A Bacillus velezensis nanomaterial composite microbial agent, its preparation method and application

Abstract

The present invention relates to the technical field of microbial control, and discloses a Bacillus velezensis nanomaterial composite microbial agent, a preparation method thereof and an application. The microbial agent of the present invention is prepared by the following steps: preparing a bacterial suspension of Bacillus velezensis; after mixing sodium alginate with the bacterial suspension, adding nano-carbon and stirring to obtain a mixed solution; adding the mixed solution into a stirred calcium chloride solution, and reacting to obtain a Bacillus velezensis microcapsule microbial agent. The microbial agent of the present invention has an antibacterial rate of >56% against Citrus brown spot pathogen, Citrus anthracnose pathogen, Citrus greasy spot and yellow spot pathogen, Banana wilt pathogen, Banana long-shaped spot pathogen; the control effect on Huanglongbing reaches 57.84%. After being irradiated by ultraviolet rays, the antibacterial ability of the microbial agent of the present invention only decreases by 4.32%, and it can play a biological control role for a long time. The growth amount of citrus seedlings treated with the microbial agent of the present invention can be increased by 220.39%, and the number of newly grown leaves can be increased by 244.67%, having a good plant growth promotion effect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial control, and more specifically, to a Bacillus velezensis nanomaterial composite bactericide and its preparation method and application. Background Art

[0002] With the continuous development of agricultural production, disease control has become one of the important means to improve crop yield and quality. Although traditional chemical pesticides have achieved remarkable results in controlling diseases, the long-term reliance on the use of chemical agents has brought a series of negative impacts such as environmental pollution, pesticide residues, and drug resistance, seriously restricting the sustainable development of agriculture. Therefore, the development of environmentally friendly and sustainable disease control technologies has become a hot topic in current agricultural research.

[0003] Biological control is a technology that uses natural biological resources to control plant diseases and has received extensive attention and application in recent years. Compared with traditional chemical control methods, biological control methods have strong environmental friendliness and can effectively reduce pesticide residues and environmental pollution. By applying specific biological agents (such as beneficial microorganisms, plant extracts, etc.), not only can the growth of pathogenic microorganisms be inhibited, but also the disease resistance of plants can be stimulated. However, the popularization and application of biological control still face some challenges, especially in maintaining the stability and activity of biological control strains.

[0004] Bacillus velezensis, as a common biological control strain, has been widely used in agricultural disease control due to its broad disease resistance spectrum and environmental adaptability. Bacillus velezensis can effectively inhibit the growth of a variety of plant pathogenic bacteria by secreting antibiotics, producing inhibitory metabolites, and symbiotic interactions with plant roots. However, the application effect of Bacillus velezensis is easily affected by the external environment, especially ultraviolet irradiation, which easily causes cell damage or inactivation, limiting its long-term application in outdoor environments. Therefore, how to effectively improve the stability of Bacillus velezensis and enhance its resistance to adverse environmental factors such as ultraviolet rays has become an important topic in the research and development of biological control technologies.

[0005] In the preparation of current slow-release bactericides, the common practice is to use materials such as sodium alginate and chitosan as embedding agents and add cross-linking agents such as CaCl2 to prepare microspheres. Sodium alginate has the advantages of being safe, non-toxic, low-cost, and mild reaction conditions. It can be used as an immobilization carrier with little damage to the embedded cells, is very suitable for immobilization operations, and the small balls prepared with sodium alginate as the embedding material have good mass transfer properties and high biological activity. Therefore, sodium alginate is often used to embed human or animal probiotics and plant growth-promoting bacteria to prepare slow-release microspheres.

[0006] However, the microspheres prepared from pure sodium alginate have poor mechanical strength, a short service life, and limited protection ability for the embedded microorganisms. Therefore, in actual research, chitosan, nanomaterials, etc. are often used as composite carriers to improve the sphericity of sodium alginate and enhance its mechanical strength, thereby improving its performance.

[0007] At present, there is no report on the use of nano-carbon sol for the preparation of sustained-release agents of Bacillus velezensis. Summary of the Invention

[0008] The present invention aims to overcome the defects of the microspheres prepared from pure sodium alginate, such as poor mechanical strength, short service life, and limited protection ability for the embedded microorganisms, and provides a composite agent of Bacillus velezensis and nanomaterials; provides a preparation method for the composite agent of Bacillus velezensis and nanomaterials;

[0009] Another object of the present invention is to provide an application of the composite agent of Bacillus velezensis and nanomaterials.

[0010] To solve the above technical problems, the technical solution of the present invention is as follows:

[0011] A composite agent of Bacillus velezensis and nanomaterials is prepared from Bacillus velezensis, sodium alginate, calcium chloride, and nano-carbon.

[0012] Further, the Bacillus velezensis is Bacillus velezensis E17-23, with the preservation number: GDMCC No. 64580, the preservation date: April 29, 2024, the preservation unit: Guangdong Provincial Microbial Culture Collection Center, and the preservation address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0013] Further, the concentration of the bacterial suspension in S1 is 10 8 ~10 9 CFU / mL.

[0014] Further, the volume ratio of sodium alginate to the bacterial suspension added in S2 is 19-20:1.

[0015] Preferably, the volume ratio of sodium alginate to the bacterial suspension added in S2 is 19:1.

[0016] Further, the particle size of nano-carbon in S2 is 10-100 nm, and the mass-volume concentration of nano-carbon in the mixed solution is 0.1-0.5%.

[0017] Preferably, the mass-volume concentration of nano-carbon in the mixed solution is 0.1%.

[0018] Further, the mass - volume concentration of sodium alginate before mixing with the bacterial suspension in S2 is 2 - 3%, and the mass concentration of calcium chloride solution before adding the mixed solution in S3 is 3 - 5%.

[0019] Preferably, the mass - volume concentration of sodium alginate before mixing with the bacterial suspension in S2 is 2%, and the mass concentration of calcium chloride solution before adding the mixed solution in S3 is 3%.

[0020] A preparation method of a Bacillus velezensis nanomaterial composite microbial agent is prepared by the following steps:

[0021] S1: Prepare a bacterial suspension of Bacillus velezensis;

[0022] S2: After mixing sodium alginate with the bacterial suspension, add nano - carbon and stir to obtain a mixed solution;

[0023] S3: Add the mixed solution into the stirred calcium chloride solution, and after reaction, obtain a Bacillus velezensis microcapsule microbial agent.

[0024] Preferably, the Bacillus velezensis is Bacillus velezensis E17 - 23.

[0025] Preferably, the preparation of the bacterial suspension of Bacillus velezensis in S1 includes the following steps: adjust the concentration of the activated Bacillus velezensis; centrifuge and discard the supernatant; resuspend with sterile water to obtain a bacterial suspension.

[0026] Preferably, shake - culture the Bacillus velezensis in LB medium for more than 10 h to activate the Bacillus velezensis.

[0027] Preferably, adjust the concentration of the activated Bacillus velezensis to OD 600 of 1.0 in LB medium.

[0028] Preferably, centrifuge at 6000 rpm for 5 min and then discard the supernatant.

[0029] Preferably, resuspend with sterile water to wash the bacterial cells, and then add sterile water to prepare a bacterial suspension.

[0030] Preferably, after stirring evenly in S2, let it stand to eliminate air bubbles.

[0031] Preferably, use a disposable sterile syringe to aspirate the mixed solution in S3 and slowly drip it into the calcium chloride solution from about 10 cm above the liquid surface.

[0032] Preferably, after reaction in S3, screen out the microcapsules and dry them to obtain the Bacillus velezensis microcapsule microbial agent.

[0033] Further, the stirring speed in S2 and S3 is 300 - 500 rpm; the reaction time in S3 is not less than 3 h.

[0034] Preferably, the stirring speed in S2 and S3 is 400 rpm; and the reaction time in S3 is 3 h.

[0035] An application of the Bacillus Velez nanomaterial composite microbial agent is used to prepare a preparation for preventing and treating plant diseases caused by citrus brown spot pathogen, citrus anthracnose pathogen, citrus yellow spot pathogen, banana wilt pathogen, and banana long spot pathogen, or a preparation for promoting plant growth.

[0036] Preferably, the plant is a citrus seedling.

[0037] Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

[0038] 1. The fungicide has a significant antibacterial effect on a variety of pathogens. The fungicide of the present invention has a significant inhibitory effect on citrus brown spot pathogen, citrus anthracnose pathogen, citrus yellow spot pathogen, banana wilt pathogen, and banana long spot pathogen, with the inhibition rate being >56%. After treatment with the fungicide, the 16S rDNA copy number of citrus Huanglongbing pathogen was significantly reduced to (1.13±0.24)×10 8 / g, and the control effect reached 57.84%. The microbial agent of the present invention has a significant inhibitory effect on various pathogens, can effectively reduce the occurrence of diseases, and reduce the use of pesticides.

[0039] 2. Improves the UV resistance of Bacillus velezensis. The present microbial agent exhibited only a 4.32% decrease in antibacterial activity after UV irradiation, a 75.84% increase in antibacterial activity compared to a non-UV-irradiated Bacillus velezensis solution. This demonstrates the agent's significant advantage in UV protection. By encapsulating the microbe in microcapsules, the present invention significantly improves the biocontrol agent's UV resistance, prolonging its shelf life in the environment and ensuring its long-term biocontrol activity.

[0040] 3. It promotes the growth of young plants. Citrus seedlings treated with the inoculant of the present invention showed a 220.39% increase in growth compared to the control group, and a 244.67% increase in the number of newly grown leaves. The microcapsule inoculant of Bacillus Velezii not only increases plant growth but also significantly increases the number of new leaves, demonstrating a favorable plant growth-promoting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the OD of Bacillus Velezii E17-23 under different concentrations of nanocarbon 600 Values (different lowercase letters indicate significant differences at the 0.01 level);

[0042] Figure 2For the antibacterial effects of different nano-carbon concentrations on Bacillus velezensis microcapsules, A is the result of plate confrontation, and B is the schematic diagram of the antibacterial circle diameter (different lowercase letters indicate significant differences at the 0.01 level);

[0043] Figure 3 For the control effect of the microbial agent on citrus leaf huanglongbing;

[0044] Figure 4 For the antibacterial effects of the microbial agent on various pathogenic bacteria, A is the result of plate confrontation, and B is the schematic diagram of the colony diameter of the pathogenic bacteria;

[0045] Figure 5 For the influence of ultraviolet irradiation on the antibacterial effect;

[0046] Figure 6 For the effect of the microbial agent on promoting the growth of sweet orange seedlings, A is the schematic diagram of the average growth amount, and B is the schematic diagram of the number of newly grown leaves. Detailed implementation manners

[0047] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0048] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0049] 1. Preparation of the culture media used in the present invention

[0050] The solid LB culture medium used in the present invention is: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, agar 15.0 g / L, pH value 7.0, sterilization conditions: 121 °C, 100 kPa, 20 min;

[0051] The liquid LB culture medium used is: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, adjust the pH value to about 7.0, and sterilize at 121 °C, 100 kPa for 20 min;

[0052] The PDA (potato dextrose agar) culture medium used is: potatoes 200 g / L, glucose 20 g / L, agar 15 g / L. First, peel the potatoes, cut them into small pieces, weigh 200 g, boil them in 1000 mL of deionized water for 20 - 30 min, filter with two layers of gauze, add deionized water to the filtrate to 1000 mL, adjust the pH value to about 7.0, add agar and glucose, boil and then dispense into conical flasks, and sterilize at 121 °C, 100 kPa for 20 min;

[0053] The NA medium used was: peptone 10 g / L, Nacl g / L, beef extract 5.0 g / L, agar powder 15 g / L, deionized water 1000 mL. Adjust the pH value to about 7.0, and sterilize at 121 °C, 100 kPa for 20 min.

[0054] Example 1

[0055] Take out the preserved Bacillus velezensis E17-23 from -80 °C, streak inoculate it on an LB plate, and culture it at 37 °C for 48 h. Pick a single colony and transfer it to a flask containing LB liquid medium for culture, and culture it in a shaker at 37 °C at 200 r·min -1 Shake and culture for 24 h. Pipette 1 mL of the bacterial solution into a triangular flask containing 40 mL of LB liquid medium for culture, and culture it in a shaker at 37 °C at 200 r·min -1 Shake and culture for 24 h, and adjust the OD of the obtained bacterial solution with a spectrophotometer 600 To about 1.0 (concentration is 10 8 ~10 9 CFU / mL) for use.

[0056] Add nano-carbon (particle size of 10 - 100 nm) to the LB liquid medium, and prepare media with nano-carbon contents of 0.1% and 0.5% respectively. Inoculate the obtained bacterial solution into the media with different nano-carbon contents according to a ratio of 1%, and culture it in a shaker at 37 °C at 200 r·min -1 Shake and culture for 12 h for use.

[0057] Example 2

[0058] Prepare 2% sodium alginate and 3% calcium chloride solutions for use. Inoculate the biocontrol bacteria into the LB medium and culture them overnight with shaking. Adjust the OD of the bacterial solution with the LB medium 600 To about 1.0 (concentration is 10 8 ~10 9 CFU / mL). Centrifuge at 6000 rpm for 5 min, discard the supernatant, resuspend with sterile water and centrifuge 3 times to wash the bacterial cells. Resuspend the bacterial cells with an equal volume of sterile water to prepare a bacterial suspension for standby (concentration is 10 8 ~10 9 CFU / mL). Mix well according to the ratio of 2% sodium alginate:bacterial suspension = 19:1, add nano-carbon sol to the mixed solution so that the final mixed solution contains 0.1% nano-carbon sol, stir evenly with a magnetic stirrer at 400 rpm, and let it stand until there are no bubbles. Use a disposable sterile syringe to suck up the above mixed solution and slowly drip it from a certain height (about 10 cm from the liquid surface) into the 3% calcium chloride solution, stir with a magnetic stirrer at 400 rpm during this period, and after reacting for 3 h, sieve out the microspheres and wash the surface with sterile water. Wait for the microcapsule microbial agent to dry and then use it.

[0059] Example 3

[0060] Prepare 3% sodium alginate and 5% calcium chloride solutions for use. Inoculate the biocontrol bacteria into LB medium and shake culture overnight. Adjust the bacterial solution to an OD 600 of about 1.0 (concentration is 10 8 ~10 9 CFU / mL) using LB medium. Centrifuge at 6000 rpm for 5 min, discard the supernatant, resuspend with sterile water and centrifuge 3 times to wash the bacterial cells. Resuspend the bacterial cells with an equal volume of sterile water to prepare a bacterial suspension for standby. Mix well according to the ratio of 3% sodium alginate:bacterial suspension = 1:1, add nano-carbon sol to the mixed solution so that the final mixed solution contains 0.1% nano-carbon sol, stir evenly with a magnetic stirrer at 300 rpm, and let stand until there are no bubbles. Use a disposable sterile syringe to suck up the above mixed solution and slowly drip it into the 5% calcium chloride solution from a certain height (about 10 cm from the liquid surface). During this period, stir with a magnetic stirrer at 400 rpm. After reacting for 3 h, screen out the microspheres and wash the surface with sterile water. Wait for the microcapsule bactericide to dry and then use it.

[0061] Comparative Example 1

[0062] The technical solution of Comparative Example 1 is similar to that of Example 1, the difference is that in Comparative Example 1, media with nano-carbon concentrations of 0%, 1.0%, 1.5%, and 2.0% are respectively prepared.

[0063] Detection method

[0064] (1) Influence of nano-carbon concentration on Bacillus velezensis E17-23

[0065] Measure the OD 600 value of Example 1 and Comparative Example 1 by a spectrophotometer;

[0066] Loading amount of Bacillus velezensis E17-23: Prepare 0.6% sodium citrate solution and sterilize it for standby. Weigh 0.1 g of each of the previously prepared microcapsules and place them in centrifuge tubes, add 1 mL of 0.6% sodium citrate solution, and dissolve at 200 rpm for 1 h. Determine the colony concentration by the dilution plating method.

[0067] (2) Antibacterial circle experiment

[0068] Heat the NA solid medium. When the NA medium cools to about 40 °C, add the pre-shaken pathogenic bacteria liquid according to a ratio of 1:100, and pour the mixed liquid into a petri dish to prepare a bacteria-containing plate. Punch holes with a 5 mm puncher. After picking out the medium with a toothpick, add 20 μL of the bacterial liquid of Example 1 and Comparative Example 1 to each hole, add 20 μL of LB medium as a control, dry it, and seal it. Place it in an incubator at 28 °C for constant temperature culture. After 24 h, observe whether a transparent antibacterial circle appears and record the results.

[0069] (3) Control effect of the compound microbial agent on citrus leaf huanglongbing

[0070] The control effect of the biocontrol bacteria on citrus huanglongbing was determined by the citrus half-leaf method. Citrus leaves with symptoms and similar morphological characteristics such as size were collected and brought back to the laboratory for storage at 4 °C for later use.

[0071] The leaf was cut in half perpendicular to the leaf vein. One half was placed in sterile water, while the other half was placed in sterile water containing 10 microcapsule microbial agents. Each treatment included 6 leaves, and the experiment was repeated 3 times. After standing at room temperature for 24 h, the DNA of the midrib of the leaf was extracted using a plant genomic DNA extraction kit (axygen) for RT-PCR.

[0072] Control effect = (content of huanglongbing bacteria in the control group - content of huanglongbing bacteria in the treatment group) / content of huanglongbing bacteria in the control group (4) Antibacterial effect of the compound microbial agent against multiple pathogenic bacteria

[0073] Prepare a PDA medium plate and inoculate the pathogenic bacteria cake (diameter 5 mm) in the center of the plate. Taking this as the center, place the microbial agent gently at the midpoints of the four sides of a square with a distance of 2.5 cm from the center according to a square layout. Using LB as a control, culture at a constant temperature of 28 °C. After 5 days, measure the colony diameter using the cross method, measure the distance between the edge of the endophytic fungus colony and the edge of the pathogenic bacteria colony, and observe whether an isolation interval is formed in the pathogenic bacteria colony. Each treatment was repeated 3 times.

[0074] (5) Growth promotion experiment of citrus (sweet orange) seedlings

[0075] Prepare the test plants, select sweet orange seedlings with similar seedling ages and growth conditions, rinse them with water, plant them in a seedling-raising cup filled with sterilized nutrient soil, and place them in an artificial climate incubator with 16 h of light and a temperature of 25 °C. After 7 days of regular management, inoculate the microbial agent. Prepare the compound microbial agent, 6 grains for each citrus seedling, and evenly sprinkle it on the ground 1 - 2 cm below the surface around the roots of the plants. Control: sterile water. Regularly measure the growth indicators of citrus seedlings.

[0076] Analysis and explanation

[0077] (1) Effect of different nano-carbon concentrations on the growth of Bacillus velezensis E17-23

[0078] As can be seen from Table 1, when the nano-carbon concentration was 0.1% and 0.5%, the OD 600 values were 0.96 ± 0.02 and 1.07 ± 0.05 respectively. Combining Figure 1Analysis shows that when the concentration of nano-carbon is 0.1% and 0.5%, the influence of nano-carbon on Bacillus velezensis E17-23 is the smallest. Too high a concentration of nano-carbon may lead to changes in the local oxidation environment, increase the generation of reactive oxygen species in bacterial cells, cause cell damage and death; it may also lead to the damage of the cell membrane, affect the material exchange inside the cell, and thus inhibit the growth of bacteria.

[0079] Table 1 Influence of different nano-carbon concentrations on the growth of Bacillus velezensis E17-23

[0080] Concentration of carbon nanotubes <![CDATA[OD value after 12 hours of cultivation 600 > 0％ 1.10±0.06 0.1％ 0.96±0.02 0.5％ 1.07±0.05 1.0％ 0.90±0.05 1.5％ 0.81±0.01 2.0％ 0.82±0.01

[0081] In addition, the addition of nano-carbon also affects the survival rate of Bacillus velezensis E17-23 in the microbial inoculum. As can be seen from Table 2, when 0.1% and 0.5% nano-carbon are added, the survival rates of strain E17-23 within 7 days are increased by 6.07% and 21.87% respectively compared with when no nano-carbon is added. The addition of nano-carbon greatly extends the storage period of the microbial inoculum.

[0082] Table 2 Influence of nano-carbon on the survival rate of strain E17-23 in the microbial inoculum

[0083]

[0084] (2) Loading amount of Bacillus velezensis E17-23

[0085] As can be seen from Table 3, when the nano-carbon concentration is 0.1%, the loading amount of Bacillus velezensis E17-23 in the unit microcapsule is the highest, reaching (1.39±0.36)×10 8 CFU / g. When the concentration is 0.5%, the loading amount slightly decreases but still reaches (1.34±0.27)×10 8 CFU / g, indicating that nano-carbon can effectively improve the embedding efficiency of microcapsules within this concentration range.

[0086] When the nano-carbon concentration is 1.0%, the loading amount drops to (1.31±0.19)×10 8 CFU / g, even lower than that of the control group without nano-carbon addition (8.23±0.75)×10 7 CFU / g. This may be because high-concentration nano-carbon leads to excessive densification of the microcapsule structure or affects the survival of the bacterial cells.

[0087] Table 3 Loading amount of Bacillus velezensis E17-23 in the unit microcapsule

[0088] Concentration of carbon nanotubes Strain loading (CFU / g) 0％ <![CDATA[(8.23±0.75)×10 7 (a)]]> 0.1％ <![CDATA[(1.39±0.36)×10 8 (b)]]> 0.5％ <![CDATA[(1.34 ± 0.27)×10 8 (b)]]> 1.0％ <![CDATA[(1.31 ± 0.19)×10 8 (b)]]> Control group 0(c)

[0089] (Different lowercase letters indicate significant differences at the 0.01 level)

[0090] (3) Influence of Different Nano-carbon Addition Amounts on the Bacteriostatic Effect of Microcapsule Bacterial Agents

[0091] Figure 2 A shows the bacteriostatic effect of different nano-carbon concentrations on the microcapsules of Bacillus velezensis. At the nano-carbon concentration of 0.1% in Example 1, the diameter of the inhibition zone reached a maximum of 1.25 ± 0.05 cm. Combining Figure 2 B analysis shows that nano-carbon at this concentration has the best effect on the structural optimization of microcapsules and the activity of strains. The bacteriostatic effect at the nano-carbon concentration of 0.5% in Example 1 is also very close, with the diameter of the inhibition zone being 1.23 ± 0.03 cm, indicating that within the range of 0.1% - 0.5%, the addition of nano-carbon maintains a relatively high level of bacteriostatic effect.

[0092] (4) Control Effect of Bacterial Agents on Huanglongbing of Citrus Leaves

[0093] As Figure 3 shown, the copy number of 16S rDNA of Citrus huanglongbing bacteria treated with sterile water was (2.68 ± 0.60) × 10 8 / g, while it was significantly reduced to (1.13 ± 0.24) × 10 8 / g after treatment with the bacterial agent, and the control effect reached 57.84%.

[0094] (5) Bacteriostatic Effect of Bacterial Agents on Multiple Pathogens

[0095] As Figure 4 shown in A, in the control group, all pathogens grew normally, covering the surface of the culture medium, showing typical colony morphology. In the petri dishes treated with the bacterial agent, the growth of pathogens was significantly inhibited, and obvious inhibition zones could be seen on some petri dishes. Combining Figure 4 B indicates that the bacterial agent has an obvious inhibitory effect on Phyllosticta citricarpa, Colletotrichum gloeosporioides, Mycosphaerella citri, Fusarium oxysporum f. sp. cubense, and Pseudocercospora musae, and the inhibition rate is > 56%.

[0096] (6) Influence of Ultraviolet Irradiation on the Bacteriostatic Effect of Microcapsule Bacterial Agents of Bacillus velezensis

[0097] The diameter of the inhibition zone of the bacterial solution of Bacillus velezensis E17 - 23 against Xanthomonas citri subsp. citri without ultraviolet irradiation was 3.08 ± 0.10 cm, and it was 2.53 ± 0.06 cm after 1 hour of ultraviolet irradiation. The diameter of the inhibition zone of the microcapsule bacterial agent against Xanthomonas citri subsp. citri without ultraviolet irradiation was 1.74 ± 0.09 cm, and it was 1.66 ± 0.09 cm after 1 hour of ultraviolet irradiation.

[0098] Taking the bacteriostatic ability of the bacterial solution of Bacillus velezensis E17 - 23 and the microcapsule bacterial agent before irradiation as 100%, calculate respectively after, as Figure 5As shown, the antibacterial ability of the Bacillus velezensis E17-23 bacterial liquid decreased significantly, only 82.16% of that before irradiation, indicating that due to the direct exposure of the bacteria to ultraviolet light, cell damage or reduced activity occurred.

[0099] In contrast, the antibacterial ability decreased less after ultraviolet irradiation, being 95.76% of that before irradiation, and there was no significant difference compared with that before irradiation after analysis, indicating that the microcapsules played a protective role on the strain, reducing the direct damage of ultraviolet light to the Bacillus velezensis E17-23 bacteria.

[0100] (7) Growth promotion effect of the Bacillus velezensis microcapsule microbial agent on citrus (sweet orange) seedlings

[0101] Figure 6 A shows the comparison of plant growth amounts (unit: cm) after treatment with the Bacillus velezensis microcapsule microbial agent and sterile water treatment. The average growth amount of the treatment group with the Bacillus velezensis microcapsule microbial agent was 3.30 ± 0.70 cm, and the average growth amount of the sterile water treatment group was 1.03 ± 0.86 cm. Figure 6 B shows the comparison of the number of newly grown leaves of plants after treatment with the Bacillus velezensis microcapsule microbial agent and sterile water treatment. The average number of newly grown leaves in the treatment group with the Bacillus velezensis microcapsule microbial agent was 5.17 ± 0.98, and the average number of newly grown leaves in the sterile water treatment group was 1.50 ± 0.84. The growth amount and the number of newly grown leaves of the plants treated with the Bacillus velezensis microcapsule microbial agent were higher than those of the sterile water treatment, indicating that the Bacillus velezensis microcapsule microbial agent of the present invention can significantly promote plant growth.

[0102] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A Bacillus velezensis nanomaterial composite microbial inoculum, characterized in that, It is prepared from Bacillus velezensis, sodium alginate, calcium chloride and nano-carbon; the mass-volume concentration of the nano-carbon is 0.1-0.5%; the Bacillus velezensis is Bacillus velezensis E17-23 with the preservation number of GDMCC No.64580; the particle size of the nano-carbon is 10-100 nm.

2. The preparation method of the Bacillus velezensis nanomaterial composite microbial inoculum according to claim 1, characterized in that, It is prepared by the following steps: S1: Prepare a bacterial suspension of Bacillus velezensis; S2: After mixing sodium alginate with the bacterial suspension, add nano-carbon and stir to obtain a mixed solution; S3: Add the mixed solution into the stirred calcium chloride solution, and after reaction, obtain the Bacillus velezensis nano-material composite bactericide.

3. The preparation method of the Bacillus velezensis nanomaterial composite microbial agent according to claim 2, characterized in that, The stirring speed in S2 and S3 is 300-500 rpm; the reaction time in S3 is not less than 3 h.

4. Use of the Bacillus velezensis nanomaterial composite microbial agent according to claim 1, characterized in that, It is used to prepare a preparation for preventing and controlling plant diseases caused by Candidatus Liberibacter asiaticus, Alternaria alternata, Colletotrichum gloeosporioides, Mycosphaerella citri, Fusarium oxysporum f. sp. cubense, or a preparation for promoting plant growth.

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