or more. Figure 1 As shown, it is shown that the Bacillus Velezii NHKJ0001 of the present application has a good antibacterial effect on the pathogen of broccoli wilt Fusarium oxysporum.

[0071] 3. Potted plant disease prevention and control test

[0072] A pot experiment was conducted. At the beginning of the experiment, broccoli seeds with full seeds were selected, and vermiculite and nutrient soil were weighed and mixed in a mass ratio of 1:1 to form a cultivation medium. 1-2 seeds were placed in each grid of the seed pot. After the seedlings grew up, they were inoculated with biocontrol bacteria (Bacillus Velezii NHKJ0001) and pathogens (Fusarium oxysporum) and their growth was observed.

[0073] On January 3, 2022, the concentration of the inoculation prepared in Example 2 was 1×10 9 cfu / ml of biocontrol bacteria, dilute the fermented liquid of biocontrol bacteria 10 times and directly irrigate the roots, 25ml for each seedling.

[0074] The pathogen Fusarium oxysporum was inoculated on January 7, and the spore concentration was 1×10 6 / ml, irrigate the roots of each seedling with 25ml.

[0075] The treatment results were observed on January 27 and the test results are shown in Table 1.

[0076] Table 1 Results of potted plant disease prevention and control test

[0077]

[0078] According to Table 1, the Bacillus Velezii NHKJ0001 in the present application has a preventive and control effect on broccoli wilt caused by Fusarium oxysporum. Figure 2 shown.

[0079] 4. Potted plant growth promotion test

[0080] At the beginning of the experiment, broccoli seeds with full seeds were selected, vermiculite and nutrient soil were weighed and mixed in a mass ratio of 1:1 to form a cultivation medium, and 1-2 seeds were placed in each grid of the seed pot. After the broccoli grew to 2 true leaves, the concentration of 1×10 9 cfu / ml of biocontrol bacteria. After 2 months of root irrigation, the stem diameter at the soil interface was recorded and photographed; the control group was irrigated with water. Statistical analysis showed that the isolated Bacillus Velezii promoted the growth of broccoli, which was manifested by thick stems, greener leaves, and a plant growth rate of 30%-50%. Figure 3 shown.

[0081] The above test results show that the Bacillus Velezii can directly inhibit the growth of pathogenic fungi in vitro, reduce the degree of harm caused by pathogens to broccoli, and promote the growth of plants.

Claims

1. Bacillus Velez Bacillus velezensis )NHKJ0001, the deposit number is CGMCC No.27608.

2. The Bacillus Velezii according to claim 1, characterized in that: The Bacillus Velez NHKJ0001 is a salt-tolerant strain.

3. A fermentation liquid, characterized in that: Obtained by fermentation of the Bacillus Velezii NHKJ0001 according to claim 1 or 2.

4. A biocontrol agent, characterized in that: The method comprises the Bacillus Velezii NHKJ0001 according to claim 1 or 2 or the fermentation broth according to claim 3.

5. Use of the Bacillus Velez NHKJ0001 according to claim 1 or 2 or the fermentation liquid according to claim 3 in preventing and controlling plant diseases caused by plant pathogens and / or promoting plant growth, characterized in that: The plant pathogen is Fusarium oxysporum ( Fusarium oxysporum ); the plant disease is broccoli wilt, and the plant is broccoli.

6. Use of the Bacillus Velez NHKJ0001 according to claim 1 or 2 or the fermentation liquid according to claim 3 in the preparation of a drug for preventing and treating broccoli wilt caused by Fusarium oxysporum and / or promoting plant growth, wherein the plant is broccoli.

7. A drug, characterized in that: It comprises the Bacillus Velezii NHKJ0001 according to claim 1 or 2, the fermentation broth according to claim 3, or the biocontrol agent according to claim 4.

8. Use of the Bacillus Velez NHKJ0001 according to claim 1 or 2, the fermentation liquid according to claim 3, or the drug according to claim 7 in the preparation of pesticides, antibacterial agents, growth promoters or fertilizers for broccoli.

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