Lotus root endophyte Bacillus Velez strain LS8 and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to a lotus root endophyte Bacillus Velez subtilis strain LS8 and its application. The present invention provides a lotus root endophyte Bacillus Velez subtilis LS8, which is deposited in the General Microbiology Center of the China Microorganism Culture Collection Committee with a deposit number of CGMCC No. 24563. The lotus root endophyte Bacillus Velez subtilis LS8 and its biocontrol agent have the characteristics of a wide range of antifungal effects and significant antagonistic effects, and can be used to prevent and control plant fungal diseases, thus having good industrial application prospects. At the same time, the lotus root endophyte Bacillus Velez subtilis strain LS8 provided by the present invention has a significant preventive effect in potted plant experiments and has great practical value for the prevention and control of crop diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a lotus root endophyte Bacillus velezensis strain LS8 and an application thereof. Background Art

[0002] Lotus root rot is caused by Fusarium oxysporum, a species of Fusarium spp. This soil-borne disease has an optimum temperature of 25-35°C and an optimal pH of around 7.2. At 28°C, the pathogen grows radially on PSA culture medium. Initially, the hyphae are white, but darken to purple-red or deep purple in later stages. Two types of conidia are produced under both natural and artificial conditions. Large conidia are sickle-shaped, tapering at both ends, slightly curved, colorless and transparent, and often have three septa. Soil-borne diseases are caused by pathogens such as fungi, bacteria, nematodes, and viruses that infect crops through the roots or stems when conditions are favorable. These diseases include damping-off, sheath blight, bacterial wilt, wilt, blight, damping-off, root rot, soft rot, root-knot nematodes, and cyst nematodes. These diseases typically infect the roots or stems of plants, causing disease in the roots and stems, or even the entire plant, resulting in significant economic losses.

[0003] Currently, soil-borne diseases are primarily controlled through the use of chemical agents. However, their long-term use can lead to a series of serious consequences, including water and soil pollution, disruption of ecological balance, pesticide residues, rampant secondary diseases, and the development of drug resistance in pathogenic microorganisms. With increasing environmental awareness, increased emphasis on food safety, and the need for ecological and environmental conservation, biodiversity protection, and sustainable agricultural development, biological control of soil-borne diseases has become a hot topic in current research and development.

[0004] Bacillus sp. is a type of spore-forming Gram-positive bacteria that lives aerobically or facultatively anaerobically and can produce endospores that are resistant to heat, drought, UV rays, and organic solvents. It is an important biological control resource. Its control mechanism is mainly: by colonizing Bacillus sp. on the roots, surface, or body of plants, it competes with pathogens for nutrients around the plants and secretes antimicrobial substances to inhibit the growth of pathogens. At the same time, it induces the plant defense system to resist the invasion of pathogens, thereby achieving the purpose of biological control. At present, some excellent Bacillus have been isolated and successfully used in the biological control of plant diseases. For example, there is a Bacillus subtilis and its fungicide, which have a good effect in preventing and controlling banana wilt; another example is a soil Bacillus that has a good effect in inhibiting soybean root rot; and another example is a Bacillus subtilis and its fungicide that have a good effect in treating peanut fruit rot. Although Bacillus biocontrol agents already exist, there are still defects such as a small number of biocontrol bacteria, a single antibacterial effect, and instability. Summary of the Invention

[0005] In view of this, the lotus root endophyte Bacillus Velez subtilis strain LS8 and its application provided by the present invention have a wide antifungal range and significant antagonistic effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides Bacillus velezensis, whose deposit number is CGMCC No.24563.

[0008] The present invention also provides the use of the above-mentioned Bacillus velezensis in the following aspects:

[0009] (I), hydrolyzing β-1,3-glycosidic bonds, cellulose, protein and / or cell walls in β-1,3-glucans; and / or

[0010] (II), inhibiting plant pathogens; and / or

[0011] (III) Control of soil-borne plant diseases; and / or

[0012] (IV), preparing cellulase, β-1,3-glucanase and / or protease; and / or

[0013] (V), preparing a product for hydrolyzing β-1,3-glycosidic bonds, cellulose, protein and / or cell walls in β-1,3-glucans; and / or

[0014] (VI), preparing products that inhibit plant pathogens; and / or

[0015] (VII) preparing products for preventing and treating soil-borne plant diseases;

[0016] The products include biocontrol agents.

[0017] In some specific embodiments of the present invention, the plant pathogens used above include one or more of Fusarium, Alteraria sp., Neocosmospora sp., Gliocladiopsis sp., Trichoderma sp., Nigrospora sp., Epicoccum sp. or Colletotrichum sp.

[0018] In some specific embodiments of the present invention, the Fusarium fungus used above includes one or more of Fusarium oxysporum f.sp.nelumbicola, Fusarium solani, Fusarium oxysporum f.sp.lini or Fusarium oxysporum f.sp.lilii.

[0019] In some specific embodiments of the present invention, the soil-borne plant diseases used above include one or more of lotus root rot, lotus root black spot, yam black skin disease, flax wilt, lily wilt, lily root rot, camphor wood disease, camphor wood trichoderma disease or honeysuckle disease.

[0020] The present invention also provides a fermentation method of the above-mentioned Bacillus velezensis, comprising inoculating the above-mentioned Bacillus velezensis into a culture medium, fermenting, and obtaining a fermentation liquid of the Bacillus velezensis;

[0021] The inoculation includes a viable count of 10 7 ~10 8 CFU / mL of the Bacillus velezensis stock solution is inoculated into the culture medium at an inoculum amount of 2% (v / v); and / or

[0022] The volume of the culture medium comprises 100 mL; and / or

[0023] The formula of the culture medium comprises: 20 g / L sucrose, 10 g / L beef extract, 10 g / L peptone, 1.25 g / L potassium dihydrogen phosphate and 1 g / L magnesium sulfate; and / or

[0024] The pH value of the culture medium is 6 to 7; and / or

[0025] The fermentation temperature includes 28°C; and / or

[0026] The shaking speed of the fermentation is 150 rpm.

[0027] In some specific embodiments of the present invention, the carbon source concentration in the culture medium in the above fermentation method is 2-3%.

[0028] In some specific embodiments of the present invention, the carbon source concentration in the culture medium in the above fermentation method is 2%.

[0029] In some specific embodiments of the present invention, the nitrogen source concentration in the culture medium in the above fermentation method is 1.5-3%.

[0030] In some specific embodiments of the present invention, the nitrogen source concentration in the culture medium in the above fermentation method is 2%.

[0031] In some specific embodiments of the present invention, the concentration of inorganic salts in the culture medium in the above fermentation method is 0.1-0.15%.

[0032] In some specific embodiments of the present invention, the concentration of inorganic salts in the culture medium in the above fermentation method is 0.125%.

[0033] In some specific embodiments of the present invention, the concentration of metal ions in the culture medium in the above fermentation method is 0.0025-0.1%.

[0034] In some specific embodiments of the present invention, the metal ion concentration in the culture medium in the above fermentation method is 0.1%.

[0035] In some specific embodiments of the present invention, the pH value of the culture medium in the above fermentation method is 5-7.

[0036] In some specific embodiments of the present invention, the pH value of the culture medium in the above fermentation method is 6-7%.

[0037] In some specific embodiments of the present invention, the carbon source of the culture medium in the above fermentation method includes one or more of glucose, sucrose, fructose, lactose, glycerol or soluble starch.

[0038] In some specific embodiments of the present invention, the carbon source of the culture medium in the above fermentation method is sucrose.

[0039] In some specific embodiments of the present invention, the nitrogen source of the culture medium in the above-mentioned fermentation method includes one or more of trypsin, beef powder, yeast extract powder, peptone + beef powder (1:1), peptone + yeast extract powder (1:1) or ammonium sulfate.

[0040] In some specific embodiments of the present invention, the nitrogen source of the culture medium in the above fermentation method is peptone + beef powder (1:1).

[0041] In some specific embodiments of the present invention, the inorganic salts contained in the culture medium in the above fermentation method include one or more of sodium chloride, calcium chloride or potassium dihydrogen phosphate.

[0042] In some specific embodiments of the present invention, the inorganic salt contained in the culture medium in the above fermentation method is potassium dihydrogen phosphate.

[0043] In some specific embodiments of the present invention, the metal ions contained in the culture medium in the above fermentation method include one or more of magnesium ions, divalent iron ions or zinc ions.

[0044] In some specific embodiments of the present invention, the culture medium in the above fermentation method contains magnesium ions.

[0045] In some specific embodiments of the present invention, the culture medium in the above fermentation method includes one or more of magnesium sulfate, ferrous sulfate or zinc sulfate.

[0046] In some specific embodiments of the present invention, the culture medium in the above fermentation method contains magnesium sulfate.

[0047] In some specific embodiments of the present invention, the inoculation amount of the inoculation in the above fermentation method is 0.1-2%.

[0048] In some specific embodiments of the present invention, the inoculation amount of the inoculation in the above fermentation method is 2%.

[0049] In some specific embodiments of the present invention, the fermentation volume in the above fermentation method is 75-150 mL.

[0050] In some specific embodiments of the present invention, the fermentation volume in the above fermentation method is 100 mL.

[0051] In some specific embodiments of the present invention, the fermentation rotation speed in the above fermentation method is 120-150 rpm.

[0052] In some specific embodiments of the present invention, the fermentation rotation speed in the above fermentation method is 150 rpm.

[0053] In some specific embodiments of the present invention, the fermentation temperature in the above fermentation method is 26-34°C.

[0054] In some specific embodiments of the present invention, the fermentation temperature in the above fermentation method is 28°C.

[0055] The present invention also provides a sterile fermentation liquid, and the preparation method thereof comprises: inoculating the above-mentioned Bacillus velezensis into a culture medium, fermenting to obtain a bacterial liquid, and filtering the bacterial liquid to obtain the sterile fermentation liquid.

[0056] The present invention also provides a biocontrol agent, comprising the above-mentioned Bacillus velezensis and acceptable adjuvants or auxiliary agents.

[0057] The present invention also provides a method for preparing the biocontrol agent, comprising inoculating the Bacillus velezensis into a culture medium, culturing with shaking at 25-30° C. for 2-4 days, and diluting the separated bacteria with water to obtain the biocontrol agent.

[0058] The culture medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 20 g / L sucrose and 5 g / L NaCl; and / or

[0059] The pH value of the culture medium is 7.2 to 7.4; and / or

[0060] The rotation speed of the oscillation is 180 r / min.

[0061] The present invention also provides a method for preparing cellulase, β-1,3-glucanase and / or protease, comprising culturing the above-mentioned Bacillus velezensis, and then isolating and obtaining the cellulase, the β-1,3-glucanase and / or the protease.

[0062] The strain and application of the present invention have the following effects:

[0063] The present invention provides a lotus root endophyte Bacillus Velez LS8 (deposited in the General Microbiology Center of the China National Committee for the Collection of Microorganisms, with a deposit number of CGMCC No. 24563) and a biocontrol agent thereof. Bacillus Velez LS8 is a Gram-positive bacterium that can produce spores and can secrete cellulase, β-1,3-glucanase and protease. The secreted β-1,3-glucanase can hydrolyze β-1,3-glucan components in the cell walls of plant fungi, and carboxymethyl cellulase can act on the fungal cell walls in conjunction with proteinase to decompose its cellulose and protein, thereby inhibiting the growth and proliferation of plant pathogenic fungi. Therefore, the LS8 of the present invention has the characteristics of a wide range of antifungal effects and significant antagonistic effects, and has good prospects for industrial application. At the same time, the lotus root endophyte Bacillus Velez LS8 provided by the present invention has a significant preventive effect in potted plant experiments and has great practical value for the prevention and control of crop diseases.

[0064] Biological Deposit Description

[0065] Biological material: LS-8, classification name: Bacillus velezensis, deposited on March 22, 2022, at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 24563.

[0066] The LS8 described in the present invention is the strain with the above-mentioned deposit number of CGMCC No.24563. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0068] Figure 1 1 shows the morphology of Bacillus velez LS8 prepared in Example 1;

[0069] Figure 2 The physiological and biochemical identification results of Bacillus velezensis LS8 prepared in Example 1 are shown; wherein A shows spore staining; B shows Gram staining; C shows catalase; D shows starch-dissolving ability; E shows indoleacetic acid IAA; F shows ammonia production ability; G shows phosphate-dissolving ability;

[0070] Figure 3 The phylogenetic tree diagram constructed based on the 16S rDNA sequence of Bacillus velezinis LS8 provided in Example 1 is shown;

[0071] Figure 4 Figures showing the inhibitory effects of Bacillus velezensis LS8 on different pathogens in Example 2; wherein, A shows black spot disease of lotus root; B shows black skin disease of yam; C shows flax wilt; D shows lily wilt; E shows lily root rot; F shows camphor wood disease; G shows camphor wood trichoderma; H shows honeysuckle disease 1; I shows honeysuckle disease 4; J shows honeysuckle disease 6; K shows lotus root rot;

[0072] Figure 5 Schematic diagram showing the experiment of controlling lotus root rot disease with Bacillus Velez LS8 in Example 3;

[0073] Figure 6 Schematic diagram showing the field experiment of controlling lotus root rot disease with Bacillus Velez LS8 in Example 4;

[0074] Figure 7 Shows the optimal carbon source screening results;

[0075] Figure 8 Shows the results of screening for the optimal nitrogen source;

[0076] Figure 9Shows the optimal inorganic salt screening results;

[0077] Figure 10 Shows the optimal metal ion screening results;

[0078] Figure 11 Shows the screening results of the optimal amount of carbon source added;

[0079] Figure 12 Shows the screening results of the optimal amount of nitrogen source;

[0080] Figure 13 Shows the screening results of the optimal amount of phosphate added;

[0081] Figure 14 Shows the screening results of the optimal addition amount of magnesium ions;

[0082] Figure 15 Shows the optimization results of the optimal pH value of the culture medium;

[0083] Figure 16 The results of optimization of the optimal inoculum size of the culture medium are shown;

[0084] Figure 17 Shows the optimization results of the most suitable volume of culture medium;

[0085] Figure 18 Shows the optimization results of the optimal rotation speed of the culture medium;

[0086] Figure 19 Shows the optimization results of the optimal fermentation temperature of the culture medium;

[0087] Figure 20A Figure 2 shows the results of the culture medium response surface optimization. The upper figure shows the effect of the interaction between fermentation temperature and liquid volume on the absorbance value, and the lower figure shows the effect of the interaction between fermentation temperature and liquid volume on the inhibition rate.

[0088] Figure 20B Figure 2 shows the results of the culture medium response surface optimization; the upper graph shows the effect of the interaction between fermentation temperature and fermentation speed on the absorbance value, and the lower graph shows the effect of the interaction between fermentation temperature and fermentation speed on the inhibition rate;

[0089] Figure 20C Figure 3 shows the results of the culture medium response surface optimization; the upper figure shows the effect of the interaction between the liquid filling volume and the fermentation speed on the absorbance value, and the lower figure shows the effect of the interaction between the liquid filling volume and the fermentation speed on the inhibition rate. DETAILED DESCRIPTION

[0090] The present invention discloses the lotus root endophyte Bacillus velezensis strain LS8 and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0091] The present invention aims to provide a lotus root endophyte Bacillus Velez LS8 and its application. The strain and its bacterial agent have broad-spectrum antibacterial properties, and the antibacterial effect is efficient and stable, and can effectively prevent and control soil-borne fungal diseases of plants.

[0092] The invention provides a lotus root endophyte Bacillus velezensis LS8, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 24563.

[0093] Preferably, the Bacillus Velezii LS8 is a Gram-positive bacterium that can produce spores and secrete cellulase, β-1,3-glucanase and protease.

[0094] Preferably, the colony edge of the Bacillus Velez LS8 is relatively neat, the surface is convex and moist, and the color is milky white and opaque.

[0095] Preferably, the Bacillus Velez LS8 can be used to prevent and control soil-borne fungal diseases of plants.

[0096] Preferably, the plant fungal diseases are lotus root rot, lotus root black spot, yam black skin disease, flax wilt, lily wilt, lily root rot, camphor wood disease and honeysuckle disease.

[0097] Preferably, the preparation method of the biocontrol agent containing Bacillus Velez LS8 comprises the following steps: inoculating Bacillus Velez LS8 into a sterilized liquid culture medium, culturing at 25-30°C, shaking on a shaker for 2-4 days, centrifuging at 12000 r / min for 5 minutes, and then diluting with sterile water to a concentration of 10 5 CFU / mL of the suspension is a biocontrol agent containing Bacillus Velez LS8.

[0098] Preferably, the liquid culture medium consists of 10 g / L tryptone, 5 g / L yeast extract, 20 g / L sucrose, and 5 g / L NaCl; and the pH value of the liquid culture medium is 7.2-7.4.

[0099] Preferably, the shaking table has an oscillation speed of 180 r / min.

[0100] Preferably, the biocontrol agent containing Bacillus Velezii LS8 is used in preventing and treating lotus root rot disease.

[0101] The Bacillus velez strain provided in the embodiments of the present invention enables the Bacillus velez and the biocontrol agent provided by the present invention to effectively prevent and control soil-borne fungal diseases of plants.

[0102] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0103] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention and to make the above-mentioned purposes, features and advantages of the embodiments of the present invention more obvious and easy to understand, the technical solutions in the embodiments of the present invention are further described in detail below with reference to the accompanying drawings:

[0104] Example 1: Preparation and identification of lotus root endophyte Bacillus velezensis LS8

[0105] 1. Preparation of Lotus Root Endophyte Bacillus Velez LS8

[0106] S01: Lotus roots were collected by multi-point sampling, washed with clean water, sterilized with 1% mercuric chloride solution for 5 minutes, rinsed three times with sterile water, sterilized with 70% ethanol for 30 seconds, rinsed three times with sterile water, and ground into powder with a mortar in a clean bench. The lotus roots in the embodiment of the present invention were taken from the seed lotus planting field in Hongzhong Village, Huashi Town, Xiangtan County;

[0107] S02: Place the ground lotus root sample into a centrifuge tube filled with sterile water and shake thoroughly to make a concentration of 10 -1 Lotus root suspension;

[0108] S03: diluting the lotus root suspension step by step to obtain lotus root suspensions of different concentrations;

[0109] S04: Select a concentration of 10 -3 , 10 -4 and 10 -5 Three gradients of soil suspension were added to NA medium plates for coating treatment and placed in an incubator at 30°C for 48 hours to obtain colonies;

[0110] S05: Select single colonies of different morphologies and streak them on a slant, and culture them for 24 hours to obtain isolated bacteria, which are then stored in a refrigerator at 4°C for future use;

[0111] S06: Use a 0.5 cm diameter puncher to inoculate the pathogenic bacteria such as yam rot and lily rot into the center of the PDA culture medium plate, and inoculate the isolated bacteria at an equal distance at the cross intersection 2.5 cm away from the center of the plate. At the same time, the plate without isolated bacteria was used as the control (CK). The experiment was repeated 3 times for each treatment and cultured at a temperature of 25°C. When the control group (CK) covered all the vessels, the size of the inhibition zone was recorded, and the one with the largest inhibition zone was selected as Bacillus Velezii LS8.

[0112] NA medium is a nutrient agar medium composed of 20 g / L agar, 10 g / L tryptone, 3 g / L beef extract, and 5 g / L NaCl. The pH of NA medium is 7.2-7.4 during preparation and sterilized for 20 minutes at 121°C. PDA medium is a potato dextrose agar medium.

[0113] 2. Identification of the lotus root endophyte Bacillus velezensis LS8:

[0114] 1. Morphological identification

[0115] The strain prepared in this example was streaked onto a NA medium plate, and then the plate was inverted and cultured at 30°C for 24 hours. The growth of the colonies on the plate was observed and recorded. The colony morphology on the plate was as follows: Figure 1 As shown in the figure, it can be seen that the colony edges of the strain prepared in this example are relatively neat, the surface is convex and moist, and the color is milky white and opaque.

[0116] The strain prepared in this example was subjected to Gram staining and spore staining using a kit, and the strain was observed and photographed under an oil immersion microscope. The Gram staining and spore staining of the strain were as follows: Figure 2 As shown, from Figure 2 As can be seen from B in the figure, after Gram staining, Bacillus velezensis LS8 is rod-shaped and blue-purple, and is a Gram-positive bacterium; Figure 2 As can be seen from Figure A, after spore staining, the cells of Bacillus Velezii LS8 appear blue and the spores appear red, which indicates that the strain provided by the present invention can produce spores.

[0117] 2. Physiological and biochemical identification

[0118] (1) Catalase test

[0119] Add 3% hydrogen peroxide dropwise to the liquid culture of the strain prepared in this example and observe immediately. If a large number of bubbles are produced, the result is positive; if no bubbles are produced, the result is negative. If the strain provided by the present invention immediately produces a large number of bubbles, the test result is positive.

[0120] (2) Oxidase test

[0121] Take a corner of clean white filter paper, dip it into a small amount of the bacterial colony prepared in this example, and add a drop of 1% dimethyl-p-phenylenediamine hydrochloride aqueous solution. Positives will immediately turn pink, and the color will gradually deepen. In this experiment, the colony did not change color, and the test result was positive.

[0122] (3) Cellulose decomposition experiment

[0123] Take a single colony of the test strain and streak it on the cellulase activity identification medium, culture it at 30℃ for 5 days, stain it with 0.1% Congo red for 30 minutes, rinse it with distilled water, and then counterstain it with 1mol / L hydrochloric acid solution for 30 minutes and rinse it to observe whether there is a transparent circle. In this experiment, a transparent circle appeared around the colony, and the test result was positive.

[0124] (4) Methyl red MR experiment

[0125] A small amount of the strain prepared in this example was inoculated onto a universal culture medium and cultured at 30°C for 3-5 days. After the culture was complete, 1 mL of the culture medium was collected and 1-2 drops of methyl red indicator were added. A positive result was bright red, a weak positive result was light red, and a negative result was yellow. In this experiment, the bacterial solution turned yellow, indicating a negative result.

[0126] (5) VP experiment

[0127] A small amount of the strain prepared in this example was inoculated onto a universal culture medium and cultured at 30°C for 4 days. After the culture was complete, 2.5 mL of the culture medium was added to 0.6 mL of a pure α-naphthol alcohol solution, followed by 0.2 mL of a 40% aqueous potassium hydroxide solution. The culture was shaken for 2-5 minutes. Positive bacteria typically immediately develop a red color. If no red color develops, the culture was placed in an incubator at room temperature or 30°C. If no red color develops within 2 hours, the result is negative. In this experiment, the culture immediately turned red, indicating a positive result.

[0128] (6) Gelatin liquefaction experiment

[0129] Take the strain prepared in this example and inoculate it into gelatin, ensuring it reaches 2 / 3 of the gelatin's depth. Incubate at 20°C for 5-7 days. Observe daily to see if the strain is liquefied. If so, the test is positive; if not, the test is negative. In this experiment, the gelatin was liquefied, resulting in a positive reaction.

[0130] (7) Nitrate reduction experiment

[0131] The strain prepared in this example was inoculated into a nitrate medium and cultured on a shaker at 30°C for 3 days. Then, 5 mL of the culture was sampled and a color developer was added according to the instructions of the kit (Nitrate Reduction Kit from Haibo Biotechnology Co., Ltd.). A yellowing reaction indicated a positive result; conversely, no color change indicated a negative result. In this experiment, the yellowing of the culture indicated a positive reaction.

[0132] (8) Hydrogen sulfide production experiment

[0133] The strain prepared in this example was inoculated into a lead acetate medium and cultured at 35°C for 24-48 hours. The results were observed. If the medium turned black, it was positive; if it did not, it was negative. In this experiment, the medium turned black, indicating a positive reaction.

[0134] (9) Citrate utilization experiment

[0135] The strain prepared in this example was streaked onto a slant of Simmons' citrate medium and incubated at 37°C for 3-7 days. If the medium is alkaline, i.e., the indicator turns blue or pink, the reaction is positive; if the medium does not change color, the reaction is negative. In this experiment, the color change of the medium indicates a positive reaction.

[0136] (10) Lecithinase activity test

[0137] Disinfect the surface of a fresh egg with 75% ethanol. Use sterile tweezers to poke a hole in the egg, pour off the egg white, and then aspirate the yolk with a sterile pipette. Add the egg to thawed NA medium cooled to approximately 50°C. Mix thoroughly, then place the mixture on an inverted plate. Inoculate the strain prepared in this example with the strain. Incubate at 30°C for 24 hours and observe. A turbid zone appears at the edge of the colony, indicating enzyme-positive results. In this experiment, a clear turbid zone at the edge of the colony indicated a positive reaction.

[0138] (11) Malonate Utilization Experiment

[0139] A 12-hour culture lawn (the strain prepared in this example) was inoculated into a malonate medium and incubated at 35°C for 24-48 hours. A change from green to blue was considered positive, while a change from green to blue was negative. In this experiment, the absence of color change in the medium indicated a negative reaction.

[0140] (12) Glucose fermentation experiment

[0141] A small amount of the strain prepared in this example was inoculated into a glucose oxidation fermentation medium. Cultured at 30°C for 3 days, the medium was observed for color changes. If no color change was observed, the culture medium was observed for another 7 days. If the culture medium turned yellow, it was considered fermentation-type. In this experiment, yellowing indicated a positive reaction.

[0142] (13) Galactose utilization experiment

[0143] The strain prepared in this example was inoculated into a galactose medium and cultured at 30°C for 2 days. Colony growth was observed. If colonies formed, the strain could utilize galactose; otherwise, it could not. In this experiment, bacterial growth indicated that the strain could utilize galactose.

[0144] (14) Arabinose utilization experiment

[0145] The strain prepared in this example was inoculated into an arabinose medium and cultured at 30°C for 2 days. Colony growth was observed. If colonies formed, the strain could utilize arabinose; otherwise, it could not. In this experiment, the strain did not grow, indicating that it could not utilize arabinose.

[0146] (15) Mannose utilization experiment

[0147] The strain prepared in this example was inoculated into a mannose medium and cultured at 30°C for 2 days. The growth of colonies was observed. If colonies formed, the strain could utilize mannose; otherwise, it could not. In this experiment, the strain grew, indicating that the strain could utilize mannose.

[0148] (16) D-fructose utilization experiment

[0149] The strain prepared in this example was inoculated into a D-fructose medium and cultured at 30°C for 2 days. Colony growth was observed. If colonies formed, the strain could utilize D-fructose; otherwise, it could not. In this experiment, bacterial growth indicated that the strain could utilize D-fructose.

[0150] (17) D-xylose utilization experiment

[0151] The strain prepared in this example was inoculated into a D-xylose medium and cultured at 30°C for 2 days. The growth of colonies was observed. If colonies formed, the strain could utilize D-xylose; otherwise, it could not. In this experiment, the strain grew, indicating that the strain could utilize D-xylose.

[0152] In summary, the results of physiological and biochemical identification are shown in Table 1; some results are shown in Figure 2 As shown in C to G.

[0153] Table 1: Physiological and biochemical characteristics of Bacillus velez strain LS8

[0154] test result test result Ammonia production + hydrogen sulfide + IAA + Lecithinase + Cellulose degradation + Oxidase + Gelatin liquefaction + Catalase + Methyl red MR - fructose + VP determination + Mannose + Nitrate reduction + Galactose + Phosphate solubilization + arabinose - Citrate reaction + Xylose + Glucose fermentation + Malonic acid utilization -

[0155] Note: +: positive reaction; -: negative reaction.

[0156] 3. 16S rDNA sequence analysis

[0157] The strain DNA was prepared using the bacterial liquid PCR method, and the specific operation steps were as follows: 25 μL of PCR reaction system, and the PCR reaction system included 12.5 μL of 2× Master Mix; 1 μL of upstream primer 27F: 5'-AGA GTT TGA TCCTGG 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 μL of ddH2O; and 1.5 μL of template DNA.

[0158] The PCR reaction conditions are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds; annealing at 53°C for 30 seconds; extension at 72°C for 1 minute; 30 cycles, extension at 72C° for 5 minutes, and storage at 4°C. The obtained PCR product was subjected to liposome gel electrophoresis analysis. From the 16S sequencing results of the biocontrol bacteria LS8, it can be seen that the DNA fragment of the strain prepared in this embodiment is about 1450bp long. The obtained PCR product was sequenced by Hunan Qingke Biotechnology Co., Ltd., and the nucleotide sequence table of the strain prepared in this embodiment is shown in the sequence table SEQ ID NO: 3. The obtained nucleotide sequence was subjected to homology sequence comparison analysis by NCBI-BLAST to obtain sequences with higher similarity. The phylogenetic tree (such as Figure 3 The 16S rDNA sequence of the strain prepared in this example has a high homology with that of Bacillus velezensis. The 16S sequence of Bacillus velezensis LS8 is shown below:

[0159]

[0160]

[0161] Based on the above morphological observation, physiological and biochemical identification and 16S rDNA sequence analysis results, it can be determined that the strain prepared in this example is Bacillus velezensis, which is named Bacillus velezensis strain LS8.

[0162] Example 2: Evaluation of the antibacterial effect of the lotus root endophyte Bacillus velezensis strain LS8

[0163] 1. Evaluation of antibacterial effect

[0164] The inhibitory effect of Bacillus Velezii LS8 on lotus root rot, lotus root black spot, yam black skin, flax wilt, lily wilt, lily root rot, camphor wood disease and honeysuckle disease was studied as examples. The specific contents are as follows:

[0165] Lotus root rot, lotus root black spot, yam black skin, flax wilt, lily wilt, lily root rot, camphor wood disease, and honeysuckle disease were used as target bacteria. A small paper disc (4 mm in diameter) soaked in a solution of Bacillus Velezii strain LS8 was placed 2.5 cm from the center of the plate. A plate without LS8 bacteria was used as a control. Each treatment was repeated 3 times. The plate was placed upside down in a 25°C incubator. When the pathogenic fungi in the control group covered the plate, the diameter of the candidate antagonistic bacteria and the diameter of the inhibition zone of the bacteria against each pathogenic fungus were measured. The antagonism index was calculated based on the inhibition zone diameter:

[0166] Inhibition rate = (diameter of inhibition zone - diameter of antagonistic bacteria) / diameter of antagonistic bacteria.

[0167] Depend on Figure 4 It can be seen that Bacillus Velez LS8 has an inhibitory effect on all of the above-mentioned plant pathogenic fungi, indicating that Bacillus Velez LS8 has a broad-spectrum antibacterial activity; from Table 2, it can be seen that the antibacterial diameter of Bacillus Velez LS8 on the above-mentioned pathogenic fungi is between 15 and 32 mm, and from the antagonism index, it can be seen that it has a good antagonistic effect on the above-mentioned pathogenic fungi, indicating that Bacillus Velez LS8 has a high efficiency antibacterial effect on the above-mentioned plant pathogenic fungi and can be used to prevent and control soil-borne plant diseases caused by the above-mentioned plant pathogenic fungi.

[0168] Table 2: Antagonistic effect of LS8 on different plant pathogenic fungi

[0169] Pathogen type Diameter of inhibition zone (mm) Inhibition rate (%) Lotus root rot 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 Yam black skin disease (Fusarium) 17.18±1.23 30.10±2.15 Flax wilt (Fusarium) 11.52±1.11 31.30±3.01 Lily wilt (Fusarium) 7.86±1.15 27.42±4.01 Lily root rot (Neocosmospora) 4.49±0.75 18.35±3.07 Camphorwood Diseases (Gliocladiopsis) 2.60±1.53 15.84±9.33 Camphor wood trichoderma (Trichoderma spp.) 19.88±0.23 43.95±0.50 Honeysuckle Disease 1 (Nigrospora) 24.26±0.76 48.85±1.52 Honeysuckle Disease 4 (Epicoccum) 3.11±0.69 16.11±3.59 Honeysuckle Disease 6 (Colletotrichum) 25.99±0.55 55.69±1.19

[0170] 2 Study on antibacterial mechanism

[0171] The Bacillus velezensis LS8 prepared in Example 1 was spot-inoculated onto plates containing colloidal chitin medium, sodium carboxymethyl cellulose medium, Poria powder medium, and skim milk agar medium, respectively. The plates were inverted and cultured in a 30°C incubator for 3 days, and the formation of transparent zones around the colonies was observed.

[0172] Results showed that Bacillus velez LS8 produced clear zones on sodium carboxymethylcellulose, Poria cocos powder, and skim milk agar media, indicating that Bacillus velez LS8 secretes cellulase, β-1,3-glucanase, and protease. 80% of the dry weight of the fungal cell wall is composed of carbohydrates, such as chitin, chitosan, glucan, cellulose, and galactan. Approximately 10% is made up of proteins and glycoproteins. Proteins include enzymes responsible for cell wall growth, specific exoenzymes, and structural proteins that cross-link polysaccharides. β-1,3-glucanase secreted by Bacillus velez LS8 hydrolyzes β-1,3-glycosidic bonds within β-1,3-glucans in the fungal cell wall. Carboxymethyl cellulase, in conjunction with proteases, acts on the fungal cell wall to decompose cellulose and protein, thereby inhibiting the growth and proliferation of plant pathogens.

[0173] Example 3: Preliminary trial of controlling lotus root rot with lotus root endophyte Bacillus velezensis LS8

[0174] 1. Preparation of biocontrol agents containing Bacillus Velez LS8

[0175] The Bacillus velezensis strain LS8 prepared in Example 1 was inoculated into a sterilized NA liquid medium (i.e., beef extract peptone liquid medium, with a ratio of 10 g / L peptone, 3 g / L beef extract, and 5 g / L NaCl, pH = 7.2-7.4), and cultured on a shaking table at 30°C for 2 days at a shaking speed of 180 r / min, followed by centrifugation at 12000 r / min for 5 minutes. The cells were taken and diluted with sterile water to a concentration of 10 5 CFU / mL of the suspension contains Bacillus Velezii LS8.

[0176] 2. Preparation of the fungal solution of Fusarium oxysporum, the pathogen of lotus root rot disease

[0177] The lotus root rot pathogen Fusarium oxysporum was inoculated into sterilized PDA liquid medium (the composition of PDA medium was 200 g peeled potato, 20 g glucose, 1000 mL sterile water, pH value was 7.2-7.4), and cultured in a shaking table at 25 ° C for 7 days with a shaking speed of 180 r / min. The bacterial solution was then diluted to 10 with sterile water. 5 CFU / mL, and the suspension of Fusarium oxysporum, the pathogen of lotus root rot disease, was obtained.

[0178] 3. Application of biocontrol agents containing Bacillus Velez LS8 in inhibiting lotus root rot

[0179] (1) The control effect of biocontrol agents containing Bacillus velezensis LS8 on lotus root rot

[0180] Test variety: Fresh lotus root (market sales)

[0181] Pathogen: Lotus root rot disease Fusarium oxysporum pathogen

[0182] Experimental treatment: Control group: Pathogens and sterile water; Treatment group: Pathogens and biocontrol agents containing Bacillus Velez LS8

[0183] The lotus roots used in the grafting screening experiment were purchased from the market. The surface of the lotus roots was washed with tap water, and the lotus roots were cut into round blocks with a diameter of about 3 cm parallel to the lotus root holes. The lotus root slices were immersed in the bacterial solution of the biocontrol bacteria for 0.5 h, and the control group was immersed in ultrapure water for 0.5 h. After drying, they were placed in a 9 cm plate containing a piece of filter paper, and 3 pieces were repeated in each plate. Use a hole puncher to inoculate the activated Fusarium oxysporum mycelium block with a diameter of 4 mm into the center of the lotus root block. Each lotus root block was inoculated with one Fusarium oxysporum mycelium block. After inoculation, the lotus root block was placed in a constant temperature incubator at 25°C and cultured until the mycelium of the control group was about to cover the lotus root block. Measure the colony diameter of the control group and the colony diameter of the experimental group. Inhibition rate = (colony diameter of the control group - colony diameter of the experimental group) / (colony diameter of the control group - mycelium block diameter)

[0184] The results are as follows Figure 5 , as shown in Table 3.

[0185] Table 3: Inhibitory effects of Bacillus Velez LS8 suspension and other screened biocontrol bacteria on lotus root rot disease

[0186] Strain name Pathogen colony diameter / mm Inhibition zone diameter Inhibition rate / % 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

[0187] Example 4: Application of Lotus Root Endophyte Bacillus Velez LS8 in Field Control of Lotus Root Corruption

[0188] 1 Materials and Methods

[0189] 1.1 Test strains

[0190] The biocontrol strain Bacillus velezensis LS8 was isolated from lotus roots and deposited in the General Microbiology Center of the China General Culture Collection Administration (CGMCC No. 24563). The first-generation strain, activated from glycerol cryopreserved tubes, was selected. Fusarium oxysporum was provided by the Hunan Plant Protection Institute.

[0191] 1.2 Test plants

[0192] The test plant is lotus, the variety is Xiangtan Cunsanlian, which was harvested in 2021 from the original seed farm of the Institute of Hemp, Chinese Academy of Agricultural Sciences.

[0193] 1.3 Culture medium and cultivation site

[0194] NB medium and PDA medium.

[0195] This experiment was carried out at the original seed farm of the Institute of Hemp, Chinese Academy of Agricultural Sciences.

[0196] 1.4 Test methods

[0197] 1.4.1 Preparation of Xianglian Plants

[0198] Lotus seeds were stored in a refrigerator at 4°C. Small holes with a diameter of 2 mm were drilled at both ends of the seeds with an electric drill in advance. The seeds were germinated in a cultivation pot filled with 20 L of clean water at a cultivation temperature of 20°C to 25°C. The water was changed once every morning and evening to prevent bacterial contamination of the water body. After 3 days of cultivation, most of the lotus seeds absorbed water and expanded, and the lotus shells cracked. After 5 days of cultivation, most of the lotus seeds germinated and were transported to the field for cultivation.

[0199] 1.4.2 Preparation of lotus root pathogens and biocontrol agent LS8

[0200] The pathogenic fungus Fusarium oxysporum was activated on PDA plates, and the mycelium was picked up from several plates and placed in sterile water at a concentration of 2×10 8 ~6×10 8 CFU / mL of Fusarium oxysporum liquid, diluted to 10 6 spare.

[0201] LS8 biocontrol bacteria were cultured in optimized culture medium and the concentration of the bacterial solution was raised to 4×10 8 , diluted to 10 6 spare.

[0202] 1.4.3 Biocontrol bacteria and pathogen inoculation

[0203] 80 lotus seeds with similar growth were selected and divided into 4 groups: blank group, biocontrol bacteria group, pathogen group and biocontrol bacteria + pathogen group. The blank group was soaked in water without treatment, and the biocontrol bacteria group was soaked in 4×10 6 CFU / mL of LS8 biocontrol bacteria for 12 h, and the pathogen group was treated with a concentration of 2×10 6 CFU / mL of Fusarium oxysporum liquid was soaked for 3 hours. The biocontrol bacteria plus pathogen group was first treated with a bacterial concentration of 4×10 6 CFU / mL of LS8 biocontrol bacteria for 12 h and then washed with clean water. The concentration of bacteria was 2×10 6 CFU / mL of Fusarium oxysporum liquid for 3 hours.

[0204] 1.4.4 Field cultivation

[0205] A cultivation experiment was conducted at the original seed field experimental base of the Institute of Bast Immunity, Chinese Academy of Agricultural Sciences. The field was divided into four 2m x 4m grids, each approximately 20cm deep. The soil between the grids was covered with plastic film, and each grid was irrigated with clean water to a depth of approximately 10cm. The grids were divided into four groups: a blank control, a biocontrol bacteria control, a pathogen control, and a biocontrol bacteria plus pathogen control. Within each grid, 20 treated lotus seeds were evenly planted in parallel in three sections, each buried approximately 3cm deep in soil. The cultivation temperature was maintained at 15°C to 25°C. Fourteen days after planting, samples were collected to record disease activity and grade the disease.

[0206] 2 Results and Analysis

[0207] The results of the biocontrol bacteria LS8 lotus field control test are shown in Table 4. Figure 6 Because Xiangtan Cunsan lotus had been cultivated for several months prior to the field experiment, the water and soil contained a high concentration of the lotus root rot pathogen. Furthermore, the leaves tested in the field experiment were floating leaves from Xiangtan lotus cultivation, which are inherently less resistant to disease. This resulted in varying degrees of infection across the groups. The average disease index (DII) in the blank group was 21.40%, with pale red lesions visible on most lotus leaves. These lesions were evenly distributed throughout the leaves, with severe cases exhibiting overall discoloration. The DII in the biocontrol group was 8.47%, demonstrating a 60.42% efficacy compared to the blank group. Lesions were primarily located along the leaf margins, with no cases of entire lotus leaves being infected. The mature leaves were the most green of the four groups, indicating healthy growth. The DII in the pathogen group was 48.68%. After treatment with the pathogen solution, newly formed pink lesions were also observed on the curled new leaves emerging from the water. Some severely affected leaves were affected by the lesions, with some of the mesophyll curling or even breaking. The disease index of the biocontrol bacteria + pathogen group was 23.41%, compared to the pathogen group, which achieved a control efficacy of 51.91%. All leaves in this group were also infected with the pathogen, with red lesions observed on each leaf. However, compared to the pathogen group, the symptoms were milder. The lesions were evenly distributed across the leaves, without forming a single, connected entity that would cause large areas of mesophyll necrosis. In some severely affected leaves, only small holes appeared in the center of the lesions. This suggests that soaking lotus seeds in the biocontrol bacteria LS8 solution before cultivation is effective in preventing and controlling lotus root rot.

[0208] Table 4: Results of the biocontrol test of LS8 in lotus fields

[0209] experimental group Disease index / % Control effect% Blank group <![CDATA[21.40±1.81 b ]]> - Biocontrol bacteria group <![CDATA[8.47±1.83 c ]]> 60.42±4.25 Pathogen group <![CDATA[48.68±3.99 a ]]> - Biocontrol bacteria + pathogen group <![CDATA[23.41±3.33 b ]]> 51.91±3.42

[0210] Example 5: Optimization of Bacillus Velez LS8 culture medium and fermentation conditions

[0211] 1 Materials and Methods

[0212] 1.1 Test strains

[0213] The biocontrol strain Bacillus velezensis LS8 was isolated from lotus roots and deposited in the General Microbiology Center of the China General Culture Collection Administration (CGMCC No. 24563). The first-generation strain, activated from glycerol cryopreserved tubes, was selected. Fusarium oxysporum was provided by the Hunan Plant Protection Institute.

[0214] 1.2 Culture medium

[0215] NB medium (g / L): peptone 10.0, sodium chloride 5.0, beef powder 3.0, pH 7.0;

[0216] Fermentation basal medium (g / L): glucose 20.0, beef extract 10.0, peptone 10.0, NaCl 5.0, natural pH;

[0217] PDA medium: potato (peeled) 200.0, glucose 20.0, agar 15.0, pH 7.0;

[0218] 1.3 Main reagents and instruments

[0219] 1.4 Test methods

[0220] 1.4.1 Seed liquid culture

[0221] The biocontrol bacteria LS8 preserved in glycerol was streaked on the LB plate. After a single colony was formed, a single colony was scraped and inoculated into NB liquid culture medium. 100 mL of liquid was added to each 250 mL shake flask. After fermentation at 28°C and 150 rpm for 24 h, the culture medium was inoculated into the experimental shake flask.

[0222] 1.4.2 Preparation of sterile fermentation broth

[0223] The preparation method of sterile fermentation liquid is the same as above. Under sterile conditions, the bacterial liquid after fermentation is repeatedly mixed and then passed through a 0.22 μm filter membrane. The obtained liquid is the sterile fermentation liquid of biocontrol bacteria LS8.

[0224] 1.4.3 Fermentation result evaluation method

[0225] 1) Detect the biomass of the fermentation broth. Using blank culture medium as a control, measure the absorbance of the fermentation broth at a wavelength of 600 nm. Repeat each treatment three times.

[0226] 2) Inhibitory effect on Fusarium oxysporum: First, a Fusarium oxysporum block with a diameter of 4 mm was inoculated into the center of the PDA culture medium. After incubation at 25°C for 1 day, a hole with an inner diameter of 6 mm was punched at a distance of 2.5 cm from the block. 100 μl of sterile fermentation liquid was inoculated into three of the holes, and 100 μl of sterile water was inoculated into one hole as a control. The culture dish was sealed and incubated at 25°C for another 3 days until the control mycelium approached the hole. The colony radius was measured to calculate the inhibition rate. Three plates were repeated for each treatment.

[0227] 1.4.4 Single-factor experiment of culture medium components

[0228] 1) Screening of the optimal carbon source

[0229] The carbon source in the basal culture medium was replaced with the following: glucose, sucrose, fructose, lactose, glycerol, and soluble starch. A blank control group was also established with no carbon source added. Two mL of activated LS8 bacterial suspension was inoculated per 100 mL of culture medium and cultured at 28°C and 150 rpm for two days. Each treatment was replicated three times.

[0230] 2) Screening of the optimal nitrogen source

[0231] Select the optimal carbon source from the screening and replace the nitrogen source in the basal culture medium with the following: tryptone, beef powder, yeast extract powder, peptone + beef powder (1:1), peptone + yeast extract powder (1:1), and ammonium sulfate. A blank control group without nitrogen source addition was established. The culture method was the same as in 4.1.

[0232] 3) Screening of optimal inorganic salt types

[0233] Select the optimal carbon and nitrogen sources screened and replace the inorganic salts in the basal culture medium with the following: sodium chloride, calcium chloride, and potassium dihydrogen phosphate. Also establish a blank control group without inorganic salts. Culture methods are the same as in 4.1.

[0234] 4) Screening of optimal metal ion species

[0235] Select the optimal carbon source, nitrogen source, and inorganic salt from the screening results, and add the following metal ions to the basal culture medium at a concentration of 0.1%: magnesium sulfate, ferrous sulfate, and zinc sulfate. Also establish a blank control group without metal ions. Culture methods are the same as in 4.1.

[0236] 1.4.5 Optimization of culture medium ratio

[0237] 1) Screening of the optimal amount of carbon source

[0238] Select the optimal culture medium components after screening, and set the optimal carbon source, i.e., sucrose concentration, to several gradients: 0%, 1%, 1.5%, 2%, 2.5%, and 3%. The culture method is the same as 4.1.

[0239] 2) Screening of the optimal amount of nitrogen source

[0240] The optimal culture medium components were selected, and the carbon source addition amount was the one optimized in 2.1. The optimal nitrogen source, i.e., peptone + beef powder (1:1), was set to several gradients: 0%, 1%, 1.5%, 2% (ck), 2.5%, and 3%. The culture method was the same as in 4.1.

[0241] 3) Screening of the optimal amount of inorganic salts

[0242] The optimal culture medium components were selected, with the carbon source addition amount being that obtained by optimization in 2.1 and the nitrogen source addition amount being that obtained by optimization in 2.2. The optimal inorganic salt concentration, potassium dihydrogen phosphate, was set to several gradients: 0%, 0.125%, 0.25%, 0.5% (ck), 0.75%, and 1%. The culture method was the same as in 4.1.

[0243] 4) Screening of the optimal amount of metal ions added

[0244] The amount of salt added was the one optimized in 2.3. The optimal metal ion concentration, magnesium sulfate, was set to several gradients: 0%, 0.025%, 0.05%, 0.1%, 0.15%, and 0.2%. The culture method was the same as in 4.1.

[0245] 5) Screening of the optimal pH value of the culture medium

[0246] Select the optimal culture medium obtained from the above optimization and add hydrochloric acid or sodium hydroxide solution to adjust the culture medium pH to 4, 5, 6, 7, 8, 9, or 10. The culture method is the same as 4.1.

[0247] 1.4.6 Optimization of culture conditions

[0248] 1) Inoculation amount

[0249] The optimized optimal culture medium was selected as the basic culture medium for the inoculum size test, and several inoculum size gradients were set: 0.01%, 0.1%, 1%, 2%, and 5%. The biomass of the biocontrol bacteria LS8 bacterial solution was determined by measuring the absorbance value of the bacterial solution at the optimal fermentation temperature obtained in 3.1, the liquid volume was 100mL / 250mL, and the speed was 150rpm. The fermentation time was 6h, 12h, and 24h. Each treatment was repeated 3 times.

[0250] 2) Liquid volume

[0251] The optimized optimal culture medium was selected as the basic culture medium for the liquid filling volume test, and several liquid filling volume gradients were set: 50 mL, 75 mL, 100 mL, 125 mL, and 150 mL. The biomass of the biocontrol bacteria LS8 liquid was determined by measuring the absorbance value of the liquid at the optimal fermentation temperature obtained in 3.1, the optimal inoculum size obtained in 3.2, 150 rpm, and a fermentation time of 24 h. Each treatment was repeated 3 times.

[0252] 3) Speed

[0253] The optimized optimal culture medium was selected as the basic culture medium for the fermentation speed test, and several liquid volume gradients were set: 120rpm, 135rpm, 150rpm, 165rpm, and 180rpm. The biomass of the biocontrol bacteria LS8 was determined by measuring the absorbance value of the bacterial solution at the optimal fermentation temperature obtained in 3.1, the optimal inoculum size obtained in 3.2, and the optimal liquid volume obtained in 3.3, and at fermentation times of 6h, 12h, and 24h. Each treatment was repeated 3 times.

[0254] 4) Fermentation temperature

[0255] The optimized optimal culture medium was selected as the fermentation temperature basal culture medium, and several fermentation temperature gradients were set: 20°C, 24°C, 28°C, 32°C, 36°C, and 40°C. Other fermentation conditions were: inoculation volume of 2%, liquid volume of 100mL / 250mL, 150rpm, fermentation time of 2d, and each treatment was repeated 3 times.

[0256] 5) Fermentation time

[0257] The optimized optimal culture medium was selected as the basic culture medium for the fermentation time test. The biomass of the biocontrol bacteria LS8 was determined by measuring the absorbance value of the culture solution at fermentation times of 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, based on the optimal fermentation temperature obtained in 3.1, the optimal inoculum size obtained in 3.2, the optimal liquid volume obtained in 3.3, and the optimal fermentation speed obtained in 3.4. Each treatment was repeated 3 times.

[0258] 1.4.7 Response Surface Optimization of Fermentation Conditions

[0259] Based on the results of the single-factor experiment, a Box-Benhnken experimental design was used within the response surface design framework to select three factors that significantly influence the antibacterial activity of the sterile fermentation broth: temperature, rotational speed, and liquid volume. Each factor was assigned three levels. Response surface analysis and variance analysis were performed using Design Expert software. A quadratic regression fit was performed on the data to obtain a multiple regression equation. The optimal value was determined within a certain range of levels to optimize the fermentation conditions. Finally, a validation experiment was conducted to test the accuracy of the model and results.

[0260] 2 Results and Analysis

[0261] 2.1 Results of single-factor experiments on culture medium components

[0262] 1) Optimal carbon source screening results

[0263] The results of the optimal carbon source screening are shown in Figure 7As shown in Table 5, CK represents the blank carbon source group. Biocontrol bacterium LS8 was able to utilize all six carbon sources and produce antibacterial substances. The sucrose group exhibited significantly higher absorbance than the other groups and the highest average inhibition rate, but showed no significant differences from the glucose, starch, and blank carbon source groups. Considering the material cost of each carbon source, sucrose was selected as the optimal carbon source for the fermentation medium of biocontrol bacterium LS8, and subsequent experiments were conducted.

[0264] Table 5 Effects of different carbon sources on LS8 growth and antibacterial substances

[0265] Carbon source type Carbon source type <![CDATA[OD 600 ]]> Inhibition rate (%) glucose glucose 0.925 6.25 sucrose sucrose 1.028 7.73 fructose Fructose 0.340 2.62 starch amylum 0.258 4.10 glycerin Glycerol 0.480 2.42 lactose lactose 0.342 2.74 CK CK 0.366 4.30

[0266] 2) Screening results of optimal nitrogen source types

[0267] The results of screening for the optimal nitrogen source are shown in Figure 8 As shown in Table 6 (corresponding data), CK is the blank nitrogen source group. Except for ammonium sulfate, the biocontrol bacteria LS8 could utilize all other nitrogen sources. The average absorbance value of the peptone + beef powder (1:1) group was the highest, but there was no significant difference from the peptone group, beef powder group, and peptone + yeast powder (1:1) group. At the same time, the average inhibition rate of the peptone + beef powder (1:1) group and the peptone + yeast powder (1:1) group was high, but there was no significant difference from the peptone group and beef powder group. Considering the cost of raw materials, peptone + beef powder (1:1) was selected as the optimal nitrogen source for the fermentation medium of the biocontrol bacteria LS8, and subsequent experiments were carried out.

[0268] Table 6 Effects of different nitrogen sources on LS8 growth and antibacterial substances

[0269] Type of nitrogen source Type of nitrogen source <![CDATA[OD 600 ]]> Inhibition rate (%) Peptone peptone 1.030 7.56 beef noodle soup 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 <![CDATA[(NH4)2SO4]]> 0.012 0.07 Blank nitrogen source CK 0.161 2.50

[0270] 3) Optimal inorganic salt type screening results

[0271] The optimal inorganic salt screening results are shown in Figure 9 As shown in Table 7 (corresponding data), CK represents the blank inorganic salt group. Biocontrol bacterium LS8 was able to utilize all four inorganic salts. The potassium dihydrogen phosphate group had the highest average absorbance value, but this was not significantly different from the sodium chloride group or the blank inorganic salt group. Furthermore, the potassium dihydrogen phosphate and potassium chloride groups had significantly higher inhibition rates than the other experimental groups. Potassium dihydrogen phosphate, with the highest absorbance value and the best antibacterial effect, was selected as the optimal inorganic salt for the fermentation medium of biocontrol bacterium LS8 for subsequent experiments.

[0272] Table 7 Effects of different inorganic salts on LS8 growth and antibacterial substances

[0273] Types of inorganic salts Types of inorganic salts <![CDATA[OD 600 ]]> Inhibition rate (%) Sodium chloride NaCl 1.207167 7.749 potassium chloride KCl 1.127 12.25 Potassium dihydrogen phosphate <![CDATA[KH2PO4]]> 1.219 14.784 calcium chloride <![CDATA[CaCl2]]> 0.585 4.507 blank CK 1.145 5.498

[0274] 4) Optimal metal ion type screening results

[0275] The optimal metal ion screening results are shown in Figure 10 As shown in Table 8 (corresponding data), CK represents the blank metal ion group. Biocontrol bacterium LS8 can utilize both ferrous sulfate and magnesium sulfate, but cannot grow in a medium containing 0.1% zinc sulfate. The magnesium sulfate group achieved the highest average absorbance value, significantly different from the other experimental groups. No significant differences in inhibition rates were observed between the magnesium sulfate, ferrous sulfate, and blank metal ion groups. Magnesium sulfate, with its highest absorbance and best inhibitory effect, was selected as the optimal metal ion for the fermentation medium of biocontrol bacterium LS8 for subsequent experiments.

[0276] Table 8 Effects of different metal ions on LS8 growth and antibacterial substances

[0277] Metal ion type <![CDATA[OD 600 ]]> Inhibition rate (%) <![CDATA[MgSO4]]> 0.994 9.391 <![CDATA[FeSO4]]> 0.811 11.773 <![CDATA[ZnSO4]]> 0.010 2.354 CK 0.892 9.941

[0278] 2.2 Culture medium ratio optimization results

[0279] 1) Screening results of optimal carbon source addition amount

[0280] The results of screening for the optimal amount of carbon source are shown in Figure 11 (See Table 9 for corresponding data) As shown, when 2% sucrose was added to the culture medium, the average absorbance value was the highest and significantly higher than that of other groups, and when 2% sucrose was added to the culture medium, the antibacterial rate was the highest and significantly higher than that of other groups. Therefore, 2% was selected as the optimal addition amount of the carbon source.

[0281] Table 9 Effects of carbon source addition on LS8 growth and antibacterial substances

[0282]

[0283]

[0284] 2) Screening results of optimal nitrogen source addition amount

[0285] The results of screening for the optimum amount of nitrogen source are shown in Figure 12 (See Table 10 for corresponding data) As shown in the table, when 2% peptone + beef powder (1:1) was added to the culture medium, the average absorbance value was the highest and significantly higher than that of other groups. When 2.5% peptone + beef powder (1:1) was added to the culture medium, the antibacterial rate was the highest, but there was no significant difference from the 1.5%, 2% and 3% groups when the addition amount was 1%. Taking into account the material cost, 2% was selected as the optimal addition amount of the nitrogen source.

[0286] Table 10 Effects of nitrogen source addition on LS8 growth and antibacterial substances

[0287] Nitrogen source addition <![CDATA[OD 600 ]]> Inhibition rate (%) 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

[0288] 3) Screening results of optimal phosphate addition amount

[0289] The results of screening for the optimal amount of phosphate are shown in Figure 13 (See Table 11 for corresponding data) As shown in the table, when 0.125% potassium dihydrogen phosphate was added to the culture medium, the average absorbance value was the highest and significantly higher than that of the other groups. When 0.125% potassium dihydrogen phosphate was added to the culture medium, the antibacterial rate was the highest and significantly different from that of the other groups. Therefore, 0.125% was selected as the optimal addition amount of inorganic salts.

[0290] Table 11 Effects of phosphate addition on LS8 growth and antibacterial substances

[0291] Phosphate addition <![CDATA[OD 600 ]]> Inhibition rate (%) 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

[0292] 4) Screening results of optimal magnesium ion addition amount

[0293] The results of screening for the optimal amount of magnesium ion addition are shown in Figure 14 (See Table 12 for corresponding data) As shown in the table, when 0.1% magnesium sulfate was added to the culture medium, the average absorbance value was the highest and significantly higher than that of the other groups. The average antibacterial rate of the group with 0.1% magnesium sulfate was the highest, but there was no significant difference between the test groups with the above-mentioned magnesium sulfate concentrations. Therefore, 0.1% was selected as the optimal addition amount of metal ions.

[0294] Table 12 Effect of magnesium ion addition on LS8 growth and antibacterial substances

[0295] Magnesium ion addition <![CDATA[OD 600 ]]> Inhibition rate (%) 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

[0296] 5) Screening results of optimal pH value of culture medium

[0297] The results of the optimization of the optimal pH value of the culture medium are shown in Figure 15 As shown in Table 13 (corresponding data), the biocontrol bacterium LS8 can grow within a pH range of 4 to 10. However, at pH 5 and 6, the absorbance was significantly lower than that of the other groups. At pH 5 and 6, the inhibition rate was significantly higher than that of the other groups. This suggests that under overly acidic or alkaline culture conditions, the biocontrol bacterium LS8 lyses, resulting in higher absorbance values ​​in the culture medium. Furthermore, since fermentation gradually lowers the pH of the culture medium, a pH of 6 to 7 was selected as the culture medium pH for the biocontrol bacterium LS8. The culture medium was prepared according to the optimized formula, and the natural pH was measured to be 6.1, which is consistent with the optimized pH value of 6 to 7.

[0298] Table 13 Effects of different pH values ​​on LS8 growth and antibacterial substances

[0299] pH <![CDATA[OD 600 ]]> Inhibition rate (%) 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

[0300] 2.3 Culture condition optimization results

[0301] 1) Optimization results of optimal inoculum size

[0302] The results of the optimization of the optimal inoculum size of the culture medium are shown in Figure 16 (See Table 14 for the corresponding data.) At 6 hours after inoculation, the absorbance values ​​of the fermentation broths with inoculum sizes of 2% and 5% were significantly higher than those of the other inoculum size test groups. At 24 hours after inoculation, the average absorbance value of the fermentation broth with an inoculum size of 0.1% was the highest among all groups, but there was no significant difference between the values ​​of the fermentation broths with an inoculum size of 0.1% and those with the 1%, 2%, and 5% groups. To prevent contamination by foreign bacteria during the initial inoculation period and considering material costs, 2% was selected as the optimal inoculum size.

[0303] Table 14 Effects of different inoculum sizes on LS8 growth

[0304]

[0305] 2) Optimization results of the optimal filling volume

[0306] The results of the optimization of the optimal volume of culture medium are shown in Figure 17 (See Table 15 for corresponding data). At 6 h of inoculation, the average absorbance value of the fermentation liquid with a liquid volume of 50 mL was the highest, which was significantly different from the liquid volumes of 100 mL, 125 mL, and 150 mL, but not significantly different from the liquid volume of 75 mL. At 24 h of inoculation, the average absorbance value of the fermentation liquid with a liquid volume of 100 mL was the highest among all groups and was significantly different from the other groups. Therefore, 100 mL was selected as the optimal liquid volume.

[0307] Table 15 Effects of different liquid volumes on the growth of LS8

[0308]

[0309] 3) Optimization results of the optimal shaker speed

[0310] The results of the optimization of the optimal rotation speed of the culture medium are shown in Figure 18 (See Table 16 for the corresponding data). At 6 h of inoculation, the average absorbance value was highest when the rotation speed was 135 rpm, but there was no significant difference from the rotation speeds of 150 rpm and 165 rpm. At 24 h of inoculation, the average absorbance value of the fermentation broth at a rotation speed of 150 rpm was the highest among all groups and was significantly different from the other groups. Therefore, 150 rpm was selected as the optimal culture speed.

[0311] Table 16 Effect of different rotation speeds on LS8 growth

[0312]

[0313]

[0314] 4) Optimum fermentation temperature test results

[0315] The results of optimization of the optimal fermentation temperature of the culture medium are shown in Figure 19(See Table 17 for corresponding data.) After 2 hours of incubation at various temperatures, the average absorbance value at 28°C was the highest, but this was not significantly different from the absorbance value at 34°C. The average inhibitory rate of the fermentation broth was the highest at 28°C, but this was not significantly different from the inhibitory rates at 26°C and 30°C. Therefore, 28°C was selected as the optimal incubation temperature.

[0316] Table 17 Effects of different temperatures on LS8 growth and antibacterial substances

[0317] temperature <![CDATA[OD 600 ]]> Antibacterial 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

[0318] 2.4 Results and analysis of response surface analysis of fermentation conditions

[0319] As can be seen from the response surface plot in Figure 20, the interaction between fermentation temperature and liquid filling volume has a significant effect on the antibacterial activity of sterile fermentation broth. As the temperature changes from low to high and the liquid filling volume changes from low to high, the inhibition zone diameter and the absorbance value of the bacterial solution tend to increase, but the difference is not significant when the temperature is 28°C and 32°C; as can be seen from the response surface plot in Figure 20, the interaction between fermentation temperature and shaker speed has a significant effect on the antibacterial activity of sterile fermentation broth. As the temperature changes from low to high and the shaker speed changes from low to high, the inhibition zone diameter and the absorbance value of the bacterial solution tend to increase, but the difference is not significant when the temperature is 28°C and 32°C; as can be seen from the response surface plot in Figure 20, the interaction between liquid filling volume and shaker speed has a significant effect on the antibacterial activity of sterile fermentation broth. As the liquid filling volume changes from high to low and the shaker speed changes from low to high, the inhibition zone diameter and the absorbance value of the bacterial solution tend to increase.

[0320] 3 Experimental Summary

[0321] The optimal culture medium for the biopesticide strain LS8 was determined to be: 20g sucrose, 10g beef extract, 10g peptone, 1.25g potassium dihydrogen phosphate, 1g magnesium sulfate, and deionized water to 1000mL, with a natural pH. The optimal culture conditions were: 2% inoculum, 100mL / 250mL liquid volume, 150rpm fermentation speed, 28°C fermentation temperature, and 2 days of fermentation.

[0322] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Bacillus velezinoffii ( Bacillus velezensis ), characterized in that, Its deposit number is CGMCC No.24563.

2. The Bacillus Velezii as claimed in claim 1 ( Bacillus velezensis ) in the following areas: (I) inhibiting plant pathogens; and / or (II) Control of soil-borne plant diseases; and / or (III) preparing products for inhibiting plant pathogens; and / or (IV) Preparation of products for the prevention and treatment of soil-borne plant diseases; The product is a biocontrol agent; The plant pathogen is a Fusarium fungus; The Fusarium fungi include Fusarium oxysporum lotus special type ( Fusarium oxysporum f.sp. nelumbicola )、Fusarium solani( Fusarium solani ), Fusarium oxysporum flammum ( Fusarium oxysporum f.sp. lini ) or Fusarium oxysporum lily-specific type ( Fusarium oxysporum f.sp. lilii ) one or more; The soil-borne plant diseases are one or more of lotus root rot, lotus root black spot, yam black skin, flax wilt, lily wilt, lily root rot, camphor wood disease, camphor wood trichoderma or honeysuckle disease.

3. The Bacillus Velezii as claimed in claim 1 ( Bacillus velezensis ) is characterized in that, comprising the Bacillus Velezii as claimed in claim 1 ( Bacillus velezensis ) is inoculated into the culture medium and fermented to obtain the Velez Bacillus ( Bacillus velezensis ) fermentation broth; The inoculation includes a viable count of 10 7 ~10 8 CFU / mL of the Velezella spp. Bacillus velezensis ) was inoculated into the culture medium at an inoculum volume of 2% (v / v); and / or The volume of the culture medium comprises 100 mL; and / or The culture medium comprises: 20 g / L sucrose, 10 g / L beef extract, 10 g / L peptone, 1.25 g / L potassium dihydrogen phosphate, and 1 g / L magnesium sulfate; and / or The pH value of the culture medium is 6-7; and / or The fermentation temperature includes 28°C; and / or The shaking speed of the fermentation comprises 150 rpm.

4. Sterile fermentation broth, characterized in that The preparation method comprises: treating the Bacillus velezensis as claimed in claim 1 ( Bacillus velezensis ) is inoculated into a culture medium, fermented to obtain a bacterial liquid, and the bacterial liquid is filtered to obtain the sterile fermentation liquid.

5. A biocontrol agent, characterized in that: comprising the Bacillus Velezii as claimed in claim 1 ( Bacillus velezensis ), and acceptable excipients or adjuvants.

6. The method for preparing the biocontrol agent according to claim 5, wherein comprising the Bacillus Velezii as claimed in claim 1 ( Bacillus velezensis ) inoculated into a culture medium, cultured at 25-30° C. with shaking for 2-4 days, and the isolated bacteria were diluted with water to obtain the biocontrol agent; The culture medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 20 g / L sucrose and 5 g / L NaCl; and / or The pH value of the culture medium is 7.2-7.4; and / or The rotation speed of the oscillation is 180 r / min.

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