Compound microbial fertilizer for preventing diseases and promoting growth and preparation method thereof

Abstract

This invention belongs to the field of biotechnology, specifically disclosing a compound microbial fertilizer for disease prevention and growth promotion, and its preparation method. The fertilizer consists of a modified biochar composite carrier, a nutrient matrix, a compound functional microbial agent, and a protective agent. The modified biochar composite carrier is formed by cross-linking biochar and sodium alginate with calcium ions to form a calcium alginate gel layer, which is then electrostatically compounded with chitosan. The compound functional microbial agent is a mixture of Bacillus amyloliquefaciens and Bacillus subtilis. The preparation method includes carrier and microbial agent pre-preparation, substrate sterilization, zoned inoculation and colonization, two-stage fermentation, post-treatment, and drying. This invention solves the problems of strain antagonism and easy loss of growth-promoting substances in compound microbial fertilizers through the synergistic effect of the modified carrier and dual-strain fermentation, possessing both highly efficient disease prevention and significant growth-promoting functions, while simultaneously realizing the resource utilization of agricultural waste.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a compound microbial fertilizer for disease prevention and growth promotion and its preparation method. Background Technology

[0002] With the transformation and upgrading of agriculture towards green and efficient practices, compound microbial fertilizers, which combine growth-promoting and disease-preventing functions, have become an important technological path to replace chemical inputs. Among them, Bacillus amyloliquefaciens and Bacillus subtilis, due to their excellent growth-promoting and disease-preventing potential respectively, are often used in the preparation of compound microbial fertilizers. Meanwhile, utilizing agricultural organic waste such as crop straw to prepare organic fertilizers is an effective means to achieve solid waste resource utilization and reduce fertilizer use while increasing efficiency.

[0003] However, existing technologies for preparing compound microbial fertilizers still face several prominent problems: First, in multifunctional strain mixing or co-fermentation modes, ecological niche competition and nutrient competition easily arise among strains, which not only inhibits the synthesis of disease-preventing active substances but also triggers antagonistic autotoxicity. Simultaneously, the growth-promoting substances produced by strain metabolism lack protective structures and are easily degraded and lost after application, leading to a significant shortening of the fertilizer's effective period. Furthermore, long-term nutrient competition among the microbial community can cause metabolic stagnation, further weakening the fertilizer's effectiveness. Second, traditional microbial carriers only possess simple adsorption capabilities and lack environmental responsiveness, making targeted slow-release of active substances impossible. The industry-standard high-temperature and moist heat sterilization process easily damages the special structure of functional carriers, causing carrier failure and decreased microbial activity. Existing carriers and fermentation processes struggle to simultaneously meet the two core requirements of long-term slow release and high viability. Finally, existing compound microbial fertilizers mostly use commercially available chemical raw materials and refined agricultural inputs, rarely utilizing agricultural and forestry organic waste such as crop straw, rice husks, and fruit shells. This not only wastes agricultural solid waste resources and pollutes the environment but also increases the raw material costs and energy consumption of the fertilizer production process.

[0004] Therefore, there is an urgent need to develop a new type of compound microbial fertilizer and its preparation process that can achieve deep synergy of strains, stable carrier function, and preparation based on agricultural solid waste. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a compound microbial fertilizer for disease prevention and growth promotion and its preparation method. The core concept is: to modify biochar through a dual reaction of ionic cross-linking and electrostatic compounding to construct a functional carrier with pH response and charge regulation capabilities; to achieve dual-strain regional colonization by combining the differences in oxygen environment in the carrier pores; and to utilize the dynamic changes in pH during fermentation to trigger the bidirectional feedback between the carrier and the microbial community, effectively avoiding the problems of strain antagonism and autotoxicity, extending the slow release period of growth-promoting substances, and realizing the high-value utilization of agricultural and forestry solid waste, thereby significantly improving the overall disease prevention and growth-promoting performance and product stability of the microbial fertilizer.

[0006] To achieve the above and other objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a compound microbial fertilizer for disease prevention and growth promotion, comprising the following components by weight: 50-80 parts of modified biochar composite carrier, 10-30 parts of nutrient matrix, 10-20 parts of compound functional microbial agent, and 1-5 parts of protective agent.

[0008] Furthermore, the protective agent is a mixture of sucrose and skim milk powder in a mass ratio of 3:1.

[0009] Furthermore, the compound functional microbial agent is prepared by mixing Bacillus amyloliquefaciens and Bacillus subtilis after seed culture at a live cell ratio of 1-1.5:1, and the preparation method is as follows:

[0010] (1) Activation and seed culture of the strains: Bacillus amyloliquefaciens ATCC23350 and Bacillus subtilis ATCC6633 were inoculated into LB liquid medium and cultured at 35℃ and 180 rpm for 16-18 hours with shaking to achieve a bacterial concentration of 0.8 × 10⁻⁶. 9 -1.2×10 9 CFU / mL, to obtain seed solution a and seed solution b, respectively;

[0011] (2) Preparation of compound bacterial solution: Mix the two seed solutions at a live bacteria ratio of 1-1.5:1 to obtain a mixed bacterial solution. Then add 5% of its volume of glycerol and 2% of its volume of trehalose as freeze-drying protectant to the mixed bacterial solution. After mixing, freeze-dry under vacuum to make solid bacterial powder, thus obtaining the compound functional bacterial agent.

[0012] Furthermore, the modified biochar composite carrier is prepared from biochar, sodium alginate, calcium chloride, and chitosan in a weight ratio of 10:2:0.5:1, and its preparation method is as follows:

[0013] (a) Biochar preparation: Agricultural organic waste such as crop straw, rice husk, and fruit shell is crushed, pyrolyzed at 500°C with limited oxygen for 3 hours, and then ground through a 100-mesh sieve to obtain biochar.

[0014] (b) Solution preparation: Sodium alginate was dissolved in sterile water to prepare a 2% (w / v) sodium alginate solution; chitosan was dissolved in 1% (v / v) glacial acetic acid solution to prepare a 1% (w / v) chitosan solution; and calcium chloride was dissolved in deionized water to prepare a 0.5 mol / L calcium chloride solution.

[0015] (c) Primary cross-linking: Biochar is added to sodium alginate solution and stirred to form a suspension. Then, calcium chloride solution is slowly added dropwise while stirring continuously to react, so that sodium alginate undergoes ionic cross-linking on the surface of biochar and at the macropore openings, forming a pH-responsive calcium alginate gel thin layer. After filtration, the free calcium ions are washed away with sterile water to obtain gel-coated biochar.

[0016] (d) Electrostatic composite: The gel-coated biochar is redispersed in sterile water, and chitosan solution is slowly added. The electrostatic interaction between the amino groups of chitosan and the remaining carboxyl groups on the surface of calcium alginate forms a polyelectrolyte composite layer, which further strengthens the gel network and introduces positive charge sites. After stirring for 30 minutes, the mixture is allowed to stand for 1 hour, filtered again, and the filter cake is washed with sterile water. The filter cake is then vacuum dried at 40°C to obtain the modified biochar composite carrier.

[0017] Furthermore, the nutrient matrix comprises a carbon source and a nitrogen source mixed in a 1:1 weight ratio, wherein the carbon source is molasses and the nitrogen source is soybean meal powder.

[0018] This invention also provides a method for preparing a compound microbial fertilizer for disease prevention and growth promotion, specifically including the following steps:

[0019] S1. Substrate sterilization treatment: The modified biochar composite carrier and nutrient matrix are mixed evenly, and the water content is adjusted to 40% (w / w) with sterile deionized water. The substrate is then sterilized by irradiation with 25-30kGy of 60Co-γ rays to avoid destroying the pH-responsive gel structure of calcium alginate-chitosan, thus obtaining a sterile fermentation substrate.

[0020] S2. Inoculation and initial colonization in designated areas: Redissolve the compound functional bacterial agent in sterile water to a concentration of 10. 9 The bacterial suspension of CFU / mL was evenly sprayed and inoculated into the fermentation substrate in step S1. The inoculation amount was 8-12 mL of bacterial suspension per 100 g of fermentation substrate. After stirring evenly, Bacillus amyloliquefaciens preferentially tended to and colonized the micro-aerobic / anoxic areas such as the macropores and cracks of biochar. Bacillus subtilis mainly attached to the flat areas on the outer surface of the carrier and the outer side of the gel layer. After static culture for 12 hours, the initial colonization was completed.

[0021] S3. First stage fermentation: The inoculated fermentation substrate was placed at 35℃ for 30 hours, and the pile was turned over every 12 hours. During this stage, Bacillus amyloliquefaciens in the macropores rapidly proliferated using the diffused molasses and soybean meal hydrolysates, and produced a large amount of organic acids (such as lactic acid and acetic acid) and indoleacetic acid (IAA) and other growth-promoting substances. The pH value of the system gradually decreased from the initial 7.0-7.2 to 5.5-5.8.

[0022] S4. Second stage fermentation: After the first stage fermentation is completed, fermentation continues at 35℃ for 48 hours, turning the pile every 8 hours. When the pH drops to 5.5-5.8, the carrier undergoes a dynamic response of charge and structure, forming a two-way feedback effect: the slightly acidic environment causes chitosan to protonate and become positively charged, electrostatically adsorbing the negatively charged Bacillus subtilis and inducing it to efficiently synthesize lipopeptide antibiotics for disease prevention; the calcium alginate gel shrinks, locking the IAA secreted by Bacillus amyloliquefaciens to achieve slow release; the protease secreted by Bacillus subtilis degrades soybean meal to generate a small molecule nitrogen source, supplying it to Bacillus amyloliquefaciens internally, and the chitosan adsorbs lipopeptides to avoid antagonistic autotoxicity, achieving synergistic effect of the two bacteria;

[0023] S5. Post-processing: After fermentation, a protective agent is added to the fermentation product, and then the mixture is mixed evenly to obtain a mixture;

[0024] S6. Low-temperature drying: The mixture obtained in step S5 is dried at a low temperature and ventilation at 40°C until the moisture content is ≤20%, and then pulverized through a 60-mesh sieve to obtain the disease-preventing and growth-promoting compound microbial fertilizer.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] (1) This invention, by designing a calcium alginate-chitosan modified biochar composite carrier and combining it with two bacterial strains, utilizes micro-distribution and pH dynamic feedback mechanisms to achieve a leap from spatial competition to synergistic enhancement of the composite bacterial community. In the early stage, the difference in micro-oxygen environment in the carrier pores guides Bacillus amyloliquefaciens and Bacillus subtilis to colonize in different areas. Bacillus amyloliquefaciens rapidly proliferates using nutrients and produces acid through metabolism, causing the pH of the system to decrease. In the later stage, the slightly acidic environment acts as a trigger signal, and the chitosan protonates and carries a strong positive charge. Electrostatic adsorption stresses Bacillus subtilis to efficiently synthesize lipopeptide antibiotics for disease prevention. Electrostatic adsorption also reduces the local lipopeptide concentration, avoiding antagonistic autotoxicity against Bacillus amyloliquefaciens. At the same time, gel contraction locks IAA to achieve slow release, and Bacillus subtilis enzymatically hydrolyzes soybean meal to feed back nitrogen to Bacillus amyloliquefaciens. Thus, a two-way feedback growth-promoting and disease-preventing system is constructed, which solves the problems of inhibited synthesis of disease-preventing substances, antagonistic autotoxicity, and easy loss of growth-promoting substances when multiple bacterial species coexist in the composite bacterial fertilizer.

[0027] (2) The preparation process of the calcium alginate-chitosan modified biochar composite carrier designed in this invention is scientific, stable, biocompatible, and highly environmentally responsive. Through a one-step cross-linking-electrostatic composite reaction, sodium alginate is first cross-linked on the surface of biochar to form a pH-responsive gel thin layer. After washing away the free calcium, chitosan is introduced to form a polyelectrolyte composite layer, which realizes the precise introduction of positive charge sites and the reinforcement of the gel network. Moreover, the substrate is sterilized by 60Co-γ-ray irradiation instead of traditional high-temperature moist heat sterilization, which avoids the destruction of the pH-responsive gel structure and is highly compatible with existing solid-state fermentation processes. This solves the problem that traditional microbial carriers cannot simultaneously achieve intelligent targeted sustained release and the survival of highly active bacterial agents.

[0028] (3) This invention utilizes the extracellular protease secreted by Bacillus subtilis to degrade soybean meal, generating small-molecule nitrogen sources that diffuse inwards. This provides an optimized carbon and nitrogen source for Bacillus amyloliquefaciens within the macropores, which in turn promotes the secondary metabolism and continuous acid production of Bacillus amyloliquefaciens, forming a chemically promoting mechanism of "nutrient mutual feedback." This solves the problem of metabolic stagnation caused by nutrient competition between the two bacteria in solid-state fermentation. In addition, the use of agricultural organic waste such as crop straw, rice husks, and fruit shells as raw materials to prepare biochar realizes the resource utilization of waste, reduces energy consumption and environmental pollution, and significantly reduces the preparation cost of compound microbial fertilizer. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0030] Figure 1 The graph shows the cumulative release rate of IAA for different microbial fertilizer samples under pH 7.0 and pH 5.5 conditions.

[0031] Figure 2 The graph shows the test results of lipopeptide antibiotic yield and antibacterial rate of different bacterial fertilizer samples. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0034] Unless otherwise specified, all materials and reagents used in the following examples and comparative examples are commercially available. The LB liquid culture medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0; food-grade sodium alginate; chitosan with a degree of deacetylation ≥85%; the nutrient matrix is ​​a 1:1 mixture of molasses and soybean meal powder by weight, wherein the molasses is beet molasses and the soybean meal powder is low-temperature defatted soybean meal pulverized through a 60-mesh sieve; and the preservative is a mixture of sucrose and skim milk powder by weight in a 3:1 ratio.

[0035] Example 1: This example provides a compound microbial fertilizer for disease prevention and growth promotion, which is composed of the following raw materials in parts by weight: 50 parts modified biochar composite carrier, 10 parts nutrient matrix, 10 parts compound functional microbial agent, and 1 part protective agent.

[0036] The preparation method of the compound functional microbial agent is as follows:

[0037] (1) Activation and seed culture of bacterial strains: Bacillus amyloliquefaciens ATCC23350 and Bacillus subtilis ATCC6633 were inoculated into LB liquid medium and cultured at 35℃ and 180 rpm for 16 hours with shaking to achieve a bacterial concentration of 0.8 × 10⁻⁶. 9 CFU / mL, to obtain seed solution a and seed solution b, respectively;

[0038] (2) Preparation of compound bacterial solution: Mix the two seed solutions at a live bacteria ratio of 1:1 to obtain a mixed bacterial solution. Then add 5 mL of glycerol and 2 mL of trehalose to 100 mL of the mixed bacterial solution as freeze-drying protectant. After mixing, freeze-dry under vacuum to obtain solid bacterial powder, thus obtaining the compound functional bacterial agent.

[0039] The method for preparing the modified biochar composite carrier is as follows:

[0040] (a) Biochar preparation: Wheat straw was crushed and then pyrolyzed at 500°C with limited oxygen for 3 hours. After cooling, it was ground through a 100-mesh sieve to obtain biochar.

[0041] (b) Solution preparation: Sodium alginate was dissolved in sterile water to prepare a 2% (w / v) sodium alginate solution, chitosan was dissolved in 1% (v / v) glacial acetic acid solution to prepare a 1% (w / v) chitosan solution, and calcium chloride was dissolved in deionized water to prepare a 0.5 mol / L calcium chloride solution.

[0042] (c) First cross-linking: 100g of biochar was added to 500mL of sodium alginate solution and stirred to form a suspension. Then, 500mL of 0.5mol / L calcium chloride solution was slowly added dropwise while stirring continuously. After that, the mixture was filtered and washed three times with sterile water to obtain gel-coated biochar.

[0043] (d) Electrostatic composite: The gel-coated biochar was redispersed in 500 mL of sterile water, and 250 mL of 1% chitosan solution was slowly added. After stirring for 30 minutes, the mixture was allowed to stand for 1 hour, filtered again, and the filter cake was washed twice with sterile water. The filter cake was placed in a vacuum drying oven at 40℃ and dried at a pressure of -0.09 MPa for 12 hours to obtain the modified biochar composite carrier.

[0044] This embodiment also provides a method for preparing a compound microbial fertilizer that prevents disease and promotes growth:

[0045] S1. Substrate sterilization treatment: Weigh 50 parts of the modified biochar composite carrier and 10 parts of the nutrient matrix prepared above, mix them evenly, adjust the water content to 40% (w / w) with sterile deionized water, and sterilize by irradiation with 25kGy of 60Co-γ rays to obtain sterile fermentation substrate.

[0046] S2. Regional inoculation and initial colonization: Redissolve the compound functional bacterial agent with sterile water to a concentration of 1×10⁻⁶. 9 The bacterial suspension of CFU / mL was evenly sprayed and inoculated into the fermentation substrate in step S1. The inoculation amount was 8 mL of bacterial suspension per 100 g of fermentation substrate. The mixture was stirred evenly and then allowed to stand for 12 hours.

[0047] S3. First stage fermentation: The inoculated fermentation substrate was placed at 35℃ for 30 hours, and the pile was turned over every 12 hours. The pH value of the system gradually decreased from 7.2 to 5.8.

[0048] S4. Second stage fermentation: Maintain 35℃ and continue fermentation for 48 hours, turning the pile every 8 hours until the pH of the system drops to 5.5;

[0049] S5. Post-processing: After fermentation, remove all fermentation products, add a protective agent to the fermentation products, and then mix them evenly to obtain a mixture;

[0050] S6. Low-temperature drying: The mixture obtained in step S5 is dried at 40°C with ventilation until the moisture content is 18%, and then pulverized through a 60-mesh sieve to obtain the compound microbial fertilizer for disease prevention and growth promotion.

[0051] Example 2: This example provides a compound microbial fertilizer for disease prevention and growth promotion, which is composed of the following raw materials in parts by weight: 65 parts modified biochar composite carrier, 20 parts nutrient matrix, 15 parts compound functional microbial agent, and 3 parts protective agent.

[0052] The preparation method of the compound functional microbial agent is as follows:

[0053] (1) Activation and seed culture of bacterial strains: Bacillus amyloliquefaciens ATCC23350 and Bacillus subtilis ATCC6633 were inoculated into LB liquid medium and cultured at 35℃ and 180 rpm for 17 hours with shaking to achieve a bacterial concentration of 1.0 × 10⁻⁶. 9 CFU / mL, to obtain seed solution a and seed solution b, respectively;

[0054] (2) Preparation of compound bacterial solution: Mix the two seed solutions at a live bacteria ratio of 1.25:1 to obtain a mixed bacterial solution. Then add 5 mL of glycerol and 2 mL of trehalose to 100 mL of the mixed bacterial solution as freeze-drying protectant. After mixing, freeze-dry under vacuum to obtain solid bacterial powder, thus obtaining the compound functional bacterial agent.

[0055] The preparation method of the modified biochar composite carrier is the same as that in Example 1.

[0056] This embodiment also provides a method for preparing a compound microbial fertilizer that prevents disease and promotes growth, specifically as follows:

[0057] S1. Substrate sterilization treatment: Weigh 65 parts of the modified biochar composite carrier and 20 parts of the nutrient matrix prepared above, mix them evenly, adjust the water content to 40% (w / w) with sterile deionized water, and sterilize by irradiation with 28kGy of 60Co-γ rays to obtain sterile fermentation substrate.

[0058] S2. Regional inoculation and initial colonization: Redissolve the compound functional bacterial agent with sterile water to a concentration of 1×10⁻⁶. 9 The bacterial suspension of CFU / mL was evenly sprayed and inoculated into the fermentation substrate in step S1. The inoculation amount was 10mL of bacterial suspension per 100g of fermentation substrate. The mixture was stirred evenly and then allowed to stand for 12 hours.

[0059] S3. First stage fermentation: The inoculated fermentation substrate was placed at 35℃ for 30 hours, and the pile was turned over every 12 hours. The pH value of the system gradually decreased from 7.1 to 5.6.

[0060] S4. Second stage fermentation: Maintain 35℃ and continue fermentation for 48 hours, turning the pile every 8 hours until the pH of the system drops to 5.5;

[0061] S5. Post-processing: After fermentation, remove all fermentation products, add a protective agent to the fermentation products, and then mix them evenly to obtain a mixture;

[0062] S6. Low-temperature drying: The mixture obtained in step S5 is dried at 40°C with ventilation until the moisture content is 16%, and then pulverized through a 60-mesh sieve to obtain the compound microbial fertilizer for disease prevention and growth promotion.

[0063] Example 3: This example provides a compound microbial fertilizer for disease prevention and growth promotion, which is composed of the following raw materials in parts by weight: 80 parts modified biochar composite carrier, 30 parts nutrient matrix, 20 parts compound functional microbial agent, and 5 parts protective agent.

[0064] The preparation method of the compound functional microbial agent is as follows:

[0065] (1) Activation of bacterial strains and seed culture: Bacillus amyloliquefaciens ATCC23350 and Bacillus subtilis ATCC6633 were inoculated into LB liquid medium and cultured at 35℃ and 180 rpm for 18 hours with shaking to achieve a bacterial concentration of 1.2×109 CFU / mL, and seed culture a and seed culture b were obtained respectively.

[0066] (2) Preparation of compound bacterial solution: Mix the two seed solutions at a live bacteria ratio of 1.5:1 to obtain a mixed bacterial solution. Then add 5 mL of glycerol and 2 mL of trehalose to 100 mL of the mixed bacterial solution as freeze-drying protectant. After mixing, freeze-dry under vacuum to obtain solid bacterial powder, thus obtaining the compound functional bacterial agent.

[0067] The preparation method of the modified biochar composite carrier is the same as that in Example 1.

[0068] This embodiment also provides a method for preparing a compound microbial fertilizer that prevents disease and promotes growth, specifically as follows:

[0069] S1. Substrate sterilization treatment: Weigh 80 parts of the modified biochar composite carrier and 30 parts of the nutrient matrix prepared above, mix them evenly, adjust the water content to 40% (w / w) with sterile deionized water, and sterilize by irradiation with 30kGy of 60Co-γ rays to obtain sterile fermentation substrate.

[0070] S2. Inoculation and initial colonization: The compound functional bacterial agent was reconstituted with sterile water to a bacterial suspension of 1×10⁹ CFU / mL, and sprayed evenly onto the fermentation substrate in step S1. The inoculation amount was 12mL of bacterial suspension per 100g of fermentation substrate. The mixture was stirred evenly and allowed to stand for 12 hours.

[0071] S3. First stage fermentation: The inoculated fermentation substrate was placed at 35℃ for 30 hours, and the pile was turned over every 12 hours. The pH value of the system gradually decreased from 7.0 to 5.5.

[0072] S4. Second stage fermentation: Maintain 35℃ and continue fermentation for 48 hours, turning the pile every 8 hours until the pH of the system drops to 5.5;

[0073] S5. Post-processing: After fermentation, remove all fermentation products, add a protective agent to the fermentation products, and then mix them evenly to obtain a mixture;

[0074] S6. Low-temperature drying: The mixture obtained in step S5 is dried at a low temperature and ventilation at 40°C until the moisture content is 15%, and then pulverized and passed through a 60-mesh sieve to obtain the compound microbial fertilizer for disease prevention and growth promotion.

[0075] Comparative Example 1: This comparative example provides a compound microbial fertilizer, which differs from Example 2 only in that: no modified biochar composite carrier is used, and pure biochar without modification is used directly. The other raw material types, amounts and preparation methods are the same as in Example 2.

[0076] Comparative Example 2: This comparative example provides a compound microbial fertilizer, which differs from Example 2 only in that the compound functional microbial agent uses only Bacillus subtilis ATCC6633 and does not use Bacillus amyloliquefaciens. The other raw materials, dosages and preparation methods are the same as in Example 2.

[0077] Comparative Example 3: This comparative example provides a compound microbial fertilizer, which differs from Example 2 only in that: the compound functional microbial agent uses only Bacillus amyloliquefaciens ATCC23350 and does not use Bacillus subtilis. The other raw materials, dosages and preparation methods are the same as in Example 2.

[0078] Comparative Example 4: This comparative example provides a compound microbial fertilizer, which differs from Example 2 only in that: without a fermentation process, the modified biochar composite carrier, nutrient matrix and compound functional microbial agent are directly and physically mixed evenly, and the types and amounts of other raw materials are the same as in Example 2.

[0079] Experimental methods:

[0080] 1. pH Response and IAA Slow-Release Performance Test: Accurately weigh 10g each of the microbial fertilizers prepared in Examples 1-3 and Comparative Examples 1-4, and place them in 200mL Erlenmeyer flasks respectively. Add 100mL of buffer solutions with pH 7.0 and pH 5.5, and place them in a constant temperature shaker at 35℃ and 150rpm. Samples were taken at 1h, 12h, 24h, 48h, 72h, and 120h. The cumulative release of indoleacetic acid (IAA) in the supernatant was determined using the Salkowski colorimetric method, and the cumulative release rate (%) was calculated. The results are as follows: Figure 1 As shown.

[0081] observe Figure 1 It was found that the release rate of the Example Group was relatively slow at pH 7.0, but increased when acidified at pH 5.5. In contrast, Comparative Example 1 showed rapid release regardless of pH, and Comparative Example 4, which did not undergo fermentation, left almost no IAA on the carrier. This also indirectly confirms the necessity of fermentation and colonization in steps S3 and S4.

[0082] 2. Lipopeptide antibiotic yield and inhibition rate test: Accurately weigh 5g each of the microbial fertilizers prepared in Examples 1-3 and Comparative Examples 1-4, add 20mL of sterile water, and extract by shaking at 150rpm for 1h. Centrifuge and collect the supernatant. The concentration of lipopeptide antibiotics (calculated as ituronin A standard) in the supernatant was detected using high-performance liquid chromatography (HPLC), and the lipopeptide yield per gram of dry weight microbial fertilizer was calculated. Simultaneously, the plate confrontation method was used, with *Fusarium wilt* as the indicator bacterium, to determine the diameter of the inhibition zone in the supernatant. The results are as follows: Figure 2 As shown.

[0083] observe Figure 2It can be seen that the lipopeptide yield and inhibition zone diameter of Examples 1-3 are significantly higher than those of the comparative examples. Comparative Example 1 was unmodified, and the chitosan was not protonated and carried a positive charge during fermentation, so it could not electrostatically adsorb the negatively charged Bacillus subtilis, resulting in a lack of stimulation for lipopeptide synthesis and the inability to be adsorbed and protected by chitosan, resulting in extremely low yield. Comparative Examples 2 and 3 used only single bacteria, and the lipopeptide yield and antibacterial effect were significantly insufficient. Comparative Example 4 did not undergo fermentation, and the bacterial cells did not proliferate and metabolize, so almost no lipopeptides were produced.

[0084] 3. Crop Growth Promotion Performance Test (Pot Experiment): Plump and uniformly sized Chinese cabbage seeds were selected for the experiment using a greenhouse pot method. Equal amounts of garden soil were placed in standard seedling pots for each group, with 5 seeds sown in each pot. After emergence, 3 seedlings of uniform growth were retained. The microbial fertilizer to be tested was mixed evenly with the garden soil according to the standard application ratio of microbial fertilizer:dry soil = 5-15g:1kg. A blank control group (garden soil only, without microbial fertilizer) was set up. The greenhouse environment temperature was 22-28℃, with natural light and uniform water and fertilizer management. The experiment lasted 25 days. After the cultivation period, the average plant height, average root length, and fresh weight per plant were measured for each group. The results are shown in Table 1.

[0085] Table 1. Growth results of bok choy

[0086]

[0087] As can be seen from the data in Table 1, the average plant height, root length, and fresh weight per plant in Examples 1-3 were significantly higher than those in the blank control and the comparative examples, demonstrating excellent growth-promoting function. Comparative Example 1, lacking the sustained-release protection of IAA by the gel layer and the induction protection of lipopeptides, showed a significant decrease in its growth-promoting and disease-preventing effects; Comparative Example 2, due to the burst release of IAA and insufficient disease-preventing ability, had limited growth-promoting effects; Comparative Example 3, lacking lipopeptide disease prevention, had roots susceptible to latent diseases, resulting in lower root length and fresh weight than the examples; Comparative Example 4, being merely a physical mixture, lacked colonization of live bacteria and produced no metabolites, thus exhibiting almost no growth-promoting effect.

[0088] 4. Disease Control Efficacy Test: Cucumber wilt was used as the soil-borne disease for testing. Cucumber seedlings of uniform growth were selected for the experiment. Garden soil mixed with the microbial fertilizers to be tested was placed in seedling trays. After transplanting the cucumber seedlings, an equal amount of cucumber wilt fungal spore suspension was poured onto the roots of each seedling. A blank control group was set up (inoculated only, without microbial fertilizer). The plants were cultured in a greenhouse at 24-30℃ and high humidity for 30 days. The disease incidence rate of each group was recorded, and the disease control efficacy was calculated using the following formula: Disease control efficacy (%) = (Incidence rate of blank control group - Incidence rate of experimental group) ÷ Incidence rate of blank control group × 100%.

[0089] Table 2. Disease control effect against Fusarium wilt

[0090]

[0091] Observing the data in Table 2, we can conclude that the control effect of the example group against cucumber wilt is much better than that of the comparative group. The control effect of comparative group 3 is the worst, and the effect of comparative group 2 is also average. This shows that the synergistic effect of the two bacteria is the key to efficient disease control. The control effects of comparative group 1 and comparative group 4 are also less than 40%, which verifies the necessity of modified carrier and fermentation process.

[0092] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0093] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A compound microbial fertilizer for disease prevention and growth promotion, characterized in that, The compound microbial fertilizer comprises the following components by weight: modified 50-80 parts of biochar composite carrier, 10-30 parts of nutrient matrix, 10-20 parts of compound functional microbial agent, and 1-5 parts of protective agent; The modified biochar composite carrier is composed of biochar, sodium alginate, calcium chloride, and chitosan in a weight ratio of 10:

2. It is prepared at a ratio of 0.5:1, and its preparation method is as follows: (a) Biochar preparation: Agricultural organic waste is crushed and then subjected to oxygen-limited pyrolysis, followed by grinding and sieving to obtain biochar; (b) Solution preparation: Dissolve sodium alginate in sterile water to prepare sodium alginate solution, dissolve chitosan in glacial acetic acid solution to prepare chitosan solution, and dissolve calcium chloride in deionized water to prepare calcium chloride solution. (c) Primary cross-linking: Biochar is added to sodium alginate solution and stirred to form a suspension. Then, calcium chloride solution is added dropwise and stirred to react. After filtration and washing, gel-coated biochar is obtained. (d) Electrostatic composite: Disperse the gel-coated biochar in sterile water, add chitosan solution, stir and let stand, filter, wash the filter cake with sterile water and vacuum dry to obtain the modified biochar composite carrier.

2. The compound microbial fertilizer for disease prevention and growth promotion according to claim 1, characterized in that, The nutrient matrix comprises a carbon source and a nitrogen source mixed in a 1:1 weight ratio.

3. The compound microbial fertilizer for disease prevention and growth promotion according to claim 2, characterized in that, The carbon source is molasses; the nitrogen source is soybean meal.

4. The compound microbial fertilizer for disease prevention and growth promotion according to claim 1, characterized in that, The compound functional microbial agent is obtained by mixing Bacillus amyloliquefaciens and Bacillus subtilis after seed culture at a live bacteria ratio of 1-1.5:

1.

5. The compound microbial fertilizer for disease prevention and growth promotion according to claim 4, characterized in that, The preparation method of the compound functional microbial agent is as follows: (1) Activation of microbial strains and seed culture: Bacillus amyloliquefaciens and Bacillus subtilis are inoculated into LB liquid culture medium respectively, and seed culture is obtained by shaking. (2) Compound microbial solution preparation: Seed culture a and seed culture b are mixed to obtain mixed microbial solution. Then, a freeze-drying protectant is added to the mixed microbial solution, and after mixing, it is freeze-dried under vacuum to make solid microbial powder, which is the compound functional microbial agent.

6. The compound microbial fertilizer for disease prevention and growth promotion according to claim 5, characterized in that, The freeze-drying protectant is glycerol and trehalose.

7. The compound microbial fertilizer for disease prevention and growth promotion according to claim 1, characterized in that, The protective agent is a mixture of sucrose and skim milk powder in a mass ratio of 3:

1.

8. A method for preparing a compound microbial fertilizer for disease prevention and growth promotion according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Substrate sterilization treatment: The modified biochar composite carrier and the nutrient matrix are mixed evenly, the moisture content is adjusted with sterile deionized water and then sterilized by irradiation to obtain the fermentation substrate; S2. Inoculation and initial colonization: The compound functional microbial agent is reconstituted with sterile water and sprayed into the fermentation substrate. After stirring evenly, it is allowed to stand for culture to complete the initial colonization. S3. First stage fermentation: Place the inoculated fermentation substrate at 35℃ for 30 hours, turning the pile over every 12 hours. S4. Second stage fermentation: After the first stage fermentation is completed, maintain 35℃ and continue fermentation for 48 hours, turning the pile over every 8 hours; S5. Post-processing: After the second stage of fermentation is completed, a protective agent is added to the fermentation product and mixed evenly to obtain a mixture; S6. Low-temperature drying: The mixture is dried in a ventilated manner, crushed and sieved to obtain the compound microbial fertilizer for disease prevention and growth promotion.

Images