CN116042489A

CN116042489A - Lotus root endophytic bacillus beijerinus strain LS8 and application thereof

Info

Publication number: CN116042489A
Application number: CN202310169878.7A
Authority: CN
Inventors: 曾粮斌; 魏林; 羊晨; 谭志坚; 易永建; 汪洪鹰; 杨媛茹; 余旺; 邢忱; 崔看; 唐炎英; 徐志德; 周金林
Original assignee: HUNAN PLANT PROTECTION INSTITUTE; Institute of Bast Fiber Crops of CAAS
Current assignee: HUNAN PLANT PROTECTION INSTITUTE; Institute of Bast Fiber Crops of CAAS
Priority date: 2023-02-27
Filing date: 2023-02-27
Publication date: 2023-05-02

Abstract

The invention relates to the technical field of biology, in particular to a bacillus subtilis strain LS8 of lotus root endophyte and application thereof. The invention provides a lotus root endophyte bacillus beleiensis LS8 which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.24563; the lotus root endophyte bacillus bailii LS8 and the biocontrol microbial inoculum thereof have the characteristics of wide antifungal range and remarkable antagonistic effect, and can be used for preventing and treating plant mycosis, thereby having good industrial application prospect. Meanwhile, the lotus root endophyte bacillus beijerinus strain LS8 provided by the invention has remarkable control effect in potting experiments, and has great practical value for crop disease control.

Description

Lotus root endophytic bacillus beijerinus strain LS8 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a bacillus subtilis strain LS8 of lotus root endophyte and application thereof.

Background

The lotus root putrefaction disease is caused by the specialization of fusarium oxysporum. The disease belongs to soil-borne diseases, the proper temperature for disease occurrence is 25-35 ℃, the optimal pH is about 7.2, under the condition of 28 ℃, bacteria on a PSA culture medium grow in a radial shape, the initial mycelium is white, the color becomes dark in the late growth period, and the color is purple or dark purple; can produce two kinds of conidium under natural condition or artificial culture condition, the large-scale conidium is sickle-shaped, and both ends are pointed, slightly bent, colorless and transparent, and most 3 diaphragms. Soil-borne diseases refer to diseases caused by the invasion of pathogens such as fungi, bacteria, nematodes and viruses from the roots or stems of crops when the conditions are appropriate, by living in the soil with the disease residues. Soil-borne diseases include paraquat, banded sclerotial blight, wilt, epidemic disease, cataplexy, root rot, soft rot, root knot nematode, cyst nematode and the like, which commonly infect plant roots or stems, thereby causing the onset of crop root stems and even whole plants, resulting in significant economic losses.

At present, soil-borne diseases are mainly prevented and controlled by adopting chemical agents, and long-term use of the chemical agents can cause a series of serious consequences such as water source soil pollution, ecological balance being destroyed, pesticide residues, rampant secondary diseases, drug resistance of pathogenic microorganisms and the like. With the enhancement of people's environmental awareness, attach importance to food safety problem and the requirement of ecological environment construction, protection biodiversity and agricultural sustainable development for biological prevention and control soil-borne disease becomes the hot spot of current research and development.

Bacillus sp is a kind of gram positive bacteria producing spores, and has the advantages of being capable of producing endospores with heat resistance, drought resistance, ultraviolet resistance and organic solvent resistance in aerobic or facultative anaerobic life, being an important biological control resource, and the control mechanism is mainly as follows: the bacillus colonizes the root, body surface or body of the plant, competes with pathogenic bacteria for nutrition around the plant, secretes antibacterial substances to inhibit the growth of the pathogenic bacteria, and induces a plant defense system to resist the invasion of the pathogenic bacteria, so that the aim of biological control is achieved. At present, part of excellent bacillus is separated and successfully applied to biological control of plant diseases, such as bacillus subtilis and bactericide thereof, and has good use effect in controlling banana wilt; if the bacillus stearothermophilus is used for inhibiting soybean root rot, the bacillus stearothermophilus has good use effect; if the bacillus subtilis and the microbial inoculum thereof have good use effect in peanut rot, although the biocontrol microbial inoculum of bacillus exists at present, the biocontrol microbial inoculum has the defects of fewer strains, single antibacterial effect and instability.

Disclosure of Invention

In view of the above, the lotus root endophyte bacillus beijerinus strain LS8 and the application thereof provided by the invention have wide antifungal range and remarkable antagonistic effect.

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

the invention provides bacillus beleidsii (Bacillus velezensis), which has a preservation number of CGMCC No.24563.

The invention also provides application of the bacillus belicus (Bacillus velezensis) in the following aspects:

(I) Hydrolyzing beta-1, 3-glycosidic linkages, cellulose, proteins and/or cell walls in beta-1, 3-glucan; and/or

(II) inhibiting plant pathogenic bacteria; and/or

(III) preventing and treating plant soil-borne diseases; and/or

(IV) preparing cellulase, beta-1, 3-glucanase and/or protease; and/or

(V) preparing a product for hydrolyzing beta-1, 3-glycosidic bonds, cellulose, proteins and/or cell walls in beta-1, 3-glucan; and/or

(VI) preparing a product that inhibits plant pathogenic bacteria; and/or

(VII) preparing a product for preventing and treating plant soil-borne diseases;

the product comprises a biocontrol agent.

In some embodiments of the invention, the phytopathogenic fungi of the above-described applications include one or more of fusarium fungi, alternaria fungi (Alteraria sp.), gibberella fungi (neospora sp.), gliocladiopsis fungi (Gliocladiopsis sp.), trichoderma sp.), nigrospora fungi (Nigrospora sp.), epicoccum fungi (Epicoccum sp.), or Colletotrichum sp.

In some embodiments of the invention, the Fusarium fungi used above include one or more of Fusarium oxysporum lotus specialization (Fusarium oxysporum f.sp.neumbicola), fusarium putrescence (Fusarium solani), fusarium oxysporum flax specialization (Fusarium oxysporum f.sp.lini), or Fusarium oxysporum lily specialization (Fusarium oxysporum f.sp.ilii).

In some embodiments of the present invention, the plant soil-borne disease for the above-described applications comprises one or more of lotus root rot, lotus root black spot, yam black skin, flax wilt, lily root rot, camphorwood disease, camphorwood mildew, or silver mountain flower disease.

The invention also provides a fermentation method of the bacillus belicus (Bacillus velezensis), which comprises the steps of inoculating the bacillus belicus (Bacillus velezensis) into a culture medium, and fermenting to obtain a fermentation liquid of the bacillus belicus (Bacillus velezensis);

the inoculation comprises the steps of counting 10 viable bacteria ⁷ ～10 ⁸ CFU/mL of stock solution of the Bacillus bailii (Bacillus velezensis) was inoculated to the medium in an inoculum size of 2% (v/v); and/or

The volume of the medium comprises 100mL; and/or

The formula of the culture medium comprises the following components: sucrose 20g/L, beef extract 10g/L, peptone 10g/L, potassium dihydrogen phosphate 1.25g/L and magnesium sulfate 1g/L; and/or

The pH value of the culture medium is 6-7; and/or

The temperature of the fermentation comprises 28 ℃; and/or

The shaking speed of the fermentation included 150rpm.

In some embodiments of the invention, the carbon source concentration in the medium in the fermentation process described above is 2-3%.

In some embodiments of the invention, the carbon source concentration in the medium in the fermentation process described above is 2%.

In some embodiments of the invention, the nitrogen source concentration in the medium in the fermentation process described above is 1.5-3%.

In some embodiments of the invention, the nitrogen source concentration in the medium in the fermentation process described above is 2%.

In some embodiments of the invention, the inorganic salt concentration in the medium in the fermentation process described above is 0.1 to 0.15%.

In some embodiments of the invention, the inorganic salt concentration in the medium in the fermentation process described above is 0.125%.

In some embodiments of the invention, the concentration of metal ions in the medium in the fermentation process described above is from 0.0025 to 0.1%.

In some embodiments of the invention, the concentration of metal ions in the medium in the fermentation process described above is 0.1%.

In some embodiments of the invention, the pH of the medium in the fermentation process described above is from 5 to 7.

In some embodiments of the invention, the pH of the medium in the fermentation process described above is between 6 and 7%.

In some embodiments of the invention, the carbon source of the medium in the fermentation process described above comprises one or more of glucose, sucrose, fructose, lactose, glycerol, or soluble starch.

In some embodiments of the invention, the carbon source of the medium in the fermentation process described above is sucrose.

In some embodiments of the invention, the nitrogen source of the medium in the above fermentation process comprises one or more of tryptone, beef meal, yeast extract meal, peptone+beef meal (1:1), peptone+yeast extract meal (1:1), or ammonium sulfate.

In some embodiments of the invention, the nitrogen source of the medium in the fermentation process described above is peptone+beef meal (1:1).

In some embodiments of the invention, the medium in the fermentation process described above contains inorganic salts including one or more of sodium chloride, calcium chloride, or potassium dihydrogen phosphate.

In some embodiments of the invention, the medium in the fermentation process described above contains an inorganic salt of potassium dihydrogen phosphate.

In some embodiments of the invention, the metal ions contained in the medium in the fermentation process described above include one or more of magnesium ions, ferrous ions, or zinc ions.

In some embodiments of the invention, the medium in the fermentation process described above contains magnesium ions.

In some embodiments of the invention, the medium in the above fermentation process comprises one or more of magnesium sulfate, ferrous sulfate, or zinc sulfate.

In some embodiments of the invention, the medium in the fermentation process described above contains magnesium sulfate.

In some embodiments of the invention, the inoculation amount of the inoculation in the fermentation process described above is 0.1-2%.

In some embodiments of the invention, the inoculum size of the inoculum in the fermentation process described above is 2%.

In some embodiments of the invention, the volume of the fermentation in the above fermentation process is 75 to 150mL.

In some embodiments of the invention, the volume of the fermentation in the above fermentation process is 100mL.

In some embodiments of the invention, the rotational speed of the fermentation in the above fermentation process is 120 to 150rpm.

In some embodiments of the invention, the rotational speed of the fermentation in the above fermentation process is 150rpm.

In some embodiments of the invention, the fermentation temperature in the above fermentation process is 26-34 ℃.

In some embodiments of the invention, the fermentation in the above fermentation process has a temperature of 28 ℃.

The invention also provides a sterile fermentation broth, the preparation method of which comprises the following steps: inoculating the bacillus belicus (Bacillus velezensis) to a culture medium, fermenting to obtain a bacterial liquid, and filtering the bacterial liquid to obtain the sterile fermentation liquid.

The invention also provides a biocontrol microbial inoculum, which comprises the bacillus beijerinckii (Bacillus velezensis) and acceptable auxiliary materials or auxiliary agents.

The invention also provides a preparation method of the biocontrol microbial inoculum, which comprises the steps of inoculating bacillus belicus (Bacillus velezensis) to a culture medium, shake culturing for 2-4 d at 25-30 ℃, and diluting the separated thalli with water to obtain the biocontrol microbial inoculum;

the formula of the culture medium comprises the following components: 10g/L tryptone, 5g/L yeast extract, 20g/L sucrose and 5g/L NaCl; and/or

The pH value of the culture medium is 7.2-7.4; and/or

The rotational speed of the oscillation comprises 180r/min.

The invention also provides a preparation method of the cellulase, the beta-1, 3-glucanase and/or the protease, which comprises the steps of culturing bacillus belgium (Bacillus velezensis) and then separating and obtaining the cellulase, the beta-1, 3-glucanase and/or the protease.

The strain and the application of the invention have the following effects:

the invention provides lotus root endophyte bacillus beleiensis LS8 (preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 24563) and a biocontrol microbial agent thereof. Bacillus belicus LS8 is gram positive bacteria, can generate spores, can secrete cellulase, beta-1, 3-glucanase and protease, the secreted beta-1, 3-glucanase can hydrolyze beta-1, 3-glucan of components in plant fungus cell walls, and carboxymethyl cellulase and protease can act on the fungus cell walls to decompose cellulose and protein, so that growth and proliferation of plant pathogenic fungi are inhibited, and therefore LS8 has the characteristics of wide antifungal range and remarkable antagonistic effect, and has good industrialized application prospect. Meanwhile, the lotus root endophyte bacillus beijerinus LS8 provided by the invention has remarkable control effect in potting experiments, and has great practical value for crop disease control.

Description of biological preservation

Biological material: LS-8, class naming: bacillus belicus (Bacillus velezensis) was deposited at the China general microbiological culture Collection center, at 22 nd year 2022, at the accession number: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing; the preservation number is CGMCC No.24563.

LS8 is the strain with the preservation number of CGMCC No.24563.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a morphology of Bacillus belicus LS8 prepared in example 1;

FIG. 2 shows the results of physiological and biochemical identification of Bacillus belicus LS8 prepared in example 1; wherein, A shows spore staining; b shows gram staining; c represents a contact enzyme; d shows starch-dissolving capacity; e represents indoleacetic acid IAA; f, the ammonia production capacity is shown; g shows the phosphorus dissolving capacity;

FIG. 3 shows a phylogenetic tree constructed from the 16S rDNA sequence of Bacillus bailii LS8 provided in example 1;

FIG. 4 is a graph showing the inhibitory effect of Bacillus belicus LS8 in example 2 on various pathogenic bacteria; wherein A shows lotus root black spot; b shows yam black skin disease; c shows flax wilt; d represents lily wilt; e shows lily root rot; f represents camphorwood disease; g shows camphorwood mould; h shows lonicera confusa disease 1; i shows honeysuckle diseases 4; j shows honeysuckle disease 6; k represents lotus root putrefaction disease;

Fig. 5 shows a schematic diagram of an experiment of preventing and treating lotus root rot by bacillus belicus LS8 in example 3;

FIG. 6 shows a schematic diagram of a field experiment for controlling lotus root rot by Bacillus bailii LS8 in example 4;

FIG. 7 shows the optimal carbon source screening results;

FIG. 8 shows the results of optimal nitrogen source screening;

FIG. 9 shows the optimal inorganic salt screening results;

FIG. 10 shows the optimal metal ion screening results;

FIG. 11 shows the results of screening for the optimum addition amount of a carbon source;

FIG. 12 shows the results of nitrogen source optimum addition screening;

FIG. 13 shows the results of screening for optimum phosphate addition;

FIG. 14 shows the results of screening for the optimum addition amount of magnesium ions;

FIG. 15 shows the results of optimal pH optimization of the culture medium;

FIG. 16 shows the results of optimal inoculum size optimization for the medium;

FIG. 17 shows the results of optimizing the optimal amount of medium;

FIG. 18 shows the results of optimal rotational speed optimization of the culture medium;

FIG. 19 shows the results of optimal fermentation temperature optimization of the culture medium;

FIG. 20A shows the results of medium response surface optimization; wherein, the upper diagram shows the influence of the interaction of the fermentation temperature and the liquid loading amount on the light absorption value, and the lower diagram shows the influence of the interaction of the fermentation temperature and the liquid loading amount on the bacteriostasis rate;

FIG. 20B shows the results of medium response surface optimization; wherein, the upper graph shows the influence of the interaction of the fermentation temperature and the fermentation rotating speed on the light absorption value, and the lower graph shows the influence of the interaction of the fermentation temperature and the fermentation rotating speed on the bacteriostasis rate;

FIG. 20C shows the results of medium response surface optimization; wherein, the interaction of the upper graph liquid loading amount and the fermentation rotating speed influences the light absorption value, and the interaction of the lower graph liquid loading amount and the fermentation rotating speed influences the bacteriostasis rate.

Detailed Description

The invention discloses a lotus root endophyte bacillus beijerinus strain LS8 and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the present disclosure. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

The invention aims to provide a lotus root endophyte bacillus beleiensis LS8 and application thereof, and the strain and the microbial inoculum thereof have broad-spectrum bacteriostasis, have high and stable bacteriostasis effects and can effectively prevent and treat plant soil-borne fungus diseases.

The invention provides a lotus root endophyte bacillus beleiensis LS8 which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.24563.

Preferably, the bacillus beleiensis LS8 is a gram positive bacterium capable of producing spores and secreting cellulases, beta-1, 3-glucanases and proteases.

Preferably, the colony edge of bacillus belicus LS8 is relatively clean, the surface is convex and moist, the color is milky white, and the bacillus belicus LS8 is opaque.

Preferably, the bacillus belicus LS8 can be used for preventing and treating soil-borne diseases of plants.

Preferably, the plant fungal diseases are lotus root rot, lotus root black spot, yam black skin, flax wilt, lily root rot, camphorwood disease and lonicera japonica disease.

Preferably, the preparation method of the biocontrol microbial agent containing bacillus belicus LS8 comprises the following steps: inoculating Bacillus bailii LS8 into sterilized liquid culture medium, culturing at 25-30deg.C, shaking for 2-4 d, centrifuging 12000r/min for 5min, diluting with sterile water to concentration of 10 ⁵ The CFU/mL suspension is prepared to obtain the biocontrol microbial inoculum containing bacillus bailii LS 8.

Preferably, the liquid culture medium consists of 10g/L tryptone, 5g/L yeast extract, 20g/L sucrose and 5g/L NaCl; the pH value of the liquid culture medium is 7.2-7.4.

Preferably, the rotation speed of shaking table vibration is 180r/min.

Preferably, the biocontrol microbial inoculum containing bacillus beijerinus LS8 is applied to preventing and treating the lotus root spoilage disease.

The bacillus belicus strain provided by the embodiment of the invention enables bacillus belicus and the biocontrol microbial inoculum thereof provided by the invention to be capable of effectively preventing and controlling plant soil-borne fungus diseases.

Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all commercially available and commercially available.

In order to better understand the technical solution in the embodiments of the present invention and make the above objects, features and advantages of the embodiments of the present invention more comprehensible, the following describes the technical solution in the embodiments of the present invention in further detail with reference to the accompanying drawings:

example 1: preparation and identification of lotus root endophyte bacillus bailii LS8

1. Preparation of lotus root endophyte bacillus beleiensis LS8

S01: collecting lotus roots by a multipoint sampling method, cleaning the collected lotus roots with clear water, sterilizing for 5min by using 1% mercuric chloride solution, rinsing 3 times by using sterile water, sterilizing for 30s by using 70% ethanol, rinsing 3 times by using sterile water, and grinding the lotus roots in an ultra-clean workbench by using a mortar, wherein the lotus roots in the embodiment of the invention are taken from a village lotus seed planting field in the flower Dan Zhen red of Xiangtan county;

S02: placing the ground lotus root sample into a centrifuge tube filled with sterile water, and sufficiently shaking to obtain a concentration of 10 ^-1 Lotus root suspension;

s03: gradually and gradiently diluting the lotus root suspension to obtain lotus root suspensions with different concentrations;

s04: selecting concentration of 10 ^-3 、10 ^-4 And 10 ^-5 Three gradient soil suspensions are added to an NA culture medium flat plate for coating treatment, and the soil suspensions are placed in a 30 ℃ incubator for culturing for 48 hours, so that bacterial colonies are obtained;

s05: selecting single bacterial colonies with different forms, marking off on an inclined plane for seed preservation, culturing for 24 hours to obtain separated bacteria, and placing the separated bacteria in a refrigerator at 4 ℃ for later use;

s06: inoculating pathogenic bacteria such as rhizoma Dioscoreae putrefying disease and Bulbus Lilii putrefying disease to the center of PDA culture medium plate with punch with diameter of 0.5cm, inoculating the isolated bacteria equidistantly at 2.5cm crisscross position from the center of plate, repeating experiment for 3 times without inoculating isolated bacteria as Control (CK), culturing at 25deg.C, recording the size of antibacterial zone when the control group (CK) is full of all vessels, and selecting Bacillus berensis LS8 with maximum antibacterial zone.

The NA culture medium is a nutrient agar culture medium, and the nutrient agar culture medium comprises 20g/L of agar, 10g/L of tryptone, 3g/L of beef extract and 5g/L of NaCl according to the concentration. The pH value of the NA culture medium is 7.2-7.4 during preparation, the NA culture medium is sterilized for 20min, and the temperature during sterilization is 121 ℃. The PDA culture medium is potato dextrose agar culture medium.

2. Identification of lotus root endophyte bacillus belgium LS 8:

1. morphological identification

The bacterial strain prepared in the embodiment is streaked on a NA culture medium flat plate, then the flat plate is inverted and cultured for 24 hours at the temperature of 30 ℃, and the growth condition of bacterial colonies on the flat plate is observed and recorded, wherein the bacterial colony morphology on the flat plate is shown as figure 1.

The strain prepared in this example was subjected to gram staining and spore staining with a kit, and the strain was observed under an oil microscope and photographed. The gram staining and spore staining of the strain are respectively shown in figure 2, and as can be seen from B in figure 2, bacillus beleiensis LS8 is rod-shaped and blue-purple after gram staining, and is gram-positive; as can be seen from A in FIG. 2, after spore staining, bacillus bailii LS8 cells appeared blue and spores appeared red, thus demonstrating that the strain provided by the present invention was able to produce spores.

2. Physiological and biochemical identification

(1) Contact enzyme assay

3% hydrogen peroxide was directly added dropwise to the liquid culture medium of the strain prepared in this example, and immediately observed. If a large number of bubbles are generated, the result is positive; if no bubble is generated, the result is negative. The strain provided by the invention immediately generates a large amount of bubbles, and the experimental result is positive.

(2) Oxidase test

A small amount of bacterial strain colonies prepared in the embodiment is dipped in one corner of white clean filter paper, one drop of 1% dimethyl p-phenylenediamine hydrochloride aqueous solution is added, and the positive person immediately turns pink and the color gradually deepens. In this experiment, the colonies did not change color, and the experimental result was positive.

(3) Cellulose decomposition experiment

Single colony of the test strain is streaked on a cellulase activity identification medium, cultured for 5 days at 30 ℃, dyed with 0.1% Congo red for 30min, washed with distilled water, counterstained with 1mol/L hydrochloric acid solution for 30min, washed, and observed whether a transparent ring exists or not. In this experiment, a transparent ring appeared around the colony, and the experimental result was positive

(4) Methyl red MR experiment

A small amount of the strain prepared in the embodiment is selected and inoculated on a general culture medium, the culture is carried out for 3 to 5 days at the temperature of 30 ℃, 1mL of culture solution is taken after the culture is finished, 1 to 2 drops of methyl red indicator are added, positive is bright red, weak positive is light red, and negative is yellow. In this experiment, the bacterial liquid turned yellow, and was therefore negative.

(5) V-P experiment

A small amount of the strain prepared in the embodiment is selected and inoculated in a general culture medium, and is cultured for 4 days at the temperature of 30 ℃, 2.5mL of culture solution is taken after the culture is finished, 0.6mL of alpha-naphthol pure alcohol solution is firstly added, 0.2mL of 40% potassium hydroxide aqueous solution is then added, the culture solution is shaken for 2-5 min, positive bacteria usually immediately show red color, if no red color appears, the culture solution is stood in a constant temperature box at room temperature or 30 ℃, and if the red color still does not appear in 2h, the culture solution can be judged as negative. In this experiment, the bacterial liquid became red immediately, so it was positive.

(6) Gelatin liquefaction experiment

The strain prepared in this example was inoculated into gelatin by puncture and the strain was located at 2/3 of the depth of gelatin. Culturing at 20 deg.c for 5-7 d. Daily observation results show that whether the strain is liquefied by bacteria or not, and if the strain is liquefied, the test is positive; if not liquefied, it is negative. In this experiment, gelatin was liquefied, so the reaction characteristic was positive.

(7) Nitrate reduction experiment

The strain prepared in this example was inoculated into a nitrate medium, shake-cultured at 30℃for 3 days, then 5mL of the culture solution was taken, and a color-developing agent was added according to the instructions of the kit (Haibo biotechnology Co., ltd., nitrate reduction kit), and the yellowing was positive, whereas the non-discoloration was negative. In this experiment, the bacterial liquid turned yellow, indicating that the reaction was positive in character.

(8) Experiment for producing hydrogen sulfide

The strain prepared in this example was inoculated in a lead acetate medium by puncture, cultured at 35℃for 24 to 48 hours, and the results were observed. If the culture medium turns black, the result is positive; and if the color is not black, the color is negative. In this experiment, the medium turned black, indicating that the reaction was positive.

(9) Citrate utilization experiments

The strain prepared in this example was streaked onto a slope of a citrate medium of Western Meng Sishi and cultured at 37℃for 3 to 7 days. If the culture medium is alkaline, the indicator is blue or pink positive; if the medium does not change color, it is negative. In this experiment, the medium was discoloured, indicating that the reaction was characterized as positive.

(10) Lecithin enzyme Activity assay

Sterilizing the surface of fresh egg with 75% ethanol, perforating the egg with sterilized forceps, removing egg white, sucking yolk with sterile suction tube, adding into NA culture medium cooled to about 50deg.C after thawing, mixing, pouring flat plate, inoculating the strain prepared in this example, culturing at 30deg.C for 24 hr, and observing. If the colony edge appears cloudy circles, the colony edge is enzyme positive. In this experiment, a clear cloudy ring appeared at the edge of the colony, indicating a positive reaction profile.

(11) Malonate utilization experiments

The bacterial lawn (the strain prepared in the embodiment) is selected and cultured for 12 hours and inoculated in malonate culture medium, and the culture medium is cultured for 24-48 hours at the temperature of 35 ℃, and the culture medium is positive when the green turns blue, and is negative otherwise. In this experiment, the medium did not change color, indicating that the reaction was negative in character.

(12) Glucose fermentation experiments

A small amount of the strain prepared in this example was selected and inoculated in a glucose oxidative fermentation medium by puncturing, and the medium was cultured at 30℃for 3 days to observe the change in color of the medium. If there is no color change, the observation is continued for 7 days, and the medium turns yellow to be fermented. In this experiment, the medium turned yellow, indicating that the reaction was positive.

(13) Galactose utilization experiment

The strain prepared in this example was inoculated into a galactose medium, and cultured at 30℃for 2 days, and colony growth was observed, and if colony formation was observed, galactose was used, otherwise, it was not used. In this experiment, the strain grew, indicating that galactose could be used by the strain.

(14) Arabinose utilization experiment

The strain prepared in this example was inoculated into an arabinose medium, and cultured at 30℃for 2 days to observe colony growth, and if colony formation was observed, arabinose could be used, otherwise, it was not used. In this experiment, the cells did not grow, indicating that the strain could not utilize arabinose.

(15) Mannose utilization experiment

The strain prepared in this example was inoculated into a mannose medium, and cultured at 30℃for 2 days to observe colony growth, and mannose was used if colonies were formed, but not otherwise. In this experiment, the strain can utilize mannose for the growth of the cells.

(16) D-fructose utilization experiment

The strain prepared in this example was inoculated into D-fructose medium, cultured at 30℃for 2 days, and colony growth was observed, and D-fructose was used if colonies were formed, but not otherwise. In this experiment, the strain can utilize D-fructose for the growth of the cells.

(17) D-xylose utilization experiments

The strain prepared in this example was inoculated into D-xylose medium, cultured at 30℃for 2 days, and colony growth was observed, and D-xylose was used if colonies were formed, otherwise, it was not used. In this experiment, the strain can utilize D-xylose for the growth of the cells.

In summary, the physiological and biochemical identification results are shown in table 1; the partial results are shown in FIG. 2C to G.

Table 1: physiological and biochemical characterization results of Bacillus bailii strain LS8

Test	Results	Test	Results
				Ammonia production	+	Hydrogen sulfide	+
IAA	+	Lecithin enzyme	+
				Cellulose degradation	+	Oxidase enzyme	+
Gelatin liquefaction	+	Contact enzyme	+
				Methyl red MR	-	Fructose	+
V-P assay	+	Mannose	+
				Nitrate reduction	+	Galactose	+
Phosphate dissolving	+	Arabinose (Arabic sugar)	-
				Citrate reaction	+	Xylose	+
Glucose fermentation	+	Malonic acid utilization	-

Note that: +: a positive reaction; -: negative reaction.

3. 16S rDNA sequence analysis

The specific operation steps of the DNA of the strain prepared by adopting the bacterial liquid PCR method are as follows: 25. Mu.L of a PCR reaction system comprising 12.5. Mu.L of a 2 XMaster Mix;1 μl of upstream primer 27F:5'-AGA GTT TGA TCC TGG CTC AG-3' (SEQ ID NO: 1); 1 μl of downstream primer 1492R:5'-GGT TAC CTT GTT ACG ACT T-3' (SEQ ID NO: 2); 9 mu L ddH ₂ O; 1.5. Mu.L of template DNA.

The PCR reaction conditions were: pre-denaturing at 94℃for 5min; denaturation at 94℃for 30s; annealing at 53 ℃ for 30s; extending at 72 ℃ for 1min;30 cycles, 72℃extension for 5min,4℃storage. The obtained PCR product was analyzed by liposaccharide gel electrophoresis, and the 16S sequencing result of the biocontrol strain LS8 revealed that the strain DNA fragment prepared in this example was about 1450bp long. The obtained PCR product was sequenced by Hunan qing Kogyo Co., ltd. And the nucleotide sequence of the strain prepared in this example is shown in the sequence table SEQ ID NO. 3. The nucleotide sequence obtained is subjected to homologous sequence comparison analysis through NCBI-BLAST to obtain a sequence with higher similarity. The phylogenetic tree (shown in FIG. 3) was constructed using MEGA6.0 software, and the 16S rDNA sequence of the strain prepared in this example had high homology with Bacillus bailii (Bacillus velezensis). The 16S sequence of Bacillus belicus LS8 is shown below:

From the above morphological observation, physiological biochemical identification and 16S rDNA sequence analysis results, it was confirmed that the strain prepared in this example was Bacillus bailii (Bacillus velezensis), which was designated Bacillus bailii strain LS8.

Example 2: evaluation of antibacterial effect of lotus root endophyte bacillus bailii strain LS8

1. Evaluation of bacteriostatic Effect

Taking lotus root rot, lotus root black spot, chinese yam black skin, flax wilt, lily root rot, camphorwood disease and honeysuckle as examples, the inhibition effect of bacillus belicus LS8 is studied, and the specific contents are as follows:

taking lotus root rot, lotus root black spot, chinese yam black skin, flax wilt, lily root rot, camphorwood disease and lonicera confusa as target bacteria, inoculating small paper sheets (the diameter of the small paper sheets is 4 mm) soaked with bacillus bailii strain LS8 bacterial liquid at a position 2.5cm away from the center of the flat plate, taking the flat plate without inoculating LS8 bacteria as a control, and repeating each treatment for 3 times. The flat plate is placed in a 25 ℃ incubator in an inverted mode, when the pathogenic fungi of the control group grow up to the flat plate, the diameter of candidate antagonistic bacteria and the diameter of a bacteriostasis circle of bacteria on each pathogenic fungi are measured, and the antagonistic index is calculated according to the diameter of the bacteriostasis circle:

Inhibition ratio = (inhibition zone diameter-antagonistic diameter)/antagonistic diameter.

As can be seen from fig. 4, bacillus belicus LS8 has an inhibitory effect on all plant pathogenic fungi, which indicates that bacillus belicus LS8 has a broad-spectrum antibacterial property; as shown in Table 2, the antibacterial diameter of Bacillus belicus LS8 against the pathogenic fungi is 15-32 mm, and the antagonistic index shows that the Bacillus belicus LS8 has good antagonistic effect against the pathogenic fungi, so that the Bacillus belicus LS8 has high-efficiency antibacterial effect against the plant pathogenic fungi, and can be used for preventing and treating plant soil-borne diseases caused by the plant pathogenic fungi.

Table 2: effect of LS8 on antagonism of different plant pathogenic fungi

Pathogenic bacteria species	Diameter of inhibition zone (mm)	Inhibition ratio (%)
			Lotus root putrefaction disease (Fusarium Oxysporum)	18.47±0.711	34.87±1.34
Lotus root black spot disease (Alternaria)	17.67±2.97	38.68±6.51
			Chinese yam black skin disease (Fusarium Fusarium)	17.18±1.23	30.10±2.15
Flax wilt (Fusarium Fusarium)	11.52±1.11	31.30±3.01
			Lily wilt (Fusarium Fusarium)	7.86±1.15	27.42±4.01
Lily root rot (Neocosmiorcora New red shell genus)	4.49±0.75	18.35±3.07
			Camphorwood disease (Gliocladiopsis)	2.60±1.53	15.84±9.33
Camphorwood mildew (Trichoderma)	19.88±0.23	43.95±0.50
			Honeysuckle disease 1 (Nigrospora Nissan)	24.26±0.76	48.85±1.52
Honeysuckle disease 4 (Epicoccum Epicoccum)	3.11±0.69	16.11±3.59
			Honeysuckle disease 6 (Colletotrichum genus)	25.99±0.55	55.69±1.19

2 study of antibacterial mechanism

Bacillus belicus LS8 prepared in example 1 was inoculated into plates containing colloidal chitin medium, carboxymethylcellulose sodium medium, poria powder medium and skimmed milk agar medium, and the plates were placed upside down in a 30℃incubator for 3d, and whether transparent rings were generated around colonies was observed.

The results show that bacillus bailii LS8 has transparent rings on sodium carboxymethyl cellulose medium, poria cocos powder medium and skim milk agar medium, which indicates that bacillus bailii LS8 can secrete cellulase, beta-1, 3-glucanase and protease. 80% of the dry weight of the fungal cell wall consists of carbohydrates such as: chitin, chitosan, dextran, cellulose, galactan, and the like. About 10% is composed of proteins including enzymes responsible for cell wall growth, specific extracellular enzymes, and structural proteins that crosslink polysaccharides, and glycoproteins. Beta-1, 3-glucosidic bond in beta-1, 3-glucan in fungal cell wall can be hydrolyzed by beta-1, 3-glucanase secreted by bacillus bailii LS8, and carboxymethyl cellulose and protease can act on fungal cell wall to decompose cellulose and protein, so that growth and proliferation of plant pathogenic fungi can be inhibited.

Example 3: initial test for preventing and treating lotus root putrefaction disease by using lotus root endophytic bacillus behenensis LS8 bacterial liquid

1. Preparation of biocontrol microbial agent containing bacillus bailii LS8

Inoculating Bacillus bailii strain LS8 prepared in example 1 into sterilized NA liquid medium (i.e. beef extract peptone liquid medium, its proportion is 10g/L peptone, 3g/L beef extract and 5g/L NaCl, pH=7.2-7.4), shake culturing at 30deg.C for 2d at shake speed of 180r/min, centrifuging at 12000r/min for 5min, collecting thallus, diluting with sterile water to concentration of 10 ⁵ The CFU/mL suspension is obtained, and bacillus belicus LS8 is contained.

2. Preparation of fusarium oxysporum fungus liquid for pathogenic bacteria of lotus root putrefying disease

Inoculating Fusarium oxysporum fungus which is pathogenic bacteria of lotus root putrefying disease into sterilized PDA liquid culture medium (PDA culture medium composition is 200g peeled potato, 20g glucose, 1000mL sterile water, pH value is 7.2-7.4), shake culturing at 25deg.C for 7d at shake speed of 180r/min, diluting the bacterial liquid to 10 with sterile water ⁵ CFU/mL to obtain the fusarium oxysporum suspension of pathogenic bacteria of lotus root putrefying disease.

3. Application of biocontrol microbial agent containing bacillus belicus LS8 in inhibition of lotus root putrefaction disease

(1) Biocontrol microbial agent containing bacillus bailii LS8 has effect of preventing and controlling lotus root putrefaction disease

Test varieties: fresh lotus root (marketing)

Pathogenic bacteria: fusarium oxysporum pathogen for lotus root putrefaction disease

Test treatment: control group: shi Bingyuan bacteria and sterile water; treatment group: shi Bingyuan bacteria and biocontrol microbial inoculum containing bacillus bailii LS8

The lotus root adopted in the tieback screening experiment is purchased from the market, the surface of the lotus root is washed by tap water, the lotus root is cut into round blocks with the diameter of about 3cm in parallel with lotus root holes, lotus root slices are placed into bacterial liquid of biocontrol bacteria to be soaked for 0.5h, a control group is placed into ultrapure water to be soaked for 0.5h, the lotus root slices are placed into 9cm plates with a piece of filter paper after being dried, and 3 lotus root slices are repeated in each plate. Inoculating activated fusarium oxysporum mycelium blocks with the diameter of 4mm to the center of a lotus root block by using a puncher, inoculating 1 fusarium oxysporum mycelium block to each lotus root block, and culturing the lotus root block in a constant-temperature incubator at 25 ℃ until the mycelium of a control group is about to grow into lotus root blocks. The colony diameters of the control group and the experimental group were measured. Antibacterial ratio= (control group colony diameter-experimental group colony diameter)/(control group colony diameter-hypha block diameter)

The results are shown in FIG. 5 and Table 3.

Table 3: inhibition of lotus root putrefaction disease by bacillus bailii LS8 bacterial suspension and other screened biocontrol bacteria

Name of the Strain	Pathogenic bacterial colony diameter/mm	Diameter of inhibition zone	Inhibition/%
				LS8	8.36±3.58	17.23±3.58	79.82±3.58
LS8-4-5	8.73±1.60	16.85±1.60	78.09±1.60
				LYM5-1	9.10±3.56	16.48±3.56	76.36±3.56
LL4-2	9.34±1.14	16.24±1.14	75.25±1.14
				Y5-5	9.63±3.98	15.95±3.98	73.92±3.98
YS-5	9.83±2.48	15.75±2.45	72.99±2.45
				LS8-5-9	10.77±3.16	14.81±3.16	68.62±3.16
LYM3-3	11.74±2.96	14.81±3.16	64.11±2.96
				Y3-5	12.21±3.38	13.37±3.38	61.96±3.38
Y6-3	13.60±3.46	11.98±3.46	55.50±3.46
				CK	25.58±3.42

Example 4: application of lotus root endophytic bacillus beijerinus LS8 bacterial liquid in field for preventing and treating lotus root putrefaction disease

1 materials and methods

1.1 test strains

The biocontrol strain bacillus beleiensis LS8 is separated from lotus root and sent to China general microbiological culture Collection center (CGMCC No. 24563), and the first generation strain obtained by the activation of a glycerol cryopreservation tube is selected. Fusarium oxysporum is provided by plant protection in Hunan province.

1.2 test plants

The plant to be tested is lotus, the variety is three lotus in Hunan, and the lotus is obtained by harvesting in 2021 of the stock market of the China academy of agricultural sciences.

1.3 Medium and cultivation sites

NB medium and PDA medium.

The test was carried out in the stock farm of China agricultural academy of sciences.

1.4 test methods

1.4.1 preparation of lotus seed plants

The lotus seeds are stored in a refrigerator at 4 ℃, small holes with the diameter of 2mm are drilled at the two ends of the lotus seeds by electric drills in advance, germination is induced in a cultivation basin filled with 20L of clear water, the cultivation temperature is 20-25 ℃, water is changed once a day in the morning and evening to prevent bacteria from polluting water, most lotus seeds absorb water and swell after 3d of cultivation, lotus shells are cracked, and most lotus seeds germinate after 5d of cultivation, and are transported to the field for cultivation.

1.4.2 preparation of pathogenic bacteria of lotus root and biocontrol bacteria LS8 microbial inoculum

Activating pathogenic bacteria Fusarium oxysporum on pda plate, scraping several plates to pick mycelium into sterile water, and preparing into concentration of 2×10 ⁸ ～6×10 ⁸ CFU/mL fusarium oxysporum liquid, dilute to 10 ⁶ And (5) standby.

Culturing LS8 biocontrol bacteria by adopting an optimized culture medium, and culturing for 2 days until the concentration of bacterial liquid is 4 multiplied by 10 ⁸ Diluted to 10 ⁶ And (5) standby.

1.4.3 biocontrol bacteria and pathogen inoculation

80 lotus seeds with basically consistent growth vigor are selected and divided into 4 groups, namely a blank group, a biocontrol bacterium group, a germ group and a biocontrol bacterium and germ group. The blank group is soaked in clear water and is not treated, and the concentration of the biocontrol bacteria group is 4 multiplied by 10 ⁶ LS8 biocontrol bacteria of CFU/mL are soaked for 12 hours, and the concentration of bacteria group is 2 multiplied by 10 ⁶ CFU/mL fusarium oxysporum bacterial liquid is soaked for 3 hours, and the concentration of the biocontrol bacteria and the bacteria added groups is 4 multiplied by 10 ⁶ The LS8 biocontrol bacteria of CFU/mL are soaked for 12 hours and then washed by clean water, and the bacteria concentration is 2 multiplied by 10 ⁶ CFU/mL fusarium oxysporum liquid is soaked for 3 hours.

1.4.4 field cultivation

The cultivation test is carried out on an original seed field test base of China national academy of sciences and hemp research institute, a field is divided into 4 square grids with the depth of 2m multiplied by 4m, each cultivation grid is about 20cm, soil between the grids is coated by a plastic film, and clear water with the depth of about 10cm is irrigated in each cultivation grid. The cultivation lattice was divided into 4 groups: blank group, biocontrol bacteria group, germ group and biocontrol bacteria plus germ group. And uniformly cultivating 20 treated lotus seeds in parallel in 3 areas in each cultivation lattice, and burying the whole lotus seeds into soil with the depth of about 3 cm. The cultivation temperature is 15-25 ℃, and after 14d of cultivation, sampling and recording diseases and grading the diseases.

2 results and analysis

The control test results of the biocontrol bacteria LS8 lotus field are shown in Table 4 and FIG. 6, as the three lotus is cultivated for several months before the field test is carried out, the water body and the soil contain a certain concentration of pathogenic bacteria of lotus rot disease, and the leaves measured by the field test are floating leaves cultivated by the lotus, so that the disease resistance is weaker, and each group is infected by diseases of different degrees. The average disease index of the blank group is 21.40%, most lotus leaves can see pale red lesions, the lesions are uniformly distributed on the whole lotus leaves, and serious whole leaves are infected and discolored; compared with a blank group, the disease index of the biocontrol bacterium group is 8.47%, the control effect of the biocontrol bacterium treatment group reaches 60.42%, the disease spots are mainly distributed on the edges of the leaves, the condition that the whole lotus leaves are infected by bacteria does not occur, the mature leaves are the most green in 4 groups, and the growth vigor is good. The disease index of the pathogenic bacteria group is 48.68%, after the leaves of the group are treated by the pathogenic bacteria liquid, newly formed pink disease spots can be observed in curled new leaves just growing out of the water surface, and part of leaves with serious disease are affected by the disease spots, and part of mesophyll curls or even breaks. The disease index of the biocontrol bacteria and germ group is 23.41%, compared with the pathogenic bacteria group, the control effect reaches 51.91%, the group of leaves are also all infected by pathogenic bacteria, red disease spots can be observed on each lotus leaf, but compared with the germ group, the disease spots are lighter in symptoms, the disease spots are uniformly distributed on the leaves, massive mesophyll necrosis is not caused by the fact that the disease spots are connected into a whole, and small holes are only formed in the center of the disease spots of the leaves with serious disease. The lotus seed is soaked in the biocontrol strain LS8 bacterial liquid before cultivation, and has a certain effect on preventing and treating the rot disease of lotus roots.

Table 4: biocontrol strain LS8 lotus field control test result

Test group	Index of disease/%	Preventing and curing effect is%
			Blank group	21.40±1.81 ^b	-
Biocontrol bacteria group	8.47±1.83 ^c	60.42±4.25
			Pathogenic bacteria group	48.68±3.99 ^a	-
Biocontrol bacterium and germ group	23.41±3.33 ^b	51.91±3.42

Example 5: bacillus bailii LS8 bacterial liquid culture medium and fermentation condition optimization

1 materials and methods

1.1 test strains

1.2 Medium

NB medium (g/L): peptone 10.0, sodium chloride 5.0, beef powder 3.0, ph 7.0;

fermentation basal medium (g/L): glucose 20.0, beef extract 10.0, peptone 10.0, naCl 5.0, pH is natural;

PDA medium: 200.0 parts of potato (peeled), 20.0 parts of glucose, 15.0 parts of agar and 7.0 parts of pH;

1.3 major reagents and instruments

1.4 test methods

1.4.1 cultivation of seed solution

And (3) streaking and inoculating biocontrol bacteria LS8 stored in glycerol in an LB plate, scraping single colony to inoculate 100mL of shaking bottled liquid per 250mL of shaking liquid after the single colony is formed, fermenting at 28 ℃ for 24 hours under the condition of 150rpm, and then inoculating the single colony into a test shaking bottle.

1.4.2 preparation of sterile fermentation broth

The preparation method of the sterile fermentation liquor is the same as that of the sterile fermentation liquor, and after the fermentation is completed, the bacteria liquor is repeatedly and uniformly mixed under the sterile condition and then passes through a filter membrane of 0.22 mu m, and the obtained liquor is the sterile fermentation liquor of the biocontrol bacteria LS 8.

1.4.3 fermentation result evaluation method

1) The biomass of the fermentation broth was measured, and the absorbance of the fermentation broth at a wavelength of 600nm was measured with a blank medium as a control, and repeated 3 times per treatment.

2) Fusarium oxysporum inhibition effect, inoculating Fusarium oxysporum blocks with the diameter of 4mm to the center of a PDA culture medium, culturing at 25 ℃ for 1d, punching at a position 2.5cm away from the blocks by a puncher with the inner diameter of 6mm, inoculating 100 μl of sterile fermentation liquor into 3 holes respectively, inoculating 100 μl of sterile water into 1 hole as a control, sealing a culture dish, continuously culturing at 25 ℃ for 3d until the control hyphae approach to the small holes, measuring the radius of a colony, calculating the bacteriostasis rate, and repeating 3 plates for each treatment.

1.4.4 Single factor test of Medium composition

1) Optimal carbon source species screening

The carbon source in the basal medium was changed to the following carbon source: glucose, sucrose, fructose, lactose, glycerol, soluble starch, and the group without carbon source was set up as a blank group. 2mL of the activated LS8 bacteria solution was inoculated per 100mL of the medium, and the culture was carried out at 28℃and 150rpm for 2d, and the treatment was repeated 3 times.

2) Optimal nitrogen source species screening

Selecting the optimal carbon source obtained by screening, and changing the nitrogen source in the basal medium into the following nitrogen source: tryptone, beef powder, yeast extract powder, peptone+beef powder (1:1), peptone+yeast extract powder (1:1), ammonium sulfate, and a group without adding nitrogen source was established as a blank test group. The cultivation method is the same as 4.1.

3) Optimal inorganic salt species screening

Selecting and screening the obtained optimal carbon source and optimal nitrogen source, and changing inorganic salts in the basic culture medium into the following inorganic salts: sodium chloride, calcium chloride, potassium dihydrogen phosphate, and no inorganic salt was used as blank test group. The culture mode is the same as 4.1.

4) Optimal metal ion species screening

Selecting and screening the obtained optimal carbon source, optimal nitrogen source and optimal inorganic salt, and adding the following metal ions into a basic culture medium: magnesium sulfate, ferrous sulfate and zinc sulfate with mass concentration of 0.1% and no metal ion group as blank test group. The cultivation method is the same as 4.1.

1.4.5 Medium ratio optimization

1) Screening of optimal addition amount of carbon source

Selecting the optimal culture medium components after screening, and setting a plurality of gradients for the optimal carbon source, namely sucrose concentration: 0%, 1%, 1.5%, 2%, 2.5%, 3%, and the same method as 4.1.

2) Screening of optimum addition amount of nitrogen source

Selecting the optimal culture medium components, optimizing the carbon source addition amount to be 2.1, and setting a plurality of gradients for the optimal nitrogen source concentration, namely peptone and beef powder (1:1): 0%, 1%, 1.5%, 2% (ck), 2.5%, 3%, and the same method as 4.1.

3) Screening of optimal addition amount of inorganic salt

Selecting the optimal culture medium components, optimizing the carbon source addition amount to be 2.1, optimizing the nitrogen source addition amount to be 2.2, and setting a plurality of gradients for the optimal inorganic salt, namely the potassium dihydrogen phosphate concentration: 0%, 0.125%, 0.25%, 0.5% (ck), 0.75%, 1%, and the same culture method as 4.1.

4) Screening of optimum addition amount of metal ions

The optimal metal ion, namely magnesium sulfate concentration, is set with a plurality of gradients by optimizing the salt addition amount to be 2.3: 0%, 0.025%, 0.05%, 0.1%, 0.15%, 0.2%, and the same method as 4.1.

5) Culture medium optimum pH value screening

The optimal culture medium obtained by the optimization is selected, and hydrochloric acid or sodium hydroxide solution is added to adjust the pH of the culture medium to 4, 5, 6, 7, 8, 9 and 10. The cultivation method is the same as 4.1.

1.4.6 optimization of culture conditions

1) Inoculum size

Selecting the optimized optimal culture medium as an inoculum size test basic culture medium, and setting a plurality of inoculum size gradients: 0.01%, 0.1%, 1%, 2%, 5%, and the optimal fermentation temperature obtained in 3.1, liquid loading amount of 100mL/250mL, 150rpm, fermentation time of 6h, 12h and 24h, and determining biomass of the biocontrol bacteria LS8 bacteria liquid by measuring bacterial liquid absorbance values, wherein each treatment is repeated for 3 times.

2) Liquid loading amount

Selecting the optimized optimal culture medium as a basal culture medium for liquid loading test, and setting a plurality of liquid loading gradients: 50mL, 75mL, 100mL, 125mL and 150mL, determining the biomass of the biocontrol strain LS8 bacterial liquid by measuring the bacterial liquid absorbance value after the optimal fermentation temperature obtained by 3.1, the optimal inoculation amount obtained by 3.2, 150rpm and the fermentation time for 24 hours, and repeating each treatment for 3 times.

3) Rotational speed

Selecting the optimized optimal culture medium as a basic culture medium for fermentation rotating speed test, and setting a plurality of liquid loading gradients: the biomass of the biocontrol bacteria LS8 bacteria liquid is determined by measuring the bacterial liquid absorbance value at the time of fermentation for 6 hours, 12 hours and 24 hours at the time of 120rpm, 135rpm, 150rpm, 165rpm and 180rpm, the optimal fermentation temperature obtained by 3.1, the optimal inoculation amount obtained by 3.2, the optimal liquid loading amount obtained by 3.3, and each treatment is repeated for 3 times.

4) Fermentation temperature

Selecting the optimized optimal culture medium as a fermentation temperature basic culture medium, and setting a plurality of fermentation temperature gradients: the other fermentation conditions were that the inoculum size was 2%, the liquid loading was 100mL/250mL, 150rpm, and the fermentation time was 2d at 20 ℃, 24 ℃, 28 ℃, 32 ℃, 36 ℃, 40 ℃, and 3 times per treatment were repeated.

5) Fermentation time

The optimized optimal culture medium is selected as a basic culture medium for fermentation time test, the biomass of the biocontrol strain LS8 bacteria liquid is determined by measuring the bacterial liquid absorbance value when the fermentation time is 6h, 12h, 24h, 48h, 72h, 96h and 120h, and the processing is repeated for 3 times in the optimal fermentation temperature obtained by 3.1, the optimal inoculation amount obtained by 3.2, the optimal liquid loading amount obtained by 3.3 and the optimal fermentation rotating speed obtained by 3.4.

1.4.7 fermentation Condition response surface optimization

According to a single factor experiment result, 3 factors which have obvious influence on the antibacterial activity of the sterile fermentation broth, namely temperature, rotating speed and liquid loading amount are selected to carry out Box-Benhnken experiment Design in response surface experiment Design, each factor takes 3 levels, design Expert software is used for carrying out response surface and variance analysis, and data are subjected to secondary regression fitting to obtain a multiple regression equation, an optimal value is obtained in a certain level range, and fermentation conditions are optimized. And finally, carrying out verification experiments on the results obtained by the experiments, and detecting the accuracy of the model and the results.

2 results and analysis

2.1 Medium composition Single factor test results

1) Optimal carbon source type screening results

The best carbon source screening result is shown in fig. 7 (corresponding data is shown in table 5), CK is a blank carbon source group, and biocontrol bacteria LS8 can utilize the above 6 carbon sources and can produce antibacterial substances. Wherein the absorbance of the sucrose group is significantly higher than that of other groups, and the average value of the bacteriostasis rate of the sucrose group is highest, but the sucrose group has no significant difference from the glucose group, the starch group and the blank carbon source group. And (3) synthesizing the material price of each carbon source, selecting sucrose as the optimal carbon source of the biocontrol strain LS8 fermentation medium, and carrying out subsequent experiments.

TABLE 5 influence of different carbon sources on LS8 growth and bacteriostatic substances

Carbon source type	Carbon source type	OD ₆₀₀	Inhibition ratio (%)
				Glucose	glucose	0.925	6.25
Sucrose	sucrose	1.028	7.73
				Fructose	Fructose	0.340	2.62
Starch	amylum	0.258	4.10
				Glycerol	Glycerol	0.480	2.42
Lactose and lactose	lactose	0.342	2.74
				CK	CK	0.366	4.30

2) Optimal nitrogen source species screening results

The optimal nitrogen source screening results are shown in fig. 8 (corresponding data are shown in table 6), CK is a blank nitrogen source group, biocontrol strain LS8 is unavailable for removing ammonium sulfate, other nitrogen sources are available, and the average value of the absorbance values measured in the peptone+beef powder (1:1) group is the highest, but the absorbance values are not significantly different from those of the peptone group, the beef powder group and the peptone+yeast powder (1:1) group. Meanwhile, the average bacteriostasis rates of the peptone and beef powder (1:1) group and the peptone and yeast powder (1:1) group are high, but the difference between the peptone and beef powder groups is not obvious, the cost of raw materials is comprehensively considered, the optimal nitrogen source of the bio-control bacteria LS8 fermentation medium is selected by the peptone and beef powder (1:1), and the subsequent test is carried out.

TABLE 6 Effect of different Nitrogen sources on LS8 growth and bacteriostatic substances

Nitrogen source species	Nitrogen source species	OD ₆₀₀	Inhibition ratio (%)
				Peptone	peptone	1.030	7.56
Beef powder	beef paste	0.923	5.98
				Yeast powder	yeast extract	0.812	6.19
Peptone + beef powder (1:1)	peptone+beef paste(1:1)	1.061	11.38
				Peptone+yeast powder (1:1)	peptone+yeast extract(1:1)	0.998	11.35
Ammonium sulfate	(NH ₄ ) ₂ SO ₄	0.012	0.07
				Blank nitrogen source	CK	0.161	2.50

3) Optimal inorganic salt species screening results

The optimal inorganic salt screening result is shown in fig. 9 (corresponding data are shown in table 7), CK is a blank inorganic salt group, biocontrol bacteria LS8 can use the 4 inorganic salts, and the average value of absorbance values of the potassium dihydrogen phosphate group is the highest, but has no obvious difference with the sodium chloride group and the blank inorganic salt group. And the antibacterial rate of the monopotassium phosphate group and the potassium chloride group is obviously higher than that of other test groups. And selecting the monopotassium phosphate with the highest light absorption value and the best antibacterial effect as the most suitable inorganic salt of the biocontrol strain LS8 fermentation medium, and carrying out subsequent experiments.

TABLE 7 influence of different inorganic salts on LS8 growth and bacteriostatic substances

Inorganic salt species	Inorganic salt species	OD ₆₀₀	Inhibition ratio (%)
				Sodium chloride	NaCl	1.207167	7.749
Potassium chloride	KCl	1.127	12.25
				Monopotassium phosphate	KH ₂ PO ₄	1.219	14.784
Calcium chloride	CaCl ₂	0.585	4.507
				Blank space	CK	1.145	5.498

4) Optimal metal ion species screening results

The optimal metal ion screening results are shown in fig. 10 (corresponding data are shown in table 8), CK is a blank metal ion group, the biocontrol strain LS8 can utilize ferrous sulfate and magnesium sulfate, and the biocontrol strain LS8 cannot grow in a zinc sulfate culture medium with the concentration of 0.1%. The average value of absorbance values of the magnesium sulfate group was highest, and was significantly different from other test groups. There was no significant difference in the bacteriostatic rate between the magnesium sulfate group, ferrous sulfate group and the blank metal ion group. And selecting magnesium sulfate with the highest light absorption value and the best antibacterial effect as the most suitable metal ion of the biocontrol strain LS8 fermentation medium, and carrying out subsequent experiments.

TABLE 8 influence of different metal ions on LS8 growth and bacteriostatic substances

Metal ion species	OD ₆₀₀	Inhibition ratio (%)
			MgSO ₄	0.994	9.391
FeSO ₄	0.811	11.773
			ZnSO ₄	0.010	2.354
CK	0.892	9.941

2.2 Medium ratio optimization results

1) Screening result of optimum addition amount of carbon source

The results of screening the optimum amount of carbon source added are shown in fig. 11 (corresponding data are shown in table 9), the average value of absorbance values is highest and significantly higher than other groups when 2% of sucrose is added to the medium, and the antibacterial rate is highest and significantly higher than other groups when 2% of sucrose is added to the medium, so that the optimum amount of carbon source added is selected to be 2%.

TABLE 9 influence of the carbon source addition on LS8 growth and bacteriostatic substances

2) Screening result of optimum addition amount of nitrogen source

The screening results of the optimum addition amount of nitrogen source are shown in fig. 12 (corresponding data are shown in table 10), the average value of absorbance values is highest when 2% of peptone and beef powder (1:1) are added in the culture medium and is obviously higher than that of other groups, the bacteriostasis rate is highest when 2.5% of peptone and beef powder (1:1) are added in the culture medium, but the difference from the groups with the addition amount of 1.5%, 2% and 3% is not obvious, and the material cost is comprehensively considered, so that the optimum addition amount with 2% of nitrogen source is selected.

TABLE 10 influence of the nitrogen source addition on LS8 growth and bacteriostatic substances

Nitrogen source addition amount	OD ₆₀₀	Inhibition ratio (%)
			0％	0.178	1.359
1％	0.552	5.100
			1.5％	1.014	6.106
2％	1.207	8.244
			2.5％	1.130	8.932
3％	1.178	6.366

3) Screening result of optimum addition amount of phosphate

The screening results of the optimum addition amount of phosphate are shown in fig. 13 (corresponding data are shown in table 11), the average value of absorbance values is highest when 0.125% of monopotassium phosphate is added to the culture medium and is significantly higher than that of other groups, and the bacteriostasis rate is highest when 0.125% of monopotassium phosphate is added to the culture medium and is significantly different from that of other groups, so that the optimum addition amount of inorganic salt is selected to be 0.125%.

TABLE 11 influence of the phosphate addition on LS8 growth and bacteriostatic substances

Phosphate addition amount	OD ₆₀₀	Inhibition ratio (%)
			0％	0.718	3.084
0.125％	1.110	8.621
			0.25％	0.849	4.523
0.5％	0.875	2.937
			0.75％	0.434	1.559
1％	0.418	1.363

4) Screening result of optimum adding amount of magnesium ions

The screening results of the optimum amount of magnesium ions are shown in fig. 14 (corresponding data are shown in table 12), and when 0.1% of magnesium sulfate is added to the culture medium, the average value of absorbance values is highest and is remarkably higher than that of other groups, and when 0.1% of magnesium sulfate is added, the average value of bacteriostasis rates of the magnesium sulfate is highest, but there is no remarkable difference from the test group to which each concentration of magnesium sulfate is added, so that the optimum amount of 0.1% of magnesium sulfate is selected as the metal ions.

TABLE 12 influence of magnesium ion addition on LS8 growth and bacteriostatic substances

Magnesium ion addition amount	OD ₆₀₀	Inhibition ratio (%)
			0％	1.602	7.03
0.025％	1.851	9.64
			0.05％	2.057	10.13
0.1％	2.252	13.99
			0.15％	1.980	8.90
0.2％	1.914	8.85

5) Culture medium optimum pH value screening result

The optimal pH value optimization result of the culture medium is shown in FIG. 15 (corresponding data are shown in Table 13), and the biocontrol strain LS8 can grow in the pH value range of 4-10. But at pH values of 5 and 6, the absorbance was significantly lower than in the other groups. When the pH value is 5 and 6, the bacteriostasis rate is obviously higher than that of other groups, and the biological control bacteria LS8 are presumed to be cracked under the culture condition of peracid or alkali, so that the absorbance value of the culture solution is higher, and the pH value of the culture medium is gradually reduced due to fermentation culture, so that the pH value of the culture medium is 6-7 and is used as the pH value of the culture medium of the biological control bacteria LS 8. And (3) preparing a culture medium according to the optimized result formula, and determining that the pH value in a natural state is 6.1, wherein the pH value meets the pH value optimized result of 6-7.

TABLE 13 influence of different pH values on LS8 growth and bacteriostatic substances

pH	OD ₆₀₀	Inhibition ratio (%)
			pH 4	1.104	4.004
pH 5	0.955	14.802
			pH 6	0.968	14.654
pH 7	1.140	8.881
			pH 8	1.140	5.599
pH 9	1.108	6.269
			pH 10	1.189	5.730

2.3 optimization of culture conditions

1) Optimizing results for optimal inoculum size

The optimum inoculum size of the culture medium is optimized as shown in FIG. 16 (corresponding data are shown in Table 14). At 6h inoculation, the absorbance values of the fermentation broths with inoculum concentration of 2% and 5% are significantly higher than those of the other inoculum concentration test groups, and at 24h inoculation, the average value of the absorbance values of the fermentation broths with inoculum concentration of 0.1% is highest in each group, but there is no significant difference from 1%, 2% and 5% groups, and 2% is selected as the optimal inoculum concentration for preventing the contamination of mixed bacteria in the initial period of inoculation and considering the material cost.

TABLE 14 Effect of different inoculum sizes on LS8 growth

2) Optimum liquid loading amount optimizing result

The optimum amount of the culture medium was optimized as shown in FIG. 17 (corresponding data are shown in Table 15). At 6h inoculation, the average value of absorbance values of the fermentation broth with the liquid loading of 50mL was highest, and the absorbance values were significantly different from those with the liquid loading of 100mL, 125mL and 150mL, but not significantly different from that with the liquid loading of 75 mL. At 24h inoculation, the average value of absorbance values of the fermentation broth with the liquid loading of 100mL was the highest for each group, and there was a significant difference from the other groups, so 100mL was selected as the optimal liquid loading.

TABLE 15 influence of different liquid loadings on LS8 growth

3) Optimum rotation speed optimization result of shaking table

The optimum rotational speed of the culture medium is optimized as shown in FIG. 18 (corresponding data are shown in Table 16). At 6h of inoculation, absorbance averages were highest at 135rpm, but not significantly different from 150rpm and 165 rpm. At 24h of inoculation, the average value of absorbance values of the fermentation broth at 150rpm was the highest for each group and had a significant difference from the other groups, so 150rpm was selected as the optimal culture speed.

TABLE 16 influence of different rotational speeds on LS8 growth

4) Optimum fermentation temperature test results

The optimum fermentation temperature optimization results of the culture medium are shown in FIG. 19 (corresponding data are shown in Table 17). After 2h incubation at each temperature, the absorbance averaged highest at 28 ℃, but was not significantly different from the absorbance at 34 ℃. At 28 ℃, the average value of the bacteriostasis rate of the fermentation broth is highest, but the difference between the bacteriostasis rate of the fermentation broth and the bacteriostasis rate of the fermentation broth is not obvious from 26 ℃ and 30 ℃, so that the optimal culture temperature of 28 ℃ is selected.

TABLE 17 Effect of different temperatures on LS8 growth and bacteriostatic substances

Temperature (temperature)	OD ₆₀₀	Bacteriostasis rate (%)
			24℃	1.320	6.25
26℃	1.767	7.63
			28℃	1.954	10.59
30℃	1.639	8.50
			32℃	1.785	5.92
34℃	1.881	6.34

2.4 fermentation Condition response surface results and analysis

The curve surface diagram of the response surface of the graph 20 shows that the interaction of the fermentation temperature and the liquid loading amount has obvious influence on the antibacterial activity of the sterile fermentation liquid, the diameter of the antibacterial ring and the absorbance value of the bacterial liquid are in a trend of increasing when the liquid loading amount is changed from low to high along with the change of the temperature from low to high, but the difference between the temperature of 28 ℃ and the temperature of 32 ℃ is not obvious; from the curve surface diagram of the response surface of FIG. 20, the influence of the interaction of the fermentation temperature and the rotation speed of the shaking table on the antibacterial activity of the sterile fermentation liquid is obvious, the diameter of the inhibition zone and the absorbance value of the bacteria liquid are in a trend of increasing when the rotation speed of the shaking table is changed from low to high along with the change of the temperature from low to high, but the difference between the temperature of 28 ℃ and the temperature of 32 ℃ is not obvious; from the curve surface diagram of the response surface of FIG. 20, the interaction of the liquid loading amount and the rotation speed of the shaking table has obvious influence on the antibacterial activity of the sterile fermentation liquid, and the diameter of the antibacterial ring and the absorbance value of the bacterial liquid tend to increase when the rotation speed of the shaking table changes from low to high along with the high liquid loading amount.

3 test knots

The optimal culture medium formula of the birth control bacteria LS8 obtained by the test is as follows: 20g of sucrose, 10g of beef extract, 10g of peptone, 1.25g of monopotassium phosphate and 1g of magnesium sulfate, and deionized water is added to 1000mL, so that the pH is natural. The optimal culture conditions are as follows: the inoculation amount was 2%, the liquid loading amount was 100mL/250mL, the fermentation speed was 150rpm, the fermentation temperature was 28℃and the fermentation time was 2d.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. Bacillus bailii (Bacillus velezensis) is characterized by having a preservation number of CGMCC No.24563.

2. The bacillus belgium (Bacillus velezensis) of claim 1 for use in:

(II) inhibiting plant pathogenic bacteria; and/or

(III) preventing and treating plant soil-borne diseases; and/or

(IV) preparing cellulase, beta-1, 3-glucanase and/or protease; and/or

(VI) preparing a product that inhibits plant pathogenic bacteria; and/or

the product comprises a biocontrol agent.

3. The use according to claim 2, wherein the phytopathogenic bacteria comprise one or more of fusarium fungi, alternaria fungi (Alteraria sp.), gibberella fungi (neocomospora sp.), gliocladiopsis fungi (Gliocladiopsis sp.), trichoderma sp.), nigrospora fungi (Nigrospora sp.), epicoccum fungi (Epicoccum sp.) or Colletotrichum sp.

4. The use according to claim 3, wherein the Fusarium fungus comprises one or more of Fusarium oxysporum lotus specialization (Fusarium oxysporum f.sp.neumbicola), fusarium putrescence (Fusarium solani), fusarium oxysporum flax specialization (Fusarium oxysporum f.sp.lini) or Fusarium oxysporum lily specialization (Fusarium oxysporum f.sp.ilii).

5. The use of claim 2, wherein the plant soil-borne disease comprises one or more of lotus root rot, lotus root black spot, yam black skin, flax wilt, lily root rot, camphorwood disease, camphorwood mildew, or lonicera confusa disease.

6. The method for fermenting bacillus beleiensis (Bacillus velezensis) according to claim 1, comprising inoculating bacillus beleiensis (Bacillus velezensis) according to claim 1 to a culture medium and fermenting to obtain a fermentation broth of bacillus beleiensis (Bacillus velezensis);

The volume of the medium comprises 100mL; and/or

The pH value of the culture medium is 6-7; and/or

The temperature of the fermentation comprises 28 ℃; and/or

The shaking speed of the fermentation included 150rpm.

7. The preparation method of the sterile fermentation broth is characterized by comprising the following steps: inoculating bacillus belicus (Bacillus velezensis) according to claim 1 into a culture medium, fermenting to obtain a bacterial liquid, and filtering the bacterial liquid to obtain the sterile fermentation liquid.

8. A biocontrol microbial agent comprising bacillus beljalis (Bacillus velezensis) according to claim 1 and acceptable adjuvants or auxiliaries.

9. The method for preparing the biocontrol microbial agent according to claim 8, which is characterized by comprising the steps of inoculating bacillus beljalis (Bacillus velezensis) according to claim 1 to a culture medium, shake-culturing for 2-4 d at 25-30 ℃, and diluting the separated microbial cells with water to obtain the biocontrol microbial agent;

The pH value of the culture medium is 7.2-7.4; and/or

The rotational speed of the oscillation comprises 180r/min.

10. A method for producing cellulase, beta-1, 3-glucanase and/or protease, comprising culturing bacillus belgium (Bacillus velezensis) according to claim 1, and then isolating and obtaining the cellulase, the beta-1, 3-glucanase and/or the protease.