Application of biochar-loaded B. subculis microbial inoculum in prevention and treatment of celery continuous cropping obstacles

Abstract

The invention discloses an application of a biochar-loaded B. subculis microbial inoculum in prevention and treatment of celery continuous cropping obstacles and a preparation method of the biochar-loaded B. subculis microbial inoculum. Belongs to the technical field of successive cropping obstacles. The problem that continuous cropping causes reduction of crop yield and soil fertility is an urgent problem to be solved in vegetable production, the research of the invention proves that the application of the biochar-loaded B.subtilis microbial agent can improve soil fertility, effectively promote growth of celery, improve soil physicochemical properties and soil enzyme activity, and the biochar-loaded B.subtilis microbial agent is expected to become a potential soil conditioner to relieve the problem of continuous cropping of soil.

Description

Technical Field

[0001] This invention relates to the field of continuous cropping obstacle technology, and more specifically to the application of biochar-loaded B. subtilis inoculant in the prevention and control of continuous cropping obstacles in celery. Background Technology

[0002] Continuous cropping, the practice of planting the same crop variety on the same land for several consecutive years, has been widely adopted in intensive agriculture to meet the growing global food demand. However, this planting method has led to serious soil degradation problems, manifested as soil acidification and hardening, increased soil-borne diseases, decreased soil fertility, and disruption of soil microbial community structure, resulting in a significant reduction in crop yield and quality. Currently, although methods such as crop rotation, intercropping, and chemical fertilization are used to alleviate the problems of continuous cropping, these methods often involve complex management processes and their effectiveness is not ideal. Therefore, there is an urgent need to develop a more cost-effective and environmentally friendly green technology to address the problems of continuous cropping.

[0003] Biochar, obtained from the pyrolysis of biomass materials under high temperature and limited oxygen conditions, is widely used in agricultural production as a soil conditioner due to its well-developed porous structure, large specific surface area, strong ion exchange capacity, and adsorption capacity. Practice has shown that applying biochar can improve the physical and chemical properties of soil and the structure of the microbial community, indirectly inhibiting the growth of pathogens. However, biochar itself cannot inhibit the growth of soil-borne pathogens. Applying biochar in combination with plant growth-promoting bacteria is more effective in inhibiting the growth of pathogens such as Fusarium oxysporum than using biochar alone. Therefore, it is necessary to develop other effective methods to inhibit the growth of soil pathogens.

[0004] Biological control is an effective method for managing soil-borne diseases. Introducing biocontrol bacteria such as *Pseudomonas aeruginosa*, slime molds, *Streptomyces*, *Bacillus subtilis*, and *Bacillus amyloliquefaciens* into the soil can effectively inhibit pathogen growth, improve soil physicochemical properties, and increase crop yield. However, the effects of using microbial agents alone are not satisfactory, mainly due to the difficulty in colonizing biocontrol bacteria in the soil and their low survival rate, which significantly limits their practical application. Immobilizing microorganisms on a carrier substrate to improve their survival rate is an effective technical strategy.

[0005] In recent years, biochar has been frequently used as a carrier to immobilize microorganisms, thereby improving their survival rate and stability in soil. Due to its large specific surface area and porous structure, biochar provides a suitable habitat for microorganisms, significantly enhancing bacterial survival and effectiveness. However, the interaction between biochar and bacteria on soil remains largely unknown. Summary of the Invention

[0006] In view of this, the present invention provides the application of biochar-loaded B. subtilis in the control of continuous cropping obstacles in celery.

[0007] This invention prepares a biochar-loaded B. subtilis inoculant as a soil conditioner, aiming to address soil degradation caused by continuous cropping of celery. By studying changes in celery growth indicators, soil physicochemical properties, and soil enzyme activity, the interaction between straw biochar and Bacillus subtilis is elucidated. The results will provide a scientific basis for the application of biochar-loaded inoculants and promote sustainable agricultural development.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] Application of biochar-loaded B. subtilis inoculant in controlling continuous cropping obstacles in celery.

[0010] Furthermore, it is used to promote celery growth, improve soil physicochemical properties, and increase soil enzyme activity.

[0011] Furthermore, promoting celery growth involves increasing the fresh weight, dry weight, plant height, stem diameter, soluble protein content, soluble sugar content, cellulose content, vitamin C content, carotenoid content, and chlorophyll content of celery.

[0012] Furthermore, the soil physicochemical properties include pH, electrical conductivity, available nitrogen, available phosphorus, available potassium, and organic matter.

[0013] Furthermore, the enzymes include catalase, urease, alkaline phosphatase, and sucrase.

[0014] Furthermore, the biochar-loaded B. subtilis inoculant is prepared by incubating and drying straw biochar and B. subtilis inoculant solution.

[0015] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0016] Continuous cropping leads to a decline in crop yield and soil fertility, which is an urgent problem to be solved in vegetable production. The research of this invention has shown that the application of biochar-loaded B. subtilis inoculant can improve soil fertility, effectively promote the growth of celery, improve soil physicochemical properties and soil enzyme activity, and is expected to become a potential soil conditioner to alleviate the problem of continuous cropping. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The XRD diffraction patterns of straw biochar and biochar loaded with B. subtilis inoculant in Example 1 of this invention are shown.

[0019] Figure 2 The images show SEM images of the cross-section (a) and longitudinal section (b) of the straw biochar in Example 1 of this invention, as well as SEM images of the surface (c) and pore interior (d) of the biochar loaded with B. subtilis inoculant. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0021] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0022] Straw biochar was purchased from Henan Yuzhongao Agricultural Technology Co., Ltd., and passed through a 2-4 mm sieve. Its physicochemical properties are shown in Table 1.

[0023] Table 1 Physicochemical properties of straw biochar

[0024]

[0025] Note: EC, electrical conductivity; AN, alkaline nitrogen; AP, available phosphorus; AK, available potassium.

[0026] The B. subtilis strain was provided by Nongben Agricultural Technology Service (Langfang) Co., Ltd.

[0027] Potassium sulfate-based compound fertilizer (N-P2O5-K2O, 15-15-15) was purchased from Zhongnong Ala Fertilizer Co., Ltd. (Qinhuangdao City, Hebei Province).

[0028] Example 1

[0029] Preparation of biochar-loaded B. subtilis inoculum

[0030] Weigh 6 grams of straw biochar into an Erlenmeyer flask and autoclave at 121°C for 20 minutes. Then add 10 mL of B. subtilis bacterial suspension (2 × 10⁻⁶) to the Erlenmeyer flask. 8 Incubate at 160 rpm for 24 h (CFU / mL) to ensure that the bacteria are fixed on the microporous structure of the straw biochar, thus preparing a biochar-loaded B. subtilis inoculum.

[0031] The characteristics of straw biochar and biochar-loaded B. subtilis were analyzed using a Hitachi Regulus 8100 scanning electron microscope (SEM, Japan), and XRD patterns were obtained using a Bruka D8 Advance diffractometer (XRD, Bruka, Germany).

[0032] XRD patterns of straw biochar and biochar-loaded B. subtilis inoculant are shown in [reference needed]. Figure 1 The XRD pattern of straw biochar showed a diffraction peak at 22°, which corresponds to the lignocellulose crystal plane of the straw biochar. No new characteristic peaks appeared in the XRD pattern of biochar loaded with *B. subtilis* inoculant, indicating that the crystalline regions of the straw biochar remained intact during the fixation process.

[0033] SEM images of straw biochar ( Figure 2 a and Figure 2 b) It exhibits the typical porous structure of biochar, with pore sizes of approximately 1–4 μm, suitable for bacterial immobilization and reproduction. For example... Figure 2 As shown in c, most B. subtilis cells were fixed on the surface of straw biochar, while a small number of cells were also observed within the pores of the straw biochar. Figure 2 d). Most bacteria on straw biochar have smooth surfaces and are morphologically active, while a few have wrinkled surfaces, which may be due to dehydration during the drying process.

[0034] Experiment 1

[0035] (1) Soil samples were taken from a farm that had been growing celery for 5 years. The sampling depth was 20 cm from the top layer. The physical and chemical properties of the soil are shown in Table 2.

[0036] Table 2. Physicochemical properties of soil

[0037]

[0038] Note: EC, electrical conductivity; AN, alkaline nitrogen; AP, available phosphorus; AK, available potassium.

[0039] (2) Experimental Design

[0040] In September 2023, celery was grown in a greenhouse in Qinhuangdao City, Hebei Province, China (119.18°E, 39.71°N). A two-factor randomized block design was used. The ceramic pots measured 32cm × 24cm × 20cm, and each pot contained 5.5kg of the aforementioned soil. Fertilization treatments included conventional fertilization (CF) and 20% reduced fertilization (RF). Soil treatments were categorized as follows:

[0041] ①CK, without any additives;

[0042] ②BC, add straw biochar;

[0043] ③BS, add B. subtilis bacterial culture (2×10) 8 CFU / mL);

[0044] ④BBS, with the addition of straw biochar and B. subtilis bacterial solution (2×10⁻⁶). 8 CFU / mL);

[0045] ⑤BIB, with added biochar-carrying B. subtilis bacteria.

[0046] All soil treatment methods are shown in Table 3.

[0047] Table 3 Soil Treatment Design

[0048]

[0049] Three concentration levels (Level 1, Level 2 and Level 3) were set for each soil treatment, as detailed in Table 4.

[0050] Table 4 Concentrations of different soil conditioners

[0051]

[0052] All soil treatments were replicated three times. Additionally, soil from celery grown continuously for 5 years served as a bare soil control (CSCK). CFCK represents the treatment combination of conventional fertilization and soil without the addition of straw biochar or B. subtilis inoculum; all other treatment combinations were represented similarly.

[0053] Before transplanting celery seedlings, 4.0g of potassium sulfate-based compound fertilizer was added to each pot as base fertilizer according to local fertilization standards, equivalent to 600kg per hectare. Two weeks later, celery seedlings with a height of 15cm were selected and planted using the five-point method on September 20, 2023. One month later, 1.2g (equivalent to 150kg / hectare, labeled CF) or 0.96g (equivalent to 120kg / hectare, labeled RF) of potassium sulfate-based compound fertilizer was applied to each pot as top dressing. Throughout the experiment, all pots were irrigated with tap water every two days, and irrigation was stopped one week before harvest.

[0054] On November 24, 2023, the celery harvest was completed. A five-point sampling method was used to collect rhizosphere soil samples from each celery plant. The collected samples were mixed and placed in sterile resealable bags as replicates, and transported to the laboratory in ice packs. The collected soil samples were air-dried, pulverized, and passed through a 0.15 mm sieve for subsequent soil property analysis. In addition, the celery roots were rinsed with tap water, and their growth characteristics and physiological properties were measured.

[0055] (3) Analysis of Celery Growth Characteristics

[0056] Plant height was measured from the base to the highest point of the plant. Stem diameter was measured using vernier calipers. After weighing the fresh weight of the plants, they were blanched at 105℃ for 15 minutes, followed by drying at 60℃ to obtain the dry weight. Chlorophyll and carotenoid contents were extracted with ethanol and then quantitatively analyzed using colorimetric methods. Cellulose content was determined using the anthrone colorimetric method. Soluble protein was determined using the Coomassie Brilliant Blue G-250 method. Vitamin C content was extracted with xylene and then quantitatively analyzed using colorimetric methods. Soluble sugar content was determined using the anthrone colorimetric method.

[0057] (4) Soil analysis

[0058] Soil electrical conductivity (EC) was measured using a conductivity meter. Soil pH was determined using a pH meter, with a soil-to-water ratio of 1:5 (w:v). Alkaline available nitrogen (AN) content in the soil was assessed using the alkaline diffusion method. Available phosphorus (AP) content was extracted with 0.5 mol / L sodium bicarbonate and then quantitatively analyzed using the molybdenum blue colorimetric method. Available potassium (AK) content was extracted with ammonium acetate and then determined using flame photometry. Soil organic matter (OM) content was digested with potassium dichromate and then determined using a UV spectrophotometer. Urease activity was determined using the sodium hypochlorite colorimetric method. Alkaline phosphatase activity was determined using the sodium phosphate colorimetric method. Catalase activity was determined using the potassium permanganate titration method. Sucrase activity was determined using the 3,5-dinitrosalicylic acid colorimetric method.

[0059] (5) Data processing

[0060] Data analysis was performed using SPSS 26.0 software. Differences among different soil treatments were assessed using two-way ANOVA. A p-value < 0.05 was considered statistically significant among treatments.

[0061] (6) Results

[0062] ① The effects of different soil treatments on celery growth

[0063] Table 5. Effects of different soil treatments on celery biomass

[0064]

[0065]

[0066] Table 6. Effects of different soil treatments on celery plant height and stem diameter

[0067]

[0068] Tables 5 and 6 show that, compared with the control (CK), the celery plant height increased significantly after the addition of straw biochar or B. subtilis inoculum. Among all soil treatments, the BIB treatment resulted in the highest celery plant height. There was no significant difference in plant height between the RFBIB and CFBIB treatments. Furthermore, level 3 showed the highest plant height among all soil treatments. Compared with the CK, the fresh weight of celery also increased significantly under different soil treatments (P<0.05), with level 3 showing the highest fresh weight. Additionally, the fresh weight of the RFBIB treatment was significantly higher than other treatments (P<0.05). The dry weight and stem diameter of celery were significantly higher than the CK treatment in all soil treatments (P<0.05), with level 3 showing the best effect. Therefore, data from level 3 were selected for subsequent analysis.

[0069] ② Effects of different soil treatments on physiological indicators of celery

[0070] Table 7 Effects of different soil treatments on physiological indicators of celery

[0071]

[0072] Table 8. Two-factor analysis of the effects of fertilization and soil treatment on physiological indicators of celery.

[0073]

[0074] Note: *P<0.05 is significant; **P<0.01 is highly significant.

[0075] Two-way ANOVA results showed that fertilization and different soil treatments significantly affected the soluble protein, soluble sugar, and cellulose content of celery (P<0.01, Table 8). In both the CF and RF groups, the soluble protein content of celery treated with soil was significantly higher than that in the CK group (P<0.05). Furthermore, significant differences existed among the different soil treatments (P<0.05). Among all treatments, the soluble protein content of celery treated with RFBIB was the highest, exceeding that of the RFCK treatment by 102.44%. The soluble sugar content of celery treated with all soils was higher than that in the CK group (P<0.05). In the CF group, there was no significant difference in soluble sugar content among the different soil treatments; however, in the RF group, the soluble sugar content of celery treated with BIB was significantly higher than that of other treatments. Similarly, the cellulose content of celery treated with all soils was significantly higher than that in the CK group (P<0.05). Among all treatments, the cellulose content of celery treated with RFBIB was the highest, increasing by 75.00% compared to the RFCK treatment.

[0076] Furthermore, fertilization and soil conditions had relatively small effects on the vitamin C, chlorophyll, and carotenoid content of celery (Table 8). Among the different soil treatments, the RFBIB treatment resulted in the highest vitamin C content in celery, but there was no significant difference between the RFBIB and CFBIB treatments (P<0.05). Chlorophyll content did not differ significantly among the CF groups, but the RFBIB treatment had the highest chlorophyll content, increasing by 21.82% compared to the RFCK treatment. The RFBIB treatment also resulted in the highest carotenoid content in celery, but there was no significant difference between the RFBIB and CFBIB treatments. These results indicate that the use of biochar-loaded B. subtilis inoculum significantly improved the quality of celery compared to the use of straw biochar or B. subtilis inoculum alone.

[0077] ③ The effects of different soil treatment methods on soil physicochemical properties

[0078] Table 9. Effects of different soil treatments on soil physicochemical properties

[0079]

[0080]

[0081] Table 10 Two-way ANOVA of the effects of fertilization and soil treatment on soil physicochemical properties

[0082]

[0083] Note: EC, electrical conductivity; AN, alkaline nitrogen; AP, available phosphorus; AK, available potassium; OM, organic matter; *P<0.05 is significant, **P<0.01 is highly significant.

[0084] Fertilization and soil treatments significantly affected soil pH, EC, AN, AP, AK, and OM content (P<0.05, Table 10). Compared with the control group (CK), different soil treatments significantly increased soil pH. Among all soil treatments, the RFBIB treatment was the most effective in improving soil pH, increasing it by 2.68% and 3.30% compared with the RFCK and CFCK treatments, respectively. The alkaline functional groups in straw biochar may neutralize acidic substances in the soil, thereby increasing pH. *B. subtilis* also improved acidic soils by improving the rhizosphere microbial community structure. Since soil electrical conductivity can affect soil fertility and crop yield by influencing water retention capacity, this experiment measured electrical conductivity. Compared with the control group (CK), the additional soil treatments significantly increased soil electrical conductivity (P<0.05), which may be due to the large specific surface area and abundant oxygen-containing active chemical groups in straw biochar. Furthermore, when B. subtilis bacterial solution is applied to the soil, extracellular electron transfer between microorganisms is promoted, which helps to improve the soil's electrical conductivity.

[0085] Different soil treatments significantly increased the contents of AN, AP, and AK (P < 0.05), which may be related to the nitrogen, phosphorus, and potassium elements contained in straw biochar itself and the ameliorative effect of B. subtilis. However, compared with the CF group, the contents of AN, AP, and AK in the RF group were relatively low, which may be related to the lower fertilizer application in the RF group. In addition, celery has a high demand for AN, AP, and AK during its growth. Furthermore, compared with the CK group, different soil treatments increased the OM content (P < 0.05). Among all soil treatments, the CFBIB treatment had the highest OM content, but there was no significant difference in OM content between the RFBIB and CFBIB treatments. Straw biochar contains aromatic carbon structures, and due to its inherent chemical stability and resistance to microbial degradation, long-term application contributes to the accumulation of soil organic carbon. In addition, B. subtilis can inhibit the growth of pathogens and reduce the incidence of diseases, which may indirectly promote the increase of soil OM content.

[0086] ④ Effects of different treatments on soil enzyme activity

[0087] Table 11 Effects of different soil treatments on soil enzyme activity

[0088]

[0089] Table 12 Two-way ANOVA of the effects of fertilization and soil treatment on soil enzyme activity

[0090]

[0091] Note: * is significant at the P<0.05 level, ** is significant at the P<0.01 level.

[0092] Two-way ANOVA showed that fertilization and soil treatment had little effect on soil catalase (Table 12). The BIB treatment showed significantly higher catalase activity than other treatments. However, there was no significant difference in catalase activity between the RFBIB and CFBIB treatments. Meanwhile, fertilization and soil treatment significantly affected the activities of urease, alkaline phosphatase, and sucrase in the soil (P<0.01, Table 12). Compared with the CK group, different soil treatments significantly increased the activities of alkaline phosphatase and sucrase (P<0.05). However, the enzyme activities in the RF group were significantly lower than those in the CF group (P<0.05), which may be related to the lower soil nutrient content in the RF group. Generally, soil nutrients (such as AN, AP, AK, and OM) are key substrates affecting enzyme activity. Therefore, the more abundant the soil nutrients, the stronger the enzyme activity. The application of straw biochar or *B. subtilis* improved the soil microbial community and altered the proportion of beneficial soil microorganisms, which in turn enhanced soil enzyme activity. Due to the interaction between the two, BIB treatment resulted in higher enzyme activity than treatment with straw biochar or B. subtilis culture alone.

[0093] Experiment 2

[0094] (1) Field trial design

[0095] This experiment was conducted on October 11, 2024, in soil samples from celery grown continuously for 5 years at Fengshuo Siji Ecological Farm, Beidaihe District, Qinhuangdao City, Hebei Province. Seven soil treatments were established, each configured in one of seven independent plots, with each plot measuring 1m × 3m = 3m². 2 The walkway is 0.3m wide. The specific handling method is as follows:

[0096] ①CK (without application of straw biochar and B. subtilis bacterial solution);

[0097] ②BC (applying straw biochar);

[0098] ③BS (apply B. subtilis bacterial solution (2×10) 8 CFU / mL);

[0099] ④BIB (applying the biochar-loaded B. subtilis agent prepared in Example 1);

[0100] ⑤BIB_20 (Apply the biochar-loaded B. subtilis inoculant prepared in Example 1, and reduce the application of chemical fertilizer by 20% when applying topdressing);

[0101] ⑥BIB_30 (Apply the biochar-loaded B. subtilis inoculant prepared in Example 1, and reduce the application of chemical fertilizer by 30% when applying topdressing);

[0102] ⑦BIB_40 (Apply the biochar-loaded B. subtilis inoculant prepared in Example 1, and reduce the application of chemical fertilizer by 40% when applying topdressing).

[0103] The specific addition concentrations for each treatment group are detailed in Table 13.

[0104] Table 13 Addition amounts for different soil treatments

[0105]

[0106] At the start of the experiment, following local traditional fertilization standards (see Table 13), potassium sulfate compound fertilizer (15-15-15, N+3P2O5+K2O≥45%) was used as base fertilizer and evenly incorporated into the soil before celery planting. Subsequently, according to the preset concentration requirements for each treatment, straw biochar and biochar-loaded B. subtilis inoculant were evenly incorporated into the soil and thoroughly mixed to ensure uniform distribution. Two weeks later, on October 23, 2024, celery seedlings approximately 15cm in height were transplanted, with 14 rows and 8 seedlings per row in each plot. One week after transplanting, B. subtilis inoculant solution was added according to the specified ratio. One month after transplanting, topdressing was applied; the specific amount of topdressing is shown in Table 13. Throughout the experiment, conventional field management practices were strictly followed. Irrigation was stopped one week before harvest, and the soil was allowed to dry.

[0107] (2) Soil sampling methods and index determination

[0108] ① Soil sampling: Using the five-point sampling method, collect soil samples from the rhizosphere of celery. Mix the soil samples from each point thoroughly to form one sample, with three replicates for each treatment. Place the collected rhizosphere soil samples into sterile sampling bags, label them, and place them in an ice box to be brought back to the laboratory. The preservation method is the same as in Experiment 1.

[0109] ② Determination of soil physicochemical properties: Same as Experiment 1.

[0110] ③ Determination of soil enzyme activity: Same as Experiment 1.

[0111] (3) Results

[0112] ① Effects of different soil treatments on soil physicochemical properties

[0113] Table 14 Effects of different soil treatments on the physicochemical properties of continuously cropped soils

[0114]

[0115] As shown in Table 14, compared with the control (CK), the soil pH values ​​of all treatment groups were significantly increased (P<0.05), indicating that straw biochar, *B. subtilis* inoculum, and biochar-loaded *B. subtilis* inoculum have significant regulatory effects on soil pH. Among them, the soil pH values ​​of the BIB_20 and BIB_30 treatment groups were the same and highest, increasing by 15.08% compared to the CK. The BIB treatment group increased by 13.10% compared to the CK, significantly higher than the BC, BS, and BIB_40 treatment groups (P<0.05). Straw biochar has the ability to adsorb acidic substances and has a high ion exchange capacity, effectively neutralizing soil acidity and thus increasing soil pH. The soil pH increase was most significant when the amount of topdressing was reduced (BIB_20 and BIB_30), possibly because appropriately reducing the amount of topdressing balanced soil nutrients and prevented the accumulation of acidic substances. However, when the amount of topdressing was reduced to 40%, the soil had less nutrients, affecting the metabolic activity of microorganisms and weakening the effect of increasing soil pH.

[0116] Compared with the control (CK), the soil EC values ​​of all treatment groups were significantly increased (P<0.05), indicating that different treatments affected soil salinity. Specifically, the BIB treatment group had the highest soil EC value, significantly higher than the CK by 20.39% (P<0.05), while the BC and BS treatment groups showed increases of 6.77% and 3.38% respectively compared to the control group. The BIB_20, BIB_30, and BIB_40 treatment groups showed significant increases of 14.30%, 8.38%, and 5.25% respectively compared to the CK. Furthermore, by rationally adjusting fertilization, it is possible to optimize soil nutrient balance, reduce the risk of soil salinity accumulation, and achieve sustainable improvement of continuously cropped soils while reducing fertilizer input.

[0117] Straw biochar, *B. subtilis* inoculum solution, and *B. subtilis* inoculum treatment with biochar all significantly increased the contents of AN, AP, and AK (P<0.05). Compared with the control (CK), the BIB treatment group had the highest contents of AN, AP, and AK in the soil, increasing by 17.68%, 32.85%, and 58.10%, respectively (P<0.05). However, with the reduction of topdressing, the contents of AP and AK in the BIB_20 and BIB_30 treatment groups gradually decreased, but were still higher than those in the BC and BS treatment groups, with significant differences. The contents of AN in the BIB_20 and BIB_30 treatment groups gradually decreased, but were still higher than those in the BC and BS treatment groups, although the differences were not significant (P<0.05). The contents of AK in the BIB_40 treatment group were lower than those in other treatment groups, but significantly higher than those in the control (CK). The contents of AN and AP in the BIB_40 treatment group were lower than those in other treatment groups, but higher than those in the control (CK). Therefore, applying straw biochar or *B. subtilis* inoculum alone resulted in a less significant increase in soil AN, AP, and AK levels compared to applying biochar-loaded *B. subtilis* inoculum. This may be due to the interaction between straw biochar and *B. subtilis* in the biochar-loaded *B. subtilis* inoculum, which is more beneficial for improving soil nutrient content. In conclusion, applying biochar-loaded *B. subtilis* inoculum can significantly increase the content of AN, AP, and AK in the soil, and has a significant effect on improving soil fertility in celery continuous cropping. Straw biochar, with its high specific surface area and porous structure, can adsorb and retain nutrients in the soil, reducing the risk of nutrient loss and thus improving the soil's fertilizer retention capacity. Its surface rich in carboxyl and hydroxyl functional groups increases the soil's cation exchange capacity (CEC), enabling the soil to more effectively adsorb and retain AN, AP, and AK. Furthermore, straw biochar provides a favorable habitat for microorganisms, promoting their growth and metabolic activities, indirectly improving soil fertility. However, while reducing the amount of topdressing can still maintain soil nutrient content to some extent, excessive reduction of chemical fertilizer application (BIB_40) directly reduces the intake of exogenous nitrogen (N), phosphorus (P) and potassium (K) in the soil, affecting the content of AN, AP and AK in the soil.

[0118] Compared with the control (CK), the soil organic matter (OM) content in all treatment groups was significantly increased (P<0.05), indicating that straw biochar, *B. subtilis* inoculum solution, and biochar-loaded *B. subtilis* inoculum significantly improved soil organic matter content. Among them, the BIB treatment group had the highest soil OM content, significantly higher than other treatment groups (P<0.05). However, with the reduction of topdressing, the soil OM content in the biochar-loaded *B. subtilis* inoculum treatment groups gradually decreased. The BIB_20 treatment group increased by 8.04% compared to the CK; the BIB_30 and BIB_40 treatment groups increased by 4.44% and 3.59% respectively compared to the CK, with no significant difference between them (P>0.05). Furthermore, the soil OM content in the BC and BS treatment groups alone increased by 4.95% and 2.77% compared to the CK, respectively. This indicates that biochar-loaded *B. subtilis* inoculum has a significant advantage over straw biochar or *B. subtilis* alone in increasing soil OM content. However, with the reduction of topdressing, the amount of available organic matter and nutrients in the soil decreases, leading to a decrease in microbial activity and affecting the accumulation and stability of organic matter. Applying biochar-loaded B. subtilis inoculant and rationally adjusting the amount of fertilizer can optimize soil organic matter content and improve continuously cropped soils.

[0119] ② Effects of different soil treatments on soil enzyme activity

[0120] Table 15 Effects of different soil treatments on enzyme activity in continuously cropped soil

[0121]

[0122] As shown in Table 15, the BC, BS, and BIB treatments significantly increased the activities of catalase, urease, alkaline phosphatase, and sucrase in the soil (P<0.05). Among them, the BIB treatment group showed the highest enzyme activity, with catalase, urease, alkaline phosphatase, and sucrase activities increasing by 22.36%, 81.82%, 65.97%, and 57.09% respectively compared to the control (P<0.05). This indicates that under normal topdressing conditions, biochar-loaded *B. subtilis* inoculant can significantly enhance soil enzyme activity, thereby improving soil fertility and microbial activity in celery continuous cropping. However, with the reduction of topdressing amount, the soil enzyme activity in the biochar-loaded *B. subtilis* inoculant treatment group gradually decreased, but remained significantly higher than that in the control (P<0.05). Furthermore, the soil enzyme activities in the BC and BS treatment groups were also increased compared to the control group, but the improvement effect was not as significant as that in the biochar-loaded *B. subtilis* inoculant treatment group. This may be related to the interaction between straw biochar and *B. subtilis* inoculant solution. The porous structure of straw biochar creates a favorable habitat for microorganisms, promoting their metabolic activities and increasing enzyme secretion. Simultaneously, the microorganisms in the biochar-loaded *B. subtilis* inoculant can decompose organic matter in the soil, further increasing enzyme synthesis and secretion. However, with reduced topdressing, the available organic matter and nutrients in the soil decrease, inhibiting microbial activity and leading to reduced enzyme synthesis and secretion efficiency. Although the reduction in topdressing weakens the effect of the biochar-loaded *B. subtilis* inoculant on enhancing soil enzyme activity to some extent, its activity is still significantly higher than the control group. This indicates that applying the biochar-loaded *B. subtilis* inoculant under reduced fertilizer use can still maintain soil fertility and microbial activity, and increase the activities of soil catalase, urease, alkaline phosphatase, and sucrase.

[0123] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0124] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of biochar-loaded B. subtilis inoculant in controlling continuous cropping obstacles in celery.

2. The application as described in claim 1, characterized in that, It is used to promote celery growth, improve soil physical and chemical properties, and increase soil enzyme activity.

3. The application as described in claim 2, characterized in that, The promotion of celery growth refers to increasing the fresh weight, dry weight, plant height, stem diameter, soluble protein content, soluble sugar content, cellulose content, vitamin C content, carotenoid content, and chlorophyll content of celery.

4. The application as described in claim 2, characterized in that, The soil physicochemical properties include pH, electrical conductivity, available nitrogen, available phosphorus, available potassium, and organic matter.

5. The application as described in claim 2, characterized in that, The enzymes include catalase, urease, alkaline phosphatase, and sucrase.

6. The application as described in any one of claims 1 to 5, characterized in that, The biochar-loaded B. subtilis inoculant is prepared by incubating and drying straw biochar and B. subtilis inoculant solution.

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