A biocontrol bacterium Bacillus Velez and its application

Abstract

The present invention discloses a biocontrol bacterium Bacillus Velez and its application. The biocontrol bacterium is classified and named as Bacillus Velez ( Bacillus velezensis ) 3-b, has been deposited in the China Center for Type Culture Collection CCTCC, its deposit number is: CCTCC NO: M2022618, deposit date: May 13, 2022. The Bacillus Velezii of the present invention Bacillus velezensis 3-b belongs to rhizosphere growth-promoting bacteria, which has a significant inhibitory effect on different pathogenic fungi such as Acer spores and Fusarium graminearum, and plays an important role in the biological control of diseases such as wheat fusarium; at the same time, the strain has a growth-promoting effect and can promote wheat growth. The strain of the present invention has simple culture conditions, is easy to preserve, is easy to industrialize, and has good development and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant protection, and in particular relates to a biocontrol bacterium Bacillus Velezii and an application thereof. Background Art

[0002] Wheat is one of the main crops in my country and is widely planted in my country. However, it will be damaged by pathogens at all stages of its growth, resulting in reduced yield and quality and toxin pollution. At present, the prevention and control of wheat diseases mainly rely on chemical control, but with the use of a large number of chemical pesticides, the shortcomings of chemical control are gradually exposed. Chemical pesticides are not easy to degrade, and are prone to residues and environmental biological hazards, destroying the ecological balance. In recent years, biological control has received widespread attention due to its safety, and new biocontrol agents are constantly being discovered.

[0003] Biocontrol agents have many advantages in wheat disease control. Since they are made from natural microorganisms, they are environmentally friendly and will not pollute the soil, water or surrounding ecological environment. Biocontrol agents are also not significantly harmful to humans and animals, and compared with chemical pesticides, they do not produce toxic residues after use. In addition, biocontrol agents have a different mechanism of action from chemical pesticides, can reduce the risk of pathogenic microorganisms developing resistance to fungicides, and have broad-spectrum fungicidal activity and persistence. In addition to effectively controlling diseases, biocontrol agents can also enhance the plant's own immunity, helping to reduce the occurrence of diseases. Summary of the invention

[0004] In view of the problem that the existing biocontrol bacteria used in crops such as wheat are still insufficient, the present invention provides a Bacillus Velezii, which can act on a variety of plant fungi and promote plant growth.

[0005] The present invention has isolated a biocontrol strain Bacillus velezine (Bacillus velezine) from a wheat-corn rotation field, which has an antagonistic effect on multiple fungi and a promoting effect on wheat growth. Bacillus velezensis )3-b, the bacteria has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M2022618, the deposit date is May 13, 2022, and the deposit address is Wuhan University, Wuhan, China.

[0006] The Bacillus Velezii of the present invention ( Bacillus velezensis ) The optimal fermentation conditions of 3-b are: LB medium for shaking culture, the shaking culture speed is 180r / min~220r / min, the culture temperature is 28℃, the initial pH is 6~7, and the fermentation time is 24~60h.

[0007] In one aspect of the present invention, the present invention provides Bacillus Velez Bacillus velezensis) 3-b in the prevention and treatment of plant fungal diseases, the fungal diseases include those caused by Fusarium graminearum ( Fusarium graminearum ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Echinopsora acerola ( Mycocentrospora acerina )、Fusarium spp.( Fusarium proliferatum ) is caused by one or more pathogens. Preferably, the disease is wheat fusarium head blight.

[0008] In one aspect of the present invention, the present invention also provides Bacillus Velez Bacillus velezensis ) 3-b and the use of fermentation broth and fermentation supernatant containing metabolites in promoting plant growth. The fermentation broth is prepared by using a culture medium to culture Bacillus velez ( Bacillus velezensis )3-b strain was fermented to obtain a large amount of Bacillus Velezii ( Bacillus velezensis ) 3-b of the bacterial solution; the fermentation supernatant is for Bacillus Velezii ( Bacillus velezensis ) After the 3-b strain is fermented and cultured, the fermentation broth is centrifuged to obtain the supernatant. Those skilled in the art will understand that both the fermentation broth and the fermentation supernatant contain a variety of metabolites produced by the strain during the fermentation process, and these metabolites are the main components for the strain to perform its functions.

[0009] In one aspect of the present invention, the present invention also discloses a method containing Bacillus Velezii ( Bacillus velezensis ) 3-b. The bacterial agent can be a liquid preparation or a powder. For the liquid preparation, it is a bacterial solution containing Bacillus Velez 3-b. The powder is a mixture of Bacillus Velez ( Bacillus velezensis ) The fermentation liquid obtained after the 3-b strain is fermented is then freeze-dried to prepare the product. During the freeze-drying process of the fermentation liquid, a freeze-drying protective agent, a buffer and other substances well known in the art may also be added.

[0010] During the growth period of plants, the roots of plants can be fertilized with Bacillus velezensis ( Bacillus velezensis ) 3-b of the bacterial agent, preferably, the bacterial solution containing Bacillus Velez 3-b is applied. In the present invention, plant growth mainly involves underground and aboveground parts, and the effect of the bacterial agent on plant growth can be measured by indicators such as root length and leaf length.

[0011] In one aspect of the present invention, due to the broad-spectrum antibacterial effect of the Bacillus Velez 3-b of the present invention, it can also be used to prevent and control fungal diseases of one or more of various other plants including wheat, rice, corn, rapeseed, and barley.

[0012] In the present invention, "Bacillus Velez 3-b", "Bacillus Velez ( Bacillus velezensis )3-b","Bacillus velezensis 3-b" all refer to the biocontrol bacterium Bacillus Velezii with the deposit number of CCTCC NO: M2022618 of the present invention. Beneficial Effects

[0013] (1) The Bacillus Velezii of the present invention ( Bacillus velezensis ) 3-b is a kind of soil bacteria that promotes growth. It has a strong inhibitory effect on bacteria such as Fusarium graminearum and plays a very important role in the biological control of plant diseases. It has a broad spectrum of antagonism against pathogens and also has the advantages of promoting plant growth.

[0014] (2) Bacillus Velezii of the present invention ( Bacillus velezensis ) 3-b can be artificially cultured, the culture conditions are simple, it is easy to preserve, it is easy to industrialize and has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The colony morphology and Gram staining results of the bacteria numbered 3-b in Example 1 are shown.

[0016] Figure 2 The phylogenetic tree of the bacteria numbered 3-b was constructed based on the concatenated gene sequences of 16S rDNA (A) and gyrA (B) in the present invention.

[0017] Figure 3 These are the results of the bacterial physiological and biochemical experiments numbered 3-b in Example 2, A is a gelatin liquefaction experiment, and B is a v-p experiment. Figure 4 is the Bacillus Velez in Example 3 ( Bacillus velezensis ) 3-b Biological characteristics study, including A phosphate solubilization ability test; B potassium solubilization ability test; C nitrogen fixation ability test; D iron love ability test.

[0018] Figure 5 is the Bacillus Velez in Example 4 ( Bacillus velezensis ) 3-b has a broad-spectrum antibacterial effect, A. Acer serrata; B. Colletotrichum gloeosporioides; C. Fusarium graminearum; D. Fusarium graminearum.

[0019] Figure 6 This is a study on the effect of Bacillus Velez 3-b on the growth of wheat seedlings in Example 5. A is a wheat morphology comparison using Bacillus Velez 3-b bacterial suspension and a blank control (CK); B is a leaf area comparison; C is a root length comparison; and C is a leaf length comparison.

[0020] Figure 7 This is a study on the protective effect of Bacillus Velez 3-b against field wheat fusarium rust in Example 6. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in the embodiments and test examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0022] Unless otherwise specified, the biochemical reagents and culture medium of the present invention are all commercially available reagents.

[0023] Example 1 Isolation and screening of Bacillus velez 3-b

[0024] A maize plant was collected from a maize-wheat rotation area in Anhui Province, and its roots were gently shaken to remove most of the loose soil that easily falls off. The soil samples were brought away from the roots of the plant using a sterile brush. Rhizosphere soil samples were collected from multiple plants of the same plant and placed in sterile bags.

[0025] The various microbial communities existing in the soil were separated by the soil dilution and coating method in the experiment. 15 grams of naturally air-dried soil was selected as the experimental sample. The specific operation was as follows: first, 150 mL of sterile water was prepared in a 250 mL conical flask for standby use. Then, 15 grams of soil was transferred into the conical flask and shaken at 180 r. min-1 for half an hour. Then, the mixed liquid was allowed to stand for 20 s to obtain a 10-fold diluted solution. Finally, the 10-fold diluted solution was subjected to a 10-fold gradient dilution to obtain a series of dilutions. 100 μL of each gradient was evenly dropped on the center of the LB plate. The coating operation should be applied with sterile equipment and ensured to be uniform. After applying, it was placed on a clean bench to be blown dry for 5 minutes. Before the LB culture medium completely absorbed the liquid substance, the culture dish cover was covered and placed in a biochemical culture device for cultivation in a dark environment at 28°C. After about 24 hours, a single colony isolate will form on the LB plate. Based on the shape and color of the colony, the required single colony can be screened from the new LB plate by using a bacterial inoculation loop, and a series of operations such as separation and purification can be performed on the colony. The screened single colony bacteria will be suspended in 15% glycerol and stored at -80°C.

[0026] A colony with high activity was screened and extracted by the plate confrontation method, numbered 3-b, and inoculated on LB medium to observe its colony morphology. The surface of 3-b colony was smooth and the edges were neat after 24 hours of culture, and the colony became wrinkled after 72 hours (see Figure 1 ) and subjected to Gram staining. If the Gram staining result is purple, it is positive (see Figure 1 ).

[0027] The 16S rDNA of strain 3-b was extracted and sequenced, and the sequencing results were compared with the nucleic acid sequence in the NCBI database. The results showed that the similarity between strain 3-b and Bacillus velez was 99.97%, and the similarity measured with specific primers was 99.53%. Figure 3 It can be seen that the phylogenetic tree of strain 3-b constructed by the neighbor-joining method using MAGE X software shows that strains with high homology to strain 3-b all belong to the genus Bacillus Velezii. Therefore, strain 3-b was named Bacillus Velezii ( Bacillus velezensis )3-b, deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M2022618, the deposit date is May 13, 2022, and the deposit address is Wuhan University, Wuhan, China.

[0028] Example 2 Bacillus Velez Bacillus velezensis ) 3-b Physiological and biochemical experimental test

[0029] Gelatin liquefaction experiment

[0030] The culture medium is: 5 g peptone; 3 g beef extract; 120 g gelatin; 1000 mL deionized water, adjust pH to 7.0-7.2, and sterilize at 121 °C with high pressure steam for 20 min. The sterilized gelatin culture medium is divided into 5 mL sterile test tubes, inoculated and cultured. At the same time, two blank controls are set up and placed in a 36 °C incubator for culture. Result observation: If there are colonies on the surface of the gelatin but there is no depression on the surface of the gelatin and it is a stable clot, it is negative, otherwise it is positive.

[0031] vp experiment

[0032] The culture medium is: 5 g peptone, 5 g glucose, 5 g potassium dihydrogen phosphate, 1000 mL deionized water, adjust the pH to 7.0-7.2, sterilize at 121 ℃ high pressure steam for 30 min, and then dispense into test tubes. Inoculate and culture: Use an inoculation loop to inoculate Bacillus Velez 3-b into the culture medium and culture at 36 ℃ for 2-6 days. If the culture medium does not change color significantly and remains light yellow or colorless, it is negative. If the culture medium turns red or pink, it is positive.

[0033] Depend on Figure 2 It can be seen that the results of the gelatin liquefaction test and the vp test were both positive, indicating that Bacillus velez 3-b may be a bacterium that can decompose gelatin and metabolize acetic acid.

[0034] Example 3 Bacillus Velez Bacillus velezensis ) 3-b Biological property test

[0035] (1) Bacillus Velez Bacillus velezensis ) Determination of phosphate solubilization capacity of 3-b

[0036] The Bacillus Velez 3-b strain was inoculated into a culture medium containing specific phosphate-solubilizing bacteria, and the appearance of a transparent phosphate-solubilizing zone around the colonies was observed. If a transparent phosphate-solubilizing zone appeared, it preliminarily indicated that the strain had the ability to solubilize phosphate.

[0037] (2) Bacillus Velez Bacillus velezensis ) 3-b Screening determination of potassium dissolving ability

[0038] Inoculate the Velez 3-b strain on a culture medium containing a specific bacterial culture medium that can dissolve potassium. Observe whether there is an oil droplet-like substance around the colony. If there is an oil droplet-like substance, it preliminarily indicates that the strain has the ability to dissolve potassium.

[0039] (3) Screening and capacity determination of nitrogen-fixing bacteria

[0040] The Velez Bacillus 3-b strain is inoculated on a specific nitrogen-fixing bacteria sucrose medium. After culturing, observe whether the three generations on the medium can survive. If they survive, it proves that Velez Bacillus 3-b has the ability to fix nitrogen.

[0041] (4) Screening and capacity determination of iron-loving bacteria

[0042] Inoculate Bacillus Velez 3-b on a specific CAS medium. After culture, observe whether there is a yellow halo around the colony. If there is a yellow halo, it preliminarily indicates that the strain has the ability to secrete siderophores.

[0043] Depend on Figure 4 The results showed that Bacillus Velez 3-b did not have the ability to solubilize phosphate and potassium, but had the ability to fix nitrogen and secrete iron carriers.

[0044] Example 4 Bacillus Velez Bacillus velezensis ) 3-b broad-spectrum antibacterial ability test

[0045] Select fresh Bacillus Velez Bacillus velezensis ) 3-b single colony, inoculate it and culture it. Take fresh mycelium blocks at the edge of the fungal colony and transfer them to the center of a clean PDA culture medium plate. Place four sterile filter paper pieces (Φ=6 mm) around each bacterial block, and then pipette 2 µL of bacterial solution onto the filter paper pieces. At the same time, use 2 µL of sterile water as a blank control. These plates were cultured in an incubator at 25°C. When the mycelium of the control group almost filled the entire plate, measure the colony diameter. Each group of experiments was repeated 3 times, and the inhibition rate was calculated and compared with other Bacillus Velez. The results are shown in the following table (1) and the antagonistic effect diagram is as follows Figure 5At the same time, the commercialized Velez Bacillus M173 strain was used as a control, and the Velez Bacillus M173 was obtained from Muen Biotechnology Co., Ltd. It can be seen from Table 1 that the Velez Bacillus ( Bacillus velezensis ) The antibacterial ability of 3-b is significantly stronger than that of the commercialized Bacillus Velezii, especially on the Acanthopanax spp. The commercialized strain control has basically no inhibitory ability, while the strain of the present invention has extremely strong inhibitory power, which shows the specificity of the strain of the present invention.

[0046] The calculation formula of the antibacterial rate of Bacillus Velez 3-b in wheat root soil is:

[0047]

[0048] Where: A refers to the diameter of the pathogen colony in the control group;

[0049] B refers to the diameter of the pathogen colony in the treatment group;

[0050]

[0051] Example 5 Study on the effect of Bacillus velez 3-b on the growth of wheat seedlings

[0052] One strain was selected from the selected single colonies of Bacillus Velez 3-b, inoculated and cultured, and the cultured single colony of Bacillus Velez 3-b was selected and inoculated into a new LB medium for culture again. Subsequently, the cultured bacterial solution was transferred to a centrifuge tube and centrifuged in a low-temperature high-speed centrifuge to remove the supernatant and leave the bacterial precipitate. Finally, the bacterial precipitate was resuspended with sterile water and then used for inoculation. 100 ml of LB liquid culture medium was added to each 250 ml triangular flask, and after high-temperature sterilization, 20 ml of bacterial suspension was inoculated. Finally, the inoculant of Bacillus Velez 3-b was obtained by culturing at 37 ° C and 160 r / min for 2-3 days. Then, the wheat was disinfected and germinated before planting, and the Bacillus Velez 3-b inoculant was irrigated to the roots of the wheat 3 days after the wheat seedlings germinated. As a control group, the same steps were used for irrigation with sterile water.

[0053] See also Figure 6The results showed that the bacterial solution of Bacillus Velez 3-b had a growth-promoting effect on the growth of wheat seedlings during the growth process. The average root length of wheat irrigated with the bacterial solution increased by 17.9%, the average length of its leaves increased by 11.9%, and the leaf area increased by 76.5%. Under the same sample conditions, wheat had a certain growth-promoting effect when irrigated with a certain amount of bacterial solution during its growth. The roots of the plants were thicker and the number of lateral roots increased significantly compared to those not irrigated with the bacterial solution, and the leaf area was significantly larger than that of the wheat not irrigated. However, after treatment with Bacillus Velez 3-b, the effect on improving the root length of wheat seedlings was limited.

[0054] Example 6 Study on the efficacy of Bacillus Velez 3-b against field wheat scab

[0055] The wheat strain for the test was Annong 203, and the experimental site was Nongcui Garden of Anhui Agricultural University. Single colonies of the test strain were picked and inoculated into LB medium and cultured in a shaking incubator at 28°C and 180 r / min for 36 hours. Two different varieties of wheat, Annong 203 and Annong 1689, were selected during the flowering period of wheat in mid-April. The bacterial solution of the test strain was evenly sprayed or injected on the surface of wheat ears of the same size and similar growth status, and then bagged. After 3 days, each wheat ear was injected with 1×10 6 10 μL of spore suspension was sprayed with sterile water as blank control, and root irrigation and spraying were used as control for two varieties of wheat. Treatment 1 was inoculated with Fusarium graminearum 3 days after spraying the bacterial solution, and treatment 2 was inoculated with Fusarium graminearum 3 days after injecting 10 μL of bacterial solution, corresponding to Figure 6 After applying Bacillus Velez 3-b, A, B, C, and D, 50 ears were randomly selected from each wheat variety 15 days later to investigate the incidence of wheat fusarium rust and record the statistical results.

[0056] The disease level is classified as follows: Level 0: no diseased ears, Level 1: the number of diseased ears ≤ 25% of the total number of ears, Level 2: the number of diseased ears accounts for 25%~50% of the total number of ears, Level 3: the number of diseased ears accounts for 50%~75% of the total number of ears, Level 4: the number of diseased ears accounts for 75%~100% of the total number of ears.

[0057] See Figure 7As shown, the control efficacy of Bacillus Velez 3-b on two different varieties of wheat applied in different ways in the field was over 60%, with the highest control efficacy reaching 81.89%. Compared with similar Bacillus Velez that have been reported so far, this strain has better control effect. For example, Bacillus Velez JS25R (CN107236693B) can reduce the disease index of wheat fusarium from 64.9% to 23.1%, with a control efficacy of 64.41%. This strain can reduce the disease index from 58.47% to 11.19%, with a control efficacy of up to 81.89%, which has significant technical advantages.

[0058] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. A biocontrol bacterium, characterized in that: The biocontrol bacteria is Bacillus Velezii ( Bacillus velezensis )3-b, deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M2022618, the deposit date is May 13, 2022, and the deposit address is Wuhan University, Wuhan, China.

2. The biocontrol bacteria according to claim 1, characterized in that: The optimal fermentation conditions of the Velez Bacillus 3-b are: shaking culture using LB medium, the shaking culture speed is 180 r / min~220 r / min, the culture temperature is 28°C, the initial pH is 6~7, and the fermentation time is 24~60 h.

3. A bacterial agent, characterized in that: The bacterial agent contains the biocontrol bacterium Bacillus Velez 3-b according to claim 1.

4. The bacterial agent according to claim 3, characterized in that The bacterial agent is a liquid preparation or a powder.

5. The bacterial agent according to claim 4, characterized in that The liquid preparation is a fermentation liquid containing the biocontrol bacterium Bacillus Velez 3-b; and the powder is a freeze-dried powder containing Bacillus Velez 3-b.

6. Use of the biocontrol bacteria according to claim 1 or the microbial agent according to claim 3 in the prevention and treatment of plant diseases, characterized in that: Fusarium graminearum Fusarium graminearum Colletotrichum gloeosporioides Colletotrichum gloeosporioides , Echinopsora cerana Mycocentrosporaacerina Fusarium Fusarium proliferatum Diseases caused by one or more of the following.

7. The use of the biocontrol bacteria or microbial agent in the prevention and treatment of plant diseases according to claim 6, characterized in that: The disease is wheat fusarium rust.

8. Use of the biocontrol bacteria according to claim 1 or the bacterial agent according to claim 3 in promoting plant growth.

9. The use of the biocontrol bacteria or microbial agent in promoting plant growth according to claim 8, characterized in that: The plants include wheat, rice, corn, rapeseed and barley.

10. The use of the biocontrol bacteria or bacterial agent in promoting plant growth according to claim 9, characterized in that: The plant is wheat.

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