Bacillus velezensis hg-11f, bacterial agent thereof, preparation method of bacterial agent, and application thereof

Abstract

The present application relates to the technical field of biological control, and in particular to Bacillus velezensis HG-11f, a bacterial agent thereof, a preparation method of the bacterial agent and application thereof, wherein the Bacillus velezensis HG-11f has been preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, on July 15, 2022, with a preservation number of CGMCC No. 25309. For yam root rot caused by Pantoea agglomerans, the control effect of the Bacillus velezensis HG-11f bacterial agent is more than 70%, and the control effect of the compound bacterial agent of the Bacillus velezensis HG-11f and Bacillus cereus BC04 is greatly improved, reaching more than 90%, thus providing a good biological control scheme and having important economic value and environmental protection benefits.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to a Bacillus belye HG-11f, its inoculum, preparation method, and application. Background Technology

[0002] According to reports, since the autumn of 2019, root rot of yams has occurred in several yam-producing areas in my country, including Henan Province. Water-soaked lesions have been frequently observed in the underground tubers of yams. Pathogen identification revealed that the pathogen is Pantotheca acuminata (Gastromycosis globulus). Pantoea agglomerating This is the first report of Bacterial Soft Rot Caused by Pantoea agglomerans on Chinese yam (Dioscorea opposita Thunb.) in China. [J]. Plant disease, 2022. Currently, the control of yam root rot mainly relies on strengthening water and fertilizer management, timely treatment of diseased plants, and spraying with chemical pesticides. However, the control effect is not significant, and chemical pesticide residues cause serious food safety and environmental pollution problems. There is an urgent need to find economical, green, and safe control measures. Microbial control utilizes naturally occurring beneficial microorganisms in the environment to control plant diseases, offering advantages such as environmental friendliness and no secondary pollution. Bacillus is a commonly used biocontrol bacterium in production, possessing strong resistance, stable colonization, strong antagonistic effects, and a good safety profile. According to the literature retrieved, Bacillus subtilis and Bacillus amyloliquefaciens are currently used to control yam root rot, but there are no reports on biological control of yam root rot caused by Pantotheca acuminata. Summary of the Invention

[0003] To address the problem of controlling root rot in yam caused by Pantotheca acuminata, this invention provides Bacillus belyssus HG-11f and its inoculum, preparation method, and application. The compound inoculum of this invention can achieve a control effect of over 90% on root rot in yam, which is significantly better than the effect of chemical control. It has great application value for the cultivation of economic crops and helps to reduce the use of chemical agents, making it green and safe.

[0004] In a first aspect, the present invention provides a Bacillus belyssus HG-11f, wherein the Bacillus belyssus ( Bacillus velezensis HG-11f has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on July 15, 2022, with accession number CGMCC No. 25309.

[0005] Secondly, the present invention provides a single-strain agent containing the above-mentioned Bacillus belye HG-11f.

[0006] Thirdly, the present invention provides an HG-11f / BC04 compound bacterial agent, comprising the aforementioned Bacillus belye HG-11f and Bacillus cereus (… Bacillus cereus Bacillus cereus BC04 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, on October 19, 2020, with accession number CGMCC No. 20907, and is disclosed in Chinese invention patent CN112342170A.

[0007] Fourthly, the present invention provides a method for preparing the above-mentioned compound microbial agent, comprising the following steps: (1) Bacillus belye HG-11f and Bacillus cereus BC04 were obtained by conventional liquid culture; (2) Mix Bacillus belye HG-11f bacterial solution and Bacillus cereus BC04 bacterial solution at a mass ratio of 1:1, inoculate the mixed bacterial solution into a fermenter, ferment for 2-3 days, and obtain HG-11f / BC04 fermentation broth; (3) Add diatomaceous earth and starch to the HG-11f / BC04 fermentation broth according to the saturated adsorption capacity, stir once every 1 hour to allow the carrier to fully adsorb the bacteria, and dry at low temperature after 24 hours to obtain HG-11f / BC04 bacterial powder. (4) Mix HG-11f / BC04 bacterial powder, stabilizer, dispersant and wetting agent in a mass ratio of 75~80:10~12:5~8:2~4, pulverize by air jet mill and pass through a 300 mesh sieve to obtain HG-11f / BC04 compound bacterial agent.

[0008] Furthermore, in step (2), the fermentation medium used for fermentation is molasses medium, and 1L of molasses medium includes 100mL of molasses, 1g of sodium chloride, 3g of urea, and water to make up to 1L.

[0009] Furthermore, the stabilizer is humic acid, the dispersant is polyvinyl alcohol, and the wetting agent is Tween 80.

[0010] Fifthly, the present invention provides the application of the above-mentioned Bacillus belye HG-11f in the prevention and control of root rot in yam.

[0011] In a sixth aspect, the present invention provides the application of the above-mentioned HG-11f inoculant or HG-11f / BC04 compound inoculant in the prevention and control of yam root rot.

[0012] Furthermore, the roots of the yam were treated with a diluted fungicide solution. The first application was made during the yam vine growth stage, and the second application was made 28 days later.

[0013] The beneficial effects of this invention are as follows: This invention isolates a strain of Bacillus belyssus HG-11f and prepares it into a microbial agent and a compound microbial agent. For yam root rot caused by Pantotheca acuminata clumps, the Bacillus belyssus HG-11f microbial agent shows a control effect exceeding 70%, while the control effect of the compound microbial agent of Bacillus belyssus HG-11f and Bacillus cereus BC04 is significantly improved, reaching over 90%. This provides an excellent biological control solution with significant economic and environmental benefits. Detailed Implementation

[0014] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0015] The Bacillus cereus BC04 used in the following examples was isolated from the Institute of Ecology, Shandong Academy of Sciences, and has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on October 19, 2020, with accession number CGMCC No. 20907. Related information is disclosed in Chinese Patent CN112342170A.

[0016] In the following examples, LB liquid medium, LB plate medium, and PDA medium were all prepared using conventional formulations and methods, and other reagents were commercially available.

[0017] Example 1 Isolation and Identification of Strains 1. Isolation of HG-11f strain Source of inoculum: Soil, collected in September 2020 from a vegetable greenhouse in Shizhong District, Jinan City, Shandong Province.

[0018] Operating steps: (1) In a clean bench, 10g of soil collected from a vegetable greenhouse in Shizhong District, Jinan City, Shandong Province was placed in 100mL of sterile physiological saline and shaken for 2 hours to fully suspend the soil and prepare a bacterial suspension.

[0019] (2) After serially diluting the bacterial suspension with sterile water, take 100 μL of each concentration of bacterial suspension dilution and spread it on LB medium plates. Incubate at 28°C for 48 h. Then pick single colonies with different morphology, size and color on the plates and streak them on LB medium plates for purification. Number them separately.

[0020] (3) Take the purified strains with different numbers and inoculate them into LB liquid medium. Ferment and culture them at 28℃ and 180rpm for 24h. Take 2mL of fermentation broth from each strain and mix it into 15mL of PDA medium. Pour the mixture into plates and wait for the PDA plates to solidify. Inoculate the center of the PDA plate with the root rot fungus of yam (i.e., Pantothecin) for inhibition co-culture. The strain with the strongest antagonistic ability against the root rot fungus of yam is obtained and is numbered HG-11f.

[0021] 2. Strain identification (1) 16S rDNA sequencing Genomic DNA was extracted from strain HG-11f and used as a template for PCR amplification using universal primers for bacterial 16S rDNA. The amplified products were recovered and sequenced.

[0022] (2) The sequences obtained from sequencing were submitted to GeneBank for homology comparison. The strain HG-11f and... Bacillus from Velez With 99.99% homology, and based on Gram staining and physiological and biochemical characteristic tests, strain HG-11f was identified as *Bacillus belyssae*. Bacillus velezensis The strain was named *Bacillus belyssus* HG-11f. It was deposited on July 15, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25309.

[0023] Example 2: Antagonistic effects of Bacillus belyssus HG-11f and Bacillus cereus BC04 on yam root rot pathogens. (1) Take the pathogen that causes root rot symptoms in yam, Pantotheca cumulus, and streak it on LB agar. After culturing at 28°C and 180 rpm for 24 h, pale yellow, rod-shaped, slender colonies with smooth edges were observed.

[0024] (2) After culturing the purified Bacillus cereus BC04 and Bacillus bereaves HG-11f strains in LB liquid medium at 28°C and 180 rpm for 24 h, 2 mL of the fermentation broth was taken from each strain and mixed into 15 mL of PDA medium. The mixture was then poured into plates to solidify and set aside for later use.

[0025] (3) Use a punch with a diameter of 0.5 cm to punch holes at the edge of the cultured yam root rot fungus colony to obtain fungal sheets for later use.

[0026] (4) Using a sterile picking needle, pick up the above-mentioned bacterial strips and place them in the center of the PDA plate in (2). At the same time, set up a PDA plate inoculated only with the bacterial strips of *Dioscorea root rot* as a control group. Each group was divided into 3 replicates. The plates were incubated at 28°C for 2-3 days, and the growth rate and colony radius of *Dioscorea root rot* were observed. After 3 days, compared with the blank control, BC04 inhibited the growth of *Dioscorea root rot* by 72.83%, and HG-11f inhibited the growth of *Dioscorea root rot* by 91.4%.

[0027] Example 3: Preparation of HG-11f bacterial agent, BCO4 bacterial agent and HG-11f / BCCO4 compound bacterial agent (1) Purified Bacillus belye HG-11f and Bacillus cereus BC04 were inoculated into LB medium and cultured at 28℃ and 180rpm for 24h to obtain HG-11f and BC04 bacterial suspensions, respectively, with bacterial concentrations of 1.2×10⁻⁶ and 1.2×10⁻⁶, respectively. 9 cfu / mL, 1×10 9 cfu / mL.

[0028] (2) Take HG-11f bacterial suspension, BC04 bacterial suspension, and a 1:1 mixture of HG-11f and BC04 bacterial suspensions, and inoculate them into six 60L mechanically stirred stainless steel fermenters. The fermentation medium is molasses medium, with each liter of molasses medium containing 100mL molasses, 1g sodium chloride, 3g urea, and water to make up to 1L. After 2 days of fermentation, HG-11f fermentation broth, BC04 fermentation broth, and HG-11f / BC04 fermentation broth were obtained, with biomass of 2.2×10⁻⁶ respectively. 10 cfu / mL, 2.0×10 10 cfu / mL, 2.10×10 10 cfu / mL.

[0029] (3) Add diatomaceous earth and starch to the three fermentation liquids in (2) according to the saturated adsorption capacity. Stir once every 1 hour to allow the carrier to fully adsorb the bacteria. After 24 hours, dry at low temperature to obtain HG-11f bacterial powder, BC04 bacterial powder and HG-11f / BC04 bacterial powder.

[0030] (4) Mix the bacterial powder, humic acid, polyvinyl alcohol and Tween 80 in a mass ratio of 78:12:7:3, pulverize them by air jet mill, and pass them through a 300-mesh sieve to obtain HG-11f bacterial agent, BC04 bacterial agent and HG-11f / BC04 compound bacterial agent respectively.

[0031] Example 4: Field trial of the effects of HG-11f single-strain agent, BC04 single-strain agent, and HG-11f / BC04 compound agent on the control of yam root rot. 1. Trial period and location: January 2019 to December 2021, Yam Planting Cooperative, Dingtao District, Heze City, Shandong Province.

[0032] 2. Test treatment (1) Take the HG-11f single bacterial agent, BC04 single bacterial agent and HG-11f / BC04 compound bacterial agent prepared in Example 3, and dilute them with water by 200 times to obtain HG-11f spray solution, BC04 spray solution and HG-11f / BC04 spray solution.

[0033] (2) The experimental field was divided into 4 treatment zones, each containing 3 plots. During the yam vine growth stage, HG-11f spray solution, BC04 spray solution, HG-11f / BC04 spray solution, and water were sprayed onto the 4 treatment zones at a rate of 6L per acre for root irrigation. A second application was performed 28 days later.

[0034] (3) During the tuber enlargement period of yam, the disease was investigated. Five sampling points were used in each plot to record the number of diseased plants and the disease level. The control effect was calculated with the water spraying area as the control. The results are shown in Table 1.

[0035] Yam root rot is classified into four levels, with the following grading standards: Level 0, no disease on the tubers; Level 1, localized water-soaked symptoms on the tubers; Level 2, water-soaked or rotten symptoms on 1 / 2 of the tubers; Level 3, water-soaked or rotten symptoms on 1 / 2 to 3 / 4 of the tubers; Level 4, water-soaked or rotten symptoms on more than 3 / 4 of the tubers.

[0036] The formula for calculating the effectiveness is as follows:

[0037]

[0038]

[0039] Table 1. Control efficacy of each treatment after two applications of medication

[0040] Note: In the same column, uppercase and lowercase letters indicate the significance of differences at the 0.01 and 0.05 levels, respectively. If the letters are the same, the difference is not significant.

[0041] As shown in Table 1, Bacillus cereus BC04 had the worst control effect on yam root rot, while Bacillus belyi HG-11f had a better control effect on yam root rot, reaching 70%; the compound Bacillus belyi HG-11f and Bacillus cereus BC04 had the best control effect on yam root rot, reaching over 90%.

[0042] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A type of Bacillus belye HG-11f, characterized in that, The bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) HG-11f has been deposited with the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, on July 15, 2022, and has been assigned the accession number CGMCC No. 25309.

2. A single-strain agent containing Bacillus belye HG-11f as described in claim 1.

3. An HG-11f / BC04 compound microbial agent, characterized in that, Contains Bacillus belyssus HG-11f and Bacillus cereus as described in claim 1. Bacillus cereus Bacillus cereus BC04 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on October 19, 2020, with accession number CGMCC No. 20907.

4. A method for preparing the HG-11f / BC04 composite microbial agent as described in claim 3, characterized in that, Includes the following steps: (1) Bacillus belye HG-11f and Bacillus cereus BC04 were obtained by conventional liquid culture; (2) Mix Bacillus belye HG-11f bacterial solution and Bacillus cereus BC04 bacterial solution at a mass ratio of 1:1, inoculate the mixed bacterial solution into a fermenter, ferment for 2-3 days, and obtain HG-11f / BC04 fermentation broth; (3) Add diatomaceous earth and starch to the HG-11f / BC04 fermentation broth according to the saturated adsorption capacity, stir once every 1 hour to allow the carrier to fully adsorb the bacteria, and dry at low temperature after 24 hours to obtain HG-11f / BC04 bacterial powder. (4) Mix HG-11f / BC04 bacterial powder, stabilizer, dispersant and wetting agent in a mass ratio of 75~80:10~12:5~8:2~4, pulverize by air jet mill and pass through a 300 mesh sieve to obtain HG-11f / BC04 compound bacterial agent.

5. The preparation method according to claim 4, characterized in that, In step (2), the fermentation medium used for fermentation is molasses medium. 1L of molasses medium includes 100mL of molasses, 1g of sodium chloride and 3g of urea, and water is added to 1L.

6. The preparation method according to claim 4, characterized in that, The stabilizer is humic acid, the dispersant is polyvinyl alcohol, and the wetting agent is Tween 80.

7. The *Bacillus belyssus* HG-11f as described in claim 1 in the prevention and control of pantothenic aerosols (Pantotheca acuminata) Pantoea agglomerans Applications related to yam root rot caused by yam.

8. The single-strain agent as described in claim 2 in controlling the spread of pantothecin (… Pantoea agglomerans Applications related to yam root rot caused by yam.

9. The HG-11f / BC04 compound microbial agent as described in claim 3, in the prevention and control of pantothecin (a type of bacteria) Pantoea agglomerans Applications related to yam root rot caused by yam.

10. The application as described in claim 8 or 9, characterized in that, The roots of the yam were treated with a diluted fungicide solution. The first application was made during the yam vine growth stage, and the second application was made 28 days later.

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