Preparation method and application of a biochar-based microbial inoculum

Abstract

The present invention provides a preparation method and application of a biochar-based microbial inoculant. The biochar-based microbial inoculant is made from Bacillus megaterium L2 with the preservation number of CCTCC NO: M2012381 and rice husk biochar. The preparation method includes the cultivation of Bacillus megaterium L2 seed liquid and liquid fermentation, the proportion mixing adsorption and static settling of biochar, and drying. The biochar-based microbial inoculant prepared by the present invention has a good effect on the prevention and control of tea tree anthracnose, effectively reduces the infection ability of tea tree anthracnose-related pathogenic bacteria, and can also effectively recruit beneficial microorganisms such as Chloroflexi, Actinobacteria and Firmicutes in the soil, further improving the prevention and control effect on tea tree pathogenic bacteria. It has great application potential in the protection and prevention of tea trees and is beneficial to the good growth of 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.

[0003] 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.

[0004] 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.

[0005] 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

[0006] Technical problem to be solved: Aiming at the above technical problems, the object of the present invention is to provide a preparation method and application of a biochar-based microbial inoculant. The biochar-based microbial inoculant is made from Bacillus megaterium L2 with a preservation number of CCTCC NO: M2012381 and rice husk biochar. The preparation method includes the seed liquid culture and liquid fermentation of Bacillus megaterium L2, the proportional mixing adsorption and static settlement of biochar, and drying. The biochar-based microbial inoculant prepared by the present invention has a good effect on the prevention and control of tea tree anthracnose, effectively reduces the infection ability of pathogens related to tea tree anthracnose, and can also effectively recruit beneficial microorganisms such as Chloroflexi, Actinobacteria, and Firmicutes in the soil, further improving the prevention and control effect on tea tree pathogens. It has great application potential in the protection and prevention of tea trees and is beneficial to the good growth of tea trees.

[0007] Technical solution: A preparation method of a biochar-based microbial inoculant includes the following steps:

[0008] S1. Bacillus megaterium L2 is subjected to seed liquid culture and liquid fermentation culture to obtain Bacillus megaterium L2 fermentation liquid;

[0009] S2. The Bacillus megaterium L2 fermentation liquid and biochar are mixed evenly and left to stand at room temperature for 7 days to obtain a base material;

[0010] S3. The base material is dried at 40 - 55 °C for 10 - 14 h to obtain the biochar-based microbial inoculant.

[0011] Further, in step S1, the preservation number of Bacillus megaterium L2 is CCTCC NO: M2012381, the preservation date is September 26, 2012, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0012] Further, the method for seed liquid culture in step S1 is: Pick and streak inoculate Bacillus megaterium L2 on an LB solid medium and culture it at 37 °C for 24 h, then pick a single colony and inoculate it into an LB liquid medium and culture it at 37 °C and 180 - 220 rpm for 12 - 14 h.

[0013] Further, the method for liquid fermentation culture in step S1 is: Centrifuge the seed liquid to obtain the cells and inoculate them into a liquid medium, and culture them at 34 °C and 200 - 220 rpm for 20 h until the concentration of the strain is (3.0 - 4.0)×10 9 CFU / mL.

[0014] Further, the composition of the liquid medium is 5 - 25 g / L of glucose, 5 - 25 g / L of yeast powder, and 10 g / L of NaCl.

[0015] Further, in the step S2, the mass ratio of the Bacillus megaterium L2 fermentation broth to the biochar is (1.75 - 2.5):1.

[0016] Application of the biochar-based microbial inoculant prepared by the method described in any one of the above in the prevention and control of tea tree diseases.

[0017] Application of the biochar-based microbial inoculant described in any one of the above in recruiting beneficial microorganisms in the rhizosphere soil of tea trees.

[0018] Further, the pathogenic bacteria of the tea tree diseases include Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2.

[0019] Beneficial effects:

[0020] 1. The Bacillus megaterium L2 provided by the present invention has good broad-spectrum antibacterial properties, can effectively prevent and control tea tree anthracnose, and has good antibacterial activity against plant pathogenic bacteria such as Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2, the pathogenic bacteria of tea blister blight, and can be applied to the biological control of pathogenic bacteria of plants such as konjac, Chinese cabbage, chili peppers, and tobacco.

[0021] 2. The biochar-based microbial inoculant prepared by the present invention can effectively reduce the infection ability of tea tree anthracnose, and the control effect on tea tree anthracnose in the field reaches 51.76%. Moreover, it can effectively recruit microorganisms such as Chloroflexi, Actinobacteria, and Firmicutes in the rhizosphere soil of tea trees, and can produce a large number of secondary metabolites with antibacterial activity, further improving the control effect of the biochar-based microbial inoculant on tea tree pathogenic bacteria, and having good application potential and research value. Description of the drawings

[0022] Figure 1 It is the antibacterial spectrum diagram of Bacillus megaterium L2 against Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2, the pathogenic bacteria of tea trees, in Example 1;

[0023] Figure 2 It is the result of the single-factor experiment on the liquid fermentation conditions of Bacillus megaterium L2;

[0024] Figure 3 It is the adsorption capacity of bamboo charcoal, rice husk charcoal, corn straw charcoal, and charcoal;

[0025] Figure 4 It is the effective viable count of bamboo charcoal, rice husk charcoal, corn straw charcoal, and charcoal;

[0026] Figure 5 The viable count of effective bacteria in Examples 2-5 and Comparative Example 1;

[0027] Figure 6 The control effects of Example 2, Comparative Example 2 and Comparative Example 3 on tea tree diseases, where A represents the incidence rate and B represents the disease index;

[0028] Figure 7 The relative abundances of rhizosphere soil microorganisms of tea trees in Example 2, Comparative Example 2 and Comparative Example 3;

[0029] Figure 8 The relative abundances of Bacillus in the rhizosphere soil of tea trees in Example 2, Comparative Example 2 and Comparative Example 3. Detailed implementation manners

[0030] The present invention will be further described below in conjunction with examples. The following examples are explanations of the present invention and the present invention is not limited to the following examples:

[0031] The bacterial strains used in the present invention: Bacillus megaterium L2 (preservation number: CCTCC NO: M2012381, preservation date: September 26, 2012, preservation 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.

[0032] Example 1

[0033] Verification test on the antibacterial activity of Bacillus megaterium L2 against tea tree pathogens

[0034] The plate control method was used to evaluate the effect of Bacillus megaterium L2 on the growth of pathogenic fungi, specifically as follows:

[0035] (1) Select tea tree pathogens: The tested pathogens are Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2;

[0036] (2) Prepare PDA solid medium: Weigh 200 g of peeled potatoes, add 1000 mL of distilled water and mix evenly. Boil gently for 30 min and filter with double-layer gauze. Then add 20 g of glucose and 20 g of agar, make up the volume to 1000 mL, and autoclave at 121 °C for 20 min for standby;

[0037] (3) Preparation of LB medium (1 L): Weigh 5 g of yeast extract, 10 g of tryptone, 10 g of sodium chloride, and 20 g of agar. Adjust the pH to 7.0 using NaOH, and autoclave at 121 °C for 20 min for later use.

[0038] (4) Preparation of Bacillus megaterium L2 bacterial solution: Pick the preserved Bacillus megaterium L2 and inoculate it onto the LB solid medium by the three-zone streaking method, and culture it at 37 °C for 24 h. Then pick a single colony of Bacillus megaterium L2 and inoculate it into 100 mL of LB liquid medium, and culture it at 37 °C and 200 rpm for 12 h to obtain the seed solution; Pipette 1 mL of the seed solution, centrifuge it at 10000 rpm for 1 min, discard the supernatant, retain the bacterial cells, then take 50 μL of sterile water to resuspend the bacterial cells, shake well, and adjust the bacterial cell suspension to a concentration of OD 600 = 0.6 for later use.

[0039] (5) Antibacterial activity test: Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2 are activated and cultured in the PDA solid medium. Place a pathogen fungal cake (diameter 0.6 cm) in the center of the PDA plate, and inoculate the Bacillus megaterium resuspended solution (OD 600 = 0.6) at a distance of 2 cm from the fungal cake on both the left and right sides of the fungal cake. Use the group without inoculating Bacillus megaterium L2 as the control group. Each treatment is repeated three times, and culture it in an incubator at 28 °C until the pathogen colony in the control group fills the culture dish. Measure the diameter of the pathogen colony and the diameter of the pathogen colony in the control group, record and take pictures, and calculate the antibacterial rate. The calculation formula is as follows:

[0040]

[0041] As Figure 1 can be seen, Bacillus megaterium L2 in Example 1 has good antibacterial activities against Clonostachys rosea 7a-w-2 and Irpex lacteus 3a-N-2, and the antibacterial rates are 78.57% and 68.76% respectively.

[0042] Example 2

[0043] A preparation method of a biochar-based microbial inoculant, comprising the following steps:

[0044] S1. Pick Bacillus megaterium L2 and streak-inoculate it onto the LB solid medium, culture it at 37 °C for 24 h, then pick a single colony and inoculate it into the LB liquid medium, and culture it at 37 °C and 200 rpm for 12 h to prepare the seed solution;

[0045] S2. Weigh 20 g of glucose, 21 g of yeast powder and 10 g of NaCl, add distilled water to make up to 1000 mL, and sterilize at 121 °C under high pressure for 20 min for standby;

[0046] S3. Pipette 6 mL of the seed solution and inoculate it into 100 mL of the liquid medium, and culture it at 34 °C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL to obtain the fermentation broth of Bacillus megaterium L2;

[0047] S4. Take 22.5 g of the fermentation broth of Bacillus megaterium L2 and 10 g of rice husk charcoal, mix them evenly, and let them stand at room temperature for 7 days to obtain the base material; S5. Dry the base material in an electrothermal constant temperature drying oven at 45 °C for 12 h to obtain the biochar-based microbial inoculant of Bacillus megaterium L2, denoted as 2.25:1.

[0048] Example 3

[0049] A preparation method of a biochar-based microbial inoculant, comprising the following steps:

[0050] S1. Pick and streak Bacillus megaterium L2 on the LB solid medium, culture it at 37 °C for 24 h, then pick a single colony and inoculate it into the LB liquid medium, and culture it at 37 °C and 200 rpm for 12 h to obtain the seed solution;

[0051] S2. Weigh 20 g of glucose, 21 g of yeast powder and 10 g of NaCl, add distilled water to make up to 1000 mL, and sterilize at 121 °C under high pressure for 20 min for standby;

[0052] S3. Pipette 6 mL of the seed solution and inoculate it into 100 mL of the liquid medium, and culture it at 34 °C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL to obtain the fermentation broth of Bacillus megaterium L2;

[0053] S4. Take 25 g of the fermentation broth of Bacillus megaterium L2 and 10 g of rice husk charcoal, mix them evenly, and let them stand at room temperature for 7 days to obtain the base material;

[0054] S5. Dry the base material in an electrothermal constant temperature drying oven at 45 °C for 12 h to obtain the biochar-based microbial inoculant of Bacillus megaterium L2, denoted as 2.5:1.

[0055] Example 4

[0056] A preparation method of a biochar-based microbial inoculant, comprising the following steps:

[0057] S1. Pick and streak Bacillus megaterium L2 on an LB solid medium, incubate at 37 °C for 24 h, then pick a single colony and inoculate it into an LB liquid medium, incubate at 37 °C and 200 rpm for 12 h to obtain a seed solution;

[0058] S2. Weigh 20 g of glucose, 21 g of yeast powder and 10 g of NaCl, add distilled water to make up to 1000 mL, and autoclave at 121 °C for 20 min for later use;

[0059] S3. Pipette 6 mL of the seed solution and inoculate it into 100 mL of the liquid medium, incubate at 34 °C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL to obtain the Bacillus megaterium L2 fermentation broth;

[0060] S4. Take 20 g of the Bacillus megaterium L2 fermentation broth and 10 g of rice husk charcoal, mix them evenly, and let stand at room temperature for 7 days to obtain a base material;

[0061] S5. Dry the base material in an electrothermal constant temperature drying oven at 45 °C for 12 h to obtain the Bacillus megaterium L2 biochar-based microbial inoculant, denoted as 2:1.

[0062] Example 5

[0063] A preparation method of a biochar-based microbial inoculant, comprising the following steps:

[0064] S1. Pick and streak Bacillus megaterium L2 on an LB solid medium, incubate at 37 °C for 24 h, then pick a single colony and inoculate it into an LB liquid medium, incubate at 37 °C and 200 rpm for 12 h to obtain a seed solution;

[0065] S2. Weigh 20 g of glucose, 21 g of yeast powder and 10 g of NaCl, add distilled water to make up to 1000 mL, and autoclave at 121 °C for 20 min for later use;

[0066] S3. Pipette 6 mL of the seed solution and inoculate it into 100 mL of the liquid medium, incubate at 34 °C and 203 rpm for 20 h until the concentration reaches 3.35×10 9 CFU / mL to obtain the Bacillus megaterium L2 fermentation broth;

[0067] S4. Take 17.5 g of the Bacillus megaterium L2 fermentation broth and 10 g of rice husk charcoal, mix them evenly, and let stand at room temperature for 7 days to obtain a base material; S5. Dry the base material in an electrothermal constant temperature drying oven at 45 °C for 12 h to obtain the Bacillus megaterium L2 biochar-based microbial inoculant, denoted as 1.75:1.

[0068] Comparative Example 1

[0069] The difference between this comparative example and Example 2 is that the ratio of Bacillus megaterium L2 and rice husk charcoal is different.

[0070] A method for preparing a biochar-based microbial agent comprises the following steps:

[0071] 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;

[0072] 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.

[0073] 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;

[0074] 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;

[0075] 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.

[0076] Comparative Example 2

[0077] The difference between this comparative example and Example 2 is that rice husk charcoal is not added.

[0078] A method for preparing Bacillus megaterium L2 fermentation broth comprises the following steps:

[0079] 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;

[0080] 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.

[0081] 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.

[0082] Comparative Example 3

[0083] 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.

[0084] Comparative Example 4

[0085] The difference between this comparative example and Example 2 is that Bacillus megaterium L2 is replaced by commercially available Bacillus megaterium.

[0086] A method for preparing a biochar-based microbial agent comprises the following steps:

[0087] 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;

[0088] S2. Weigh 20g glucose, 21g yeast powder and 10g NaCl, add distilled water to 1000mL, and sterilize under high pressure at 121℃ for 20min;

[0089] 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;

[0090] 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.

[0091] Performance Testing

[0092] (1) Single factor experiment optimization of liquid fermentation conditions of Bacillus megaterium L2

[0093] 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:

[0094] Single-factor experiment settings: carbon source types (maltose, lactose, sucrose, soluble starch, glucose), carbon source addition amounts (5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L), nitrogen source types (urea, tryptone, peptone, yeast extract, ammonium sulfate), nitrogen source addition amounts (5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L), inoculation amounts (1%, 2%, 4%, 6%, 8%, 10%), fermentation temperatures (28 °C, 31 °C, 34 °C, 37 °C, 40 °C), and rotation speeds (140 rpm, 160 rpm, 180 rpm, 200 rpm, 220 rpm);

[0095] Determination of viable cell count: The bacterial solution was serially diluted, and three dilution concentrations of 10 -5 , 10 -6 , 10 -7 were selected. 0.2 mL of the diluted solution was pipetted into an LB solid medium for plate coating, and the colony count was statistically analyzed after culturing at 28 °C for 24 h.

[0096] As can be seen from Figure 2 , for carbon source types: In the optimization of liquid fermentation conditions, five carbon sources, namely maltose, lactose, sucrose, soluble starch, and glucose, were screened. Among the 5 carbon sources, glucose had the best utilization effect, and the viable cell count could reach 1.25×10 8 CFU / mL, while soluble starch had the worst utilization effect, and the viable cell count was 1.2×10 7 CFU / mL. The effects of the 5 carbon source types on the viable cell count in the fermentation broth were significantly different (p < 0.05). Therefore, glucose was selected as the carbon source for the screening test of the addition amount.

[0097] Carbon source addition amount: Five addition amount gradients of 5 - 25 g / L were selected for screening. Among the five carbon source addition amounts, 20 g / L had the best fermentation effect, and the viable cell count could reach 1.15×10 9 CFU / mL, while 5 g / L had the worst fermentation effect, and the viable cell count was 4.8×10 8 CFU / mL. The effects of the 5 carbon source addition amounts on the viable cell count in the fermentation broth were significantly different (p < 0.05). Therefore, 20 g / L was selected as the optimal carbon source addition amount.

[0098] Nitrogen source types: Five nitrogen sources, namely urea, tryptone, peptone, yeast extract, and ammonium sulfate, were screened. Among the five nitrogen sources, yeast extract had the best utilization effect, and the viable cell count could reach 1.85×10 8 CFU / mL, while urea had the worst utilization effect, and the viable cell count was 5.65×10 7 CFU / mL. The effects of the 5 nitrogen source types on the viable cell count in the fermentation broth were significantly different (p < 0.05). Therefore, yeast extract was selected as the optimal nitrogen source for the screening test of the addition amount.

[0099] Nitrogen source addition amount: Five addition amount gradients of 5 - 25 g / L were selected for screening. Among the five nitrogen source addition amounts, the fermentation effect was the best at 20 g / L, and the viable cell count could reach 3.2×10 8 CFU / mL. The fermentation effect was the worst at 5 g / L, and the viable cell count was 1.4×10 8 CFU / mL. The effects of the 5 carbon source addition amounts on the viable cell count in the fermentation broth were significantly different (p < 0.05). Therefore, 20 g / L was selected as the optimal nitrogen source addition amount.

[0100] Inoculum size of bacterial liquid: Six inoculum sizes of 1%, 2%, 4%, 6%, 8%, and 10% were selected for screening. The results showed that the fermentation effect was the best at the inoculum size of 6%, and the viable cell count was 2.5×10 8 CFU / mL. The fermentation effect was the worst at 1%, and the viable cell count was 1.47×10 8 CFU / mL. The effects of the six inoculum sizes of bacterial liquid on the viable cell count in the fermentation broth were significantly different (p < 0.05). Therefore, 6% was selected as the optimal inoculum size of bacterial liquid.

[0101] Fermentation temperature: Five fermentation temperatures of 28℃, 31℃, 34℃, 37℃, and 40℃ were selected for screening. The results showed that the fermentation effect was the best at 31℃, and the viable cell count was 1.7×10 8 CFU / mL. The fermentation effect was the worst at 40℃, and the viable cell count was 7.1×10 7 CFU / mL. The effects of the five fermentation temperatures on the viable cell count in the fermentation broth were significantly different (p < 0.05). Therefore, 31℃ was selected as the optimal fermentation temperature.

[0102] Rotation speed: Five rotation speeds of 140 rpm, 160 rpm, 180 rpm, 200 rpm, and 220 rpm were selected for screening. The experimental results showed that the fermentation effect was the best at 200 rpm, and the viable cell count was 1.4×10 9 CFU / mL. The fermentation effect was the worst at 140 rpm, and the viable cell count was 1.5×10 8 CFU / mL. The effects of the five rotation speeds on the viable cell count in the fermentation broth were significantly different (p < 0.05). Therefore, 200 rpm was selected as the optimal rotation speed.

[0103] (2) Optimization of the liquid fermentation conditions of Bacillus megaterium L2 by Plackett - Burman test

[0104] Based on the optimization of the single - factor test of the liquid fermentation conditions of Bacillus megaterium L2, five factors were selected to design the Plackett - Burman test, as follows:

[0105] Plackett - Burman test factor settings: Select the main effect factors from A (carbon source addition amount), B (nitrogen source addition amount), C (inoculum size), D (rotation speed), and E (fermentation temperature). Select an experimental design with N = 12, and each factor selects two levels: high level "1" and low level "-1". Using the viable cell count as the response value, establish the functional relationship between the response value and the independent variables to determine the optimal experimental conditions. The factor levels of the Plackett - Burman test are shown in Table 1.

[0106] Table 1 Factor levels of Plackett - Burman test

[0107]

[0108] Table 2 Plackett - Burman test design table and its response values

[0109]

[0110] As can be seen from Table 2, the first - order regression equation model fitted from the Plackett - Burman test results is: Y = 0.9654 - 0.0053A + 0.6093B - 0.0717C - 0.3856D + 0.3059E (p = 0.0031), indicating that this regression equation is extremely significant. Moreover, the p - values of B nitrogen source addition amount (p = 0.0007), D rotation speed (p = 0.0066), and E temperature (p = 0.0179) are all less than 0.05, and the coefficients are all positive values, indicating that the nitrogen source addition amount, rotation speed, and temperature have a significant correlation with the viable cell count of the bacterial liquid. However, the p - values of A carbon source addition amount and C inoculum size are both greater than 0.05, indicating that the carbon source addition amount and inoculum size have no significant effect on the cell number of Bacillus megaterium L2 fermentation broth. Thus, it can be seen that increasing the nitrogen source addition amount, rotation speed, and temperature step by step while keeping other factor levels unchanged can increase the viable cell count of Bacillus megaterium L2 fermentation broth.

[0111] (3) Optimization of the steepest ascent test for the liquid fermentation conditions of Bacillus megaterium L2

[0112] Based on the results of the single - factor test and the Plackett - Burman test for the liquid fermentation conditions of Bacillus megaterium L2, select three main effect factors, namely the nitrogen source addition amount, rotation speed, and temperature, which have a significant impact on the viable cell count of the liquid fermentation of Bacillus megaterium, for the steepest ascent test. The factor design table of the steepest ascent test is shown in Table 3.

[0113] Table 3 Factor design table of the steepest ascent test

[0114] Number B Nitrogen source addition amount (g / L) D Rotation speed (rpm) E Temperature (°C) 1 12 170 26 2 15 180 28 3 18 190 30 4 21 200 32 5 24 210 34 6 27 220 36

[0115] Table 4 Steepest ascent test design and results

[0116] Test number B: Yeast powder content D: Rotation 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

[0117] As can be seen from 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 viable count in the bacterial liquid is the largest, reaching 3.213×10 9 CFU / mL.

[0118] (4) Optimization of the liquid fermentation conditions of Bacillus megaterium L2 by response surface experiment

[0119] Based on the results of the Plackett-Burman experiment and the steepest ascent experiment, the Box-Behnken experimental design method was used to optimize the liquid fermentation conditions of Bacillus megaterium L2. The optimal experimental points of the nitrogen source addition amount, temperature, and rotation speed in the results of the steepest ascent experiment were selected as the "0" level, and the selections on the left and right of the optimal experimental points in the results of the steepest ascent experiment were used as the "-1" and "1" levels. Taking the viable count as the response value, the factor levels of the Box-Behnken experiment are shown in Table 5, and 17 response analysis experiments were designed by Design Expert software. The experimental design and results are shown in Table 6.

[0120] Table 5 Factor levels of the Box-Behnken experiment

[0121]

[0122] Table 6 Box-Behnken experimental design and results

[0123]

[0124] Table 7 Analysis of the results of the Box-Behnken experiment

[0125]

[0126]

[0127] Using Design Expert software to conduct multiple regression fitting analysis on the data in Table 6, a regression equation with the viable count 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 can be seen from Table 7, the p value of the regression equation model of the response surface optimization result is <0.0001, indicating that the equation has a good fitting degree, and the lack-of-fit term p = 0.1322 > 0.05, indicating that the model does not lack fit and the selection is reasonable; at the same time, in the regression equation, A, B, AB, BC, A 2 、B2 and C 2 are all significant influencing factors, and the determination coefficient R 2 = 0.9878, R 2 Adj = 0.9721, indicating that the model has a good fitting degree, and there is a high correlation between the predicted value and the actual value of the model. Moreover, through the analysis of Design-Expert 13 software, the optimal conditions for the liquid fermentation of Bacillus megaterium L2 are as follows: the nitrogen source addition amount is 21 g / L, the rotation speed is 203 rpm, and the temperature is 34 °C. The predicted value of the viable cell count in the bacterial liquid is 3.324×10 9 CFU / mL. After experimental verification, the actual value of the viable cell count in the bacterial liquid is measured to be 3.35×10 9 CFU / mL, showing no significant difference from the predicted value, indicating that the model has a high degree of fitting with the actual situation, and the optimal conditions for the liquid fermentation of Bacillus megaterium L2 are stable and feasible.

[0128] (5) Screening of biochar carriers for microbial inoculants

[0129] Determination of biochar adsorption capacity: Select bamboo charcoal, rice husk charcoal, corn straw charcoal, and charcoal as alternative carriers. Weigh 5.00 g of biochar and put it into a sealed bag. Use a pipette to add 1 mL of water to the bag successively, and then add water again after thorough stirring. During the process of adding water, pay attention to observing the morphology of the carrier to ensure that it is moist but does not leave water droplets. At the same time, record the maximum water absorption of each carrier for comparison.

[0130] Determination of viable cell count: Pipette 8.8 mL of Bacillus megaterium L2 bacterial liquid and mix it evenly with 10 g of biochar. Let it stand at room temperature for 7 days, and then put it into an electrothermal constant temperature drying oven at 45 °C for 12 h. After the carbon-bacteria complex is thoroughly dried, measure the viable cell count in the microbial inoculant for comparison.

[0131] From Figure 3 and Figure 4 it can be seen that the adsorption capacity and viable cell count of rice husk charcoal are higher than those of bamboo charcoal, charcoal, and corn straw charcoal. Moreover, after the Bacillus megaterium L2 microbial inoculant prepared with rice husk charcoal is placed for 7 days, the decline rate of the viable cell count is the smallest, reaching 3.12×10 9 CFU / g, indicating that rice husk charcoal has good adsorption capacity and has little impact on the survival rate of Bacillus megaterium L2. Therefore, rice husk charcoal is selected as the carrier of the microbial inoculant.

[0132] (6) Viable cell counts of Examples 2-5 and Comparative Example 1

[0133] From Figure 5It can be seen that the viable count 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 the Bacillus megaterium L2 bacterial liquid to rice husk biochar is 2.25:1, the effective viable count of the biochar-based microbial inoculant is the largest, which is 4.18×10 9 CFU / g.

[0134] (7) Field control efficacy tests of Example 2, Comparative Example 2, Comparative Example 3, and Comparative Example 4

[0135] Field control efficacy tests in tea gardens were carried out using Example 2, Comparative Example 2, and Comparative Example 3, as follows:

[0136] The test site was selected in the Miaozi Tian area of Bijie City. The test tea garden was a densely planted sloping tea garden with the variety of Fuding Dabaicha and three-year-old tea seedlings. A total of 4 treatments were set up in the test (T1: blank treatment, T2: rice husk biochar, T3: biochar-based microbial inoculant, T4: Bacillus megaterium L2 fermentation broth, T5: commercially available Bacillus megaterium biochar-based microbial inoculant). The application rates of the T3, T4, and T5 groups were all 100 g / mu, arranged in a randomized block design with 3 replicates, a total of 15 plots, and the plot area was 30m 2 ²; At the same time, 30 days after applying the medicine, the disease incidence of the tea leaves in each treatment was investigated. 50 leaves were investigated for each treatment. According to the tea leaf disease grading table (see Table 8) and the calculation formula, the incidence rate, disease index, and control effect of tea anthracnose in each treatment were investigated.

[0137] Table 8 Tea leaf disease grading table

[0138] Disease level value Percentage of lesion area in the whole leaf area Grade 0 No lesion Grade 1 <5 Grade 3 6-25 Grade 5 26-50 Grade 7 51-75 Grade 9 >76

[0139]

[0140] Table 9 Field control effect of biochar-based microbial inoculant on tea anthracnose

[0141]

[0142] Note: Different lowercase letters after the data in the same column indicate significant differences between treatments (p<0.05).

[0143] From Table 9 and Figure 6It can be seen that in the treatment area where the biochar-based microbial inoculant prepared in Example 2 was applied, the incidence and disease index of tea anthracnose in tea plants were significantly lower than those in Comparative Example 2 and Comparative Example 3. The control effect on tea anthracnose was 51.76±0.93%, while in Comparative Example 2, the control effect of the fermentation broth of Bacillus megaterium L2 on tea anthracnose was 45.65±0.48%. In addition, the control effect of the commercially available Bacillus megaterium biochar-based microbial inoculant on tea anthracnose was 39.86±1.38%, which was significantly lower than that of the biochar-based microbial inoculant and the fermentation broth of Bacillus megaterium L2 (p<0.05). This indicates that the biochar-based microbial inoculant prepared by the co-compounding of Bacillus megaterium L2 and rice husk biochar can improve the control effect of tea anthracnose in tea plants.

[0144] (8) Rhizosphere soil microbial diversity of tea plants treated in Example 2, Comparative Example 2 and Comparative Example 3

[0145] Using the 4 grouped treatment areas set in the (7) field control effect test, soil samples were collected from the sunny slope of the tea garden based on the five-point sampling method, divided into 0 days, 30 days, and 60 days, and the three periods were respectively labeled as "F", "S", and "T"; Soil sample sampling method: Taking the main trunk of the tea plant as the core, soil samples at 20 cm were taken. When sampling, the surface fallen leaves and humus were first removed, and the drilling depth was 20 cm. Each sampling point formed a mixed soil sample, and each soil sample needed to collect about 100 g. After removing impurities such as sand and gravel and withered leaves from the soil samples, microbial amplicon sequencing was carried out to analyze the microbial diversity in the soil samples.

[0146] It can be seen from Figure 7 that T1 in the figure represents the blank treatment, T2 represents rice husk charcoal (Comparative Example 3), T3 represents the biochar-based microbial inoculant (Example 2), and T4 represents the fermentation broth of Bacillus megaterium L2 (Comparative Example 2). Compared with Comparative Example 2 and Comparative Example 3, in the treatment area where the biochar-based microbial inoculant prepared in Example 2 was applied, the relative abundances of Chloroflexi, Actinobacteriota, and Proteobacteria in soil samples ST3 and TT3 were the highest. Especially, the relative abundance of Actinobacteriota reached the maximum level (21.78±2.49%) in soil sample TT3. According to research reports, the predatory Chloroflexi group Herpetosiphon in Chloroflexi shows varying degrees of killing effects on various pathogenic bacteria and has the ability to synthesize antibacterial active molecules. Moreover, Actinobacteriota can produce different types of antibacterial secondary metabolites (such as lipopeptides, macrocyclic lipids, and polyenes) and has broad-spectrum antibacterial activity.

[0147] In addition, Figure 8The results showed that Firmicutes was also significantly enriched in different samples. Further analysis was carried out on the Bacillus microorganisms enriched in different soil samples. From Figure 7 it can be seen that in the samples treated for 30 days, the relative abundances of Bacillus cellulasensis, Bacillus thermolactis, and Bacillus strypoxylicola were the highest in ST3, while Bacillus infernus was significantly enriched in the TT3 sample, with a relative abundance of 0.01±0.002%. In summary, these Bacillus microorganisms can produce antibacterial metabolites and have good antibacterial activities against the pathogenic fungi and bacteria of tea anthracnose in tea plants, which can further improve the control effect of the biochar-based microbial inoculant on tea plants.

[0148] As described above, it is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Application of biochar-based microbial inoculum in preventing and controlling tea tree disease pathogens Clonostachys rosea f. catenulata and Irpex lacteus, characterized in that, The preparation method of the biochar-based microbial inoculum comprises the following steps: S1. Bacillus megaterium L2 is subjected to seed liquid culture and liquid fermentation culture to obtain a Bacillus megaterium L2 fermentation broth; S2. The Bacillus megaterium L2 fermentation broth and biochar are mixed evenly and left 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 h to obtain the biochar-based microbial inoculum; The preservation number of the Bacillus megaterium L2 is CCTCC NO: M2012381, the preservation date is September 26, 2012, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The application according to claim 1, wherein The method for culturing the seed liquid in step S1 is as follows: Pick and streak Bacillus megaterium L2 on an LB solid medium and culture it at 37 °C for 24 h, then pick a single colony and inoculate it into an LB liquid medium, and culture it at 37 °C and 180 - 220 rpm for 12 - 14 h.

3. The application according to claim 1, characterized in that, The method of liquid fermentation culture in step S1 is as follows: The cells are obtained by centrifuging the seed solution and inoculated into a liquid medium, and cultured at 34 °C and 200 - 220 rpm for 20 h until the concentration of the strain is (3.0 - 4.0)×10 9 CFU / mL.

4. The application according to claim 3, wherein The composition of the liquid medium is 5 - 25 g / L of glucose, 5 - 25 g / L of yeast powder, and 10 g / L of NaCl.

5. The application according to claim 1, characterized in that, In step S2, the mass ratio of the Bacillus megaterium L2 fermentation broth to the biochar is (1.75 - 2.5):1.

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