Bacillus velezensis nano-material complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microbial control, and discloses a bacillus velezensis nano-material complex microbial inoculant as well as a preparation method and application thereof. The microbial inoculum is prepared by the following steps: preparing a bacterial suspension of bacillus velezensis; mixing sodium alginate with the bacterial suspension, adding nanocarbon, and stirring to obtain a mixed solution; and adding the mixed solution into a stirred calcium chloride solution, and reacting to obtain the bacillus velezensis microcapsule fungicide. The bacteriostasis rates of the fungicide for citrus brown spot, citrus colletotrichum gloeosporioides, citrus lipolytic macular blight, banana fusarium wilt and banana long leaf spot bacteria are all greater than 56%; the prevention and treatment effect on the Huanglongbing reaches 57.84%. After ultraviolet irradiation, the bacteriostatic ability of the microbial agent is reduced by only 4.32%, and the microbial agent can play a biological prevention and control role for a long time. The growth amount of citrus seedlings treated by the microbial inoculum can be increased by 220.39%, the number of newly grown leaves can be increased by 244.67%, and the microbial inoculum has a good plant growth promoting effect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial control, and more specifically, to a Bacillus Velez nanomaterial composite bacterial agent and a preparation method and application thereof. 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, long-term reliance on the use of chemical agents has also brought about a series of negative effects such as environmental pollution, pesticide residues, and drug resistance, which seriously restricts the sustainable development of agriculture. Therefore, the development of environmentally friendly and sustainable disease control technology has become a hot topic in current agricultural research.

[0003] Biological control is a technology that uses natural biological resources to control plant diseases. It has received widespread attention and application in recent years. Compared with traditional chemical control methods, biological control methods are more environmentally friendly and can effectively reduce pesticide residues and environmental pollution. By applying specific biological agents (such as beneficial microorganisms, plant extracts, etc.), it can not only inhibit the growth of pathogenic microorganisms, but also stimulate the disease resistance of plants. However, the promotion and application of biological control still faces some challenges, especially in the stability and activity maintenance of biological control strains.

[0004] Bacillus subtilis is a common biological control bacteria species, which is widely used in agricultural disease control because of its broad disease resistance spectrum and environmental adaptability. Bacillus subtilis can effectively inhibit the growth of various plant pathogens by secreting antibiotics, producing inhibitory metabolites, and symbiotically interacting with plant roots. However, the application effect of Bacillus subtilis is easily affected by the external environment, especially ultraviolet radiation, which can easily cause cell damage or inactivation, limiting its long-term application in outdoor environments. Therefore, how to effectively improve the stability of Bacillus subtilis 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 technology.

[0005] At present, the common practice in the preparation of sustained-release bacterial agents is to use sodium alginate, chitosan and other materials as embedding agents and add CaCl2 and other cross-linking agents to prepare microspheres. Sodium alginate has the advantages of safety, non-toxicity, low price, mild reaction conditions, etc. It can be used as an immobilization carrier with little damage to embedded cells, which is very suitable for immobilization operations. In addition, the microspheres prepared with sodium alginate as the embedding material have good mass transfer and high biological activity. Therefore, sodium alginate is often used to embed human or animal probiotics and plant growth-promoting bacteria to prepare sustained-release microspheres.

[0006] However, microspheres prepared from pure sodium alginate have poor mechanical strength, short service life, and limited ability to protect embedded microorganisms. Therefore, in actual research, chitosan, nanomaterials, etc. are often added as composite carriers to improve the sphericity of sodium alginate, increase its mechanical strength, and thus improve its performance.

[0007] At present, there is no report on the use of nanocarbon sol for the preparation of sustained-release bacterial agents of Bacillus Velezii. Summary of the invention

[0008] The present invention provides a Bacillus Velez nanomaterial composite bacterial agent to overcome the defects of poor mechanical strength, short service life, and limited protection ability for embedded microorganisms of microspheres prepared from pure sodium alginate in the prior art; and provides a preparation method of the Bacillus Velez nanomaterial composite bacterial agent;

[0009] Another object of the present invention is to provide an application of a Bacillus Velez nanomaterial composite bacterial agent.

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

[0011] A Bacillus Velez nanomaterial composite bacterial agent is prepared from Bacillus Velez, sodium alginate, calcium chloride and nanocarbon.

[0012] Furthermore, the Bacillus velezensis is Bacillus velezensis E17-23, deposit number: GDMCC No.64580, deposit date: April 29, 2024, deposit unit: Guangdong Provincial Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City.

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

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

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

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

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

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

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

[0020] A method for preparing a Bacillus Velez nanomaterial composite bacterial agent is prepared by the following steps:

[0021] S1: preparing a bacterial suspension of Bacillus Velez;

[0022] S2: After mixing sodium alginate and bacterial suspension, add nanocarbon and stir to obtain a mixed solution;

[0023] S3: adding the mixed solution into the stirred calcium chloride solution to obtain the Bacillus Velez microcapsule agent after reaction.

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

[0025] Preferably, the preparation of the bacterial suspension of Bacillus Velez subtilis in S1 comprises the following steps: adjusting the concentration of the activated Bacillus Velez subtilis; discarding the supernatant after centrifugation; and resuspending with sterile water to obtain a bacterial suspension.

[0026] Preferably, the Bacillus Velez subtilis is shake-cultured in the LB medium for more than 10 hours to activate the Bacillus Velez subtilis.

[0027] Preferably, the concentration of the activated Bacillus Velez is adjusted to OD 600 is 1.0.

[0028] Preferably, the supernatant is discarded after centrifugation at 6000 rpm for 5 min.

[0029] Preferably, after the bacteria are resuspended in sterile water to wash the bacteria, sterile water is added to prepare a bacterial suspension.

[0030] Preferably, S2 is stirred evenly and then allowed to stand to eliminate bubbles.

[0031] Preferably, in S3, a disposable sterile syringe is used to absorb the mixed liquid, and the mixed liquid is slowly added dropwise to the calcium chloride solution from a distance of about 10 cm from the liquid surface.

[0032] Preferably, after the reaction in S3, the microcapsules are screened out and dried to obtain the Bacillus Velez microcapsule agent.

[0033] Furthermore, the stirring speed in S2 and S3 is 300-500 rpm; and 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 bacterial agent is used to prepare a preparation for preventing and controlling plant diseases caused by citrus brown spot pathogen, citrus anthracnose pathogen, citrus fat spot yellow spot pathogen, banana wilt pathogen, and banana long spot pathogen, or a preparation for promoting plant growth.

[0036] Preferably, the plants are citrus seedlings.

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

[0038] 1. The fungicide has significant antibacterial effect on various pathogens. The fungicide of the present invention has significant inhibitory effect on citrus brown spot pathogen, citrus anthracnose pathogen, citrus fat spot yellow spot pathogen, banana wilt pathogen and banana long spot pathogen, and the inhibition rate is >56%. After being treated 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 a variety of pathogens, can effectively reduce the occurrence of diseases, and reduce the use of pesticides.

[0039] 2. Improve the resistance of Bacillus Velez to ultraviolet rays. The antibacterial ability of the bacterial agent of the present invention decreases by only 4.32% after ultraviolet irradiation. Compared with the Bacillus Velez bacterial solution without ultraviolet irradiation, the antibacterial ability is improved by 75.84%, showing the significant advantage of the bacterial agent of the present invention in ultraviolet protection. The present invention significantly improves the resistance of biocontrol bacteria to ultraviolet rays through the embedding of microcapsules, prolongs its effective period in the environment, and ensures that it can play a long-term biological control role.

[0040] 3. It has a promoting effect on the growth of plant seedlings. The growth of citrus seedlings treated with the bacterial agent of the present invention increased by 220.39% compared with the control group, and the number of new leaves increased by 244.67%. The Velez subtilis microcapsule bacterial agent of the present invention not only increases the growth of plants, but also significantly increases the number of new leaves, and has a good 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 2The antibacterial effect of different nanocarbon concentrations on Bacillus Velez microcapsules, A is the result of plate confrontation, B is a schematic diagram of the diameter of the inhibition zone (different lowercase letters indicate significant differences at the 0.01 level);

[0043] Figure 3 The control effect of the fungicide on citrus leaf Huanglongbing;

[0044] Figure 4 The antibacterial effect of the microbial agent on various pathogens, A is the plate confrontation result, and B is a schematic diagram of the diameter of the pathogen colony;

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

[0046] Figure 6 The effect of the bacterial agent on promoting the growth of orange seedlings. A is a schematic diagram of the average growth amount, and B is a schematic diagram of the number of newly grown leaves. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

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

[0050] The solid LB 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 7.0, sterilization conditions: 121°C, 100 kPa, 20 min;

[0051] The liquid LB medium used was: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, pH was adjusted to about 7.0, and sterilized at 121°C, 100 kPa, for 20 min;

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

[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, pH value was adjusted to about 7.0, and sterilized at 121°C, 100 kPa, for 20 min.

[0054] Example 1

[0055] Take out the stored Bacillus Velezii E17-23 from -80℃, streak it on LB plate, and culture it at 37℃ for 48h. Pick a single colony and culture it in LB liquid medium at 37℃ and 200r / min. -1 Incubate in a shaker for 24 hours. Pipette 1 mL of bacterial solution into a flask containing 40 mL of LB liquid medium and culture at 37°C, 200 r·min -1 The culture was shaken in a shaker for 24 h, and the obtained bacterial solution was adjusted by spectrophotometer to OD 600 To about 1.0 (concentration of 10 8 ~10 9 CFU / mL) for later use.

[0056] Nanocarbon (particle size of 10-100 nm) was added to LB liquid culture medium to prepare culture medium with nanocarbon content of 0.1% and 0.5%, respectively. The obtained bacterial liquid was inoculated into culture medium with different nanocarbon content at 1%, and the culture medium was incubated at 37℃ and 200 r·min -1 Incubate the culture in a shaker for 12 h and set aside.

[0057] Example 2

[0058] Prepare 2% sodium alginate and 3% calcium chloride solution for later use. Inoculate the biocontrol bacteria into LB medium and shake overnight. Use LB medium to adjust the bacterial solution to OD 600 About 1.0 (concentration 10 8 ~10 9 CFU / mL). Centrifuge at 6000 rpm for 5 min, discard the supernatant, resuspend with sterile water and centrifuge three times to wash the cells. Resuspend the cells with an equal volume of sterile water to prepare a bacterial suspension for later use (concentration of 10 8 ~10 9 CFU / mL). Mix the mixture in a ratio of 2% sodium alginate: bacterial suspension = 19:1, add nanocarbon sol to the mixed solution, so that the final mixed solution contains 0.1% nanocarbon sol, stir evenly with a magnetic stirrer at 400rpm, and let it stand until there are no bubbles. Use a disposable sterile syringe to absorb the above mixture, and slowly add it dropwise to a 3% calcium chloride solution from a certain height (about 10cm from the liquid surface), stirring with a magnetic stirrer at 400rpm. After reacting for 3h, sieve out the microspheres and wash the surface with sterile water. Wait for the microcapsule bacterial agent to dry and set aside.

[0059] Example 3

[0060] Prepare 3% sodium alginate and 5% calcium chloride solution for later use. Inoculate the biocontrol bacteria into LB medium and shake overnight. Use LB medium to adjust the bacterial solution to OD 600 About 1.0 (concentration 10 8 ~10 9 CFU / mL). Centrifuge at 6000rpm for 5min, discard the supernatant, resuspend with sterile water and centrifuge 3 times to wash the bacteria. Resuspend the bacteria with an equal volume of sterile water to prepare a bacterial suspension for use. Mix according to the ratio of 3% sodium alginate: bacterial suspension = 1:1, add nanocarbon sol to the mixed solution, so that the final mixed solution contains 0.1% nanocarbon sol, stir evenly with a magnetic stirrer at 300rpm, and let it stand until there are no bubbles. Use a disposable sterile syringe to absorb the above mixture, and slowly add it dropwise to 5% calcium chloride solution from a certain height (about 10cm from the liquid surface), stirring with a magnetic stirrer at 400rpm during the reaction. After reacting for 3h, sieve out the microspheres and wash the surface with sterile water. Wait for the microcapsule bacterial agent to dry and set aside.

[0061] Comparative Example 1

[0062] The technical scheme of Comparative Example 1 is similar to that of Example 1, except that the culture medium in Comparative Example 1 is configured with nanocarbon concentrations of 0%, 1.0%, 1.5%, and 2.0%, respectively.

[0063] Detection Methods

[0064] (1) Effect of nanocarbon concentration on Bacillus velezensis E17-23

[0065] The OD of Example 1 and Comparative Example 1 was measured by spectrophotometer. 600 value;

[0066] Velez Bacillus E17-23 loading amount: Prepare 0.6% sodium citrate solution and sterilize for later use. Weigh 0.1g of each microcapsule prepared in the above article and place it in a centrifuge tube, add 1mL of 0.6% sodium citrate solution, and dissolve at 200rpm for 1h. The colony concentration was determined by the dilution coating method.

[0067] (2) Inhibition zone test

[0068] Heat the NA solid culture medium, wait until the NA culture medium cools to about 40°C, add the pathogenic bacteria solution that has been shaken in advance at a ratio of 1:100, pour the mixed solution into the culture dish, and prepare the bacterial plate. Punch holes with a 5mm puncher, pick out the culture medium with a toothpick, add 20μL of the bacterial solution of Example 1 and Comparative Example 1 to each well, add 20μL of LB culture medium to the control, blow dry, and seal. Put it in a 28°C incubator for constant temperature culture, observe whether a transparent inhibition zone appears after 24h, and record the results.

[0069] (3) Control effect of composite bacterial agent on citrus leaf Huanglongbing

[0070] The citrus half-leaf method was used to determine the control effect of biocontrol bacteria on citrus Huanglongbing. Citrus leaves with symptoms, similar size and other morphological characteristics were collected and brought back to the laboratory and stored at 4℃ for use.

[0071] The leaves were cut into two halves perpendicular to the veins. One half was placed in sterile water, while the other half was placed in sterile water containing 10 microcapsule inoculants. Each treatment included 6 leaves, and the experiment was repeated 3 times. After standing at room temperature for 24 hours, the plant genomic DNA extraction kit (axygen) was used to extract the midrib DNA for RT-PCR.

[0072] Control effect = (Huanglongbing bacteria content in the control group - Huanglongbing bacteria content in the treatment group) / Huanglongbing bacteria content in the control group (4) Antibacterial effect of composite bacterial agent on various pathogens

[0073] Prepare a PDA culture medium plate, inoculate the pathogen cake (5mm in diameter) in the center of the plate. With this as the center, place the mild bacterial agent at the midpoint of the four sides of the square 2.5cm away from the center according to the square layout. Take LB as the control, culture at a constant temperature of 28℃, and use the cross method to measure the colony diameter after 5 days, measure the distance between the edge of the endophytic fungal colony and the edge of the pathogen colony, and observe whether the pathogen colony forms an isolation zone. Repeat 3 times for each treatment.

[0074] (5) Citrus (orange) seedling growth promotion experiment

[0075] Prepare test plants, take orange seedlings of similar age and growth conditions, rinse with clean water, plant them in a seedling cup filled with sterilized nutrient soil, and place them in an artificial climate incubator with a light intensity of 16 hours and a temperature of 25°C. After 7 days of routine management, inoculate with bacterial agents. Prepare a composite bacterial agent, 6 per citrus seedling, and evenly spread it 1 to 2 cm below the ground surface around the root system of the plant. Control: sterile water. Regularly measure the growth indicators of citrus seedlings.

[0076] Analysis

[0077] (1) Effects of different nanocarbon concentrations on the growth of Bacillus velezensis E17-23

[0078] It can be seen from Table 1 that when the nanocarbon concentration is 0.1% and 0.5%, OD 600 The values ​​were 0.96±0.02 and 1.07±0.05 respectively. Figure 1Analysis shows that when the concentration of nanocarbon is 0.1% and 0.5%, nanocarbon has the least effect on Bacillus Velez E17-23. Too high a concentration of nanocarbon may lead to changes in the local oxidative environment, increase the generation of reactive oxygen species in bacterial cells, and cause cell damage and death; it may also cause damage to the cell membrane, affect the exchange of substances in the cell, and inhibit the growth of bacteria.

[0079] Table 1 Effects of different nanocarbon concentrations on the growth of Bacillus velezensis E17-23

[0080] Nanocarbon concentration <![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 nanocarbon also affected the survival rate of Bacillus Velez E17-23 in the inoculum. As shown in Table 2, when 0.1% and 0.5% nanocarbon were added, the survival rate of strain E17-23 within 7 days increased by 6.07% and 21.87% compared with the absence of nanocarbon. The addition of nanocarbon greatly improved the storage period of the inoculum.

[0082] Table 2 Effect of nanocarbon on the survival rate of strain E17-23 in bacterial agent

[0083]

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

[0085] As shown in Table 3, at 0.1% nanocarbon concentration, the loading amount of Bacillus Velezii E17-23 per microcapsule was the highest, reaching (1.39±0.36)×10 8 CFU / g, when the concentration was 0.5%, the loading capacity decreased slightly, but still reached (1.34±0.27)×10 8 CFU / g, indicating that nanocarbon within this concentration range can effectively improve the encapsulation efficiency of microcapsules.

[0086] When the nanocarbon concentration was 1.0%, the loading amount decreased to (1.31±0.19)×10 8 CFU / g, even lower than the control group without nanocarbon addition (8.23±0.75)×10 7 CFU / g. This may be because the high concentration of nanocarbon leads to excessive density of the microcapsule structure or affects the survival of bacteria.

[0087] Table 3 Loading amount of Bacillus Velez E17-23 in unit microcapsules

[0088] Nanocarbon concentration Strain load (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) Effect of different nanocarbon addition amounts on the antibacterial effect of microencapsulated bacterial agents

[0091] Figure 2 A shows the antibacterial effect of different nanocarbon concentrations on Bacillus velez microcapsules. In Example 1, at a nanocarbon concentration of 0.1%, the maximum diameter of the antibacterial zone reached 1.25±0.05cm. Figure 2 B analysis shows that this concentration of nanocarbon has the best effect on the structural optimization of microcapsules and the activity of strains. The antibacterial effect of 0.5% nanocarbon concentration in Example 1 is also very close, with an antibacterial zone diameter of 1.23±0.03cm, indicating that within the range of 0.1% to 0.5%, the addition of nanocarbon maintains a high level of antibacterial effect.

[0092] (4) Effect of bacterial agents on the control of citrus leaf Huanglongbing

[0093] like Figure 3 As shown in the figure, the 16S rDNA copy number of citrus Huanglongbing disease treated with sterile water was (2.68±0.60)×10 8 / g, but it was significantly reduced to (1.13±0.24)×10 8 / g, the control effect reached 57.84%.

[0094] (5) Antibacterial effect of the agent on various pathogens

[0095] like Figure 4 As shown in A, in the control group, all pathogens grew normally, covering the surface of the culture medium and showing typical colony morphology. In the culture dishes treated with the microbial agent, the growth of pathogens was significantly inhibited, and obvious inhibition zones were visible on some culture dishes. Figure 4 B indicates that the fungicide has obvious inhibitory effect on citrus brown spot pathogen, citrus anthracnose pathogen, citrus yellow spot pathogen, banana wilt pathogen and banana long spot pathogen, and the inhibition rate is more than 56%.

[0096] (6) Effect of ultraviolet irradiation on the antibacterial effect of microencapsulated Bacillus velezensis

[0097] The diameter of the inhibition zone of Bacillus Velez E17-23 bacterial liquid against citrus canker bacteria without UV irradiation was 3.08±0.10cm, and the diameter of the inhibition zone after 1 hour of UV irradiation was 2.53±0.06cm. The diameter of the inhibition zone of the microcapsule bacterial agent against citrus canker bacteria without UV irradiation was 1.74±0.09cm, and the diameter of the inhibition zone after 1 hour of UV irradiation was 1.66±0.09cm.

[0098] The antibacterial capacity of Bacillus Velez E17-23 solution and microcapsule bacterial agent before irradiation was set as 100%, and the calculation results were as follows: Figure 5As shown, the antibacterial ability of Bacillus Velez E17-23 culture liquid was significantly reduced after irradiation, only 82.16% of that before irradiation, indicating that the cells were damaged or their activity was reduced due to direct exposure of the bacteria to ultraviolet rays.

[0099] In contrast, the antibacterial ability decreased less after ultraviolet irradiation, which was 95.76% of that before irradiation. Analysis showed no significant difference from that before irradiation, indicating that the microcapsules protected the strain and reduced the direct damage of ultraviolet rays to the Bacillus Velez E17-23 bacteria.

[0100] (7) The effect of microcapsule of Bacillus Velez on the growth promotion of citrus (orange) seedlings

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

[0102] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled 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 list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A Bacillus Velez nanomaterial composite bacterial agent, characterized in that: The invention is prepared from Bacillus Velez, sodium alginate, calcium chloride and nano-carbon.

2. The Bacillus Velez nanomaterial composite bacterial agent according to claim 1, characterized in that: The Bacillus Velez is Bacillus Velez E17-23, and its deposit number is GDMCC No.64580.

3. A method for preparing the Bacillus Velez nanomaterial composite bacterial agent according to any one of claims 1 to 2, characterized in that: Prepared by the following steps: S1: preparing a bacterial suspension of Bacillus Velez; S2: After mixing sodium alginate and bacterial suspension, add nanocarbon and stir to obtain a mixed solution; S3: adding the mixed solution into the stirred calcium chloride solution, and obtaining the Bacillus Velez nanomaterial composite bacterial agent after reaction.

4. The method for preparing the Bacillus Velez nanomaterial composite bacterial agent according to claim 3, characterized in that: The stirring speed in S2 and S3 is 300-500 rpm; the reaction time in S3 is not less than 3 hours.

5. An application of the Bacillus Velez nanomaterial composite bacterial agent according to any one of claims 1 to 2, characterized in that: Used for preparing preparations for preventing and controlling plant diseases caused by citrus brown spot pathogen, citrus anthracnose pathogen, citrus yellow spot pathogen, banana wilt pathogen, and banana long spot pathogen, or preparations for promoting plant growth.

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