Preparation method and application of straw biochar bacterium-carrying agent granular fertilizer

Abstract

The invention discloses a preparation method and application of a straw biochar bacterium-carrying agent granular fertilizer. Belongs to the technical field of carbon-loaded microbial agents. The preparation method comprises the following steps: incubating biochar and a bacterial liquid to prepare a carbon-loaded bacterial agent, adding auxiliary materials, and granulating to obtain the straw biochar-loaded bacterial agent granular fertilizer. The straw biochar bacterium-carrying agent granular fertilizer disclosed by the invention has remarkable effects on promoting the growth of celery, improving the quality of the celery and optimizing a soil microbial community structure, has a certain cumulative effect and stability in long-term application, and provides a new idea for relieving continuous cropping obstacles of the celery.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of carbon carrier agent, more particularly to a preparation method and application of a straw biochar carrier agent granular fertilizer. BACKGROUND

[0002] Celery is a two-year green leafy vegetable of the Apiaceae family. Celery is not only delicious, but also has high medicinal and nutritional value, and is favored by people. With the continuous improvement of people's healthy diet consciousness, the market demand for celery is increasing year by year, and the cultivation area in various places is also expanding year by year. In recent years, the cultivation methods and varieties of celery have also shown a trend of diversification, but the diseases and pests have become more and more serious, especially the continuous cropping obstacles caused by continuous cropping, such as slow growth, weak growth, poor stress resistance, low yield, and poor quality.

[0003] The main reason for the occurrence of continuous cropping obstacles is soil acidification, soil hardening, soil biological degradation, and soil-borne disease aggravation, which is a kind of soil biological obstacle caused by continuous cropping of crops and microorganisms in the soil. In production, techniques such as crop rotation, soil disinfection, increasing the amount of fertilizer application, or spraying pesticides are usually selected for prevention and control. However, these methods are not only complex to operate, but long-term application may also cause greater ecological environmental problems and biological safety problems.

[0004] Biochar is a substance generated by pyrolysis of agricultural waste such as straw, rice husk, and mushroom dregs under low-temperature limited oxygen or anaerobic conditions. Due to its porous structure, large specific surface area, rich organic carbon and mineral composition, biochar has become an effective soil conditioner, and its effect on improving acid soil caused by continuous cropping is particularly significant, but it is difficult to inhibit plant pathogens when used alone. There are also reports on the use of biochar as a carrier material to load biocontrol agents to improve soil properties. However, due to the complex and variable soil environment, the survival rate of biocontrol agents in soil is low, and it is difficult to achieve sustained improvement effect.

[0005] In summary, how to provide a carbon carrier agent for continuously and effectively preventing and controlling celery soil continuous cropping obstacles is a problem that needs to be solved by those skilled in the art. SUMMARY

[0006] Therefore, the present application provides a preparation method and application of a straw biochar carrier agent granular fertilizer.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] A preparation method of a straw biochar carrier agent granular fertilizer, comprising the following steps:

[0009] (1) Preparation of biochar

[0010] The corn stalks are pyrolyzed in an atmosphere furnace, crushed and sieved for use;

[0011] (2) Preparation of B. subtilis bacterial solution

[0012] After the B. subtilis strain is activated, it is cultured to the logarithmic phase, and the bacterial solution concentration is adjusted for use;

[0013] (3) Preparation of straw biochar carrier agent granular fertilizer

[0014] (31) After the biochar and B. subtilis bacterial solution are incubated and dried, the biochar carrier agent is obtained;

[0015] (32) The biochar carrier agent is mixed with auxiliary materials to granulate.

[0016] Further, in the step (1), the corn stalks are pyrolyzed in an atmosphere furnace at 500-650°C for 2-4h.

[0017] Further, in the step (2), the bacterial solution concentration is 2×10 9 CFU / mL.

[0018] Further, in the step (31), the mass-volume ratio of the biochar and B. subtilis bacterial solution is 30-50g: 6-9mL.

[0019] Further, in the step (32), the specific parameters of the incubation are as follows: 30°C on a shaker, 120rpm for 18-24h.

[0020] Further, the auxiliary materials include corn starch, peanut meal and clay;

[0021] The mass ratio of the biochar carrier agent, corn starch, peanut meal and clay is 50: 9-12: 21-28: 10-20.

[0022] The straw biochar carrier agent granular fertilizer prepared by the above preparation method is applied in the prevention and control of the continuous cropping obstacles of celery.

[0023] Further, it is used to improve the growth parameters, physiological indexes and microbial community structure of celery.

[0024] Further, the growth parameters include fresh weight, dry weight, plant height and stem diameter.

[0025] Further, the physiological indexes include soluble protein content, soluble sugar content, cellulose content, vitamin C content, carotenoid content and chlorophyll content.

[0026] According to the above technical solution, compared with the prior art, the beneficial effects obtained by the present application are:

[0027] The present application prepares a charcoal carrier microbial agent by incubating biochar with microbial liquid, and then adds excipients to granulate to obtain a straw biochar carrier microbial agent granular fertilizer. The straw biochar carrier microbial agent granular fertilizer of the present application has a significant effect in promoting the growth of celery, improving the quality of celery, and optimizing the structure of soil microbial community, and has a certain cumulative effect and stability in long-term application, providing a new idea for relieving the continuous cropping obstacles of celery. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

[0029] Figure 1 XRD diffraction patterns of biochar and biochar carrier microbial agent in experiment 1 of the present application;

[0030] Figure 2 SEM images of the cross section (a) and longitudinal section (b) of biochar, and SEM images of the surface (c) and pore inside (d) of biochar carrier microbial agent in experiment 1 of the present application;

[0031] Figure 3 Dilution curves of soil bacteria (a) and fungal communities (b) under different soil treatments in experiment 2 of the present application;

[0032] Figure 4 NMDS analysis of microbial communities in soil under different soil treatments in experiment 2 of the present application: weighted UniFrac distance of bacteria (a) and Bray-Curtis distance of fungi (b); when stress < 0.2, the figure has a certain explanatory meaning; when stress < 0.1, it can be considered as a good order; when stress < 0.05, it has very good representativeness;

[0033] Figure 5 Relative abundance of bacteria (a) and fungi (b) at the door level under different soil treatments in experiment 2 of the present application. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0035] The required reagents in the present application are conventional experimental reagents, which are purchased from a commercial channel. The experimental methods not mentioned are conventional experimental methods, which are not described here.

[0036] The B.subtilis strain in the following embodiments is provided by Nongben Agricultural Technology Service (Langfang) Co., Ltd.

[0037] Embodiment 1

[0038] (1) Preparation of biochar

[0039] After the corn stalks are picked, the surface soil is washed with tap water, and the corn stalks are mechanically crushed after natural air drying. Then, the corn stalks are placed in a crucible and pyrolyzed in an argon atmosphere furnace at 500℃ for 2h. The obtained sample is crushed and sieved through a 2mm sieve for standby use.

[0040] (2) Preparation of B.subtilis bacterial solution

[0041] One ring of B.subtilis strain is inoculated in LB liquid medium from a preserved slant, and shaken and cultured at 30℃ on a shaker until the logarithmic growth phase. The bacterial solution is adjusted to a concentration of 2×10 9 CFU / mL for standby use.

[0042] (3) Preparation of straw biochar carrier agent granular fertilizer

[0043] B.subtilis bacterial solution 6mL is added to a flask containing 50mL of sterile water, shaken, and then 50g of biochar is added. Then, the flask is placed in a shaker at 30℃ and incubated at 120rpm for 24h. The precipitate is collected by centrifugation and dried in an oven at 45℃ to obtain the biochar carrier agent.

[0044] The straw biochar carrier agent granular fertilizer is prepared using a granulation device. First, 50g of biochar carrier agent, 12g of corn starch, 28g of peanut meal and 10g of clay are thoroughly mixed, and then 30mL of deionized water is added to knead the above-mentioned materials into a ball. The ball is placed in a granulation device and pressed into a cylindrical shape with a diameter of 0.5cm and a length of 1cm. After natural air drying, the product is ready for use.

[0045] Embodiment 2

[0046] (1) Preparation of biochar

[0047] The corn stalks after picking corn were washed with tap water to remove the soil on the surface, and then were mechanically crushed after natural air drying. Subsequently, the corn stalks were placed in a crucible, pyrolyzed in an atmosphere furnace at 650°C for 4h with argon gas, and then were crushed and sieved through a 2mm sieve for later use.

[0048] (2) Preparation of B. subtilis bacterial solution

[0049] One loop of B. subtilis strain was inoculated into LB liquid medium, and was cultured on a shaker at 30°C until the logarithmic growth phase. The bacterial solution was adjusted to a concentration of 2 x 10 9 CFU / mL for later use.

[0050] (3) Preparation of corn stalk biochar bacterial inoculant granular fertilizer

[0051] B. subtilis bacterial solution 9mL was added to a flask containing 75mL of sterile water, and then 30g of biochar was added after shaking. Subsequently, the flask was placed on a shaker at 30°C, and was incubated at 120rpm for 18h. The precipitate was collected by centrifugation, and was dried in an oven at 40°C to obtain the biochar bacterial inoculant.

[0052] The corn stalk biochar bacterial inoculant granular fertilizer was prepared using a granulation device. First, 50g of biochar bacterial inoculant, 9g of corn starch, 21g of peanut meal, and 20g of clay were mixed thoroughly, and then were kneaded into a ball with 30mL of deionized water. The ball was placed in the granulation device to be pressed and formed into a cylindrical particle with a diameter of 0.5cm and a length of 1cm. After natural air drying, the particle was ready for use.

[0053] Example 3

[0054] (1) Preparation of biochar

[0055] The corn stalks after picking corn were washed with tap water to remove the soil on the surface, and then were mechanically crushed after natural air drying. Subsequently, the corn stalks were placed in a crucible, pyrolyzed in an atmosphere furnace at 550°C for 3h with argon gas, and then were crushed and sieved through a 2mm sieve for later use.

[0056] (2) Preparation of B. subtilis bacterial solution

[0057] One loop of B. subtilis strain was inoculated into LB liquid medium, and was cultured on a shaker at 30°C until the logarithmic growth phase. The bacterial solution was adjusted to a concentration of 2 x 10 9 CFU / mL for later use.

[0058] (3) Preparation of corn stalk biochar bacterial inoculant granular fertilizer

[0059] A flask with 60 mL of sterile water was added with 7 mL of B. subtilis bacterial solution, and 40 g of biochar was added after shaking. Then the flask was placed in a shaker at 30°C, 120 rpm for 20 h, and the precipitate was collected by centrifugation and dried in an oven at 42°C to obtain the biochar carrier.

[0060] The straw biochar carrier granular fertilizer was prepared using a granulation device. First, 50 g of biochar carrier, 10 g of corn starch, 25 g of peanut meal, and 15 g of clay were mixed thoroughly, and then 30 mL of deionized water was added to knead the materials into a ball. The ball was placed in a granulation device for compression molding to obtain cylindrical particles with a diameter of 0.5 cm and a length of 1 cm. After natural air drying, the particles were ready for use.

[0061] Experiment 1

[0062] The surface morphology of the biochar and biochar carrier prepared in Example 1 was characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM).

[0063] As can be seen from Figure 1 , at a diffraction angle of 22°, a relatively wide diffraction peak appeared, corresponding to the (002) crystal plane of lignocellulose, indicating that the biochar retained the crystalline structure characteristics of lignocellulose during preparation. In the biochar carrier sample, no new characteristic peaks were observed, indicating that the original crystalline regions of the biochar were not destroyed during immobilization.

[0064] As can be seen from Figure 2 a and Figure 2 b, the biochar has a rich porous structure with a pore size of about 1-4 μm. This porous structure provides good conditions for the immobilization and proliferation of bacteria. The high porosity and large specific surface area of the biochar make it an ideal microorganism immobilization carrier. As can be seen from Figure 2 c and Figure 2 d, B. subtilis was successfully immobilized on the surface of the biochar Figure 2 c), and some cells even entered the pores of the biochar Figure 2 d. In addition, most of the cells were observed to be in an elliptical rod-like structure, with smooth cell surfaces and full morphology, indicating good activity. A few cells had wrinkles on the surface, which may have been caused by dehydration during the drying process.

[0065] Experiment 2

[0066] (1) Design of pot experiment

[0067] A 5-year-old soil from a continuous celery field was used as a sample, and celery was planted in pots.

[0068] The soil treatments were as follows:

[0069] ① blank control (CK, continuous cropping soil, no addition of biochar and bacterial liquid);

[0070] ② application of biochar (BC) prepared in Example 1;

[0071] ③ application of B. subtilis bacterial liquid (2 x 10 9 CFU / mL) (BS);

[0072] ④ application of straw biochar bacterial inoculant granular fertilizer (BIB) prepared in Example 1;

[0073] The specific addition amount is shown in Table 1, and each treatment is repeated 3 times.

[0074] At the beginning of the test, the soil was mixed with vermiculite at a volume ratio of 3:1, and then 5.5 kg was weighed, and the biochar (BC) / straw biochar bacterial inoculant granular fertilizer (BIB) was mixed with the soil according to the amount in Table 1, and then loaded into a 32 cm x 24 cm x 20 cm pottery pot. Add 4.0 g of compound fertilizer (40 kg / acre) as basic fertilizer to each pot. Select celery seedlings with a height of about 15 cm, and use the five-point method to plant 5 seedlings in each pot. After the seedlings are acclimated for one week, add B. subtilis bacterial liquid (BS) to the soil according to the amount in Table 1. After one month of planting, apply 1.2 g of compound fertilizer (10 kg / acre) to each pot for fertilization; the BIB treatment group increases one group of treatment, and the amount of fertilizer is 0.96 g per pot (8 kg / acre), that is, the RFBIB treatment group. During the test, irrigate 1000 mL of tap water per pot every 2 days, and carry out conventional field management. Stop watering one week before harvesting celery and carry out drying treatment.

[0075] This test is continuously planted for two crops, that is, the second crop continues to use the soil of the first crop, and no other additional treatment is carried out.

[0076] Table 1 Soil treatment (amount of 5.5 kg of potting soil)

[0077]

[0078] (2) Celery sampling method and index determination

[0079] At the harvest stage, 3 plants with uniform growth were selected from each pot, and the whole plant was dug out. 9 plants were collected in each treatment group. The plant height was measured with a tape measure; the stem diameter was measured with an electronic vernier caliper; the fresh weight of the plant was weighed with an analytical balance; then it was placed in a 105°C oven for 30 min, and then dried at 70°C to constant weight, and finally the dry weight was measured; the soluble protein content was detected by Coomassie Brilliant Blue G-250 staining method; the soluble sugar and cellulose contents were determined by anthrone colorimetry; the vitamin C (VC) content was determined by xylene extraction colorimetry; and the carotenoid and chlorophyll contents were determined by ethanol immersion colorimetry. The above determination methods were carried out according to the "Plant Physiology Experiment Guide" edited by Cai Yongping.

[0080] (3) Soil sampling method and index determination

[0081] According to the five-point sampling method, the celery rhizosphere soil was collected, and the soil samples at each point were mixed uniformly to serve as one sample, with 3 replicates for each treatment. The collected samples were placed in sterile sampling bags, labeled and placed in an ice-packed incubator. The soil samples collected were sent to the laboratory as soon as possible for microbial community analysis.

[0082] Genomic DNA was extracted from the soil samples according to the kit instructions, and the V3+V4 region of bacterial 16S rRNA gene and fungal ITS region were amplified for PCR, respectively. The amplification products were sent to Meiji Biotechnology Co., Ltd. for sequencing using the Illumina MiSeq sequencing platform.

[0083] The sequencing data processing flow is as follows: First, the obtained PE (Paired-End) reads are split by sample. Then, based on the sequencing quality, the double-end reads are quality controlled and filtered to remove low-quality reads and adapter sequences. Next, according to the overlap relationship between double-end reads, splicing is performed to generate optimized data after quality control. Then, sequence noise reduction methods are used to process the optimized data to remove noise and amplicon errors in the sequencing process, thereby obtaining accurate OTUs (Operational Taxonomic Units) representative sequences and their abundance information. Based on the OTUs representative sequences and their abundance information, further community diversity analysis and species composition analysis are carried out. The dilution curve is used to indicate whether the sequencing data volume is large enough, and the Non-metric Multidimensional Scaling (NMDS) is used to show the species composition difference between samples. According to the community Bar chart, the relative abundance of each dominant species in the sample is obtained.

[0084] (4) Data analysis

[0085] Data processing and statistical analysis were completed by Microsoft Excel 2010 and SPSS Statistics 26.0, in which Excel was used for data collation and preliminary processing, and SPSS was used for one-way ANOVA. Further multiple comparisons used Duncan method, with a significance level set at P < 0.05, and significant differences were indicated by different lowercase letters. Plotting was completed by Origin 2021 software. R language (version 3.3.1) was used to draw dilution curves, NMDS plots, and community Bar plots.

[0086] (5) Results

[0087] ①Effects of different soil treatments on growth indexes of continuous celery

[0088] As shown in Table 2, there were significant differences in fresh weight of two crops of celery among different soil treatment groups (P < 0.05). In the first crop of celery, the fresh weight of the RFBIB treatment group was the highest, reaching 69.96 g / plant, which was significantly higher than that of other treatment groups (P < 0.05), and was 3.53 times that of the CK group, indicating that it had the best promoting effect on celery growth. In the second crop of celery, the fresh weight of the RFBIB treatment group was still the highest, reaching 121.33 g, which was significantly higher than that of other treatment groups (P < 0.05), and was 2.16 times that of the CK group. The BIB treatment group was second, and was 2.09 times that of the CK group. In addition, the fresh weight of celery in the first and second crops of the BIB treatment group was significantly higher than that of the BC and BS treatment groups (P < 0.05), indicating that the application of straw biochar carrier agent granular fertilizer to soil had a more significant promoting effect on celery growth.

[0089] As shown in Table 2, compared with the CK, the dry weight of two crops of celery in different soil treatment groups was significantly improved (P < 0.05). In the first crop of celery, the dry weight of celery in the RFBIB treatment group was 6.97 g / plant, which was significantly higher than that of other treatment groups (P < 0.05), and the BIB treatment group was second (P < 0.05). In the second crop of celery, the dry weight of celery in the RFBIB treatment group was still the highest, reaching 17.64 g / plant, which was significantly higher than that of other treatment groups (P < 0.05). Compared with the CK, the dry weight of celery in the first and second crops of the RFBIB treatment group was 4.17 times and 2.40 times, respectively, which was significantly higher than that of the BC and BS treatment groups (P < 0.05). This result indicated that the application of straw biochar carrier agent granular fertilizer had a more obvious promoting effect on celery growth than the application of biochar or B. subtilis bacterial solution alone, and the effect of reduced fertilization combined with straw biochar carrier agent granular fertilizer on improving celery yield was better.

[0090] From Table 2, compared with CK, the plant height of two crops of celery in different soil treatment groups was significantly improved (P<0.05). In the first crop of celery, the plant height of the RFBIB treatment group was the highest, reaching 46.75 cm, which was 1.59 times that of the CK group (P<0.05), but there was no significant difference compared with the BIB treatment group. In the second crop of celery, the plant height of the RFBIB treatment group was 1.49 times that of the CK group, which was significantly higher than that of other treatment groups (P<0.05).

[0091] From Table 2, in terms of stem diameter, the RFBIB treatment group had a significantly higher improvement effect on the first and second crops of celery than other treatment groups (P<0.05), which was 1.81 times and 1.93 times that of the CK group, respectively.

[0092] Table 2 Effect of different soil treatments on the growth parameters of continuous cropping celery

[0093]

[0094] Biochar can provide a better environment for plant growth by improving the physical properties of soil. At the same time, as a carrier of microorganisms, biochar can significantly improve the activity and diversity of microorganisms in the soil. In this test, the application of straw biochar carrier microbial agent granular fertilizer significantly improved the fresh weight, dry weight, plant height and stem diameter of celery, and the improvement effect was significantly higher than that of the treatment groups of biochar and B. subtilis bacterial liquid alone, indicating that the interaction of biochar and B. subtilis bacterial liquid has a more significant promoting effect on the growth of celery, and has a significant advantage in improving the growth of celery and relieving the continuous cropping obstacles.

[0095] In this test, two crops of celery were planted, and the RFBIB treatment group significantly promoted the growth of celery. Biochar can improve soil structure, enhance aeration and water retention, and provide a good growth environment for celery roots. At the same time, B. subtilis bacterial liquid can decompose organic matter, release nutrients, and improve soil fertility, so as to reduce the amount of chemical fertilizers and avoid the inhibitory effect of excessive nutrients on the growth of celery, thereby promoting the growth of celery. Especially in the first crop of celery, the effect is better, indicating that it has strong adaptability and stability in improving continuous cropping soil.

[0096] ②Effect of different soil treatments on the physiological indicators of continuous cropping celery

[0097] As shown in Table 3, in the first crop of celery planting, the soluble protein content of the RFBIB treatment group was the highest, reaching 2.90 mg / g, which was significantly higher than that of other treatment groups (P < 0.05), and increased by 211.83% compared with CK. The soluble protein content of the BIB treatment group was 2.62 mg / g, which was significantly higher than that of other treatment groups. In the second crop of celery planting, the soluble protein content of the RFBIB treatment group was 2.24 mg / g, which was still significantly higher than that of all other treatment groups (P < 0.05).

[0098] As shown in Table 3, in the first crop of celery planting, the soluble sugar content of the RFBIB treatment group increased by 123.08% compared with CK (P < 0.05), and in the second crop data, it reached 382.05%, with more obvious promotion effect (P < 0.05).

[0099] As shown in Table 3, in the first crop and the second crop of celery planting, the cellulose content of the RFBIB treatment group was significantly higher than that of the control group (P < 0.05). In the first crop of celery planting, the cellulose content of the RFBIB treatment group increased by 60.34% compared with CK (P < 0.05), and in the second crop of celery, the promotion effect was more obvious, reaching 307.08% (P < 0.05).

[0100] As shown in Table 3, in the first crop of celery planting, the VC content and carotenoid content of the RFBIB treatment group were 21.04 mg / 100 g and 0.47 mg / g, respectively, which were significantly higher than those of CK, BC and BS treatment groups (P < 0.05), and had no significant difference with BIB treatment group (P > 0.05). In the second crop of celery planting, the VC content and carotenoid content of BIB and RFBIB treatment groups were significantly higher than those of other treatment groups (P < 0.05), but the content of BIB treatment group was significantly lower than that of RFBIB treatment group (P < 0.05), indicating that reduced fertilization was beneficial to improve the VC content and carotenoid content of celery.

[0101] As shown in Table 3, in the first crop and the second crop of celery planting, the chlorophyll content of the RFBIB treatment group was significantly higher than that of the control group. In the first crop of celery, the chlorophyll content of the RFBIB treatment group increased by 243.59% compared with CK (P < 0.05), and in the second crop of celery, it increased by 181.82% (P < 0.05). In addition, the chlorophyll content of the RFBIB treatment group was significantly higher than that of BC, BS and BIB treatment groups in both crops of celery (P < 0.05). This result showed that the RFBIB treatment group had a significant promoting effect on the accumulation of chlorophyll content in celery.

[0102] Table 3 of the systematic evaluation shows that, compared with the control (CK), the BC, BS, BIB, and RFBIB treatment groups showed significantly higher contents of soluble protein, soluble sugar, cellulose, vitamin C, carotenoids, and chlorophyll, demonstrating strong adaptability and sustainability in two consecutively planted celery crops, indicating a more significant growth-promoting effect. The BIB and RFBIB treatment groups showed higher values ​​for all indicators than the other treatment groups. The addition of biochar significantly improved soil porosity, water retention, and aeration, thereby optimizing soil structure. Simultaneously, biochar, as a carrier for microorganisms, significantly enhanced the activity and diversity of microorganisms in the soil. *B. subtilis* bacterial solution, as a rhizosphere growth-promoting bacterium, secretes plant hormones (such as indoleacetic acid, cytokinins, and gibberellins) and other organic compounds, thereby promoting nutrient absorption and enhancing the physiological metabolism of celery. It is possible that the interaction between biochar and *B. subtilis* bacterial solution promoted celery growth. In conclusion, the RFBIB treatment group showed higher values ​​for all physiological indicators than the other treatment groups in both crops of celery, indicating a better growth-promoting effect on celery.

[0103] Table 3 Effects of different soil treatments on physiological parameters of continuously cropped celery

[0104]

[0105] ③ Effects of different treatments on soil alpha diversity

[0106] Fifteen soil samples from five treatment groups planted with the first crop of celery were sequenced using Illumina. A total of 13,977 bacterial OTUs were detected, covering 3,213 species, 1,269 genera, 629 families, 383 orders, 161 classes, and 48 phyla; 3,128 fungal OTUs were also detected, covering 1,047 species, 642 genera, 298 families, 127 orders, 60 classes, and 17 phyla. Dilution curves were constructed based on the sequencing results. Figure 3 As can be seen, the Shannon curves for bacteria and fungi tend to be flat, indicating that the amount of sequencing data is sufficient to reflect most of the microbial diversity information in the samples. Microbial cultures were performed on samples from different soil treatments, and the results are shown in Table 4.

[0107] Table 4. Effects of different soil treatments on soil culturable microorganisms

[0108]

[0109] As shown in Table 4, the number of cultivable bacteria, actinomycetes and fungi in the BIB treatment group was the largest; among them, the number of bacteria and fungi was significantly higher than that in other treatment groups (P<0.05), and the number of actinomycetes had no significant difference with that in the RFBIB treatment group. This shows that the application of straw biochar carrier agent granular fertilizer in soil promotes the growth of soil microorganisms.

[0110] (4) Beta diversity analysis of different treatment soils

[0111] In this experiment, NMDS method was used to analyze the composition of soil bacteria and fungi community, and the results are shown in Table 4. Figure 4 Among them, the distance between bacterial community samples was calculated by weighted UniFrac algorithm, and the distance between fungal community samples was calculated by Bray-Curtis algorithm to explore the differences in community composition between different treatment groups. As shown in Table 4, Figure 4 a, the bacterial community composition of different treatment groups was significantly different (R=0.67, P=0.001), and the stress value was 0.059, which could be considered as a good order. The results of NMDS analysis of bacterial community showed that the application of biochar, B. subtilis liquid, straw biochar carrier agent granular fertilizer affected the composition of soil bacterial microbial community. Especially the BIB treatment group was obviously separated from other treatment groups, indicating that the bacterial community composition was significantly different from other treatment groups. For fungal community Figure 4 b), BC, BS, BIB and RFBIB treatment groups were obviously separated from CK treatment group, reflecting that the fungal community composition was significantly different between different treatment groups (R=0.96, P=0.001, stress=0.059). This further shows that the application of straw biochar carrier agent granular fertilizer affects the composition of soil microbial community, changes the composition of soil bacteria and fungi community, and indirectly affects the properties of soil.

[0112] (5) Analysis of soil species composition of different treatments

[0113] Figure 5 The relative abundance of soil bacterial phylum and fungal phylum under different treatments is shown in Table 4. Figure 5As can be seen from Table 2, the top nine bacterial phyla with the highest relative abundance in all samples were Proteobacteria, Chloroflexi, Actinobacteriota, Acidobacteriota, Firmicutes, Bacteroidota, Gemmatimonadota, Myxococcota and Cyanobacteria, with a total relative abundance of 92.8% to 94.74%. In the CK treatment group, the relative abundance of Proteobacteria was 17.94%, while in the BIB and RFBIB treatment groups, the relative abundance of Proteobacteria was 19.55% and 19.00% respectively, indicating that the application of straw biochar carrier granular fertilizer in soil promoted the growth and enrichment of Proteobacteria. Related studies have shown that Proteobacteria plays a key role in soil ecosystems, including participating in nitrogen cycling, decomposing organic matter and promoting plant growth, and an increase in its relative abundance helps to improve soil fertility and improve soil microbial community composition. The relative abundance of Chloroflexi in the BIB and RFBIB groups was 18.72% and 18.79% respectively, which was significantly higher than that in the control group. Related studies have shown that Chloroflexi bacteria can decompose complex organic matter, improve soil fertility, promote plant growth and effectively alleviate the problem of soil nutrient imbalance under continuous cropping. In addition, Actinobacteriota has the ability to decompose complex organic matter, and the relative abundance in the BIB and RFBIB treatment groups was 17.40% and 17.07% respectively, which was higher than that in the CK group (16.93%), which may be due to the application of straw biochar carrier granular fertilizer increasing the organic matter content of the soil. Compared with the CK treatment group, the relative abundance of Acidobacteriota was higher in other treatment groups, while the relative abundance of Bacteroidota was higher in other treatment groups. This may be related to the massive proliferation of Acidobacteriota bacteria, while the growth of Bacteroidota bacteria was inhibited. In addition, the relative abundance of Bacteroidota in the CK treatment group was lower than that in other treatment groups, which may be due to the inhibition of the growth of certain beneficial bacteria by the acidic environment of the soil.

[0114] By Figure 5It can be seen that in all soil samples, the phylum with the highest relative abundance in the fungal kingdom is Ascomycota, and the highest relative abundance in the BIB treatment group is 72.52%. Ascomycota can form a symbiotic relationship with plants, enhance the ability of plants to absorb water and mineral elements, and thus promote plant growth. This may be that the application of straw biochar carrier granular fertilizer in soil increases the relative abundance of Ascomycota, optimizes the abundance of soil-related microbial communities, and provides a more favorable growth environment for plants, thereby promoting the growth of plants. Therefore, after applying the straw biochar carrier granular fertilizer, the growth of Proteobacteria, Chloroflexi and Actinobacteriota in the soil is promoted, the growth of Acidobacteriota is inhibited, the soil microbial community structure is changed, the degree of soil acidification is reduced, the decomposition of organic matter is promoted, and the like, thereby alleviating the continuous cropping obstacles and creating a healthy soil environment for plant growth.

[0115] Experiment 3

[0116] (1) Field test design

[0117] The test was carried out in the soil of the continuous cropping of celery for 5 years in Fengsuo Four-Season Ecological Farm in Beidaihe District, Qinhuangdao City, Hebei Province on October 11, 2024. The test was set up with 7 soil treatments, which were arranged in 7 independent plots, and each plot had an area of 1m x 3m = 3m 2 , and the walking path was 0.3m wide. The specific treatment methods are as follows:

[0118] ① CK, (without applying biochar and bacterial liquid);

[0119] ② BC (applying the biochar prepared in Example 1);

[0120] ③ BS (applying B. subtilis bacterial liquid (2 x 10 9 CFU / mL));

[0121] ④ BIB (applying the straw biochar carrier granular fertilizer prepared in Example 1);

[0122] ⑤ BIB_20 (applying the straw biochar carrier granular fertilizer prepared in Example 1, reducing the application of chemical fertilizer by 20% during topdressing);

[0123] ⑥ BIB_30 (applying the straw biochar carrier granular fertilizer prepared in Example 1, reducing the application of chemical fertilizer by 30% during topdressing);

[0124] ⑦ BIB_40 (applying the straw biochar carrier granular fertilizer prepared in Example 1, reducing the application of chemical fertilizer by 40% during topdressing).

[0125] The specific addition concentration of each treatment group is shown in Table 5.

[0126] Table 5 Different soil treatment addition amount

[0127]

[0128] At the beginning of the experiment, according to the local traditional fertilization standard (see Table 5), potassium sulfate compound fertilizer (15-15-15, N+3P2O5+K2O≥45%) was used as base fertilizer and uniformly turned into the soil before planting celery. Subsequently, according to the concentration requirements of each treatment, biochar and straw biochar inoculant granular fertilizer were uniformly turned into the soil and mixed well with the soil to ensure uniform distribution in the soil. Two weeks later, on October 23, 2024, celery seedlings with a height of about 15 cm were selected for transplanting, 14 rows were planted in each plot, and 8 plants were planted in each row. After transplanting, B. subtilis liquid was added according to the proportion, and one month after transplanting, topdressing was performed, and the specific topdressing amount is shown in Table 5. During the entire experiment, strict routine field management measures were followed. One week before harvest, irrigation was stopped and drying was performed.

[0129] (2) Celery sampling method and index determination

[0130] Sampling method: At the harvest period, according to the soil treatment type, 10 celery plants with uniform growth were selected for each treatment group, and the whole plant was dug out.

[0131] The determination method of growth index and physiological index is the same as that of Experiment 2.

[0132] (3) Results

[0133] ① Effect of different soil treatments on the growth index of continuous celery

[0134] Table 6 Effect of different soil treatments on the growth parameters of continuous celery

[0135]

[0136] From Table 6, compared with CK, different soil treatments significantly improved the fresh weight, dry weight, plant height and stem diameter of celery (P<0.05). Among them, the promotion effect of BIB_20 treatment group was the most obvious, and the fresh weight, dry weight, plant height and stem diameter were increased by 263.08%, 207.59%, 34.82% and 64.04% (P<0.05) compared with CK group. The BIB treatment group was second, and the fresh weight, dry weight, plant height and stem diameter were increased by 213.72%, 175.19%, 25.08% and 35.11% (P<0.05) compared with CK. In addition, the indicators of celery in BIB_30 and BIB_40 treatment groups were lower than those in BIB_20 treatment group, but they were significantly higher than those in CK (P<0.05). This shows that soil application of straw biochar carrier agent granular fertilizer can obviously promote the growth of celery, and still has strong promoting effect under the condition of reducing chemical fertilizer.

[0137] B. subtilis can form a symbiotic relationship with plant roots, improving the plant's absorption and utilization efficiency of nutrients. In addition, B. subtilis can secrete extracellular enzymes to decompose complex organic matter in the soil, releasing nutrients such as nitrogen, phosphorus, potassium that can be absorbed by plants, optimizing plant growth conditions. In addition, the application of biochar can improve the absorption efficiency of nutrients by plants, and promote photosynthesis by adjusting the stomatal conductance and photosynthetic efficiency of plants, thereby improving the growth of plants. However, when the fertilizer reduction is more than 20%, i.e. BIB_30 and BIB_40 treatment groups, although the growth indicators of celery are still significantly higher than those of CK, the promotion effect is weakened. This may be related to the insufficient supply of nutrients caused by the reduction of fertilizer amount. Celery has a large demand for nutrients such as nitrogen, phosphorus and potassium during growth, and the reduction of fertilizer amount may lead to insufficient supply of soil nutrients, thereby limiting the further growth of celery.

[0138] ②Effects of different soil treatments on physiological indicators of continuous cropping celery

[0139] Table 7 Effects of different soil treatments on physiological indicators of continuous cropping celery

[0140]

[0141]

[0142] As shown in Table 7, compared with CK, the soluble protein, soluble sugar and chlorophyll contents of celery were significantly improved by different soil treatments (P<0.05). Among them, the BIB_20 treatment group had the most obvious improvement effect, and the soluble protein, soluble sugar and chlorophyll contents were increased by 40.32%, 90.18% and 173.58% respectively compared with CK. The BIB treatment group was the second, and the soluble protein, soluble sugar and chlorophyll contents were increased by 31.45%, 76.00% and 150.94% respectively compared with CK. Although the improvement effect of BIB_30 and BIB_40 treatment groups was smaller than that of BIB_20 treatment group, it was still significantly higher than that of CK (P<0.05). This shows that under the condition of normal fertilization and 20% reduction of topdressing, the straw biochar microbial inoculant granular fertilizer has the most obvious effect on the improvement of soluble protein, soluble sugar and chlorophyll contents of celery. This may be attributed to the interaction between biochar and B. subtilis. On the one hand, biochar improves the physical properties of soil, such as increasing soil porosity, adjusting soil pH and enhancing soil water and fertilizer retention capacity, to provide a more superior environment for plant growth. On the other hand, B. subtilis promotes nutrient uptake and enhances the physiological metabolism of celery by secreting plant hormones (such as indole acetic acid) and organic compounds. This mechanism of interaction between biochar and microorganisms provides a comprehensive promotion effect on the growth and physiological metabolism of celery.

[0143] As shown in Table 7, compared with CK, the cellulose, VC and carotenoid contents of celery were significantly improved by different soil treatments (P<0.05). Among them, the cellulose, VC and carotenoid contents of celery in the BIB_20 treatment group were significantly higher than those in other treatment groups, and were increased by 11.97%, 34.01% and 30.95% respectively compared with CK (P<0.05). The cellulose, VC and carotenoid contents of celery in the BIB, BIB_30 and BIB_40 treatment groups were significantly higher than those in the CK group, but significantly lower than those in the BIB_20 treatment group. This shows that there is an optimal range for the influence of reducing topdressing amount on the physiological indicators of celery.

[0144] In summary, the interaction between biochar and B. subtilis promotes the growth of celery and improves its nutritional quality, further proving that applying straw biochar microbial inoculant granular fertilizer can improve the quality of celery.

[0145] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0146] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a granular biochar inoculant of straw, characterized in that, The preparation method comprises the following steps: (1) preparation of biochar Put corn stalks in an atmosphere furnace for pyrolysis, crush and sieve for use; (2) preparation of B.subtilis bacterial liquid Activate B.subtilis bacteria and culture to logarithmic phase, and adjust the concentration of the bacterial liquid for use; (3) preparation of straw biochar bacterial carrier granular fertilizer (31) incubate and dry the biochar and B.subtilis bacterial liquid to obtain a biochar bacterial carrier; (32) mix the biochar bacterial carrier with auxiliary materials to granulate.

2. The method of claim 1, wherein, In the step (1), the corn stalks are put in an atmosphere furnace at 500-650℃ for pyrolysis for 2-4h.

3. The method of claim 1, wherein, The concentration of the bacterial solution in step (2) is 2 x 10 9 CFU / mL.

4. The method of claim 1, wherein, In the step (31), the mass-volume ratio of the biochar and B.subtilis bacterial liquid is 30-50g:6-9mL.

5. The method of claim 1, wherein, In the step (32), the specific parameters of incubation are as follows: 30℃, 120rpm, 18-24h.

6. The method of claim 1, wherein, The auxiliary materials include corn starch, peanut meal and clay; The mass ratio of the biochar bacterial carrier, corn starch, peanut meal and clay is 50:9-12:21-28:10-20.

7. Application of the straw biochar bacterial carrier granular fertilizer prepared by the preparation method of any one of claims 1-6 in prevention and treatment of celery continuous cropping obstacles.

8. Use according to claim 7, wherein the compound is ###0002### The application is used for improving the growth parameters, physiological indexes and microbial community structure of celery.

9. Use according to claim 8, wherein the compound is ###0002### The growth parameters include fresh weight, dry weight, plant height and stem diameter.

10. The use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The physiological indexes include soluble protein content, soluble sugar content, cellulose content, vitamin C content, carotenoid content and chlorophyll content.

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