Biochar agent based on Bacillus atrophaeus A126 and its preparation method and application

Abstract

The present invention relates to the technical field of microbial agents, and discloses a biochar agent based on Bacillus atrophaeus A126, and a preparation method and application thereof. The preparation method comprises the steps of: inoculating Bacillus atrophaeus A126 into a BU culture medium and culturing for 72 to 120 hours to obtain a BU fermentation product; mixing the BU fermentation product and nutrients with pine biochar to obtain a first product; mixing the first product with a mixed powder of a suspension of Bacillus atrophaeus A126 and pine biochar to obtain a second product; mixing the second product with a mixed solution of the BU fermentation product and sodium alginate to obtain a third product; adding the third product to a calcium chloride solution to obtain a biochar agent; wherein the deposit number of Bacillus atrophaeus A126 is CGMCC NO.32444. The biochar agent prepared by the present invention has good and stable antibacterial effect, and can significantly reduce the incidence and disease index of potato scab after use.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and in particular to a biochar agent based on Bacillus atrophaeus A126, and a preparation method and application thereof. Background Art

[0002] Potato scab is caused by pathogenic Streptomyces, which seriously affects the appearance quality and commercial value of tubers, causing huge economic losses to the global potato industry. As an environmentally friendly method, biological control avoids environmental pollution and human health problems caused by the use of chemical agents, and is a hot topic in plant disease control research. In recent years, a large number of studies have found that a variety of microorganisms have significant control effects on potato scab. Among them, the most representative is Bacillus. Several species of Bacillus are regarded as the most promising biological control agents because of their safety, reliability, strong environmental adaptability, and ability to produce a variety of antibacterial compounds. However, the development of biological control agents faces many problems, the first of which is the antibacterial ability. Different fermentation conditions (such as pH, temperature, carbon and nitrogen sources) lead to the instability of the antibacterial ability of biological control agents. At the same time, production cost issues are also an important factor affecting the implementation of biological control. Secondly, direct application of biocontrol bacteria as biological control agents will face a variety of difficult situations, including biological factors such as microbial competition, predation by natural enemies and some abiotic factors such as nutrient deficiency, unsuitable soil pH and temperature, which will lead to unstable control effects of biocontrol bacteria in the field.

[0003] Therefore, the existing technology still needs to be improved and developed. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a biochar agent based on Bacillus atrophaeus A126 and a preparation method and application thereof, aiming to solve the problem of unstable field biological control effect of existing potato scab.

[0005] The technical solution of the present invention is as follows:

[0006] In the first aspect, a method is provided based on Bacillus atrophaeus ( Bacillus atrophaeus ) A method for preparing a biochar agent of A126, the preparation method comprising the steps of:

[0007] Bacillus atrophaeus A126 was inoculated into BU medium and cultured for 72-120 hours to obtain BU fermentation product;

[0008] mixing the BU fermentation product and nutrients to obtain a mixture;

[0009] The mixture is mixed with pine wood biochar and pelletized to obtain a first product;

[0010] The first product is mixed with a mixed powder of a suspension of Bacillus atrophaeus A126 and pine biochar, so that the mixed powder is loaded on the first product to obtain a second product;

[0011] mixing the second product with a mixed solution of BU fermentation product and sodium alginate, so that the mixed solution is coated on the second product, to obtain a third product;

[0012] adding the third product into a calcium chloride solution to obtain the biochar agent;

[0013] The BU medium is prepared with water and includes: 0.3 g / L magnesium sulfate heptahydrate, 5 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate trihydrate, 5 g / L sodium chloride, 50 g / L brown sugar and 4 g / L urea;

[0014] The nutrients are prepared with water, including: 0.3 g / L magnesium sulfate heptahydrate, 5 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate trihydrate, 5 g / L sodium chloride, 500 g / L brown sugar and 40.6 g / L urea;

[0015] The Bacillus atrophaeus A126 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC NO.32444.

[0016] Specifically, the present invention uses the Bacillus atrophaeus A126 with excellent antagonistic properties obtained in the early stage as a biocontrol bacterium for potato scab. In order to provide it with a suitable living space and sufficient nutrients when used, a preparation method of a biochar agent based on Bacillus atrophaeus A126 is designed. The biochar agent prepared by the preparation method includes three layers: inner, middle and outer. The inner layer is the core nutrient layer, which is a mixture of BU fermentation products, nutrients and pine biochar; wherein, the BU fermentation product is obtained by culturing Bacillus atrophaeus A126 in BU culture medium, and contains a variety of antagonistic substances produced after fermentation, such as lipopeptides, polyketide compounds and volatiles, etc. These antagonistic substances can inhibit the utilization of nutrients by scab pathogens; the nutrients provide Bacillus atrophaeus A126 with rich nutrients for its growth. The middle layer is the active bacteria layer, which is a mixture of Bacillus atrophaeus A126 and pine biochar. Bacillus atrophaeus A126 can use the nutrients in the inner layer to continue to grow and reproduce, and the antagonistic effect is enhanced with the help of pine biochar. The outer layer is the microcapsule layer, which is made of sodium alginate and calcium chloride, and plays a role of slow release and protection.

[0017] Among them, BU medium is obtained after optimization based on Bacillus atrophaeus A126 in the present invention, and has better types and ratios of carbon and nitrogen sources. Bacillus atrophaeus A126 is cultured using BU medium, and the obtained BU fermentation product has stronger antibacterial ability. The difference between the nutrient and BU medium is that the carbon and nitrogen source concentration of the nutrient is 10 times that of the carbon and nitrogen source of the BU medium, and the higher concentration of carbon and nitrogen sources provides rich and continuous nutrition for Bacillus atrophaeus A126. Pine biochar is obtained based on the screening of Bacillus atrophaeus A126 and scab pathogens in the present invention. It promotes the growth and antibacterial effect of Bacillus atrophaeus A126, and has a weak ability to promote the growth of scab pathogens. It is an excellent carrier for Bacillus atrophaeus A126 when used as a biocontrol bacterium for the prevention and control of potato scab.

[0018] According to a preferred technical solution, in the mixture, the volume ratio of BU fermentation product to nutrients is 1:1.8-2.2.

[0019] According to a preferred technical solution, in the first product, the volume mass ratio of the mixture to the pine biochar is 1:1.2-1.8.

[0020] According to a preferred technical solution, in the mixed powder, the volume mass ratio of the Bacillus atrophaeus A126 suspension and the pine wood biochar is 1:1.2-1.8.

[0021] In the preferred technical solution, the concentration of Bacillus atrophaeus A126 in the suspension is 10 8 ~10 9 cfu / mL.

[0022] According to a preferred technical solution, in the second product, the mass ratio of the first product to the mixed powder is 6 to 8:1.

[0023] According to a preferred technical solution, the mixed solution is obtained by mixing the BU fermentation product and a sodium alginate solution with a mass concentration of 4-6%, wherein the volume ratio of the BU fermentation product to the sodium alginate solution with a mass concentration of 4-6% is 1:0.8-1.2.

[0024] According to a preferred technical solution, the mass concentration of the calcium chloride solution is 1.8-2.2%.

[0025] In a preferred technical solution, when Bacillus atrophaeus A126 is inoculated into BU medium and cultured for 72 to 120 hours, the culture temperature is 18 to 37°C; further preferably, the culture temperature is 28 to 37°C.

[0026] In a preferred technical solution, the pH of the BU medium is 5-10; further preferably, the pH of the BU medium is 6-8.

[0027] In a second aspect, a biochar agent based on Bacillus atrophaeus A126 is provided, wherein the biochar agent is prepared by the preparation method described in the first aspect.

[0028] In a third aspect, there is provided a use of the biochar agent as described in the second aspect in the prevention and treatment of potato scab.

[0029] Beneficial effects: In the early stage of the present invention, Bacillus atrophaeus A126 with excellent antagonistic properties was isolated from the soil. By optimizing the culture conditions, the fermentation products of Bacillus atrophaeus A126 had better antibacterial ability, and pine biochar was screened as a suitable microbial carrier, thereby preparing a biochar agent based on Bacillus atrophaeus A126. The biochar agent has good and stable antibacterial effect and significant prevention and control effect. When it was applied to the prevention and control of potato scab, the incidence rate was 44.6%, significantly lower than 79.2% of the positive control group; the disease index was 0.138, significantly lower than 0.300 of the positive control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a graph showing the effects of different pH and temperature ranges on the growth of Bacillus atrophaeus A126 and scab pathogens.

[0031] Figure 2 This is a graph showing the effects of different carbon and nitrogen sources on the growth of Bacillus atrophaeus A126 and scab pathogens.

[0032] Figure 3 It is the experimental verification of the prediction results of response surface methodology and the effect of BU culture medium on the growth of Bacillus atrophaeus A126 and scab pathogens.

[0033] Figure 4 This is a graph showing the effect of different biochars on the antibacterial ability of Bacillus atrophaeus A126.

[0034] Figure 5 These are electron microscope images of peach wood biochar and pine wood biochar before and after the adsorption of Bacillus atrophaeus A126.

[0035] Figure 6 This is a graph showing the effects of peach wood biochar and pine wood biochar on the growth of Bacillus atrophaeus A126 and scab pathogens.

[0036] Figure 7 This is a test result graph of the inhibition area of ​​the fermentation liquid of Bacillus atrophaeus A126 obtained by adding peach wood biochar and the fermentation liquid of Bacillus atrophaeus A126 obtained by adding pine wood biochar.

[0037] Figure 8 This is a photo of biochar.

[0038] Fig. 9This is the result of the field experiment of biochar agent. DETAILED DESCRIPTION

[0039] The present invention provides a biochar agent based on Bacillus atrophaeus A126 and a preparation method and application thereof. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described below through specific embodiments.

[0040] Example 1

[0041] In this example, the optimal culture conditions and inoculation carriers of Bacillus atrophaeus A126 were screened to prepare a biochar agent based on Bacillus atrophaeus A126, and its effect in the prevention and treatment of potato scab was verified, as follows:

[0042] 1. Plant materials, strains and culture media

[0043] The potato variety used in this example was "Feureta", purchased from the Potato Research Institute of Dingxi City, Gansu Province, China. S. scabies P139) was isolated from potato scab lesions in the experimental field of the Potato Center Farm of Inner Mongolia University. Bacillus atrophaeus ) A126 was isolated from the potato rhizosphere soil of the farm of the South Campus of Inner Mongolia University. It was deposited in the General Microbiology Center of the China Culture Collection Administration on October 31, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.32444.

[0044] The scab pathogen was taken out from the -80℃ ultra-low temperature freezer, thawed, and 20μL was taken out to revive and isolate a single colony in Gause No. 1 solid culture medium. A single colony was picked and cultured in Gause No. 1 liquid culture medium at room temperature for 3 to 5 days. The obtained scab pathogen culture was used for subsequent experimental research.

[0045] The Bacillus atrophaeus A126 was taken out from the -80°C ultra-low temperature freezer, thawed, and 20 μL was taken out to revive in NA medium and isolate a single colony. A single colony was picked and cultured in NB medium at 28°C overnight until the OD value was 0.5-0.8. The obtained Bacillus atrophaeus A126 culture was used for subsequent experimental studies.

[0046] The culture medium involved was prepared with deionized water and had the following composition:

[0047] Gao's solid culture medium No. 1: soluble starch 20.0g / L, sodium chloride (NaCl) 0.5g / L, potassium nitrate (KNO3) 1.0g / L, potassium hydrogen phosphate trihydrate (K2HPO4·3H2O) 0.5g / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.5g / L, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.01g / L, agar 20g / L.

[0048] Gao's liquid culture medium No. 1: soluble starch 20.0 g / L, sodium chloride (NaCl) 0.5 g / L, potassium nitrate (KNO3) 1.0 g / L, dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O) 0.5 g / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.5 g / L, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.01 g / L.

[0049] NA medium: tryptone 10 g / L, sodium chloride (NaCl) 0.5 g / L, beef extract 3 g / L, agar 20 g / L.

[0050] NB medium: tryptone 10 g / L, sodium chloride (NaCl) 0.5 g / L, beef extract 3 g / L.

[0051] 2. Optimization of culture conditions of Bacillus atrophaeus A126

[0052] (1) Optimal pH and temperature range of strains

[0053] pH and temperature are important regulatory parameters in the process of microbial fermentation. This experiment evaluated the growth of Bacillus atrophaeus A126 and scab pathogens under different pH and temperature conditions, providing data support for subsequent optimal carbon and nitrogen source screening experiments.

[0054] ① pH optimization and temperature optimization of Bacillus atrophaeus A126

[0055] (i) pH optimization experiment

[0056] The culture of Bacillus atrophaeus A126 was inoculated into NB medium with different pH ranges (4, 5, 6, 7, 8, 9, 10, 11) at a 1% (v / v) inoculation rate and cultured at 28°C and 200 rpm for 48 h. During this period, samples were taken every 4 h and their OD values ​​were measured at a wavelength of 600 nm. The results are shown in Figure 1 As shown in A. Figure 1As shown in Figure A, Bacillus atrophaeus A126 can grow in NB medium with a pH of 5-10; when the pH is 6-8, the concentration of Bacillus atrophaeus A126 is significantly higher than that in other pH ranges; and when the pH is 4 or 11, there is no obvious increasing trend in the concentration during the entire experimental period, indicating that the growth of Bacillus atrophaeus A126 is inhibited.

[0057] Take 30 μL of the fermentation liquid of Bacillus atrophaeus A126 treated with different pH ranges and cultured for 48 hours, and add it to the Oxford cup in the culture dish pre-coated with scab pathogens. After the bacterial liquid dries, take out the Oxford cup so that the strain can grow freely. After culturing at 28℃ for 3 to 5 days, use imageJ to observe and measure the size of the antibacterial area. The results are as follows: Figure 1 As shown in B, different lowercase letters represent P <0.05. Figure 1 As shown in Figure B, after culturing for 48 h under the condition of pH 5-9, the inhibitory area of ​​Bacillus atrophaeus A126 fermentation broth against scab pathogens had no significant difference, both of which were 2.5 cm 2 When the pH is 10, the inhibition area is significantly reduced. P <0.05; when the pH is 4 or 11, there is no antibacterial effect.

[0058] (ii) Temperature optimization experiment

[0059] The culture of Bacillus atrophaeus A126 was inoculated into NB medium (pH=7) at a 1% (v / v) inoculation rate, and placed in an oscillating incubator at different temperature ranges (18℃, 23℃, 28℃, 32℃, 37℃) and cultured at 200rpm for 48h. During this period, samples were taken every 4h and their OD values ​​were measured at 600nm. The results are shown in Figure 1 As shown in C. Figure 1 From Figure C, we can see that the optimal culture temperature range of Bacillus atrophaeus A126 is 28~37℃.

[0060] Take 30 μL of the fermentation liquid of Bacillus atrophaeus A126 after being treated at different temperature ranges and cultured for 48 hours, and add it to the Oxford cup in the culture dish pre-coated with scab pathogens. After the bacterial liquid dries, take out the Oxford cup so that the strain can grow freely. After culturing at 28℃ for 3 to 5 days, use imageJ to observe and measure the size of the antibacterial area. The results are as follows: Figure 1 As shown in D, different lowercase letters represent P <0.05. Figure 1 As shown in Figure D, after culturing at different temperatures for 48 h, there was no significant difference in the inhibition area of ​​Bacillus atrophaeus A126 fermentation broth against scab pathogens.

[0061] ② pH optimization and temperature optimization of scab pathogens

[0062] (i) pH optimization experiment

[0063] The scab pathogen culture was inoculated into Gao's No. 1 liquid medium with different pH ranges (4, 5, 6, 7, 8, 9, 10, 11) at a 1% (v / v) inoculation rate and cultured at 28°C and 200 rpm for 96 hours. During this period, samples were taken every 12 hours, and the samples were broken by a sterile small wall-breaking machine for 30 seconds. Then, gradient dilutions were performed and the bacterial colony-forming units (CFU) on the plates were counted after culturing at 28°C for 2 to 3 days. The results are as follows: Figure 1 As shown in E. Figure 1 As shown in Figure E, the pathogens of scab can grow in Gao's No. 1 liquid medium with a pH range of 4 to 11. Although there are differences in the growth rate at the early stage, after 96 hours of culture, the concentration of the pathogens of scab exceeded 10 6 cfu / mL; from the perspective of culture speed, pH 5-9 is the optimal growth condition range for scab pathogens.

[0064] (ii) Temperature optimization experiment

[0065] The scab pathogen culture was inoculated into Gao's No. 1 liquid medium at a 1% (v / v) inoculation rate, and placed in incubators at different temperature ranges (18°C, 23°C, 28°C, 32°C, and 37°C) and cultured at 200rpm for 96h. During this period, samples were taken every 12h, and after sampling, they were broken by a sterile small wall-breaking machine for 30 seconds, followed by gradient dilution and spot plating. After culturing at 28°C for 2 to 3 days, the bacterial colony forming units (CFU) on the plates were counted. The results are as follows: Figure 1 As shown in F. Figure 1 It can be seen from Figure 5 that within the tested temperature range, the growth rate of scab pathogens increased with the increase of temperature; at 37°C, the growth rate of scab pathogens was the fastest and entered the stable period after 24 hours of culture; except for 18°C, the final concentrations of scab pathogens under other temperature conditions reached the maximum and remained consistent, and all exceeded 10 6 cfu / mL.

[0066] (2) Effects of different carbon and nitrogen sources on the growth of Bacillus atrophaeus A126 and scab pathogens

[0067] In this experiment, different carbon and nitrogen sources were added to the mineral salt medium (MSS) to study the effects of different carbon and nitrogen sources on the growth of Bacillus atrophaeus A126 and scab pathogens. The mineral salt medium was prepared with deionized water and the ingredients included: magnesium sulfate heptahydrate (MgSO4·7H2O) 0.3g / L, potassium dihydrogen phosphate (KH2PO4) 5.0g / L, potassium dihydrogen phosphate trihydrate (K2HPO4·3H2O) 5.0g / L and sodium chloride (NaCl) 5.0g / L.

[0068] ① Screening of the best carbon and nitrogen sources for Bacillus atrophaeus A126

[0069] (i) Optimal carbon source screening experiment

[0070] 2.0 g / L sodium nitrate (NaNO3) and 1.0 g / L ammonium sulfate ((NH4)2SO4) were added to the MSS medium as standard nitrogen sources, and four carbon sources to be tested were added at a concentration of 10 g / L: glucose, brown sugar, bran and corn flour. During inoculation, the culture of Bacillus atrophaeus A126 was inoculated into the liquid culture medium of different carbon sources at an inoculation rate of 1% (v / v), and then cultured at 28°C, pH=7, and 200 rpm. Since the experimental conditions were based on the optimal temperature and pH conditions, the sampling time of Bacillus atrophaeus A126 was shortened to sampling every 2 hours, and gradient dilutions were performed. After overnight culture, the bacterial colony forming units (CFU) on the plate were counted. The entire experiment lasted for 24 hours. The results are as follows Figure 2 As shown in A. Figure 2 As shown in Figure 1, the four different carbon sources had significant effects on the growth of Bacillus atrophaeus A126. Among them, when brown sugar and bran were used as carbon sources, the growth rate of Bacillus atrophaeus A126 was the highest, and the final concentrations reached 1.38×10 9 cfu / mL and 1.03×10 9 cfu / mL, significantly higher than other carbon sources, P <0.05; when corn flour was used as the carbon source, the final concentration of Bacillus atrophaeus A126 was the lowest, only 4.2×10 7 cfu / mL. Therefore, brown sugar and bran were the best carbon sources for Bacillus atrophaeus A126.

[0071] Take 30 μL of the culture of Bacillus atrophaeus A126 treated with different carbon sources and cultured for 48 hours, and drop it into the Oxford cup in the culture dish pre-coated with scab pathogens. After the bacterial solution dries, take out the Oxford cup so that the strain can grow freely. After culturing at 28℃ for 3 to 5 days, use imageJ to observe and measure the size of the inhibition area. The results are as follows Figure 2 As shown in B, different lowercase letters represent P<0.05. Figure 2 As shown in Figure B, after 48 h of culture under different carbon sources, the inhibition area of ​​Bacillus atrophaeus A126 fermentation broth was the largest when brown sugar was used as the carbon source, which was 4.48 cm 2 , significantly higher than other carbon sources, P <0.05; when bran and corn flour were used as carbon sources, the inhibition area of ​​Bacillus atrophaeus A126 fermentation liquid was the smallest, which was 2.61cm 2 and 1.79cm 2 .

[0072] (ii) Screening experiment for optimal nitrogen source

[0073] 10 g / L of glucose was added to the MSS medium as a standard carbon source, and five nitrogen sources to be tested were added at a concentration of 3 g / L: sodium nitrate, ammonium sulfate, urea, peptone and beef extract. During inoculation, the culture of Bacillus atrophaeus A126 was inoculated into liquid culture media with different nitrogen sources at an inoculation rate of 1% (v / v), and then cultured at 28°C, pH=7, and 200 rpm. Since the experimental conditions were based on the optimal temperature and pH conditions, the sampling time of Bacillus atrophaeus A126 was shortened to sampling every 2 hours, and gradient dilutions were performed. After overnight culture, the bacterial colony forming units (CFU) on the plate were counted. The entire experiment lasted for 24 hours. The results are as follows Figure 2 As shown in C. Figure 2 As shown in Figure C, when beef extract and peptone were used as nitrogen sources, the growth rate of Bacillus atrophaeus A126 was the highest, and the final concentrations were 2.05×10 9 cfu / mL and 2.16×10 9 cfu / mL, significantly higher than other nitrogen sources, P When sodium nitrate, ammonium sulfate and urea were used as nitrogen sources, the growth rate of Bacillus atrophaeus A126 was low, but it could eventually reach a higher concentration, which was 8.42×10 8 cfu / mL, 5.67×10 8 cfu / mL and 7.17×10 8 cfu / mL.

[0074] Take 30 μL of the culture of Bacillus atrophaeus A126 treated with different nitrogen sources and cultured for 48 hours, and drop it into the Oxford cup in the culture dish pre-coated with scab pathogens. After the bacterial solution dries, take out the Oxford cup so that the strain can grow freely. After culturing at 28℃ for 3 to 5 days, use imageJ to observe and measure the size of the inhibition area. The results are as follows Figure 2 As shown in D, different lowercase letters represent P <0.05. Figure 2 It can be seen from Figure D that after 48 h of culture under different nitrogen sources, there was no significant difference in the inhibition area of ​​Bacillus atrophaeus A126 fermentation broth against scab pathogens; among them, the inhibition area of ​​Bacillus atrophaeus A126 fermentation broth was slightly larger when urea and ammonium sulfate were used as nitrogen sources, which were 3.74 cm 2 and 3.44cm 2 .

[0075] ② Screening of the best carbon and nitrogen sources for scab pathogens

[0076] (i) Optimal carbon source screening experiment

[0077] 2.0 g / L sodium nitrate (NaNO3) and 1.0 g / L ammonium sulfate ((NH4)2SO4) were added to the MSS medium as standard nitrogen sources, and four carbon sources to be tested were added at a concentration of 10 g / L: glucose, brown sugar, bran and corn flour. During inoculation, the scab pathogen culture was inoculated into the liquid culture medium of different carbon sources at an inoculum amount of 1% (v / v), and then cultured at 28°C, pH=7, and 200 rpm. After 96 hours of cultivation, the culture was broken by a sterile small wall breaker for 30 seconds, followed by gradient dilution and spot plating, and CFU counts were performed 2 to 3 days later. The results are as follows Figure 2 As shown in E, different lowercase letters represent P <0.05. Figure 2 As shown in Figure E, when brown sugar and bran were used as carbon sources, the final concentration of scab pathogens was the highest, both reaching 10 7 cfu / mL, indicating that brown sugar and bran are the best carbon sources for scab pathogens; when glucose and corn flour are used as carbon sources, the final concentration of scab pathogens is lower, but can still reach 10 6 cfu / mL.

[0078] (ii) Screening experiment for optimal nitrogen source

[0079] 10 g / L of glucose was added to the MSS medium as a standard carbon source, and five nitrogen sources to be tested were added at a concentration of 3 g / L: sodium nitrate, ammonium sulfate, urea, peptone and beef extract. During inoculation, the scab pathogen culture was inoculated into the liquid culture medium with different nitrogen sources at an inoculum amount of 1% (v / v), and then cultured at 28°C, pH=7, and 200 rpm. After 96 hours of culture, the culture was broken by a sterile small wall breaker for 30 seconds, followed by gradient dilution and spot plating, and CFU counts were performed 2 to 3 days later. The results are as follows Figure 2 As shown in F, different lowercase letters represent P <0.05. Figure 2It can be seen from Figure F that when beef extract, sodium nitrate and peptone were used as nitrogen sources, the final concentration of scab pathogens was 10 6 cfu / mL, among which peptone was the highest and was the best nitrogen source for scab pathogens. When ammonium sulfate was used as the nitrogen source, the final concentration of scab pathogens was relatively low, at 1.89×10 4 cfu / mL; when urea was used as the nitrogen source, the final concentration of scab pathogens was the lowest, only 6×10 1 cfu / mL, significantly lower than the initial concentration of 10 3 cfu / mL, P <0.05, indicating that scab pathogens cannot use urea as a nitrogen source for growth.

[0080] In summary, brown sugar and bran are the best carbon sources for the growth of Bacillus atrophaeus A126 and scab pathogens, and brown sugar is most conducive to improving the antagonism of Bacillus atrophaeus A126 to scab pathogens. At the same time, beef extract and peptone are the best nitrogen sources for the growth of Bacillus atrophaeus A126 and scab pathogens, while urea is most conducive to improving the antagonism of Bacillus atrophaeus A126 and is least conducive to the growth of scab pathogens. Therefore, in the subsequent fermentation condition optimization experiment, brown sugar and urea were selected as carbon and nitrogen sources.

[0081] (3) Optimization of fermentation conditions of Bacillus atrophaeus A126 using response surface methodology

[0082] Based on the previous single-factor experimental results, the pH was controlled at 6-8, the temperature was set at 32°C, and the Box-Behnken experimental design was used to determine the optimal carbon source, nitrogen source concentration and fermentation time. Brown sugar content (1-5% (w / v)), urea content (0.1-1% (w / v)) and fermentation time (24-96 hours) were selected as the three factors of the study. Each factor was set at three levels, where -1 represented the lowest value, 0 represented the middle value, and 1 represented the highest value. The center point was repeated 5 times, and the response value was the inhibition area of ​​the fermentation broth of Bacillus atrophaeus A126. There were 17 groups of experiments in total, and the entire experiment was repeated 5 times. The experimental results are shown in Table 1.

[0083] Table 1 Box-Behnken test design and inhibition area

[0084]

[0085] The response surface test results were fitted using multiple regression analysis, and the quadratic regression equation was obtained: Inhibition area (Y) = 5.43516 + 0.81305A-0.54735B + 0.895725C-0.0542AB + 0.6139AC-0.485255A 2-0.222905B 2 -0.682705C 2 +0.38555A 2 B. Among them, A, B, and C are brown sugar concentration, urea concentration, and fermentation time, respectively. The p value of the regression equation model is 0.0049, indicating that the model is significantly reliable. The correction coefficient of the model is 0.8101, showing a good fitting effect. The analysis results show that the order of factors affecting the antibacterial ability is: fermentation time > brown sugar concentration > urea concentration. The response surface method analysis was performed using Design Expert 13 software, and the optimal fermentation conditions were obtained as follows: brown sugar 5% (w / v), urea 0.406336% (w / v), and time 95.9998h. Under these conditions, the theoretical value of the antibacterial area is 6.64cm 2 .

[0086] (4) Verification of fermentation conditions of Bacillus atrophaeus A126

[0087] In order to simplify the operation, the experimental conditions were set as brown sugar concentration 5% (w / v), urea concentration 0.4% (w / v), and fermentation time 96 hours. In this embodiment, the optimized culture medium is recorded as Brown sugar Urea medium (BU) medium, and the composition of BU medium is: MSS medium + brown sugar 50g / L + urea 4g / L. The composition of the control culture medium is: MSS medium + brown sugar 10g / L + urea 3g / L.

[0088] The culture of Bacillus atrophaeus A126 was inoculated into BU medium at a rate of 1% (v / v), and then cultured at 32°C and 200rpm for 96 hours, which was recorded as the BU group; the culture of Bacillus atrophaeus A126 was inoculated into the control medium at a rate of 1% (v / v), and then cultured at 32°C and 200rpm for 96 hours, which was recorded as the control group. Take 30μL of the fermentation liquid of Bacillus atrophaeus A126 from the BU group and the control group, and add them dropwise into the Oxford cup on the culture dish pre-coated with scab pathogens. After the bacterial liquid dries, take out the Oxford cup so that the strain can grow freely. After culturing at 28°C for 3 to 5 days, observe and measure the size of the inhibition area. The experimental results are as follows: Figure 3 As shown in A and B, different lowercase letters represent P <0.05. Figure 3 As shown in Figures A and B, the fermentation liquid of Bacillus atrophaeus A126 in the BU group had a larger inhibition area against scab pathogens, with an average inhibition area of ​​6.94 cm 2 , significantly higher than the control group of 4.67cm 2 , P <0.05, and the predicted value is 6.64cm 2The results show that the model fits well and the optimized BU medium has better antibacterial ability due to its higher carbon-nitrogen ratio.

[0089] In addition, this example studies the effect of BU medium on the growth of Bacillus atrophaeus A126 and scab pathogens.

[0090] For Bacillus atrophaeus A126, the culture of Bacillus atrophaeus A126 was inoculated into BU medium at a 1% (v / v) inoculation rate, which was recorded as the BU group; the culture of Bacillus atrophaeus A126 was inoculated into the control medium at a 1% (v / v) inoculation rate, which was recorded as the control group. Samples were taken every 2 hours, and gradient dilutions were plated. After overnight culture, the bacterial colony forming units (CFU) on the plate were counted. The experiment lasted for 24 hours. The results are as follows: Figure 3 As shown in C. Figure 3 As shown in Figure 3, in the early stage of culture, the growth rate of Bacillus atrophaeus A126 in BU medium was significantly lower than that in the control medium; in the stable period, the final concentrations of both reached 10 9 cfu / mL.

[0091] For scab pathogens, the scab pathogen culture was inoculated into BU medium at a 1% (v / v) inoculation rate, recorded as the BU group; the scab pathogen culture was inoculated into the control medium at a 1% (v / v) inoculation rate, recorded as the control group. Samples were taken every 12 hours, and after sampling, they were broken by a sterile small wall breaker for 30 seconds, and then gradient dilutions were plated. CFU counts were performed 2 to 3 days later. The experiment lasted for 96 hours. The results are as follows: Figure 3 As shown in D. Figure 3 As shown in Figure 3, in the early stage of culture, the growth rate of scab pathogens in BU medium was significantly lower than that in control medium; in the stable period, the final concentrations of both reached 10 7 cfu / mL.

[0092] 3. Preparation of biochar matrix and its effect on Bacillus atrophaeus A126

[0093] (1) Preparation of biochar

[0094] The material and preparation temperature of biochar will affect its performance. In this example, 10 types of biochar were selected for research, including wheat straw biochar (350℃ and 600℃), corn straw biochar (350℃ and 600℃), oat grass biochar (350℃ and 600℃), potato plant biochar (350℃ and 600℃), peach wood biochar (800℃) and pine wood biochar (500℃). Among them, wheat straw biochar (350℃ and 600℃), corn straw biochar (350℃ and 600℃), oat grass biochar (350℃ and 600℃) and potato plant biochar (350℃ and 600℃) were purchased from Cuilin Biotechnology Co., Ltd., Guangzhou, China. These biochars were made by sintering the raw materials at 350℃ and 600℃ in a muffle furnace (model: split SRJX-10-13) in an oxygen-free environment for 7~8 hours. 8h later; peach wood biochar was purchased from Longxin Water Purification Materials Sales Department, Gongyi City, Henan Province, China. The biochar was made by burning peach wood in an industrial carbonization furnace at 800℃ for 3 hours without oxygen; pine wood biochar was homemade, using pine wood boards discarded from the construction site, heated to 500℃ at a rate of 7~13℃ / min in a horizontal carbonization furnace (DLTH-1000), carbonized for 3 hours and kept warm for 2 hours, then crushed and sieved with 100 mesh. All biochars were sterilized at 121℃ for 20 minutes before the experiment, sterilized twice in a row, and dried in an oven at 80℃.

[0095] (2) Biochar screening

[0096] In order to find an inoculation carrier suitable for Bacillus atrophaeus A126, 10 kinds of biochar were added to the culture medium at a concentration of 5% (w / v), and then inoculated with Bacillus atrophaeus A126 for cultivation. By observing the changes in the antagonistic ability of Bacillus atrophaeus A126 under different biochar conditions, the effects of different biochars on the antibacterial ability of Bacillus atrophaeus A126 were evaluated. The experiment adopted the culture medium formula ratio used in the previous single factor. Considering that biochar would affect the pH of the culture medium, sodium chloride was used instead of mineral salts. The final mixed culture medium composition was: water + brown sugar 10g / L + urea 3g / L + sodium chloride 5g / L + biochar 50g / L. The Bacillus atrophaeus A126 culture was inoculated into the mixed culture medium containing different biochars at an inoculation rate of 1% (v / v), and cultured at 28°C and 200rpm for 60 hours. Then 30μL of the cultured product was taken and dropped into the Oxford cup on the culture dish pre-coated with scab pathogens. After the bacterial solution is dry, remove the Oxford cup so that the strain can grow freely. After culturing at 28°C for 3 to 5 days, observe and measure the size of the inhibition area. The experimental results are shown in Table 2 and Figure 4 As shown in Table 2 and Figure 4It can be seen that the antibacterial ability of the fermentation liquid of Bacillus atrophaeus A126 added with wheat straw biochar, corn straw biochar, potato plant biochar and oat grass biochar was poor, and the antibacterial area was 0-2.5 cm 2 In contrast, the fermentation broth of Bacillus atrophaeus A126 with pine biochar and peach biochar showed stronger antibacterial ability, with the antibacterial area reaching 6.06±0.41cm 2 and 5.28±0.81cm 2 , which was significantly higher than that of other biochar treatment groups. P <0.05. Therefore, pine biochar and peach biochar are ideal inoculation carriers for Bacillus atrophaeus A126.

[0097] Table 2 Effects of different biochars on the antibacterial ability of Bacillus atrophaeus A126

[0098]

[0099] Note: Different lowercase letters in Table 2 represent P The significant difference was <0.05.

[0100] (3) Determination of the physical and chemical properties and adsorption efficiency of biochar and electron microscopy observation

[0101] In this example, the physicochemical properties and adsorption efficiency of pine biochar and peach biochar were measured, and the experimental results are shown in Table 3. In the determination of physicochemical properties, the pH value was measured by mixing the biochar with deionized water in a ratio of 1:10; the specific surface area, pore size and pore volume were measured using a rapid specific surface and pore size distribution analyzer (ASAP 2020, USA); the carbon, nitrogen and hydrogen contents were analyzed by an elemental analyzer (ELEMETA VARIO MACRO CUBE, Germany).

[0102] In the determination of adsorption efficiency, 1 g of biochar was mixed with 20 mL of Bacillus atrophaeus A126 suspension (10 9 cfu / mL), placed in a shaker for 2 hours, and then left to stand for 6 hours to separate the biochar from the suspension of Bacillus atrophaeus A126. 100 μL of the supernatant was diluted in gradients and then plated. After culturing for 1 day, the bacterial colony forming units (CFU) on the plate were counted. The results are shown in Table 3. The biochar adsorption efficiency was calculated by the following equation:

[0103] ;

[0104] Wherein, CFUa and CFUb are the concentration of the initial Bacillus atrophaeus A126 suspension and the concentration of the supernatant, respectively.

[0105] Table 3 Physical and chemical properties and adsorption efficiency of biochar

[0106]

[0107] In order to explore the microstructural changes before and after the adsorption of Bacillus atrophaeus A126 on biochar, scanning electron microscopy observations were also performed in this example. First, the biochar before and after adsorption was fixed in glutaraldehyde at 4°C overnight, and washed three times with 0.1M phosphate buffer (PBS, pH=7.2), each time for 15 minutes. Then, it was dehydrated with a series of ethanol with gradually increasing concentrations (30%, 50%, 70%, 80%, 90% and 100%), each time for 15 minutes, and finally freeze-dried for 48 hours. Observation was carried out using a scanning electron microscope (regulus 8100, Hitachi, Japan) at 5kV, and the results are as shown Figure 5 shown. Figure 5 In the figure, A is the image before peach wood biochar adsorption, B is the image before pine wood biochar adsorption, C is the image after peach wood biochar adsorption, and D is the image after pine wood biochar adsorption. Figure 5 As shown in Table 3, the surface of peach wood biochar is rough and complex and porous, and these porous structures are significantly beneficial to increase its specific surface area and pore volume; in contrast, the surface of pine wood biochar is relatively smooth and has fewer pore structures. After adsorption, a large number of Bacillus atrophaeus A126 are attached to the inside and outside of the pores of peach wood biochar, and the adsorption efficiency reaches 97.6%; in contrast, the number of pores of pine wood biochar is relatively small, but a large number of Bacillus atrophaeus A126 are also attached to its surface, and the adsorption efficiency is 93.6%; this shows that both biochars have strong adsorption capacity, but the adsorption capacity of peach wood biochar is slightly better than that of pine wood biochar.

[0108] (4) Evaluation of biochar growth-promoting ability

[0109] Biochar has potential effects in disease control, such as promoting the growth of pathogens, etc. Therefore, this example evaluated the effects of pine biochar and peach biochar on the growth of Bacillus atrophaeus A126 and scab pathogens.

[0110] For Bacillus atrophaeus A126, pine biochar and peach biochar were mixed with NB medium at a ratio of 1:80 (w / v), and then the culture of Bacillus atrophaeus A126 was inoculated into NB medium containing pine biochar or peach biochar at a ratio of 1% (w / v). NB medium without biochar was used as a control. The culture was cultured at a temperature of 28°C and an oscillation speed of 200rpm for 24h. During this period, samples were taken every 2 hours and gradient dilutions were plated. After overnight culture, the bacterial colony forming units (CFU) on the plate were counted. The results are shown in Figure 2. Figure 6 As shown in A. Figure 6As shown in Figure A, there was no significant difference in the logarithmic phase with or without the addition of biochar; but after entering the stable phase, the bacterial concentration in the NB medium with the addition of pine biochar and peach biochar was significantly higher than that in the control group, indicating that both biochars can promote the growth of Bacillus atrophaeus A126.

[0111] For scab pathogens, pine biochar and peach biochar were mixed with water at a ratio of 1:20 (w / v) and cultured with shaking for 6 hours. Then, 30 μL of pine biochar or peach biochar water suspension was added to the Oxford cup on the culture dish pre-coated with scab pathogens. After culturing in an incubator at 28°C for 3 to 5 days, the growth of scab pathogens around the biochar in the culture dish was observed and the size of the growth promotion area was measured. The results are as follows: Figure 6 As shown in B and C, different lowercase letters represent P <0.05. Figure 6 From B and C, we can see that the growth area of ​​pine wood biochar on scab pathogens is 1.67 cm 2 , while the growth promotion area of ​​peach wood biochar is as high as 8.95cm 2 , which is about 5 times that of pine biochar, indicating that peach biochar has a significantly stronger growth-promoting effect on scab pathogens than pine biochar. P <0.05, which means that peach wood biochar significantly promoted the growth of pathogens.

[0112] (5) Effect of biochar on the antibacterial ability of Bacillus atrophaeus A126 fermentation broth

[0113] Since the BU fermentation broth (i.e., the fermentation broth of Bacillus atrophaeus A126 obtained by culturing with the optimized BU medium) needs to directly contact with biochar in the subsequent process of preparing biochar inoculant, this experiment evaluated the effects of pine biochar and peach biochar on the antibacterial ability of BU fermentation broth.

[0114] Pine biochar and peach biochar were mixed with BU fermentation broth at a ratio of 1:20 (w / v) to obtain mixed cultures. After the mixed culture was shaken and mixed in a shaker at 28°C and 200rpm for 2 hours, 30μL of the mixed culture was dripped into the Oxford cup on the culture dish coated with scab pathogens. After the bacterial solution was dried, the Oxford cup was removed to facilitate the free growth of the strain. After culturing at 28°C for 3 to 5 days, the size of the inhibition area was observed and measured. The results are as follows: Figure 7 As shown, different lowercase letters represent P <0.05. Figure 7 It can be seen that the effects of the two biochars mixed with BU fermentation broth on the inhibition area of ​​scab pathogens were significantly different; among them, the inhibition area of ​​the mixed culture with pine biochar was 6.28 cm 2, which is significantly higher than the antibacterial area of ​​3.87 cm in the mixed culture with peach wood biochar. 2 , P <0.05. This indicates that peach wood biochar reduces the antibacterial ability of BU fermentation liquid, and the antibacterial effect of pine wood biochar as an inoculation carrier is better than that of peach wood biochar. Subsequently, pine wood biochar was selected for granulation of bacterial agents and field experiment verification.

[0115] 4. Preparation, application and evaluation of biochar agents

[0116] (1) Preparation of biochar agent

[0117] In order to provide Bacillus atrophaeus A126 with a suitable living space and sufficient nutrients when used as a biocontrol bacterium, this embodiment provides a biochar agent based on Bacillus atrophaeus A126. The biochar agent of this embodiment includes three layers: inner, middle and outer. The inner layer is the core nutrient layer, which is a mixture of BU fermentation products, nutrients and biochar; wherein, the BU fermentation product is obtained by culturing Bacillus atrophaeus A126 in BU culture medium, and contains a variety of antagonistic substances produced after fermentation, such as lipopeptides, polyketide compounds and volatiles, etc. These antagonistic substances can inhibit the utilization of nutrients by pathogens; nutrients provide rich nutrition for Bacillus atrophaeus A126 for its growth. The middle layer is the live bacteria layer, which is a mixture of Bacillus atrophaeus A126 and biochar. Bacillus atrophaeus A126 can use the nutrients in the inner layer to continue to grow and reproduce. The outer layer is a microcapsule layer, which is prepared from sodium alginate and calcium chloride, and plays a role of sustained release and protection.

[0118] The preparation process of biochar agent is as follows:

[0119] ①Inner layer

[0120] (i) Dissolve 40.6 g of urea in 1 L of MSS medium, add 500 g of brown sugar, mix and dissolve at 60°C, sterilize at 121°C for 20 min, and cool for use to obtain nutrients.

[0121] (ii) The culture of Bacillus atrophaeus A126 was inoculated into BU medium at an inoculum rate of 1% (v / v) and cultured for 96 hours to obtain a BU fermentation product.

[0122] (iii) The BU fermentation product and nutrients are mixed in a ratio of 1:2 (v / v) to form a mixture, and then the pine biochar is mixed with the mixture in a ratio of 1:1.5 (w / v), and the mixture is prepared into particles by a small particle preparation machine to obtain the inner nutrient layer of the biochar agent, i.e., the first product.

[0123] ②Middle level

[0124] (i) Resuspend Bacillus atrophaeus A126 in the logarithmic growth phase in MSS medium to a concentration of about 10 9 cfu / mL, and a suspension of Bacillus atrophaeus A126 was obtained.

[0125] (ii) The suspension of Bacillus atrophaeus A126 was mixed with sterilized pine wood biochar at a ratio of 1:1.5 (v / w) to form a mixed powder that can adhere to the inner layer particles.

[0126] (iii) mixing the first product with the mixed powder at a ratio of 7:1 (w / w) so that the surface of the first product is evenly loaded with the mixed powder containing Bacillus atrophaeus A126, thereby obtaining a second product.

[0127] ③Outer layer

[0128] (i) The BU fermentation product is mixed with sterilized 5% (w / v) sodium alginate at a ratio of 1:1 (v / v), and the mixture is coated on the second product to obtain a third product.

[0129] (ii) The third product is quickly turned into a sterilized 2% (w / v) calcium chloride solution using a glass rod to form a sodium alginate microcapsule wall with sustained-release and protective effects, thereby obtaining a biochar agent.

[0130] Biochar agent form Figure 8 As shown. Figure 8 It can be seen that the biochar agent is an elliptical or round particle, and the particle size increases gradually from the inner layer to the outer layer, which are 8.26±0.86mm, 9.66±1.25mm and 10.34±1.07mm respectively. The pH value of the biochar agent is between 6 and 8, among which the pH values ​​from the inner layer to the outer layer are 6.82, 6.71 and 7.26 respectively.

[0131] (2) Field experiment of biochar agent

[0132] The field experiment was carried out in the experimental field of the Potato Center Farm of Inner Mongolia University in mid-May 2024. There were 5 treatment groups in this experiment: ① blank control; ② pine wood biochar; ③ pine wood biochar + scab pathogen; ④ biochar agent + scab pathogen; ⑤ scab pathogen. The experiment adopted a completely randomized block design, with each treatment group allocated an 80cm×80cm square, 6 potato plants planted in each square, and each treatment group had 5 replicates, totaling 30 squares.

[0133] A single colony of scab pathogen was selected and cultured in Gao's No. 1 liquid medium at 28°C and 200 rpm for 3-5 days to obtain a scab pathogen culture. Subsequently, 300 μL of the scab pathogen culture was spread on Gao's No. 1 solid medium and cultured in an incubator at 28°C for about 10 days. After the culture was completed, the culture medium was transferred to a wall breaking machine, and distilled water was added for breaking. The concentration of the scab pathogen was finally adjusted to about 10 6 cfu / mL, as the inoculum of scab pathogens. The first inoculation operation was carried out in the winter of the previous year of the experiment (i.e., November 20, 2023), and a concentration of 10 was applied at a dosage of 2L / square. 6 cfu / mL of scab pathogen inoculum. In May 2024, a second irrigating was carried out before potato planting, with the same concentration and volume of scab pathogen inoculum as the first. The blank control used only water, and the application rates of the other treatments were as follows: 350g / square of pine biochar and 700g (wet weight) / square of biochar inoculant. Potato tubers were harvested at 120 days, and the incidence and disease index of tubers were evaluated.

[0134] The severity of tuber scab is divided into five levels according to the percentage of lesion area: level 0 = no symptoms, level 1 <5%, level 2 = 5-12.5%, level 3 = 12.5-25%, level 4 = 25-50%, and level 5 >50%.

[0135] The formula for calculating the incidence of potato scab is:

[0136] ;

[0137] The disease index (DI) of potato scab is calculated according to the following formula:

[0138] ;

[0139] Where i is the representative value of each level (from 0 to 5), and N is the number of diseased potatoes at each level.

[0140] The experimental results are as follows Fig. 9 As shown, different lowercase letters represent P <0.05. Fig. 9 As shown in Figure A, the scab disease incidence rate of the positive control treatment group ⑤ with only scab pathogens added was 79.2%, and the scab disease incidence rate of the treatment group ④ with scab pathogens and biochar was 44.6%, which was significantly lower than that of the positive control treatment group ⑤. P <0.05; while the scab disease incidence rate of treatment group ③ with added scab pathogens and pine wood biochar was 70.9%, which was only slightly lower than that of the positive control group ⑤, and did not show an ideal control effect. Fig. 9As shown in Figure B, the potato disease index of the positive control treatment group ⑤ with only scab pathogens added was 0.300, and the potato disease index of the treatment group ④ with scab pathogens and biochar was 0.138, which was significantly lower than that of the positive control group. P <0.05. In summary, the biochar agent of the present invention performs well in the prevention and treatment of potato scab and has the potential to be used as a biological control agent.

[0141] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Based on Bacillus atrophaeus ( Bacillus atrophaeus ) A method for preparing a biochar agent of A126, characterized in that: The preparation method comprises the steps of: Bacillus atrophaeus A126 was inoculated into BU medium and cultured for 72-120 hours to obtain BU fermentation product; mixing the BU fermentation product and nutrients to obtain a mixture; The mixture is mixed with pine wood biochar and pelletized to obtain a first product; The first product is mixed with a mixed powder of a suspension of Bacillus atrophaeus A126 and pine biochar, so that the mixed powder is loaded on the first product to obtain a second product; mixing the second product with a mixed solution of BU fermentation product and sodium alginate, so that the mixed solution is coated on the second product, to obtain a third product; adding the third product into a calcium chloride solution to obtain the biochar agent; The BU medium is prepared with water and consists of the following ingredients: 0.3 g / L magnesium sulfate heptahydrate, 5 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate trihydrate, 5 g / L sodium chloride, 50 g / L brown sugar and 4 g / L urea; The nutrient is prepared with water and consists of the following ingredients: 0.3 g / L magnesium sulfate heptahydrate, 5 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate trihydrate, 5 g / L sodium chloride, 500 g / L brown sugar and 40.6 g / L urea; The Bacillus atrophaeus A126 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC NO.32444.

2. The preparation method according to claim 1, characterized in that: In the mixture, the volume ratio of BU fermentation product to nutrients is 1:1.8-2.

2.

3. The preparation method according to claim 1, characterized in that: In the first product, the volume mass ratio of the mixture to the pine biochar is 1:1.2~1.

8.

4. The preparation method according to claim 1, characterized in that: In the mixed powder, the volume mass ratio of the Bacillus atrophaeus A126 suspension to the pine wood biochar is 1:1.2-1.

8.

5. The preparation method according to claim 1, characterized in that: In the suspension of Bacillus atrophaeus A126, the concentration of Bacillus atrophaeus A126 is 10 8 ~10 9 cfu / mL.

6. The preparation method according to claim 1, characterized in that: In the second product, the mass ratio of the first product to the mixed powder is 6 to 8:

1.

7. The preparation method according to claim 1, characterized in that: The mixed solution is obtained by mixing the BU fermentation product and a sodium alginate solution with a mass concentration of 4-6%, wherein the volume ratio of the BU fermentation product to the sodium alginate solution with a mass concentration of 5% is 1:0.8-1.

2.

8. The preparation method according to claim 1, characterized in that: The mass concentration of the calcium chloride solution is 1.8-2.2%.

9. A biochar agent based on Bacillus atrophaeus A126, characterized in that: The biochar agent is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the biochar agent according to claim 9 in the prevention and treatment of potato scab.

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