Bacillus velezensis with antibacterial activity and application thereof

Abstract

The invention discloses bacillus velezensis with antibacterial activity and application of the bacillus velezensis, and belongs to the technical field of biological prevention and control of crop diseases. The bacillus velezensis FD-001 is preserved in the China General Microbiological Culture Collection Center on November 15, 2023, and the preservation number of the bacillus velezensis FD-001 is CGMCC (China General Microbiological Culture Collection Center) No. 29016. According to the invention, a bacillus velezensis FD-001 strain is separated from corn, a plate confrontation test proves that the bacillus velezensis FD-001 strain can effectively antagonize various corn pathogenic fungi, and the screened strain FD-001 has a light broad-spectrum antibacterial property; in combination with pot experiments, it is proved that the strain can effectively control corn stalk rot, and a safe and reliable biological product is provided for prevention and treatment of corn diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control of crop diseases, and particularly relates to Bacillus velezine with antibacterial activity and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Corn is an important food crop and cash crop in my country's agricultural production. It has a wide range of applications and can create rich benefits. However, soil-borne diseases have always existed in the planting and growth process of corn. Among them, corn stalk rot is a soil-borne disease that seriously threatens corn production, causing serious lodging and yield loss. It is widely distributed and has serious harm. At present, the prevention and control of this disease mainly adopts chemical seed dressing agents and increased application of potassium fertilizers, but the prevention effect is generally poor, and most of them only work at 30%. In addition, the long-term use of chemical seed dressing agents and chemical fertilizers has had varying degrees of adverse effects on the soil ecological environment.

[0004] Biological control has become a research hotspot in recent years because of its environmental friendliness, safety, reliability, cost savings and efficiency improvements. It has broad application prospects, but the existing technology lacks new strains that can effectively control corn stalk rot. Summary of the invention

[0005] The purpose of the present invention is to provide a Bacillus Velez subtilis with antibacterial activity and its application to solve the problems existing in the above-mentioned prior art. The Bacillus Velez subtilis FD-001 provided by the present invention can effectively antagonize a variety of corn pathogenic fungi, effectively control corn stalk rot, and provide a safe and reliable biological product for corn disease prevention and control.

[0006] To achieve the above object, the present invention provides the following solutions: In a first aspect of the present invention, there is provided a Bacillus Velezii ( Bacillus velezensis ) FD-001 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 15, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.29016.

[0007] The second aspect of the present invention provides a bacterial agent, which comprises the strain of Bacillus Velezii FD-001 or its fermentation liquid.

[0008] Furthermore, the concentration of Bacillus Velezii FD-001 in the fermentation broth is 1×10 8 ~1×10 10cfu / mL. Furthermore, the preparation method of the fermentation broth is as follows: after activating the Velez Bacillus FD-001 in MRS medium, adjusting the bacterial cell density of the bacterial suspension, and then inoculating the activated Velez Bacillus FD-001 in MRS medium for expansion culture to obtain a Velez Bacillus FD-001 concentration of 1×10 8 ~1×10 10 cfu / mL of fermentation broth.

[0009] Preferably, the inoculation amount of the activated Bacillus Velezii FD-001 is 1-3%.

[0010] The third aspect of the present invention provides a use of the Bacillus Velezii FD-001 or the bacterial agent in the preparation of a drug for inhibiting the growth of corn pathogenic fungi.

[0011] Furthermore, the corn pathogenic fungi include Monosporus zeae, Fusarium zeae, Fusarium graminearum, Fusarium moniliforme, Pythium spp., Colletotrichum graminearum and Cephalosporium zeae.

[0012] The fourth aspect of the present invention provides a use of the Bacillus Velez FD-001 or the bacterial agent in the preparation of a drug for preventing and treating corn stalk rot.

[0013] The fifth aspect of the present invention also provides a method for preventing and controlling corn stalk rot, which comprises applying the Bacillus Velez FD-001 or the bacterial agent to corn planting soil.

[0014] The sixth aspect of the present invention further provides a drug for preventing and treating corn stalk rot, wherein the drug comprises the Bacillus Velez subtilis FD-001 or the bacterial agent.

[0015] The present invention has the following beneficial effects: The present invention isolates a strain of Bacillus Velezii ( Bacillus velezensis ) FD-001 strain. The plate confrontation test proved that the Velez subtilis FD-001 strain can effectively antagonize a variety of corn pathogenic fungi. The strain FD-001 obtained by screening has broad-spectrum antibacterial properties. Combined with the pot experiment, it was confirmed that the strain can effectively control corn stalk rot, providing a safe and reliable biological product for the prevention and control of corn diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The inhibition rate of CNZD-007 strain against 7 corn pathogenic fungi. DETAILED DESCRIPTION

[0017] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0018] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0019] The following examples are only used to illustrate the embodiments of the present invention and do not constitute any limitation to the present invention. Those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the present invention, and these modifications all fall within the scope of protection of the present invention.

[0020] Example 1 Isolation, identification and preservation of strain FD-001 1. Isolation and identification of strains Place the cleaned corn in a sterilized beaker, add 150 mL of sterile water, and shake at 100 r / min for 2 h. Dilute the suspension culture to 10 -1 , 10 -2 , 10 -3 After that, 150 μL was spread on the MRS solid plate and inverted and statically cultured at 37°C for 72 h. A single colony was picked and streaked onto a new MRS solid plate. The monoclonal strain obtained was streaked for the second time to obtain the pure strain FD-001. After growing on MRS medium at 37°C for 3 days, the colonies formed were small, white, round, moist, with a smooth surface, and short rod-shaped cells.

[0021] The method of direct amplification of 16S rDNA from bacteria was adopted. The PCR amplification primers used were 16S rDNA bacterial universal primers, specifically 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 2), 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 3). 25 μL PCR reaction system: DNA template 1 μL, dNTP (10 mM) 3 μL, primers (10 mM) 1 μL each, 10xPCR Buffer 7 μL, MgSO4 (50 mM) 2 μL, Taq enzyme (5U μL -1)0.5 μL, ultrapure water 9.5 μL. PCR reaction conditions: 95℃ pre-denaturation for 5 min, enter thermal cycle: 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles; 72℃ final extension for 10 min. After amplification, take 5 μL of the amplified sample and perform electrophoresis detection on 1% agarose gel at 60 V. Recover the target DNA fragment and send it to the sequencing company for sequencing. The sequencing results are as follows (SEQ ID No. 1):

[0022] The above spliced ​​sequences were compared by BLAST in the NCBI database, and combined with the morphological identification results, it was confirmed that strain FD-001 was Bacillus velezinsis ( Bacillus velezensis ).

[0023] 2. Deposit of strain FD-001 The strain FD-001 was named Bacillus velezinsis ( Bacillus velezensis ), which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 15, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.29016.

[0024] Example 2 Antibacterial Effect of Bacillus Velezii FD-001 on Pathogenic Fungi Preparation of Bacillus Velez FD-001 bacterial suspension: After the screened Bacillus Velez was activated in MRS medium, the OD of the bacterial suspension was adjusted. 600nm =0.5, inoculated into 100 mL of MRS medium at a 2% inoculum for expansion culture, and cultured at 37°C for 2 days to obtain fresh bacterial liquid. Then, centrifuge the FD-001 bacterial liquid at 4000 r / min for 20 min, discard the waste liquid, resuspend the precipitate with sterile water, centrifuge again at 4000 r / min for 20 min, discard the waste liquid, add sterile water, and adjust the fermentation liquid OD 600 Adjust the value to OD 600 =1 for spare.

[0025] The FD-001 strain was cultured against various maize pathogenic fungi (Monospora maydis Diplodia maydis )、Fusarium sorghum-specific type( Fusarium roseum f.sp.cerealis graminearum ( F. graminearum )、Fusarium moniliforme( F. moniliforme )、Pythium( Pythium spp. ), Colletotrichum graminearum ( Coletotrichum graminicolum ) and Cephalosporium maydis ( Cephalosporium maydis) were stored in the biological laboratory of Zhongdao Ecological Environment Engineering (Huimin) Co., Ltd.) for antagonism test. The activated corn pathogenic fungus cake (d=6 mm) was inoculated in the center of the MRS culture medium, and two perpendicular straight lines were drawn with the center point of the culture medium as the intersection point. The position 20 mm away from the center intersection was marked on the straight line, and a sterile filter paper was placed on it; 10 μL of FD-001 strain fermentation liquid was dripped on the sterile filter paper, and after standing for 30 min, it was placed in an incubator at 28°C for inverted culture. Each treatment was repeated three times. After 4 days of culture, the pathogen colony diameters of the treatment group and the control group were measured to determine the antagonistic effect of Bacillus Velez FD-001 strain. The results are shown in Figure 1 and Table 1.

[0026] Note: The formula is: Inhibition rate (%) = (colony radius of control group - colony radius of treatment group) / colony radius of control group × 100; The values ​​in the table are mean ± SD; ** indicates that there is a significant difference between the treatment group and the control group at the 1% level after t-test.

[0027] Example 3 Control Effect of Bacillus Velezii FD-001 on Corn Stalk Rot 1. Preparation of Bacillus Velezii Inoculants and Pathogens The bacterial solution of Bacillus Velezii FD-001 prepared in Example 2 was concentrated, freeze-dried and crushed to obtain a bacterial count of 1×10 8 cfu / g solid inoculum; Preparation of pathogen inoculum: soak corn kernels overnight, cook them in water, crush the corn husks, and divide them into Erlenmeyer bottles. Then inoculate PDA liquid culture medium with Fusarium graminearum at a ratio of 0.5% (v / w), and culture them in a light incubator for 7 days to obtain the inoculum - Fusarium corn kernel culture.

[0028] 2. Pot Plant Test Potted test was conducted at the seedling stage of Zhengdan 958 maize. Before planting, 5 g / pot of Bacillus sibiricum FD-001 was added to the soil holes. The treatment without adding the bacterium was used as blank control. There were 5 plants in each pot and 5 pots in each treatment. When the average emergence rate reached 50%, the emergence rate was counted. The height of maize seedlings was measured at the 3-leaf stage. All potted plants were inoculated with 10 g of Fusarium corn kernel culture by root wounding method. The disease situation was recorded after 7 days, and the disease index and control effect were calculated. The incidence rate (%) = number of diseased plants / total number of plants investigated × 100; disease index = (number of diseased plants × representative value of the disease level) / (total number of plants × representative value of the most serious disease level) × 100; control effect = (control disease index - treatment disease index) / control disease index × 100%.

[0029] The disease level survey standard for stem rot in the seedling stage is as follows: Level 0: disease-free; Level 1: normal growth of the above-ground and underground parts, a small amount of lesions can be seen on the roots, the area accounts for less than 1 / 4 of the total root surface area, and the root group is white with brown; Level 2: the growth of the above-ground and underground parts is obviously hindered, the leaf color becomes lighter, the lateral roots are few and short, there are no hairy roots, the lesions are continuous, accounting for 1 / 4~1 / 2 of the total root surface area, and the root group is white and brown; Level 3: the growth of the above-ground and underground parts is extremely abnormal, the above-ground parts can be seen to be green and yellow, the lateral roots are extremely small, the lesions account for 1 / 2~3 / 4 of the total root surface area, and the root group is brown with white; Level 4: germination but no seedlings emerge, almost suffocated to death, the lesions account for more than 3 / 4 of the total root surface area, and the roots are brown.

[0030] As can be seen from Table 2, Bacillus Velez FD-001 can effectively reduce the incidence and disease index of corn stalk rot caused by Fusarium graminearum, and has a significant biocontrol effect.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus Velezii ( Bacillus velezensis )FD-001, It is characterized in that It was deposited in the General Microbiology Center of China Microorganism Culture Collection on November 15, 2023, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.29016.

2. A bacterial agent, It is characterized in that The bacterial agent comprises the strain of Bacillus Velezii FD-001 according to claim 1 or its fermented bacterial liquid.

3. The bacterial agent according to claim 2, It is characterized in that The concentration of Bacillus Velez FD-001 in the fermentation broth was 1×10 8 ~1×10 10 cfu / mL.

4. The bacterial agent according to claim 3, It is characterized in that The preparation method of the fermentation bacterial liquid is as follows: after activating the Velez Bacillus FD-001 in an MRS medium, adjusting the bacterial cell density of the bacterial suspension, and then inoculating the activated Velez Bacillus FD-001 in an MRS medium for expansion culture to obtain a Velez Bacillus FD-001 with a concentration of 1×10 8 ~1×10 10 cfu / mL of fermentation broth.

5. The bacterial agent according to claim 4, It is characterized in that The inoculation amount of activated Bacillus Velezii FD-001 is 1~3%.

6. Use of the Bacillus Velez FD-001 according to claim 1 or the bacterial agent according to any one of claims 2 to 5 in the preparation of a drug for inhibiting the growth of corn pathogenic fungi.

7. The use according to claim 6, It is characterized in that The corn pathogenic fungi include Monosporus maydis, Fusarium zeae, Fusarium graminearum, Fusarium moniliforme, Pythium spp., Colletotrichum graminearum and Cephalosporium maydis.

8. Use of the Bacillus Velez FD-001 according to claim 1 or the bacterial agent according to any one of claims 2 to 5 in the preparation of a drug for preventing and treating corn stalk rot.

9. A method for preventing and controlling corn stalk rot, It is characterized in that The Bacillus Velez subtilis FD-001 described in claim 1 or the bacterial agent described in any one of claims 2 to 5 is applied to corn-growing soil.

10. A drug for preventing and treating corn stalk rot, It is characterized in that The method comprises the Bacillus Velezii FD-001 according to claim 1 or the bacterial agent according to any one of claims 2 to 5.

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