CN117701467A

CN117701467A - Bacillus bailii SF18-3 and application thereof, biocontrol microbial inoculum and preparation method and application thereof

Info

Publication number: CN117701467A
Application number: CN202311841725.9A
Authority: CN
Inventors: 王辉; 孙晓东; 刘丽; 于舒怡; 关天舒; 李柏宏; 邹春蕾; 郑丽娇; 王海欧
Original assignee: Liaoning Academy of Agricultural Sciences
Current assignee: Liaoning Academy of Agricultural Sciences
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-03-15

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus beijerinckii SF18-3 and application thereof, a biocontrol microbial agent and a preparation method and application thereof. The bacillus beleiensis SF18-3 with the preservation number of CCTCC NO: M2023736 is separated from animal excrement to obtain the bacillus beleiensis SF18-3 with a good inhibition effect on bacterial alternaria, and the bacillus beleiensis SF18-3 has obvious disease degree of pepper bacterial alternaria and has wide application prospect.

Description

Bacillus bailii SF18-3 and application thereof, biocontrol microbial inoculum and preparation method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to bacillus beijerinckii SF18-3 and application thereof, a biocontrol microbial agent and a preparation method and application thereof.

Background

Bacterial leaf spot of vegetables mainly infects peppers, tomatoes and other various solanaceae crops, seedlings, leaves, stems and fruits can be ill, but the disease is common with leaves. When the leaf blade is damaged, yellow halo is arranged around the disease spot, water stain spots are initially generated, the yellow green color is generated, the disease spot is round or irregular after the disease spot is gradually enlarged, the edge is dark brown, slightly raised, the central color is light, slightly recessed, the surface is rough like scab, the disease spot can be connected into large irregular plaque, and the leaf tip and the leaf margin become yellow and dry, break, perforation and fallen leaves when serious. The later stage can cause the perforation of the blade, and the blade is easy to fall off. Bacterial Spot disease etiology is Xanthomonas campestris pv.vesica (Doi dge), which is currently classified into four types, namely Xanthomonas euvesicatoria, xanthomonas vesicatoria, xanthomonas gardneri, xanthomonas perforans. Through many years of investigation, many large-scale seedling raising enterprises or individual seedling raising sites in China have occurrence, the incidence rate of the disease reaches more than 40 percent, the disease is serious, the yield loss caused by the disease per year reaches 10 to 20 percent, the yield loss caused by the disease per year reaches more than 80 percent, even seedlings cannot be sold, and the economic loss is very serious.

The prevention and treatment of bacterial spot disease of vegetables on production still takes chemical agents as main agents, however, the frequent use of single pesticides leads to the increase of disease resistance, insensitivity to novel pesticides, increased pesticide residues of vegetables and serious fruit pollution. Especially, the problems that the bacterial diseases of vegetables are difficult to prevent and diagnose and the medicine is applied blindly are more remarkable in production, so that the disease control period is delayed, and various pesticides of vegetable farmers are mixed by themselves, so that the food and the environmental safety are seriously endangered. The biological control is safe and effective to crops and environment, is an excellent alternative way for controlling vegetable diseases and insect pests by chemical pesticides, and has important significance for safe and green control of vegetables.

At present, biocontrol bacteria with control effect on vegetable bacterial spot disease include: bacillus amyloliquefaciens (Bacillus amyloliqufaciens), bacillus subtilis (B.subtilis), pseudomonas syringae (Pseudomonas syringae), pseudomonas putida (P.putida), and Pseudomonas fluorescens (P.fluoscens). Bacillus is used as an important biocontrol resource, and the prevention and treatment of bacterial spot disease by Bacillus bailii have not been reported yet.

Disclosure of Invention

In order to solve the problems, the invention provides bacillus beliensis SF18-3, application thereof, biocontrol microbial inoculum, preparation method thereof and application thereof, and the bacillus beliensis SF18-3 with better inhibition effect on bacterial spot pathogens is obtained by separating from animal excrement, and has obvious disease degree of pepper bacterial spot diseases and wide application prospect.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides bacillus belicus (Bacillus velezensis) SF18-3 which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2023736 at the month of 05 and 11 of 2023.

The invention also provides application of the bacillus belicus SF18-3 in preventing and treating pepper bacterial spot disease.

The invention also provides a preparation method of the biocontrol microbial inoculum, which comprises the following steps:

inoculating the seed solution containing the bacillus beijerinckii SF18-3 in the fermentation culture solution for fermentation to obtain the biocontrol microbial agent.

Preferably, the fermentation conditions include: the time is 12-96 h, the temperature is 10-35 ℃, and the rotating speed is 100-180 rpm.

Preferably, the fermentation conditions include: the time was 72 hours, the temperature was 25℃and the rotational speed was 140rpm.

Preferably, the viable count of bacillus bailii SF18-3 in the seed solution is 1×10 ⁷ CFU/mL。

Preferably, the fermentation medium comprises NB liquid medium, the pH of which is 7.0.

The invention also provides the biocontrol microbial inoculum prepared by the preparation method.

The invention also provides application of the biocontrol microbial inoculum in preventing and treating pepper bacterial spot disease.

Preferably, the application comprises: the biocontrol microbial inoculum is applied 1 time before or at the initial stage of the onset of the bacterial spot disease of the capsicum by adopting a stem and leaf spray method after dilution, and then applied 1 time after 7 d.

The beneficial effects of the invention are as follows:

the bacillus belicus SF18-3 with a good inhibition effect on bacterial spot germs is obtained by separating from animal excrement, and the bacterial strain has obvious morbidity degree of pepper bacterial spot germs and has wide application prospect.

Drawings

FIG. 1 is a preliminary screening of SF18-3 strain;

FIG. 2 is SF18-3 colony characterization;

FIG. 3 shows SF18-3 cell microscopic morphology;

FIG. 4 is a partial physiological and biochemical characterization;

FIG. 5 shows SF18-316S rDNA amplification results;

FIG. 6 is a clustering analysis of SF18-3 with known sequences on the network;

FIG. 7 shows PCR detection of SF18-3 lipopeptides gene;

FIG. 8 shows the effect of fermentation time on SF18-3 production of bacteriostatic substances;

FIG. 9 shows the effect of fermentation temperature on SF18-3 production of bacteriostatic substances;

FIG. 10 shows the effect of different rotational speeds on SF18-3 production of bacteriostatic substances;

FIG. 11 is a graph showing the effect of initial pH on SF18-3 production of bacteriostatic substances;

FIG. 12 shows the pathogenic bacteria inhibitory effect of different concentrations of SF18-3 fermentation broth;

FIG. 13 is a SF18-3 inoculant field pot control test.

Description of biological preservation

Bacillus belicus SF18-3, latin Bacillus velezensis, was deposited at China center for type culture Collection (CCTCC NO: M2023736) at 20/11/2023, and was deposited at university of Wuhan, china.

Detailed Description

inoculating the seed solution containing the bacillus beijerinckii SF18-3 in a fermentation medium for fermentation to obtain the biocontrol microbial inoculum.

In the present invention, the conditions of the fermentation preferably include: the time is 12-96 h, the temperature is 10-35 ℃, and the rotating speed is 100-180 rpm; more preferably, the method comprises: the time was 72 hours, the temperature was 25℃and the rotational speed was 140rpm. In the present invention, the viable count of Bacillus bailii SF18-3 in the seed liquid is preferably 1X 10 ⁷ CFU/mL. In the present invention, the fermentation medium preferably comprises NB liquid medium, and the pH of the fermentation medium is preferably 7.0.

In the present invention, the application preferably includes: the biocontrol microbial inoculum is applied 1 time before or at the initial stage of the onset of the bacterial spot disease of the capsicum by adopting a stem and leaf spray method after dilution, and then applied 1 time after 7 d. In the present invention, the dilution factor of the biocontrol agent is preferably 10 to 50 times.

The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.

Example 1

1. Preparation of pathogenic bacteria and bacterial suspension

Pathogenic bacteria of bacterial spot disease of Capsici fructus (hereinafter referred to as pathogenic bacteria) are obtained by inoculating purified pathogenic bacteria strain to NA plate at 25deg.C, standing for 72 hr, scraping thallus under aseptic condition with aseptic inoculating loop, and preparing into 1×10 with aseptic water ⁷ cfu/mL of bacterial suspension, and is prepared on site.

2. Separation and screening of biocontrol bacteria

(1) Separation

73 parts of plant rhizosphere soil and 4 kinds of decomposed animal manure are collected from main production areas of vegetables and other crops in Liaoning province, such as 13 areas of new people, law bank, malus, anshan mountain, cucurbit island and the like. Weighing 5.0g of soil/manure respectively, putting into 45mL of sterile water containing glass beads, oscillating at 140rpm for 10min, and standing for 20min, which is 10 ^-1 Taking 10 of soil diluent ^-1 1mL of the soil dilution was added to 9mL of sterile water, which was 10 ^-2 Diluting solution, sequentially preparing 10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 Serial dilutions of soil. Take 10 ^-5 、10 ^-6 、10 ^-7 Three gradient soil dilutions of 100 μl each were spread on NA medium, cultured at 28 ℃ for 2-3 d for isolation of beneficial bacteria; take 10 ^-4 、10 ^-5 、10 ^-6 100 mu L of each soil dilution is respectively coated on a Ma Dingshi culture medium and a Gao I culture medium, and cultured for 5-7 d at 28 ℃ for separating beneficial fungi from actinomycetes. The beneficial microorganisms with different plate characteristics were purified and stored at 4℃for further use.

Preparation of beneficial microbial suspensions: and (3) preparing a pathogenic bacteria suspension 1.

(2) Screening

The method of transparent ring with toxic medium is utilized. 5mL (1×10) of the prepared bacterial suspension of the pathogenic bacteria in step 1 ⁷ CFU/mL) 1mL was poured into 45mL NA medium dried at 45℃to make 1X 10-containing ⁶ Toxin-containing plates of CFU/mL cells. The isolated beneficial microorganisms are made into a bacterial dish by a sterile puncher under the aseptic condition, and the bacterial dish is placed in the center of a toxic flat plate, and the result is observed after 4-5 d culture at 28 ℃ and is repeated for 3 times. Finally, 1 strain of biocontrol bacteria with inhibition effect on pathogenic bacteria is obtained and named SF18-3 (figure 1).

3. Identification of biocontrol bacteria SF18-3

(1) Morphological observation

The strain SF18-3 is a positive bacterium with spores as a result of gram staining. The colonies were milky white, non-moist, matt, opaque, wrinkled and uneven in edges (fig. 2, 3).

(2) Physiological and biochemical test

SF18-3 methyl red positive, peroxidase, oxidase weak positive, V-P, nitrate reduction, indole production were all negative (FIG. 4).

(3) 16S rDNA sequence analysis

Extracting genome of biocontrol bacteria by CTAB method, subjecting the extracted product to 1.2% agarose gel electrophoresis, and measuring OD by ultraviolet spectrophotometer _260/280 The values were checked for concentration and purity. PCR amplification uses a Beijing Ding Guo prosperous 2X Taq PCR MasterMix kit, products are subjected to 1.5% agarose gel electrophoresis, sol is recovered by using NEP025-1 gel cutting, and the products are sequenced by Beijing Ding Guo prosperous biotechnology Limited company. The 16s rDNA sequences of the obtained bacterial strains were aligned with known sequences on the net and subjected to cluster analysis using BioEdit and MEGA 7.0. SF18-3 was finally determined to be Bacillus bailii (Bacillus velezensis) (FIGS. 5, 6).

PCR system: mix 12.5. Mu.L, DNA 1.0. Mu.L, primers 0.5. Mu.L each, sterile distilled water to 25. Mu.L.

PCR procedure: pre-denaturation at 94℃for 3min, denaturation at 94℃for 30sec, annealing at 55℃for 30sec, elongation at 72℃for 1min,30cycles, elongation at 72℃for 5min.

4. Biocontrol bacterium SF18-3 bacteriostasis mechanism

(1) SF18-3 extracellular enzyme Activity assay

The level of lysozyme (Lys), protease (Protease), chitinase (Chitinase), xylanase (Xylanase), cellulase (CE) and chicken acetyl CoA (A-CoA) in SF18-3 fermentation stock was determined by a double antibody sandwich method. The results of the experiments show that the protease and xylanase activities produced during SF18-3 fermentation are the primary antibacterial substances, followed by chitinase and acetyl-CoA (Table 1).

TABLE 1 enzymatic Activity in SF18-3 fermentation broths

Project	Activity(s)	Deviation of	Unit (B)
				Protease enzyme	536.1	6.28	U/L
Chitinase	131.8	3.00	IU/L
				Xylanase enzyme	564.2	8.92	U/L
Lysozyme	55.79	1.11	IU/L
				Cellulase enzymes	64.18	1.69	U/L
Acetyl-coa	135.0	1.57	IU/L

(2) SF18-3 lipopeptides gene detection

Lipopeptides are a class of secondary metabolites produced by bacillus and have an inhibitory effect on a variety of diseases. The research develops the lipopeptide synthesis gene related to the biological control of the strain SF18-3 for PCR detection, and the lipopeptide specific primers and the PCR programs are shown in tables 2 and 3. The test results showed that SF18-3 strain amplified iturin synthase gene (ITMA), fengypin synthase gene (FENB), antimycosin synthase gene (MYCB), nitropyrrolin synthesis regulatory gene (PRNC) and gamboge pyocin synthesis regulatory gene (PLTC) (FIG. 7).

TABLE 2SF18 lipopeptides specific primers

TABLE 3PCR reaction procedure

5. Optimization of pathogenic bacteria inhibition conditions by biocontrol bacteria SF18-3

And a bacteriostasis test is carried out by adopting a toxic medium oxford cup method, so that the influence of different fermentation conditions (fermentation time, temperature, pH and revolution) on SF18-3 to produce bacteriostasis substances is optimized.

(1) Preparation of toxic plates

5mL (1×10) of the prepared bacterial suspension of the pathogenic bacteria in step 1 ⁷ CFU/mL) 1mL was poured into 45mLNA medium which was dried to 45℃to give a composition containing 1X 10 ⁶ The CFU/mL toxic plate of the thallus is prepared first.

NA medium: 10.0g of peptone, 3.0g of beef powder, 5.0g of sodium chloride and 18.0g of agar powder.

(2) Preparation of biocontrol strain SF18-3 suspension

The SF18-3 strain is inoculated on an NA plate and is subjected to static culture for 72 hours at 25 ℃, and is picked up by an inoculating loop to be sterilized water under the aseptic condition to prepare 1 multiplied by 10 ⁷ CFU/mL mother liquor, and is prepared on site.

(3) Influence of fermentation time on SF18-3 production of bacteriostatic substances

The pipette aspirates SF18-3 bacterial suspension (1X 10) ⁷ CFU/mL) 1mL was inoculated into a 250mL Erlenmeyer flask containing 150mLNB medium, cultured at 140rpm at 25℃for 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 200. Mu.L of SF18-3 broth treated at different times was injected into a sterile oxford cup placed on a toxic medium NA plate, cultured at 25℃for 5d at constant temperature, and the antibacterial zone width was measured for 3 replicates.

The test results show (FIG. 8) that the antibacterial active substances of SF18-3 on pathogenic bacteria strains are reduced after being increased to 96 hours along with the prolonged culture time: 72h is an inflection point for generating antibacterial substances, 0-72 h of antibacterial active substances show an obvious rising trend, and 72-84 h of antibacterial active substances slowly rise and then have a descending trend. The bacteriostasis bandwidth reaches 0.95cm at 72h. According to the test result, 72h is selected as the optimal fermentation time.

(4) Influence of fermentation temperature on SF18-3 production of antibacterial substances

The pipette aspirates SF18-3 bacterial suspension (1X 10) ⁷ CFU/mL) 1mL was inoculated into a 250mL Erlenmeyer flask containing 150mLNB medium, incubated at 10℃at 15℃at 20℃at 25℃at 30℃at 35℃at 40℃in a shaking table for 72 hours, and SF18-3 broth from different treatments was placed in an oxford cup (200. Mu.L) placed on a toxic medium NA plate, incubated at 25℃for 5 days at constant temperature, and the antibacterial zone width was measured and repeated 3 times.

The temperature gradient test results show (figure 9) that the activity of SF18-3 antibacterial substances rises rapidly along with the temperature rise in the range of 10-25 ℃, and the transparent circle changes little at 25-35 ℃. According to the test results, 25℃was chosen as the optimal fermentation temperature.

(5) Influence of shaking table rotational speed on SF18-3 to produce antibacterial substances

The pipette aspirates SF18-3 bacterial suspension (1X 10) ⁷ CFU/mL) 1mL was inoculated into a 250mL Erlenmeyer flask containing 150mLNB medium, cultured at 25℃for 72 hours in shaker 100rpm, 120rpm, 140rpm, 160rpm, 180rpm, 200rpm, and constant temperature (pH 7.0), the SF18-3 broth treated differently was placed in an oxford cup (200. Mu.L) placed on a plate of toxic medium NB, and incubated at 25℃for 5 days at constant temperature, and the antibacterial zone width was measured for 3 replicates.

The rotational speed test results show (figure 10) that between 100rpm and 160rpm, the antibacterial active substances increase along with the increase of the rotational speed of the shaking table, 140rpm is the inflection point of the width of the antibacterial band, the antibacterial band width is 1.07cm, and the change amplitude of the antibacterial band width is small after the rotational speed is higher than 140rpm to 160rpm, and then the antibacterial band width slowly decreases. According to the test results, 140rpm was selected as the optimal fermentation speed.

(6) Influence of initial pH on SF18-3 production of bacteriostatic substances

Preparing liquid culture medium with different initial pH values of 2, 3, 4, 5, 6, 7, 8, 9, and 10NB, inoculating 1mL of SF18-3 bacterial suspension (1×10) ⁷ CFU/mL) was incubated at 140rpm in a 250mL Erlenmeyer flask containing 150mL of the culture solution at constant temperature in a 25℃shaker for 72 hours, and the different treated SF18-3 fermentation broth was placed in an oxford cup (200. Mu.L) placed on a toxic medium NA plate, incubated at constant temperature for 5 days at 25℃and the zone width was measured for 3 replicates.

The acid-base gradient test result shows (figure 11) that the width of the antibacterial zone increases remarkably from pH 3 until the antibacterial effect is best at pH 7, the width is 0.98cm, and then the antibacterial effect decreases sharply; the antibacterial effect of pH 7-pH 8 is not changed greatly, and after the antibacterial effect is larger than pH 9, the antibacterial effect is obviously reduced. Based on the test results, the fermentation broth was finally selected to have an optimal initial pH of 7.0.

In summary, the test results show that the optimal combination of fermentation conditions with optimal antibacterial effect is as follows: NB medium is used as a substrate, the optimal initial pH value is 7.0, the temperature is 25 ℃, the rotation speed of a shaking table is 140rpm, and the fermentation time is 72 hours.

6. Indoor antibacterial effect of SF18-3 fermentation liquor with different concentrations on pathogenic bacteria

Adopts a toxic medium transparent ring method.

(1) Preparation of toxic plates

(2) Preparation of SF18-3 bacterial solutions with different concentrations

The pipette aspirates SF18-3 bacterial suspension (1X 10) ⁷ CFU/mL) 1mL was inoculated into a 250mL Erlenmeyer flask containing 150mLNB medium, and after culturing at 140rpm for 72 hours at 25℃the solution was 1 Xsolution, which was then stepwise diluted to 10-fold, 50-fold, 100-fold, 500-fold and 1000-fold, and prepared as it is.

(3) Indoor bacteriostasis test

The SF18-3 bacteria liquid with different concentrations is placed in an oxford cup (200 mu L) which is placed in a toxic medium NA flat plate, is placed in a constant temperature culture for 5d at 25 ℃, and the width of a bacteria inhibition zone is measured and repeated for 3 times.

From the results of the test, the results of the test showed (Table 4, FIG. 12) that the 1 Xfungus liquid had the best antibacterial effect, the width of the transparent ring was 0.97cm, the 10 Xfungus liquid was 0.75cm, and the rest were 100 Xfungus liquid, 500 Xfungus liquid and 1000 Xfungus liquid in this order.

TABLE 4 inhibitory effect of SF18-3 at various concentrations on pathogenic bacteria

Note that: a. b and c are 0.05 level difference analysis.

7. Biocontrol bacterium SF18-3 has effect of preventing and controlling bacterial spot disease of capsicum in field pot culture

(1) Preparation of SF18-3 bacterial liquid with different concentrations

Based on the test result of 6, SF18-31 Xbacterial liquid, 10 Xbacterial liquid, 50 Xbacterial liquid, 100 Xbacterial liquid and 500 Xbacterial liquid are prepared for field potting test, and the method is the same as 6 (2).

(2) Test treatment

The test was run with 8 treatments, 3 replicates per treatment, see in particular table 5.

TABLE 5 field control effect of SF18-3 on bacterial spot disease of capsicum

Note that: 1.0.3% tetramycin aqueous solution, liaoning micro engineering Co., ltd; 46% copper hydroxide water dispersible granule 50 g/mu, duPont, U.S.A.

Level difference analysis of 0.05 for a, b, c, d.

(3) Method and period of administration

The test is carried out when 8-10 leaves of the pepper seedlings are selected. The application method comprises the following steps: stem and leaf spraying and drug application period: spraying onto capsicum leaf at early stage or early stage of disease spot, and administering 2 times after 7 days for 2 times.

(4) Investigation and prevention effect calculation method

The investigation was performed on day 10 after the 2 nd administration. According to disease grading standards, plant leaf disease conditions are investigated part by part, total leaf numbers, leaf numbers and disease grades of each disease grade are recorded, disease grades and corresponding symptom descriptions are shown in table 6, and Disease Indexes (DI) are calculated.

TABLE 6 identification of disease grade index for bacterial Spot disease of Capsicum annuum

Grade of illness	Description of disease symptoms
		0	Whole leaf no disease spot
1	The disease spots occupy less than 1/20 of the leaf area
		2	The disease spots occupy less than 1/20 to 1/10 of the leaf area
3	The disease spots occupy less than 1/10 to 1/4 of the leaf area
		4	The area of the disease spots is within 1/4 to 1/2 of the leaf area
5	The disease spots occupy more than 1/2 of the leaf area

The condition index (DI) calculation is calculated according to the following formula:

wherein: DI: index of condition;

s: representative values of each level;

n: the number of leaves at each stage;

s: the highest representative value of the onset;

n: the total leaf number was investigated.

(5) Test results

The control effects of SF18-31 Xbacterial liquid, 10 Xbacterial liquid, 50 Xbacterial liquid, 100 Xbacterial liquid and 500 Xbacterial liquid on pepper bacterial spot disease are respectively 70.23%, 67.21%, 65.58%, 53.95% and 41.40%; the control effect of the control medicament of 57.5 ml/mu of tetramycin aqua and 50 g/mu of 46% copper hydroxide water dispersible granule on bacterial spot disease of capsicum is 71.40% and 76.05% respectively. At the level of f=0.05, the prevention and treatment effects of SF18-31×fungus liquid, 10×fungus liquid, and 50×fungus liquid on diseases are not significantly different, and the three are significantly different from 100×fungus liquid and 500×fungus liquid. The SF18-31 Xbacterial liquid, 10 Xbacterial liquid, 50 Xbacterial liquid and the control agent have no obvious difference in treatment and control effects of 57.5 ml/mu of 0.3% tetramycin aqueous solution, and the 100 times liquid and 500 times liquid have obvious differences in treatment and control effects of 57.5 ml/mu of 0.3% tetramycin aqueous solution; the treatment of 50 g/mu of SF18-31 Xbacterial liquid and 46% copper hydroxide water dispersible granule has no obvious difference on disease control effect, and the other 4SF18-3 bacterial liquid concentration treatments and the treatment of 50 g/mu of 46% copper hydroxide water dispersible granule have obvious difference (figure 13).

Based on the above test results, the use of the concentration is recommended: SF18-310 x-50 x bacterial liquid, and a drug administration method: stem and leaf spraying method, preventing and curing period: the application is carried out in the early stage or the early stage of disease onset, and the application times are as follows: the drug was applied 2 times throughout the growth period, 7 days apart.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. Bacillus bailii (Bacillus velezensis) SF18-3 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2023736 at the month 11 of 2023.

2. Use of bacillus belgium SF18-3 according to claim 1 for controlling bacterial blotch of capsicum.

3. The preparation method of the biocontrol microbial agent is characterized by comprising the following steps:

inoculating the seed solution containing bacillus belicus SF18-3 according to claim 1 into a fermentation medium for fermentation to obtain the biocontrol microbial agent.

4. A method of preparation according to claim 3, wherein the fermentation conditions comprise: the time is 12-96 h, the temperature is 10-35 ℃, and the rotating speed is 100-180 rpm.

5. The method according to claim 4, wherein the fermentation conditions include: the time was 72 hours, the temperature was 25℃and the rotational speed was 140rpm.

6. The method according to claim 3, wherein the viable count of Bacillus bailii SF18-3 in the seed solution is 1X 10 ⁷ CFU/mL。

7. The method of claim 3, wherein the fermentation medium comprises NB liquid medium, and the pH of the fermentation medium is 7.0.

8. A biocontrol microbial agent prepared by the method of any one of claims 3-7.

9. The use of the biocontrol microbial agent of claim 8 for preventing and treating bacterial spot disease of capsicum.

10. The application according to claim 9, characterized in that it comprises: the biocontrol microbial inoculum is applied 1 time before or at the initial stage of the onset of the bacterial spot disease of the capsicum by adopting a stem and leaf spray method after dilution, and then applied 1 time after 7 d.