Preparation method and application of biochar-based microbial agent

Abstract

The invention provides a preparation method and application of a biochar-based microbial inoculant, the biochar-based microbial inoculant is prepared from bacillus megatherium L2 with the preservation number of CCTCC NO: M2012381 and rice hull biochar, and the preparation method of the biochar-based microbial inoculant comprises the steps of bacillus megatherium L2 seed liquid culture and liquid fermentation, biochar proportion mixing adsorption, standing and drying. The biochar-based microbial agent prepared by the invention has a good effect of preventing and treating the tea tree anthracnose, effectively reduces the infection ability of tea tree anthracnose related pathogenic bacteria, can also effectively recruit beneficial microorganisms such as chloromentholum, actinomycelum and thick-wall mycolum in soil, further improves the prevention and treatment effect on the tea tree pathogenic bacteria, and has a good application prospect in the field of prevention and treatment of tea tree anthracnose. The method has huge application potential in protection and prevention of the tea trees and is beneficial to good growth of the tea trees.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microbial control, and specifically relates to a preparation method and application of a biochar-based microbial agent. Background Art

[0002] Tea (Camellia sinensis) is widely consumed because it is rich in flavonoids (catechins), purine alkaloids (theobromine and caffeine) and l-theanine ketones. However, with the continuous expansion of tea planting, the occurrence of plant diseases and insect pests is becoming more and more serious, seriously affecting the yield and quality of tea. In particular, tea anthracnose and tea cake disease are the main diseases of tea leaves. After being infected, the tea leaves are damaged, causing serious economic losses to tea production. For decades, the prevention and control of plant diseases such as tea cake disease and anthracnose in agricultural production mainly rely on chemical pesticides. However, the increasing problem of fungicide resistance and the public's concern about the harmful effects of chemicals have prompted researchers to explore and develop environmentally friendly chemical substitutes or supplements. Among them, antimicrobial active molecules and antagonistic microorganisms are widely used in pest control research. Therefore, biological control has attracted more and more attention due to its good biological control effect, non-toxic and harmless, no pollution, and not easy to cause pests and diseases to develop resistance, and has become an important means of preventing and controlling tea diseases. Bacillus megaterium L2 is a strain with independent intellectual property rights in our laboratory. It was obtained from the rhizosphere of tobacco and has a strong inhibitory effect on a variety of pathogenic fungi and bacteria. For example, it has a preventive and therapeutic effect on plant diseases such as konjac soft rot, Ralstonia solanacearum, Agrobacterium tumefaciens and Sclerotium uniformis. It is considered to be a biocontrol strain with great research value. Biochar is an environmentally friendly material with broad application prospects. It can be used as a soil conditioner to promote crop growth and increase yields by increasing soil organic matter content, improving soil structure, and improving soil water and fertilizer retention capacity. At the same time, in the development of slow-release fertilizers, biochar can effectively reduce the loss and waste of chemical fertilizers and improve fertilizer utilization by adsorbing and slowly releasing nutrients. Studies have shown that by preparing microbial agents with biochar and Bacillus bacterial liquid, compared with microbial liquid alone, biochar-based microbial agents can more efficiently improve the plant growth promotion and soil pollution remediation effects of bacteria. Therefore, mixing biochar with biocontrol microbial liquid to prepare microbial agents has broad application prospects in the agricultural field and plant disease control. Summary of the invention

[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a preparation method and application of a biochar-based microbial agent, wherein the biochar-based microbial agent is made of Bacillus megaterium L2 with a deposit number of CCTCC NO: M2012381 and rice husk biochar, and the preparation method thereof includes Bacillus megaterium L2 seed liquid culture and liquid fermentation, biochar proportional adsorption and standing, and drying. The biochar-based microbial agent prepared by the present invention has a good effect on the prevention and treatment of tea anthracnose, effectively reduces the infection ability of pathogens related to tea anthracnose, and can also effectively recruit beneficial microorganisms such as Chloroflexi, Actinomycetes and Firmicutes in the soil, further improving the prevention and treatment effect of tea pathogens, and has great application potential in the protection and prevention of tea trees, which is conducive to the good growth of tea trees.

[0004] Technical solution: A method for preparing a biochar-based microbial agent, comprising the following steps: S1. Bacillus megaterium L2 was subjected to seed culture and liquid fermentation to obtain Bacillus megaterium L2 fermentation broth; S2. The fermentation liquid of Bacillus megaterium L2 and the biochar were mixed evenly and allowed to stand at room temperature for 7 days to obtain a base material; S3. The base material is dried at 40-55°C for 10-14 hours to obtain a biochar-based microbial agent. Furthermore, the deposit number of Bacillus megaterium L2 in step S1 is CCTCC NO: M2012381, the deposit date is September 26, 2012, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Furthermore, the method for culturing the seed liquid in step S1 is: picking Bacillus megaterium L2 and streaking it into LB solid culture medium, culturing it at 37° C. for 24 hours, then picking a single colony and inoculating it into LB liquid culture medium, culturing it at 37° C. and 180-220 rpm for 12-14 hours. Furthermore, the method of liquid fermentation culture in step S1 is: the seed liquid is centrifuged to obtain bacterial cells and inoculated into liquid culture medium, and cultured at 34°C and 200-220rpm for 20h until the bacterial concentration is (3.0-4.0)×10 9 CFU / mL. Furthermore, the liquid culture medium is composed of 5-25 g / L glucose, 5-25 g / L yeast powder, and 10 g / L NaCl. Furthermore, in step S2, the mass ratio of the Bacillus megaterium L2 fermentation broth to the biochar is (1.75-2.5):1. Application of the biochar-based microbial agent prepared by any of the methods described above in preventing and controlling tea tree diseases. The use of the biochar-based microbial agent described in any of the above items in recruiting beneficial microorganisms in the rhizosphere soil of tea trees. Furthermore, the pathogens of the tea tree diseases include Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2. Beneficial effects: 1. The Bacillus megaterium L2 provided by the present invention has good broad-spectrum antibacterial activity, can effectively prevent and treat tea tree anthracnose, and has good antibacterial activity against plant pathogens such as Clonostachys rosea 7a-w-2 and Irpexlacteus 3a-N-2, the pathogens of tea cake disease, and can be used for biological control of pathogens of plants such as konjac, cabbage, pepper, and tobacco. 2. The biochar-based microbial agent prepared by the present invention can effectively reduce the infection ability of tea tree anthracnose, and the prevention and control rate of tea tree anthracnose in the field reaches 51.76%. It can also effectively recruit microorganisms such as Chloroflexi, Actinomycetes and Firmicutes in the rhizosphere soil of tea trees, and can produce a large number of secondary metabolites with antibacterial activity, further improving the prevention and control effect of biochar-based microbial agents on tea tree pathogens, and has good application potential and research value. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The antibacterial spectrum of Bacillus megaterium L2 in Example 1 against tea tree pathogens Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2; Figure 2 The results of single factor experiment on liquid fermentation conditions of Bacillus megaterium L2; Figure 3 The adsorption capacity of bamboo charcoal, rice husk charcoal, corn straw charcoal and wood charcoal; Figure 4 The effective viable bacteria count of bamboo charcoal, rice husk charcoal, corn straw charcoal and wood charcoal; Figure 5 The effective viable counts of Examples 2-5 and Comparative Example 1; Figure 6 The control effects of Example 2, Comparative Example 2 and Comparative Example 3 on tea tree diseases, wherein A represents the incidence rate and B represents the disease index; Figure 7 The relative abundance of rhizosphere soil microorganisms of tea plants in Example 2, Comparative Example 2 and Comparative Example 3; Figure 8 The relative abundance of Bacillus in the rhizosphere soil of tea trees in Example 2, Comparative Example 2 and Comparative Example 3. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: The bacterial species used in the present invention: Bacillus megaterium L2 (deposit number: CCTCCNO: M2012381, deposit date: September 26, 2012, deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province), Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2 were isolated, identified and preserved by the Biotechnology Laboratory of Guizhou University. Example 1 Verification test of antibacterial activity of Bacillus megaterium L2 against tea plant pathogens The plate control method was used to evaluate the effect of Bacillus megaterium L2 on the growth of pathogenic fungi, as follows: (1) Selection of tea plant pathogens: The tested pathogens were Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2; (2) Preparation of PDA solid medium: weigh 200 g of peeled potatoes, add 1000 mL of distilled water and mix well, boil over low heat for 30 min and filter through double-layer gauze, then add 20 g of glucose and 20 g of agar, make up to 1000 mL, sterilize under high pressure at 121°C for 20 min, and set aside; (3) Prepare LB medium (1 L): weigh 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and 20 g agar, adjust the pH to 7.0 with NaOH, and sterilize by autoclave at 121°C for 20 min. (4) Preparation of Bacillus megaterium L2 bacterial solution: Select the preserved Bacillus megaterium L2 and inoculate it on LB solid culture medium using the three-zone streak method and culture it at 37°C for 24 hours. Then select a single bacterial colony of Bacillus megaterium L2 and inoculate it into 100 mL LB liquid culture medium. Culture it at 37°C and 200 rpm for 12 hours to obtain seed solution. Take 1 mL of seed solution and centrifuge it at 10000 rpm for 1 minute to discard the supernatant, retain the bacteria, and then take 50 μL of sterile water to resuspend the bacteria. Shake it thoroughly and adjust the concentration of the bacterial resuspension to OD 600 =0.6, standby; (5) Antibacterial activity test: Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2 were activated and cultured in PDA solid medium. The fungal cake (diameter 0.6 cm) was placed in the center of the PDA plate, and a Bacillus megaterium resuspension solution (OD 600 =0.6), the group without Bacillus megaterium L2 was used as the control group, each treatment was repeated three times, and the culture was carried out in a 28°C incubator until the pathogen colonies in the control group filled the culture dish, the diameters of the pathogen colonies and the control group were measured, recorded and photographed, and the inhibition rate was calculated. The calculation formula is as follows: Depend on Figure 1 It can be seen that the Bacillus megaterium L2 in Example 1 has good antibacterial activity against Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2, and the antibacterial rates are 78.57% and 68.76%, respectively. Example 2 A method for preparing a biochar-based microbial agent comprises the following steps: S1. Pick Bacillus megaterium L2 and streak it on LB solid medium, culture it at 37°C for 24h, then pick a single colony and inoculate it into LB liquid medium, culture it at 37°C and 200rpm for 12h to obtain seed solution; S2. Weigh 20 g glucose, 21 g yeast powder and 10 g NaCl, add distilled water to make up to 1000 mL, and sterilize under high pressure at 121°C for 20 min. S3. Take 6 mL of seed solution and inoculate it into 100 mL of liquid culture medium. Cultivate it at 34°C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL, and the fermentation broth of Bacillus megaterium L2 was prepared; S4. Take 22.5g of Bacillus megaterium L2 fermentation liquid and 10g of rice husk charcoal, mix them evenly, and let them stand at room temperature for 7 days to obtain a base material; S5. The base material is dried in a 45°C electric constant temperature drying oven for 12 hours to obtain a Bacillus megaterium L2 biochar-based microbial agent, recorded as 2.25:1. Example 3 A method for preparing a biochar-based microbial agent comprises the following steps: S1. Pick Bacillus megaterium L2 and streak it on LB solid medium, culture it at 37°C for 24h, then pick a single colony and inoculate it into LB liquid medium, culture it at 37°C and 200rpm for 12h to obtain seed solution; S2. Weigh 20 g glucose, 21 g yeast powder and 10 g NaCl, add distilled water to make up to 1000 mL, and sterilize under high pressure at 121°C for 20 min. S3. Take 6 mL of seed solution and inoculate it into 100 mL of liquid culture medium. Cultivate it at 34°C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL, and the fermentation broth of Bacillus megaterium L2 was prepared; S4. Take 25g of Bacillus megaterium L2 fermentation broth and 10g of rice husk charcoal and mix well and let stand at room temperature for 7 days to obtain a base material; S5. The base material was dried in a 45°C electric constant temperature drying oven for 12 hours to obtain Bacillus megaterium L2 biochar-based microbial agent, recorded as 2.5:1. Example 4 A method for preparing a biochar-based microbial agent comprises the following steps: S1. Pick Bacillus megaterium L2 and streak it on LB solid medium, culture it at 37°C for 24h, then pick a single colony and inoculate it into LB liquid medium, culture it at 37°C and 200rpm for 12h to obtain seed solution; S2. Weigh 20 g glucose, 21 g yeast powder and 10 g NaCl, add distilled water to make up to 1000 mL, and sterilize under high pressure at 121°C for 20 min. S3. Take 6 mL of seed solution and inoculate it into 100 mL of liquid culture medium. Cultivate it at 34°C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL, and the fermentation broth of Bacillus megaterium L2 was prepared; S4. Take 20g of Bacillus megaterium L2 fermentation broth and 10g of rice husk charcoal and mix well, and let stand at room temperature for 7 days to obtain a base material; S5. The base material was dried in an electric constant temperature drying oven at 45°C for 12 hours to obtain Bacillus megaterium L2 biochar-based microbial agent, recorded as 2:1. Example 5 A method for preparing a biochar-based microbial agent comprises the following steps: S1. Pick Bacillus megaterium L2 and streak it on LB solid medium, culture it at 37°C for 24h, then pick a single colony and inoculate it into LB liquid medium, culture it at 37°C and 200rpm for 12h to obtain seed solution; S2. Weigh 20 g glucose, 21 g yeast powder and 10 g NaCl, add distilled water to make up to 1000 mL, and sterilize under high pressure at 121°C for 20 min. S3. Take 6 mL of seed solution and inoculate it into 100 mL of liquid culture medium. Cultivate it at 34°C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL, and the fermentation broth of Bacillus megaterium L2 was prepared; S4. Take 17.5g of Bacillus megaterium L2 fermentation liquid and 10g of rice husk charcoal, mix them evenly, and let them stand at room temperature for 7 days to obtain a base material; S5. The base material is dried in a 45°C electric constant temperature drying oven for 12 hours to obtain a Bacillus megaterium L2 biochar-based microbial agent, recorded as 1.75:1. Comparative Example 1 The difference between this comparative example and Example 2 is that the ratio of Bacillus megaterium L2 and rice husk charcoal is different. A method for preparing a biochar-based microbial agent comprises the following steps: S1. Pick Bacillus megaterium L2 and streak it on LB solid medium, culture it at 37°C for 24h, then pick a single colony and inoculate it into LB liquid medium, culture it at 37°C and 200rpm for 12h to obtain seed solution; S2. Weigh 20 g glucose, 21 g yeast powder and 10 g NaCl, add distilled water to make up to 1000 mL, and sterilize under high pressure at 121°C for 20 min. S3. Take 6 mL of seed solution and inoculate it into 100 mL of liquid culture medium. Cultivate it at 34°C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL, and the fermentation broth of Bacillus megaterium L2 was prepared; S4. Take 15g of Bacillus megaterium L2 fermentation broth and 10g of rice husk charcoal and mix well, and let stand at room temperature for 7 days to obtain a base material; S5. The base material was dried in an electric constant temperature drying oven at 45°C for 12 hours to obtain Bacillus megaterium L2 biochar-based microbial agent, recorded as 1.5:1. Comparative Example 2 The difference between this comparative example and Example 2 is that rice husk charcoal is not added. A method for preparing Bacillus megaterium L2 fermentation broth comprises the following steps: S1. Pick Bacillus megaterium L2 and streak it on LB solid medium, culture it at 37°C for 24h, then pick a single colony and inoculate it into LB liquid medium, culture it at 37°C and 200rpm for 12h to obtain seed solution; S2. Weigh 20 g glucose, 21 g yeast powder and 10 g NaCl, add distilled water to make up to 1000 mL, and sterilize under high pressure at 121°C for 20 min. S3. Take 6 mL of seed solution and inoculate it into 100 mL of liquid culture medium. Cultivate it at 34°C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL, and the fermentation broth of Bacillus megaterium L2 was obtained. Comparative Example 3 The difference between this comparative example and Example 2 is that no Bacillus megaterium L2 fermentation liquid is added, and only rice husk charcoal is used. Comparative Example 4 The difference between this comparative example and Example 2 is that Bacillus megaterium L2 is replaced by commercially available Bacillus megaterium. A method for preparing a biochar-based microbial agent comprises the following steps: S1. Pick commercially available Bacillus megaterium and streak it on LB solid medium, culture it at 37°C for 24h, then pick a single colony and inoculate it into LB liquid medium, culture it at 37°C and 200rpm for 12h to prepare seed solution; S2. Weigh 20g glucose, 21g yeast powder and 10g NaCl, add distilled water to 1000mL, and sterilize under high pressure at 121℃ for 20min; S3. Take 6 mL of seed solution and inoculate it into 100 mL of liquid culture medium. Cultivate it at 34°C and 203 npm for 20 h until the concentration reaches 3.35 × 10 9 CFUmL, commercially available Bacillus megaterium fermentation broth was prepared; S4. Take 22.5g of commercially available Bacillus megaterium fermentation liquid and 10g of rice husk charcoal and mix them evenly, let them stand at room temperature for 7 days to obtain a base material; S5. The base material is dried in a 45°C electric constant temperature drying oven for 12h to obtain a commercially available Bacillus megaterium biochar-based microbial agent. Performance Testing (1) Single factor experiment optimization of liquid fermentation conditions of Bacillus megaterium L2 Using LB medium as the basic medium, the inoculation volume of 2%, the fermentation temperature of 28°C, the rotation speed of 150rpm, and the culture time of 20h as the basic culture conditions, and the number of viable bacteria in the bacterial liquid as the evaluation index, the single factor optimization of the liquid fermentation conditions of Bacillus megaterium L2 was carried out, as follows: Single factor experimental setting: carbon source type (maltose, lactose, sucrose, soluble starch, glucose), carbon source addition amount (5g / L, 10g / L, 15g / L, 20g / L, 25g / L), nitrogen source type (urea, tryptone, peptone, yeast powder, ammonium sulfate), nitrogen source addition amount (5g / L, 10g / L, 15g / L, 20g / L, 25g / L), inoculation amount (1%, 2%, 4%, 6%, 8%, 10%), fermentation temperature (28℃, 31℃, 34℃, 37℃, 40℃) and rotation speed (140rpm, 160rpm, 180rpm, 200rpm, 220rpm); Determination of viable bacterial count: dilute the bacterial solution in a gradient manner, and select a dilution of 10 -5 , 10 -6 , 10 -7 For the three dilution concentrations, 0.2 mL of the dilution solution was taken and placed into LB solid culture medium for plate spreading. The number of colonies was counted after incubation at 28°C for 24 h. Depend on Figure 2 It can be seen that the types of carbon sources: Liquid fermentation conditions were optimized. First, five carbon sources, maltose, lactose, sucrose, soluble starch and glucose, were screened. Among the five carbon sources, glucose had the best utilization effect, and the number of viable bacteria could reach 1.25×10 8 CFU / mL, soluble starch utilization was the worst, with a viable bacterial count of 1.2×10 7 CFU / mL. The effects of the five carbon sources on the number of viable bacteria in the fermentation broth were significantly different (p<0.05), so glucose was selected as the carbon source for the addition amount screening test. Carbon source addition amount: Five addition amount gradients of 5-25g / L were selected for screening. Among the five carbon source addition amounts, 20g / L had the best fermentation effect, and the number of viable bacteria could reach 1.15×10 9 CFU / mL, the fermentation effect of 5g / L was the worst, with a viable bacterial count of 4.8×10 8 The effects of the five carbon source additions on the number of viable bacteria in the fermentation broth were significantly different (p<0.05), so 20 g / L was selected as the optimal carbon source addition. Types of nitrogen sources: Urea, tryptone, peptone, yeast powder, and ammonium sulfate were screened. Yeast powder had the best utilization effect among the five nitrogen sources, and the number of viable bacteria could reach 1.85×10 8 CFU / mL, urea utilization was the worst, with a viable bacterial count of 5.65×10 7 The effects of the five nitrogen sources on the number of viable bacteria in the fermentation broth were significantly different (p<0.05), so yeast powder was selected as the optimal nitrogen source for addition screening test. Nitrogen source addition amount: Five addition amount gradients of 5-25g / L were selected for screening. Among the five nitrogen source addition amounts, 20g / L had the best fermentation effect, and the number of viable bacteria could reach 3.2×10 8 CFU / mL, the fermentation effect of 5g / L was the worst, with a viable bacterial count of 1.4×10 8 The effects of the addition of five carbon sources on the number of viable bacteria in the fermentation broth were significantly different (p<0.05), so 20 g / L was selected as the optimal nitrogen source addition amount. Inoculation volume of bacterial solution: Six inoculation volumes of 1%, 2%, 4%, 6%, 8%, and 10% were selected for screening. The results showed that the fermentation effect was best with an inoculation volume of 6%, and the number of viable bacteria was 2.5×10 8 CFU / mL, 1% had the worst fermentation effect, with a viable bacterial count of 1.47×10 8 The effects of the six bacterial inoculum sizes on the number of viable bacteria in the fermentation broth were significantly different (p<0.05), so 6% was selected as the optimal bacterial inoculum size. Fermentation temperature: Five fermentation temperatures of 28°C, 31°C, 34°C, 37°C, and 40°C were selected for screening. The results showed that the fermentation effect at 31°C was the best, with a viable count of 1.7×10 8 CFU / mL, the fermentation effect at 40°C was the worst, with a viable count of 7.1×10 7 The effects of the five fermentation temperatures on the number of viable bacteria in the fermentation broth were significantly different (p<0.05), so 31°C was selected as the optimal fermentation temperature. Rotation speed: Five rotation speeds of 140rpm, 160rpm, 180rpm, 200rpm and 220rpm were selected for screening. The experimental results showed that the fermentation effect at 200rpm was the best, with a viable bacterial count of 1.4×10 9 CFU / mL, the fermentation effect at 140 rpm was the worst, with a viable bacterial count of 1.5×10 8 The effects of the five rotation speeds on the number of viable bacteria in the fermentation broth were significantly different (p<0.05), so 200 rpm was selected as the optimal rotation speed. (2) Optimization of liquid fermentation conditions of Bacillus megaterium L2 by Plackett-Burman test Based on the optimization of single factor experiment on liquid fermentation conditions of Bacillus megaterium L2, five factors were selected to design the Plackett-Burman experiment, as follows: Plackett-Burman experimental factor setting: the main effect factors were screened from A (carbon source addition amount), B (nitrogen source addition amount), C (inoculation amount), D (rotation speed), and E (fermentation temperature), and an experimental design of N=12 was selected. Each factor selected two levels: high level "1" and low level "-1". The number of viable bacteria was used as the response value, and the functional relationship between the response value and the independent variable was established to determine the optimal experimental conditions. The Plackett-Burman experimental factor levels are shown in Table 1. Table 1 Plackett-Burman test factor level table Table 2 Plackett-Burman test design table and its response values As shown in Table 2, the linear regression equation model obtained by fitting the results of the Plackett-Burman test is: Y = 0.9654-0.0053A + 0.6093B-0.0717C-0.3856D + 0.3059E (p = 0.0031), indicating that the regression equation is extremely significant, and B nitrogen source addition p = 0.0007, D speed p = 0.0066, E temperature p = 0.0179 are all less than 0.05, and the coefficients are all positive, indicating that the nitrogen source addition, speed and temperature have a significant correlation with the number of viable bacteria in the bacterial liquid, but the p values ​​of A carbon source addition and C inoculum are both greater than 0.05, indicating that the carbon source addition and inoculum have no significant effect on the number of bacteria in the fermentation liquid of Bacillus megaterium L2. It can be seen that the nitrogen source addition, speed and temperature gradually increase, and the levels of other factors remain unchanged, which can increase the number of viable bacteria in the fermentation liquid of Bacillus megaterium L2. (3) Optimization of the steepest ramp test for liquid fermentation of Bacillus megaterium L2 Based on the results of single factor test and Plackett-Burman test on liquid fermentation conditions of Bacillus megaterium L2, three main effect factors, namely nitrogen source addition, rotation speed and temperature, which have a significant impact on the number of viable bacteria in liquid fermentation of Bacillus megaterium, were selected for the steepest climbing test. The design table of the steepest climbing test factors is shown in Table 3. Table 3 Design table of steepest climbing test factors serial number B Nitrogen source addition amount (g / L) D Speed ​​(rpm) Temperature(℃) 1 12 170 26 2 15 180 28 3 18 190 30 4 21 200 32 5 24 210 34 6 27 220 36 Table 4 Steepest climbing test design and results Test No. B: Yeast powder content D: Speed E: Temperature <![CDATA[Viable cell count (×10 9 CFU / mL)]]> 1 12 170 26 2.59 2 15 180 28 2.56 3 18 190 30 2.06 4 21 200 32 3.21 5 24 210 34 2.32 6 27 220 36 2.97 As shown in Table 4, when the nitrogen source addition amount (yeast powder content) is 21 g / L, the rotation speed is 200 rpm, and the temperature is 32 °C, the number of viable bacteria in the bacterial solution is the largest, reaching 3.213 × 10 9 CFU / mL. (4) Optimization of liquid fermentation conditions of Bacillus megaterium L2 by response surface experiment Based on the results of Plackett-Burman test and steepest climbing test, the Box-Behnken experimental design method was used to optimize the liquid fermentation conditions of Bacillus megaterium L2. The optimal test points of nitrogen source addition, temperature and rotation speed in the steepest climbing test results were selected as the "0" level, and the left and right selections of the optimal test points in the steepest climbing test results were selected as the "-1" and "1" levels. The number of viable bacteria was used as the response value. The Box-Behnken experimental factor levels are shown in Table 5. 17 response analysis experiments were designed by Design Expert software. The experimental design and results are shown in Table 6. Table 5 Box-Behnken test factor level table Table 6 Box-Behnken experimental design and results Table 7 Analysis of Box-Behnken test results The Design Expert software was used to perform a multivariate regression fitting analysis on the data in Table 6, and the regression equation with the number of viable bacteria as the response value was obtained: Y = 3.27 + 0.15A + 0.2359B + 0.0091C + 0.2392AB + 0.1043AC + 0.3425BC - 0.7625A 2 -0.9558B 2 -0.2157C 2 ; As shown in Table 7, the p of the regression equation model of the response surface optimization result is <0.0001, indicating that the equation has a good fit, and the lack of fit term p = 0.1322> 0.05, indicating that the model is not lack of fit and the selection is reasonable; at the same time, A, B, AB, BC, A in the regression equation 2 , B 2 , C 2 All of them are significant influencing factors, and the determination coefficient R 2 =0.9878, R 2 Adj =0.9721, indicating that the model fits well and the model prediction value has a high correlation with the actual value. The Design-Expert 13 software analysis showed that the optimal conditions for liquid fermentation of Bacillus megaterium L2 were 21 g / L nitrogen source addition, 203 rpm rotation speed, and 34 °C temperature. The predicted value of the number of live bacteria in the bacterial liquid was 3.324 × 10 9 CFU / mL, the actual value of the viable bacterial count in the bacterial solution was verified by experiments to be 3.35×10 9CFU / mL, which had no significant difference from the predicted value, indicating that the model had a high degree of fit with the actual value and the optimal conditions for liquid fermentation of Bacillus megaterium L2 were stable and feasible. (5) Screening of biochar carriers for microbial agents Determination of biochar adsorption capacity: bamboo charcoal, rice husk charcoal, corn straw charcoal and charcoal were selected as alternative carriers. 5.00 g of biochar was weighed and put into a sealed bag. 1 mL of water was added to the bag one by one using a pipette. After sufficient stirring, water was added again. During the process of adding water, attention was paid to the morphology of the carrier to ensure that it was moist but no water droplets were left. At the same time, the maximum amount of water absorbed by each carrier was recorded for comparison. Determination of effective viable bacteria count: 8.8 mL of Bacillus megaterium L2 bacterial solution was mixed evenly with 10 g of biochar, placed at room temperature for 7 days, and then placed in a 45°C electric constant temperature drying oven for drying for 12 hours. After the carbon-bacteria complex was fully dried, the effective viable bacteria count in the microbial agent was determined for comparison. Depend on Figure 3 and Figure 4 It can be seen that the adsorption capacity and effective viable bacteria count of rice husk charcoal are higher than those of bamboo charcoal, wood charcoal, and corn straw charcoal. In addition, the effective viable bacteria count of Bacillus megaterium L2 microbial inoculant prepared by rice husk charcoal decreased the least after being placed for 7 days, reaching 3.12×10 9 CFU / g, indicating that rice husk charcoal has good adsorption capacity and has no significant effect on the survival rate of Bacillus megaterium L2, so rice husk charcoal was selected as the carrier of microbial agent. (6) Number of viable cells in Examples 2-5 and Comparative Example 1 Depend on Figure 5 It can be seen that the number of live bacteria in the biochar-based microbial inoculants prepared in Examples 2-5 is significantly higher than that in Comparative Example 1. In particular, when the ratio of Bacillus megaterium L2 bacterial solution to rice husk charcoal is 2.25:1, the effective number of live bacteria in the biochar-based microbial inoculants is the largest, which is 4.18×10 9 CFU / g. (7) Field control efficacy test of Example 2, Comparative Example 2, Comparative Example 3 and Comparative Example 4 The tea garden field prevention effect test was carried out using Example 2, Comparative Example 2 and Comparative Example 3, as follows: The experimental site was selected in Miaozitian area of ​​Bijie City. The experimental tea garden was a densely planted sloping tea garden. The variety was Fuding Dabai, three-year-old tea seedlings. There were four treatments in the experiment (T1: blank treatment, T2: rice husk biochar, T3: biochar-based microbial agent, T4: Bacillus megaterium L2 fermentation liquid, T5: commercially available Bacillus megaterium biochar-based microbial agent). The dosage of T3, T4 and T5 groups was 100g / mu, and the random block arrangement was repeated 3 times, with a total of 15 plots, and the plot area was 30m 2At the same time, 30 days after the application of the pesticide, the disease situation of each treatment tea was investigated. 50 leaves were investigated for each treatment. According to the tea disease classification table (see Table 8) and the calculation formula, the incidence rate, disease index and control effect of anthracnose in each treatment tea were investigated. Table 8 Tea disease classification table Disease level The area of ​​lesions as a percentage of the entire leaf area (%) Level 0 No disease spots Level 1 <5 Level 3 6-25 Level 5 26-50 Level 7 51-75 Level 9 >76 Table 9 Field control effect of biochar-based microbial agents on tea anthracnose Note: Different lowercase letters after the data in the same column indicate significant differences among treatments (p<0.05). From Table 9 and Figure 6 It can be seen that in the treatment area where the biochar-based microbial agent prepared in Example 2 was applied, the incidence rate and disease index of tea anthracnose of tea trees were significantly lower than those in Comparative Example 2 and Comparative Example 3, and the control effect of tea anthracnose was 51.76±0.93%, while the control effect of tea anthracnose using Bacillus megaterium L2 fermentation broth in Comparative Example 2 was 45.65±0.48%. In addition, the control effect of commercially available Bacillus megaterium biochar-based microbial agent on tea anthracnose was 39.86±1.38%, which was significantly lower than that of biochar-based microbial agent and Bacillus megaterium L2 fermentation broth (p<0.05), indicating that the biochar-based microbial agent prepared by compounding Bacillus megaterium L2 with rice husk biochar can improve the control effect of tea anthracnose on tea trees. (8) Microbial diversity of rhizosphere soil of tea trees treated in Example 2, Comparative Example 2 and Comparative Example 3 The four grouping treatment areas set in the field efficacy test (7) were used to collect soil samples on the sunny slope of the tea garden based on the five-point sampling method, which were divided into 0 days, 30 days, and 60 days. The three periods were marked as "F", "S", and "T" respectively. The soil sample sampling method was as follows: the main trunk of the tea tree was taken as the core, and the soil sample was taken at 20 cm. The surface fallen leaves and humus were removed first during sampling. The drilling depth was 20 cm. Each sampling point formed a mixed soil sample. About 100 g of each soil sample was collected. After removing impurities such as sand, gravel, and dead leaves from the soil samples, microbial amplicon sequencing was performed to analyze the microbial diversity in the soil samples. Depend on Figure 7It can be seen that T1 in the figure represents blank treatment, T2 represents rice husk charcoal (Comparative Example 3), T3 represents biochar-based microbial agent (Example 2), and T4 represents Bacillus megaterium L2 fermentation broth (Comparative Example 2). Compared with Comparative Examples 2 and 3, in the area treated with the biochar-based microbial agent prepared in Example 2, the relative abundance of Chloroflexi, Actinobacteriota and Proteobacteria in soil samples ST3 and TT3 was the highest, especially the relative abundance of Actinobacteria in soil sample TT3 reached the maximum level (21.78±2.49%). According to research reports, the predatory Chloroflexi group Herpetosiphon in Chloroflexi exhibited varying degrees of killing effects on a variety of pathogenic bacteria, had the ability to synthesize antibacterial active molecules, and Actinobacteria were able to produce different types of antibacterial secondary metabolites (such as lipopeptides, large inner ring lipids and polyenes) and had broad-spectrum antibacterial activity. also, Figure 8 The results showed that Firmicutes were also significantly enriched in different samples, and further analysis was conducted on the enriched Bacillus microorganisms in different soil samples. Figure 7 It can be seen that among the samples treated for 30 days, Bacillus cellulasensis, Bacillus thermolactis and Bacillustrypoxylicola had the highest relative abundance in ST3, while Bacillus infernus was significantly enriched in TT3 samples, with a relative abundance of 0.01±0.002%. In summary, these Bacillus microorganisms can produce antibacterial metabolites, which have good antibacterial activity against pathogenic fungi and bacteria of tea anthracnose, and can further improve the control effect of biochar-based microbial agents on tea trees. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a biochar-based microbial agent, characterized in that: The following steps are involved: S1. Bacillus megaterium L2 was subjected to seed culture and liquid fermentation to obtain Bacillus megaterium L2 fermentation broth; S2. The fermentation liquid of Bacillus megaterium L2 and the biochar were mixed evenly and allowed to stand at room temperature for 7 days to obtain a base material; S3. The base material is dried at 40-55°C for 10-14 hours to obtain a biochar-based microbial agent.

2. The method for preparing a biochar-based microbial agent according to claim 1, characterized in that: The deposit number of Bacillus megaterium L2 in step S1 is CCTCC NO: M2012381, the deposit date is September 26, 2012, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

3. The method for preparing a biochar-based microbial agent according to claim 1, characterized in that: The method for culturing the seed liquid in step S1 is: picking Bacillus megaterium L2 and streaking it into LB solid culture medium, culturing it at 37° C. for 24 hours, then picking a single colony and inoculating it into LB liquid culture medium, culturing it at 37° C. and 180-220 rpm for 12-14 hours.

4. The method for preparing a biochar-based microbial agent according to claim 1, characterized in that: The method of liquid fermentation culture in step S1 is: the seed liquid is centrifuged to obtain bacterial cells and inoculated into liquid culture medium, and cultured at 34°C and 200-220rpm for 20h until the bacterial concentration is (3.0-4.0)×10 9 CFU / mL.

5. The method for preparing a biochar-based microbial agent according to claim 4, characterized in that: The liquid culture medium is composed of 5-25 g / L glucose, 5-25 g / L yeast powder, and 10 g / L NaCl.

6. The method for preparing a biochar-based microbial agent according to claim 1, characterized in that: In the step S2, the mass ratio of Bacillus megaterium L2 fermentation liquid to biochar is (1.75-2.5):

1.

7. Use of the biochar-based microbial agent prepared according to the method according to any one of claims 1 to 6 in preventing and controlling tea tree diseases.

8. Use of the biochar-based microbial agent according to any one of claims 1 to 6 in recruiting beneficial microorganisms in the rhizosphere soil of tea trees.

9. The use according to claim 7, characterized in that: The pathogenic bacteria of the tea tree diseases include Rhodospirillaria rubrum and Albugo sphaerocephala.

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