Preparation method of carbon-based microbial fertilizer capable of reducing vegetable clubroot morbidity

Abstract

The invention relates to a preparation method of a carbon-based microbial fertilizer, and belongs to the technical field of agricultural microbial fertilizers. The method comprises the following steps: inoculating a bacillus firmus TB-7 strain into an NA liquid culture medium, and culturing for 24 hours under the condition of a constant-temperature shaking table at 37 DEG C to obtain a strain fermentation solution; sterilizing the biochar for 60 minutes under the condition of 121 DEG C; mixing the fermentation liquor with the sterilized biochar according to the mass ratio of 1: 2, and regulating the water content to 30%, so that the content of effective viable bacteria in the mixture is not less than 3 * 10 CFU / g; and culturing the mixture at a constant temperature of 37 DEG C for 24 hours to obtain the carbon-based microbial fertilizer. The culture medium components of the TB-7 strain are optimized through a single factor experiment and an orthogonal experiment, and the thallus activity and yield of the TB-7 strain are improved. The obtained bacterial fertilizer has the characteristics of high bacterial content and stable preparation conditions, and can be applied as a base fertilizer in the Chinese cabbage planting process. The invention provides the preparation method of the carbon-based microbial fertilizer, which is simple and convenient in preparation process, strong in strain adaptability and high in carrier stability.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of microbial technology, and particularly relates to a method for preparing carbon-based microbial fertilizer that can reduce the incidence of clubroot disease in Chinese cabbage. Background Technology

[0002] Cruciferous vegetables have significant economic value in China, but clubroot is a major disease affecting their growth. *Platycodon brassicae*, the pathogen of clubroot, is an obligate parasite that primarily damages the roots of cruciferous crops, causing clubroot disease. This leads to abnormal swelling of the roots, resulting in nodular masses of varying sizes, hindering nutrient and water absorption. Later, leaves turn yellow and wither, and in severe cases, the entire plant dies. Dormant spores of *Platycodon brassicae* can survive in the soil for up to 17 years or more, making clubroot control difficult, severely impacting farmers' economic benefits, and hindering the sustainable development of the cruciferous vegetable industry. Currently, clubroot control mainly relies on agricultural and chemical methods. Agricultural control methods include crop rotation, artificially altering the soil environment, strengthening field management, rational fertilization, and loosening the soil through cultivation; however, these methods are slow-acting, time-consuming, and have limited effectiveness. Chemical control is currently the most widely used control measure. Pesticides such as fluazinam, cyazofamid, and chlorothalonil are fast-acting, short-lasting, and highly effective in controlling clubroot. However, long-term use of chemical pesticides can lead to numerous problems, including pesticide resistance, environmental pollution, and food safety issues. From the perspective of sustainable and green ecological development, clubroot control must explore other effective and environmentally friendly methods. Biological control has advantages such as zero pollution and high safety, and therefore has significant research value and application prospects.

[0003] Based on the above analysis, the existing technologies have the following problems and shortcomings: Clubroot disease in Chinese cabbage is currently mainly controlled through agriculture and chemical pesticides. Agricultural control involves artificially altering the soil environment and strengthening field cultivation management, but the control effect is not significant and takes a long time. Chemical control mainly involves manually spraying pesticides to suppress clubroot disease, which easily leads to pesticide resistance, kills beneficial microorganisms in the soil, reduces soil fertility, and severely reduces Chinese cabbage yield.

[0004] During the search, the closest prior art example to this invention is the patent "Bio-organic Fertilizer and its Preparation Method and Application" (application number CN114751792A). This document discloses a bio-organic fertilizer containing a specific strain (WSWFJ44, belonging to Bacillus subtilis). Its formula includes polyglutamic acid, fulvic acid, various organic residues (such as marigold flower residue and rapeseed meal), and decomposing microbial inoculants, and is used to prevent and control root-knot nematode disease, while promoting crop root regrowth and improving yield and quality.

[0005] Existing technical problems:

[0006] 1. This literature uses a solid organic matrix (plant residue) composite microbial agent method, without involving the carbon-based microbial fertilizer system of "biochar + TB-7 strain", nor does it optimize the requirements of microbial liquid impregnation, humidity adjustment and high bacterial content based on biochar carrier.

[0007] 2. This system uses the Bacillus subtilis WSWFJ44 strain, lacking comparative data on culture medium optimization, fermentation conditions, and significant increases in bacterial count for specific strains (such as Bacillus subtilis TB-7). This means that the existing technology does not offer the advantages of using TB-7 and carbon-based carriers in terms of microbial fertilizer efficacy, preservation stability, and industrial application. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a method for preparing carbon-based microbial fertilizer that can reduce the incidence of clubroot disease in Chinese cabbage.

[0009] This invention is achieved through a method for preparing a carbon-based microbial fertilizer that can reduce the incidence of clubroot disease in Chinese cabbage, comprising the following steps:

[0010] Step 1: Inoculate TB-7 strain into NA liquid medium and culture at 37°C in a constant temperature shaker for 24 h to obtain the fermentation broth of the strain.

[0011] Step two: Sterilize the biochar at 121℃ for 60 minutes. Add the prepared bacterial solution to the sterilized biochar to prepare 5 kg of carbon-based bio-fertilizer, with a bacterial count of 3 × 10⁻⁶. 8 With a concentration of CFU·g⁻¹ or higher, adjust the moisture content to 30%, place it in an incubator, and incubate at a constant temperature of 37℃ for 24 hours to obtain carbon-based microbial fertilizer.

[0012] Furthermore, the culture medium was optimized as follows: 3.0 g beef extract, 5 g corn flour, 5.0 g NaCl, 10.0 g peptone, 1000 mL water, pH 7.2–7.5. This invention utilizes single-factor experiments and orthogonal experiments to optimize the culture medium components and distribution ratios of the *Bacillus stolonifer* strain. Compared to the unoptimized state, the effective bacterial count of the strain increased by 10.5 times, providing a theoretical basis for its development and field application, and laying the foundation for subsequent optimization of fermentation conditions and its application in production.

[0013] Furthermore, the carbon-based microbial fertilizer is a mixture of Bacillus stolonifera fermentation broth and biochar in a 1:2 ratio. This parameter was determined through multiple experiments in this invention, and the carbon-based microbial fertilizer formulated according to this parameter exhibits stable effective viable bacteria count and field application effects.

[0014] Furthermore, Bacillus stolonifera was screened by detecting the inhibition rate of germination of clubroot spores in Chinese cabbage. It is more direct and targeted than currently used strains and can effectively inhibit the germination of clubroot spores.

[0015] Furthermore, apply 50 g of carbon-based microbial fertilizer as base fertilizer to each cabbage plant at the time of planting, and perform other field management as in conventional production, without using fungicides.

[0016] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0017] The difficulty in overcoming the problems and shortcomings of existing technologies lies in the fact that clubroot disease has become a serious disease affecting the yield and quality of cruciferous vegetables. *Cladophora brassicae* is an obligate live parasite, difficult to culture in vitro, making it extremely challenging to obtain artificially purified cultures for screening biocontrol agents against clubroot disease. Microorganisms with antagonistic effects against clubroot disease in cruciferous vegetables mainly include *Bacillus*, *Trichoderma*, *Streptomyces*, and *Lysobacterium*, but most are still in the experimental stage. Therefore, to better control clubroot disease, it is urgent to find new antagonistic bacteria for its control. Thus, developing microbial fertilizers that can effectively control clubroot disease in Chinese cabbage while also improving the soil microecological environment and promoting Chinese cabbage yield is a key challenge in the biological control of this crop.

[0018] The TB-7 strain (CGMCC NO: 35108) screened in this invention has been identified as *Bacillus stolonifer*, a member of the genus *Bacillus*. Currently, there are no reports on its application in the control of clubroot disease. Compared with *Bacillus subtilis* XF-1, which is already in practical use, both are *Bacillus* species. However, they differ in their screening methods: *Bacillus subtilis* cannot be artificially cultured, while the XF-1 strain was screened through a pathogen indicator plate confrontation test, which has certain limitations. The prerequisite for clubroot disease is the germination of dormant spores; the TB-7 strain (*Bacillus stolonifer*) screened in this invention by detecting the inhibition rate of dormant spore germination is more direct and targeted.

[0019] Statistics show that clubroot disease has occurred in more than 10 prefectures and cities (49 counties and districts) in Yunnan Province, with a total affected area of ​​over 2 million mu (approximately 133,333 hectares). It has become a major factor affecting the yield of Chinese cabbage and other cruciferous vegetables in Yunnan. The *Bacillus stolonifera* in the carbon-based microbial fertilizer used in this invention, through optimization of the fermentation culture components, can secrete bacteriocins to inhibit the germination of dormant clubroot spores, effectively reducing the incidence of clubroot disease in Chinese cabbage and increasing its yield.

[0020] To improve the adaptability of the strains, this invention used cabbage plants in vegetable greenhouses in Sanbao Township, Qilin District, Qujing City, Yunnan Province, where the incidence of clubroot disease in the entire crop was 60%~70%, as well as the rhizosphere soil of the cabbage plants, as soil samples for screening biocontrol strains. This was done to improve the tolerance of the strains and make the prepared inoculant more suitable for the soil environment in which cabbage is grown.

[0021] This invention provides a carbon-based microbial fertilizer that reduces the incidence of clubroot disease in Chinese cabbage. The fertilizer contains a fermentation broth of *Bacillus stolonifera* strain, a probiotic that secretes various auxins and antibacterial substances during its growth. Clubroot pathogens cannot be artificially cultured; previously used strains were screened through plate confrontation tests with indicator bacteria. However, clubroot disease development requires the germination of dormant spores, thus limiting the effectiveness of previously screened strains in controlling the disease. This invention, by detecting the inhibition rate of dormant spore germination, utilizes *Bacillus stolonifera* strains, which are more direct and targeted. Furthermore, this invention mixes the *Bacillus stolonifera* fermentation broth with biochar at a 1:2 ratio. This parameter was determined through multiple experiments, and the carbon-based microbial fertilizer formulated according to this parameter exhibits stable effective viable bacteria counts and field application effects. Biochar, rich in functional groups, porous, and with a large specific surface area, is a high-quality soil conditioner. When applied to the field with biochar loaded with Bacillus thuringiensis, it promotes the growth and development of Chinese cabbage, improves its agronomic traits, and has a good control effect on clubroot disease, significantly reducing its incidence and mortality. Furthermore, compared with traditional chemical control, the application of this carbon-based microbial fertilizer can regulate the soil microecological environment, increase the number of beneficial microorganisms in the soil, and reduce the number of pathogens, thereby reducing the occurrence of Chinese cabbage diseases, reducing the amount of chemical fertilizers and pesticides used, and achieving the effect of biological control and increased yield. It is a green, safe, and high-quality microbial fertilizer.

[0022] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: Many achievements have been made in screening biocontrol bacteria for the prevention of clubroot disease, but only one biological pesticide, Bacillus subtilis, is registered in my country for the prevention and control of clubroot disease (http: / / www.icama.org.cn / zwb / index). This biochar-based fertilizer can effectively reduce the incidence of clubroot disease in Chinese cabbage, increase the yield per mu of Chinese cabbage, and increase the economic benefits of farmers.

[0023] (2) The technical solution of the present invention solves the technical problem that people have long wanted to solve but have never been able to succeed in: the clubroot fungus cannot be artificially cultured, and other strains currently used (such as XF-1) are screened by the pathogen indicator bacteria plate confrontation test, which has certain limitations; the prerequisite for the onset of clubroot disease is the germination of dormant spores, and the strong Bacillus strain screened by the present invention by detecting the germination inhibition rate of dormant spores is more direct and targeted.

[0024] (3) The technical solution of the present invention overcomes technical bias:

[0025] In the production of Bacillus organic fertilizer, the scientific combination of microbial fermentation broth and biochar can effectively reduce the infection rate of clubroot disease in Chinese cabbage, improve the soil, and promote the yield of Chinese cabbage, achieving a complementary effect.

[0026] In existing technologies, methods to reduce the infection rate of clubroot disease in Chinese cabbage mainly rely on chemical control and planting disease-resistant varieties. However, these methods have the following technical problems:

[0027] 1) Environmental impact: Chemical control (such as fluazinam suspension) involves spraying pesticides to inhibit or kill diseases. It is fast-acting and has a short duration of effect. However, long-term application of pesticides can cause certain damage to the soil structure and ecological environment. Therefore, it is not advisable to apply them in excessive amounts.

[0028] 2) Stability of effect: Planting disease-resistant varieties, that is, using disease-resistant varieties to prevent the occurrence of clubroot. However, their physiological races are complex and variable. After years of promotion, the resistance of resistant varieties can easily be lost, and the disease resistance is unstable, which is affected by the genetic background of the variety and environmental conditions.

[0029] Technological advancements:

[0030] This invention represents a significant technological advancement by using fermentation broth of robust spore-forming strains added to biochar to produce microbial fertilizer, thereby reducing the incidence of clubroot disease in Chinese cabbage.

[0031] 1) Environmental friendliness: The carbon-based microbial fertilizer used in the method of this invention reduces the use of chemical pesticides, is environmentally friendly, reduces the risk of pollution to soil and water, and meets the requirements for green, environmentally friendly and safe vegetable planting.

[0032] 2) Targeted biocontrol effect of clubroot: The Bacillus stolonifera in this invention was screened by detecting the inhibition rate of clubroot dormant spores in Chinese cabbage. It is more direct and targeted than the strains currently used on the market and can effectively inhibit the germination of clubroot dormant spores.

[0033] 3) Cost-effectiveness: The carbon-based microbial fertilizer method has lower costs and is easy to scale up, resulting in higher economic benefits compared to traditional chemical reagents and breeding methods.

[0034] 4) Optimization of fermentation culture components of the strain: The performance of the strain itself, the appropriate culture medium and the culture conditions of the strain can all affect the fermentation level of Bacillus. In this invention, the culture medium components and the distribution ratio of each group of Bacillus stolonifer strain were optimized by using single-factor experiments and orthogonal experiments. Compared with the unoptimized case, the effective bacterial count of the strain was increased by 10.5 times, which improved the stability of this invention in the prevention and control of clubroot disease in Chinese cabbage.

[0035] Through these technological advancements, this invention provides a more environmentally friendly, economical, and effective solution for reducing the incidence of clubroot disease in Chinese cabbage. Attached Figure Description

[0036] Figure 1 This is a flowchart of a method for preparing carbon-based microbial fertilizer that can reduce the incidence of clubroot disease in Chinese cabbage, provided in an embodiment of the present invention.

[0037] Figure 2 These are the experimental results of Bacillus thymotherum provided in the embodiments of the present invention in actual production. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] The China General Microbiological Culture Collection Center (CGMCC) issued an Acceptance Notice (Receipt) and a Viability Report for the preservation of biological materials in patent proceedings. The documents show that the client is Zhou Xiaogang from the Yunnan Academy of Agricultural Sciences, and the submitted biological material, numbered TB-7, was identified as *Cytobacillus firmus*.

[0040] The biological material was officially accepted and registered by CGMCC on July 4, 2025, with the registration number CGMCC No. 35108. According to the preservation regulations, the material is to be preserved for thirty years, with the possibility of extension upon request before the expiration date. The document clearly states that the viability of the material was tested on the same day, and the result was "viable".

[0041] This invention is based on and improves upon the screening scheme of the XF-1 strain developed by Professor He Yueqiu's team. By observing the germination inhibition rate of the strain against clubroot diseased cabbage buds, a more intuitive and accurate method was used to screen for Bacillus TB-7, which has a good inhibitory function against clubroot disease in cabbage. Combined with biochar, a microbial fertilizer capable of reducing the incidence of clubroot disease in cabbage was obtained. The specific method is as follows: Step 1, the TB-7 strain was inoculated into NA liquid medium and cultured at 37°C in a shaker for 24 hours to obtain the fermentation broth of the strain.

[0042] Step two: Sterilize the biochar at 121℃ for 60 minutes. Add the prepared bacterial solution to the sterilized biochar to prepare 5 kg of carbon-based bio-fertilizer, with a bacterial count of 3 × 10⁻⁶. 8 With a concentration of CFU·g⁻¹ or higher, adjust the moisture content to 30%, place it in an incubator, and incubate at a constant temperature of 37℃ for 24 hours to obtain carbon-based microbial fertilizer.

[0043] The culture medium in step one consisted of 3.0 g beef extract, 5 g corn flour, 5.0 g NaCl, 10.0 g peptone, and 1000 mL water, with a pH of 7.2–7.5. This invention utilizes single-factor experiments and orthogonal experiments to optimize the culture medium components and distribution ratios of the *Bacillus stolonifer* strain. Compared to the unoptimized result, the effective bacterial count of the strain increased by 10.5 times, providing a theoretical basis for its development and field application, and laying the foundation for subsequent optimization of fermentation conditions and its application in production.

[0044] The carbon-based microbial fertilizer mentioned in step two is a mixture of TB-7 fermentation broth and biochar at a ratio of 1:2. This parameter was determined through multiple experiments in this invention, and the carbon-based microbial fertilizer prepared according to this parameter has a stable number of effective live bacteria and stable field application effects.

[0045] This invention provides a carbon-based microbial fertilizer that can reduce the incidence of clubroot disease in Chinese cabbage. The fertilizer contains 3×10⁻⁶ Bacillus stolonifera. 8 CFU·g -1 The moisture content is 30%.

[0046] A cabbage growing base aimed to increase cabbage yield and quality, thereby improving the economic benefits of cabbage cultivation, by reducing the incidence of clubroot disease. Therefore, they chose to use the aforementioned microbial inoculant for treatment.

[0047] 1) Preparation of fermentation broth for bacterial strains:

[0048] The TB-7 strain was inoculated into NA liquid medium and cultured in a constant temperature shaker at 37°C for 24 h to obtain the fermentation broth of the strain.

[0049] 2) Preparation of carbon-based microbial fertilizer:

[0050] The biochar was sterilized at 121℃ for 60 minutes. The prepared fermentation broth was added to the sterilized biochar to prepare 5 kg of carbon-based bio-fertilizer, with a bacterial count of 3 × 10⁻⁶. 8 CFU·g -1 Adjust the moisture content to 30%, place it in an incubator, and incubate at 37℃ for 24 hours to obtain carbon-based microbial fertilizer.

[0051] 3) Applied to cabbage cultivation:

[0052] In a Chinese cabbage planting base, Chinese cabbage seeds were sown in fields infected with clubroot disease. Three treatments were set up, with each treatment replicated three times. Each replicate had 30 seedlings, for a total of 90 seedlings per treatment. Treatment 1 involved applying 50 g of carbon-based microbial fertilizer per Chinese cabbage plant as basal fertilizer, with other field management as in conventional production, and no fungicides used. Treatment 2 involved conventional basal fertilizer, but with the application of 500 times diluted 50% fluazinam suspension to control clubroot disease; Treatment 3 served as a blank control (CK).

[0053] 4) Effectiveness Evaluation:

[0054] Forty-five days after planting, the cabbages were pulled up by the roots and the soil around the roots was washed off with clean water. The incidence and disease index of the plants were investigated. The results showed that the treatment with carbon-based microbial fertilizer significantly reduced the incidence of clubroot disease in the cabbages, achieving the expected results.

[0055] The carbon-based microbial fertilizer and its *Bacillus stolonifera* strain provided in this invention, which can reduce the incidence of clubroot disease in Chinese cabbage, have not yet been reported for application in clubroot disease control. Compared to *Bacillus subtilis* XF-1, which is already in practical use, both are *Bacillus* strains, but their screening methods differ: *Bacillus subtilis* cannot be artificially cultured, while the XF-1 strain was screened through a pathogen indicator bacteria plate confrontation test, which has certain limitations; the prerequisite for clubroot disease is the germination of dormant spores, and the *Bacillus stolonifera* strain screened by this invention through detecting the germination inhibition rate of dormant spores is more direct and targeted. Furthermore, *Bacillus* can produce round or oval spores, exhibiting strong stress resistance and environmental adaptability, being acid-resistant and heat-resistant. The antibacterial substances produced by *Bacillus* have certain biocontrol and growth-promoting effects, enabling them to adapt more quickly to the soil environment of Chinese cabbage cultivation, and the biocontrol effect of the microbial fertilizer on clubroot disease can be observed in a short period.

[0056] like Figure 1 As shown in the embodiment of the present invention, the method for preparing carbon-based microbial fertilizer that can reduce the incidence of clubroot disease in Chinese cabbage includes the following steps:

[0057] Step 1: Inoculate TB-7 strain into NA liquid medium and culture at 37°C in a constant temperature shaker for 24 h to obtain the fermentation broth of the strain.

[0058] Step two: Sterilize the biochar at 121℃ for 60 minutes. Add the prepared bacterial solution to the sterilized biochar to prepare 5 kg of carbon-based bio-fertilizer, with a bacterial count of 3 × 10⁻⁶. 8 With a concentration of CFU·g⁻¹ or higher, adjust the moisture content to 30%, place it in an incubator, and incubate at a constant temperature of 37℃ for 24 hours to obtain carbon-based microbial fertilizer.

[0059] The culture medium in step one consisted of 3.0 g beef extract, 5 g corn flour, 5.0 g NaCl, 10.0 g peptone, and 1000 mL water, with a pH of 7.2–7.5. This invention utilizes single-factor experiments and orthogonal experiments to optimize the culture medium components and distribution ratios of the *Bacillus stolonifer* strain. Compared to the unoptimized result, the effective bacterial count of the strain increased by 10.5 times, providing a theoretical basis for its development and field application, and laying the foundation for subsequent optimization of fermentation conditions and its application in production.

[0060] The carbon-based microbial fertilizer mentioned in step two is a mixture of TB-7 fermentation broth and biochar at a ratio of 1:2. This parameter was determined through multiple experiments in this invention, and the carbon-based microbial fertilizer prepared according to this parameter has a stable number of effective live bacteria and stable field application effects.

[0061] This invention provides a carbon-based microbial fertilizer that can reduce the incidence of clubroot disease in Chinese cabbage. The fertilizer contains 3×10⁻⁶ Bacillus stolonifera. 8 CFU·g -1 The moisture content is 30%. Currently, there are no reports on the application of the *Bacillus stolonifer* used in the control of clubroot disease. The development of clubroot disease requires the germination of dormant spores. The *Bacillus stolonifer* strain screened in this invention by detecting the inhibition rate of dormant spore germination is more direct and targeted than the already practically applied *Bacillus subtilis* XF-1. Furthermore, *Bacillus stolonifer* can produce round or oval spores, exhibiting extremely strong resistance and environmental adaptability. It is acid-resistant, heat-resistant, and can survive in various adverse environments such as low oxygen, ultraviolet radiation, electromagnetic radiation, and certain chemical reagents. The antibacterial substances produced by *Bacillus stolonifer* have certain biocontrol and growth-promoting effects and are harmless to humans and animals, and are often used as biocontrol bacteria in field production.

[0062] This invention verifies the application of Bacillus thuringiensis in actual production through field trials. The experimental results are shown in […]. Figure 2 See Table 1.

[0063] Table 1 Results of the efficacy test of each treatment against clubroot disease

[0064]

[0065] Note: Data in the table are mean ± standard deviation. p ≤ 0.05. The same letters in the table indicate no significant difference. The same applies below.

[0066] Table 2 Fresh weight of individual plants in the aboveground parts for each treatment

[0067]

[0068] Table 3 Average plant height of aboveground parts for each treatment

[0069]

[0070] Compared with the water treatment group, both TB-7 bacterial solution and 500-fold dilution of 50% fluazinam suspension reduced the incidence and disease index of clubroot disease in Chinese cabbage. The field control efficacy of TB-7 bacterial solution was 43.76%, and that of 500-fold dilution of 50% fluazinam was 78.61%. While investigating the disease incidence in Chinese cabbage, the aboveground fresh weight and plant height of the three treatments were also investigated. The results showed that the average aboveground fresh weight of Chinese cabbage plants treated with TB-7 carbon-based microbial fertilizer reached 131.067 g / plant, and the average plant height was 20.62 cm, equivalent to a yield of 397.128 kg / mu. The water treatment was the second best, with an average aboveground fresh weight of 91.312 g / plant, an average plant height of 18.33 cm, and a yield of 269.82 kg / mu. The lowest average fresh weight of the above-ground parts was observed with the 500-fold dilution of 50% fluazinam treatment, at 74.807 g / plant; the average plant height was 17.76 cm; and the equivalent yield was 229.563 kg / mu (see Tables 2 and 3). These conclusions indicate that TB-7 carbon-based microbial fertilizer can not only alleviate clubroot disease to some extent but also increase the yield of Chinese cabbage plants, demonstrating its practical application value.

[0071] Example 1

[0072] Preparation method of carbon-based microbial fertilizer to reduce the incidence of clubroot disease in vegetables: Bacillus stolonifera TB-7 strain was inoculated into NA liquid medium containing 3.0 g beef extract, 5.0 g corn flour, 5.0 g NaCl, 10.0 g peptone, and 1000 mL distilled water. The medium was cultured in a shaker at 37℃ for 24 hours to obtain a bacterial concentration of 1.2 × 10⁻⁶. 9The fermentation broth was prepared at a concentration of CFU / mL. Biochar, pulverized to an 80-mesh sieve, was then autoclaved at 121°C for 60 minutes and cooled. The resulting fermentation broth was mixed with the sterilized biochar at a mass ratio of 1:2, and the moisture content was adjusted to 30%. After thorough mixing, the mixture was incubated at 37°C for 24 hours. The effective viable bacteria count of the finished carbon-based microbial fertilizer was determined to be 3.6 × 10⁻⁶. 8 CFU / g.

[0073] The microbial fertilizer prepared by the above method was applied to Chinese cabbage cultivation at a rate of 50 g per plant. After application, the incidence of clubroot disease in Chinese cabbage decreased by more than 40%.

[0074] Example 2

[0075] Using rice husk charcoal as a biochar raw material can reduce the incidence of clubroot disease while increasing the yield and quality of Chinese cabbage. Following the method in Example 1, *Bacillus thuringiensis* TB-7 was cultured in an optimized NA medium (3.5 g beef extract, 6.0 g corn flour, 5.0 g NaCl, 12 g peptone, 1000 mL water, pH 7.3), achieving an effective bacterial count of 1.0 × 10⁻⁶ in the fermentation broth. 9 CFU / mL. Rice husk charcoal was selected as the biochar raw material, sterilized at 121℃ for 60 minutes, and then cooled for later use. The fermentation broth and biochar were mixed at a mass ratio of 1:2, the humidity was adjusted to 30%, and the mixture was placed in a constant temperature incubator for 24 hours to obtain the finished carbon-based microbial fertilizer with a viable cell count of 3.2 × 10⁻⁶ CFU / mL. 8 CFU / g.

[0076] In field trials of Chinese cabbage prepared by the above method, when 50 g of the microbial fertilizer was applied as a basal fertilizer to each plant, the plants showed better growth and individual quality than those that did not receive the above-mentioned carbon-based microbial fertilizer. The average plant height increased by 12%, and the yield increased by 15%.

[0077] Example 3

[0078] When charcoal chips are used as a biochar source, in addition to reducing the incidence of clubroot disease in Chinese cabbage, application at the early stage of planting can also promote root development and improve the growth and disease resistance of Chinese cabbage. Following the method in Example 1, *Bacillus stolonifer* strain TB-7 was inoculated into conventional NA liquid medium and cultured in a shaker at 37°C for 24 hours, yielding a bacterial count of 9.5 × 10⁻⁶. 8 The fermentation broth contained CFU / mL of biochar. The biochar prepared from sawdust charcoal was sterilized at 121℃ for 60 minutes and then set aside. The fermentation broth and sterilized biochar were mixed at a 1:2 ratio, humidified to 30%, and incubated at 37℃ for 24 hours. The resulting microbial fertilizer had a viable count of 3.8 × 10⁻⁶. 8 CFU / g.

[0079] When applied to areas severely affected by clubroot disease in Chinese cabbage, the disease index was significantly reduced, the root system was well-developed, and the leaves above ground were thick and green.

[0080] Example 4

[0081] Using straw charcoal as a biochar source can improve the stability and reproductive capacity of the TB-7 strain, resulting in a longer-lasting fertilizer effect after the application of carbon-based microbial fertilizer. This promotes the growth of cabbage plants while continuously reducing the incidence of clubroot disease. Following Example 1, fermentation in conventional NA medium for 24 hours yielded a bacterial count of 1.0 × 10⁻⁶ cells in the fermentation broth. 9 CFU / mL. After sterilizing the straw charcoal at 121℃ for 1 hour, it was mixed with the fermentation broth at a 1:2 mass ratio. The humidity was adjusted to 30%, and the mixture was incubated at a constant temperature for 24 hours to obtain carbon-based microbial fertilizer with a viable bacterial count of 3.5 × 10⁻⁶. 8 CFU / g.

[0082] In a cabbage cultivation experiment, applying 50 g of fertilizer per plant resulted in significantly better plant growth than the control group, with an average yield increase of 12% per plant and a 35% decrease in disease incidence. This example demonstrates that straw charcoal, as a carrier, can also ensure the stable reproduction and functional performance of TB-7.

[0083] Example 5

[0084] Through the optimization of the culture scheme and culture medium formulation of strain TB-7 in Examples 1-4, and the screening of biochar sources, the optimal carbon-based microbial fertilizer scheme was obtained through the inhibition rate experiment of clubroot dormant spore germination. Following the method in Example 1, strain TB-7 was inoculated into a corn flour substitute medium (8.0 g corn flour, 2.5 g beef extract, 5.0 g NaCl, 9 g peptone, 1000 mL water, pH 7.4) and cultured at 37°C for 24 hours, with a fermentation broth concentration of 1.1 × 10⁻⁶. 9 CFU / mL.

[0085] Bamboo charcoal was selected as the biochar carrier and sterilized at 121℃ for 60 minutes. The fermentation broth was mixed with the sterilized bamboo charcoal (1:2), the moisture content was adjusted to 30%, and the mixture was incubated for 24 hours. The resulting biofertilizer had an effective viable bacteria count of 3.4 × 10⁻⁶. 8 CFU / g. This microbial fertilizer significantly reduced the incidence of clubroot disease in pot experiments, and the number of detectable microbial communities in the soil remained stable.

[0086] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a carbon-based microbial fertilizer, characterized in that, Includes the following steps: (1) Inoculate Bacillus TB-7 strain into liquid culture medium and culture to obtain fermentation broth; (2) Mix the fermentation broth with sterilized biochar to make a carbon-based mixture; (3) The mixture is cultured at a constant temperature to obtain carbon-based microbial fertilizer. (4) The proportions, humidity and selection of carbon-based materials in carbon-based microbial fertilizers are specified.

2. The preparation method according to claim 1, characterized in that, In step (1), the culture conditions are 24 hours of culture in a constant temperature shaker at 37℃.

3. The preparation method according to claim 1, characterized in that, The liquid culture medium is NA liquid culture medium, and its components, in the following mass ratio, include: 3 g beef extract, 5 g corn flour, 5 g NaCl, 10 g peptone, 1000 mL distilled water, with a pH value of 7.2 to 7.

5.

4. The preparation method according to claim 3, characterized in that, The components and proportions of the culture medium were optimized through single-factor experiments and orthogonal experiments.

5. The preparation method according to claim 1, characterized in that, The biochar was sterilized at 121°C for 60 minutes.

6. The preparation method according to claim 1, characterized in that, The fermentation broth and biochar are mixed in a mass ratio of 1:2, the moisture content of the mixture is controlled at 30%, and the effective viable bacteria count of the microbial fertilizer is not less than 3 × 10⁻⁶. 8 CFU / g.

7. A carbon-based microbial fertilizer, characterized in that, It is composed of biochar and Bacillus TB-7 strain, with the biochar serving as a carrier, and the microbial fertilizer contains a surviving microbial community.

8. The carbon-based microbial fertilizer as described in claim 7, characterized in that, The effective viable bacteria count of the microbial fertilizer is not less than 3 × 10⁻⁶. 8 CFU / g, moisture content is 30%.

9. A method for applying carbon-based microbial fertilizer in Chinese cabbage cultivation, characterized in that, The microbial fertilizer is applied to the rhizosphere soil as a base fertilizer during cabbage planting.

10. The application method as described in claim 9, characterized in that, Applying 50 g of microbial fertilizer to each cabbage plant can reduce the incidence of clubroot disease and promote plant growth.

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