Preparation method and application of anti-cadmium growth-promoting bacteria biochar microbial agent
By preparing cadmium-resistant growth-promoting biochar inoculant, and combining Burkholderia cepacia P10 with biochar, the problems of chili pepper growth and cadmium accumulation in cadmium-contaminated soil were solved, achieving the effect of significantly promoting chili pepper growth and reducing cadmium content under high cadmium stress.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUIZHOU UNIV
- Filing Date
- 2026-02-12
- Publication Date
- 2026-06-12
AI Technical Summary
In the existing technology, the application of Burkholderia cepacia P10 in the field of cadmium-contaminated soil remediation has not been fully studied, and the adsorption and fixation capacity of biochar in cadmium-contaminated soil remediation has not been fully utilized, resulting in the failure to effectively alleviate the impact of cadmium pollution on pepper growth and fruit quality.
A cadmium-resistant growth-promoting biochar agent was prepared by combining Burkholderia cepacia P10 with biochar. The preparation method included biochar pretreatment, strain activation, and biochar addition steps to form a cadmium-resistant growth-promoting biochar agent with a high number of viable bacteria. This agent was then used in cadmium-contaminated soil to promote pepper growth and reduce cadmium accumulation.
Within the soil cadmium stress concentration range of 10-70 mg/kg, it significantly promoted the growth and development of chili peppers and reduced the accumulation of cadmium in the soil, plants, and fruits. Especially under high cadmium stress, it effectively reduced the cadmium content by 21.00%, 54.35%, and 45.45%.
Smart Images
Figure CN122181545A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural microbial technology, and provides a method for preparing cadmium-resistant growth-promoting biochar agent and its application. Background Technology
[0002] Due to its high toxicity, easy migration, and difficulty in degradation, soil cadmium pollution not only affects the quality of agricultural products and the safety of the ecological environment, but also restricts the sustainable development of agriculture. Soil heavy metal cadmium pollution not only poisons plants and soil microorganisms, but also disrupts the balance of the ecosystem.
[0003] Currently, phytorheic dermatitis-promoting bacteria (PGPR) co-remediation technology has attracted widespread attention due to its environmental friendliness and low cost. The strains of PGPR strains reported for co-remediation of heavy metal-contaminated soil mainly include Bacillus sp., Pseudomonas sp., Rhizobium sp., Sphingomonas sp., and Microbacterium sp. However, research and patent reports on Burkholderia cepacia combined with biochar in the remediation of cadmium-contaminated soil are still lacking.
[0004] Biochar, as a novel soil conditioner, has a large specific surface area, rich pore structure, and strong adsorption capacity. It can retain available cadmium and other substances in the soil through precipitation, complexation, electrostatic attraction, and cation exchange, and can adsorb heavy metals in the soil and improve the soil's physical and chemical properties.
[0005] Burkholderia cepacia P10 was isolated by the inventor from the rhizosphere soil of tea trees. This strain has been reported in the inventor's patent application (patent application number 201910267582.2, patent title: A strain of Burkholderia cepacia P10 and its application). The accession number of Burkholderia cepacia P10 is CCTCCNO:M 2019172, and it was deposited at the China Center for Type Culture Collection on March 8, 2019. This patent discloses that Burkholderia cepacia P10 has the functions of dissolving inorganic phosphorus, producing ACC deaminase, and secreting siderophores, which can promote peanut growth. However, the study did not investigate the strain's cadmium resistance and its effect on improving the cadmium tolerance of chili peppers.
[0006] This invention has found that preparing cadmium-resistant and growth-promoting biochar agent from Burkholderia cepacia P10 can significantly improve the resistance of chili peppers in cadmium-polluted environments, promote the growth and development of chili peppers, and reduce the accumulation of cadmium in soil, plants, and fruits. Summary of the Invention
[0007] The technical problem to be solved by the present invention is to provide a method for preparing an anti-cadmium growth-promoting bacterial biochar agent and its application. This agent combines the anti-cadmium growth-promoting properties of Burkholderia cepacia P10 with the adsorption and fixation capacity of biochar, which can promote the growth of peppers in areas with high cadmium background values and reduce cadmium accumulation.
[0008] To solve the above technical problems, the present invention adopts the following technical solution: A method for preparing a cadmium-resistant and growth-promoting biochar agent, the preparation method comprising the following steps: (1) Biochar pretreatment: After passing the biochar through a 30-50 mesh sieve, wash it with deionized water 2-4 times, each time using 150-250 mL of deionized water. Then soak it in 0.1 mol / L HCl solution and stir continuously for 0.8-1.5 h. Wash it with deionized water until the pH is 6.8-7.2. Dry it at 35-45℃ to constant weight to obtain pretreated biochar for later use. (2) Activation of bacterial strain: 45-55 μL of Burkholderia cepacia P10 bacterial suspension preserved in glycerol was inoculated into 45-55 mL of LB liquid medium and activated overnight at 28-32℃; 0.9-1.1 mL of activated bacterial suspension was transferred into 45-55 mL of fresh LB liquid medium and cultured at 28-32℃ with shaking at 140-160 rpm for 18-20 h until the logarithmic growth phase was reached, and the OD was adjusted. 600 A value of 0.9-1.1 indicates the presence of P10 activated bacterial suspension. (3) Adding biochar: Add the pretreated biochar to fresh LB liquid medium at a ratio of 0.5-2 g: 50 mL. After autoclaving, inoculate with 0.8-1.2 mL of P10 activated bacterial suspension. Fix at 28-32℃ and 140-160 rpm for 18-22 h with shaking. Centrifuge at 6000 rpm for 14-16 min, discard the supernatant, and wash the precipitate 2-3 times with 8-12 mL of deionized water each time. After natural drying, obtain the cadmium-resistant growth-promoting bacterial biochar agent. The viable count of Burkholderia cepacia P10 strain in the cadmium-resistant growth-promoting bacterial biochar agent is 0.98 × 10⁻⁶. 15 -1.08×10 15cfu / g。 In step (1) above, the biochar pretreatment is as follows: after passing the biochar through a 40-mesh sieve, it is washed three times with deionized water, each time using 200 mL of deionized water. Then, it is soaked in 0.1 mol / L HCl solution and stirred continuously for 1 hour. After washing with deionized water until the pH is 7.0, it is dried at 40°C to constant weight to obtain pretreated biochar for later use.
[0009] Specifically, in step (1) above, the biochar is coconut shell biochar.
[0010] In step (2) above, the strain is activated as follows: 50 μL of *Burkholderia cepacia* P10 culture preserved with glycerol is inoculated into 50 mL of LB liquid medium and activated overnight at 30°C; 1 mL of the activated culture is transferred to 50 mL of fresh LB liquid medium and cultured at 30°C and 150 pm on a shaker for 18-20 h until the logarithmic growth phase is reached, and the OD is adjusted. 600 The value is 1.0 (viable bacteria count is 1.0 × 10⁻⁶). 9 P10 activated bacterial suspension was obtained by (cfu / mL).
[0011] Specifically, in step (2) above, the LB liquid culture medium is prepared by adding 10.0g of tryptone, 5.0g of yeast extract and 10.0g of sodium chloride to 1000mL of distilled water, adjusting the pH to 7.0-7.2, and sterilizing at 121℃ for 20min.
[0012] In step (3) above, biochar is added: the pretreated biochar is added to fresh LB liquid medium at a ratio of 1g:50mL. After autoclaving at 121℃ for 20min, 1mL of P10 activated bacterial suspension is added. The mixture is then shaken and fixed at 30℃ and 150rpm for 20h, followed by centrifugation at 6000rpm for 15min. The supernatant is discarded, and the precipitate is washed twice with 10mL of deionized water each time. After natural drying, the cadmium-resistant growth-promoting bacterial biochar agent is obtained. The viable count of Burkholderia cepacia P10 strain in the cadmium-resistant growth-promoting bacterial biochar agent is 0.98×10⁻⁶. 15 -1.08×10 15cfu / g。 Specifically, in step (3) above, the LB liquid culture medium is prepared by adding 10.0g of tryptone, 5.0g of yeast extract and 10.0g of sodium chloride to 1000mL of distilled water, adjusting the pH to 7.0-7.2, and sterilizing at 121℃ for 20min.
[0013] Specifically, in step (3) above, the viable count of Burkholderia cepacia P10 strain in the prepared cadmium-resistant growth-promoting biochar agent is 1.03 × 10⁻⁶. 15 cfu / g.
[0014] The aforementioned application of cadmium-resistant growth-promoting biochar in the preparation of chili growth promoters shows that cadmium-resistant growth-promoting biochar can be used in areas with high soil cadmium background values to promote chili growth and development, improve fruit yield and quality, and reduce cadmium accumulation in soil, plants and fruits.
[0015] The aforementioned application method for the cadmium-resistant growth-promoting biochar agent is to dilute it with water and apply it to the roots of the chili pepper plant at a rate of 0.01g per plant, once every 15 days.
[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses Burkholderia cephalosporin P10 to prepare a cadmium-resistant growth-promoting biochar agent. Under soil cadmium stress concentrations of 10-70 mg / kg, it significantly promotes the growth and development of chili peppers and reduces cadmium accumulation in the soil, plants, and fruits. Even at a soil cadmium stress concentration of 70 mg / kg, the effective cadmium content in the soil after application of the cadmium-resistant growth-promoting biochar agent decreased by 21.00%, while the cadmium content in plant roots, stems, and fruits decreased by 54.35%, 44.71%, and 45.45%, respectively. This indicates that the application of the cadmium-resistant growth-promoting biochar agent significantly reduced the transport and accumulation of cadmium in the soil to the roots and stems, thereby effectively reducing cadmium accumulation in the fruits.
[0017] 2. The preparation process of the cadmium-resistant and growth-promoting bacterial biochar agent of the present invention is simple and easy to operate, which is conducive to industrial promotion. Attached Figure Description
[0018] Figure 1 For Cd 2+ Growth curves of strain P10 under concentration stress; Figure 2 For different concentrations of Cd 2+ The amount of siderophores produced by strain P10 under stress; Figure 3 For different concentrations of Cd 2+ The amount of EPS produced by strain P10 under stress; Figure 4 The amount of cadmium adsorbed in different parts of the culture medium of strain P10; Figure 5 For different concentrations of Cd 2+ Scanning electron microscope and transmission electron microscope images of strain P10 under stress; Figure 6 The microstructure of biochar inoculant under a scanning electron microscope; Figure 7 The effects of biochar inoculants on chili peppers at different growth stages under different concentrations of cadmium stress; Figure 8 The effects of different concentrations of biochar inoculants on pepper fruits under cadmium stress. Detailed Implementation
[0019] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.
[0020] The following examples use the following method for preparing LB liquid culture medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1000 mL distilled water, pH 7.0-7.2, sterilized at 121°C for 20 min; The coconut shell biochar was purchased from Tanno Environmental Protection Materials Co., Ltd. in Pingdingshan City, Henan Province.
[0021] Example 1: Preparation method of cadmium-resistant and growth-promoting biochar agent (1) Biochar pretreatment: After passing coconut shell biochar through a 40-mesh sieve, wash it three times with deionized water, each time using 200 mL of deionized water. Then soak it in 0.1 mol / L HCl solution and stir continuously for 1 h. Wash it with deionized water until the pH is 7.0. Dry it at 40℃ to constant weight to obtain pretreated biochar for later use. (2) Activation of bacterial strain: 50 μL of Burkholderia cepacia P10 bacterial suspension preserved with glycerol was inoculated into 50 mL of LB liquid medium and activated overnight at 30°C; 1 mL of activated bacterial suspension was transferred into 50 mL of fresh LB liquid medium and cultured at 30°C with shaking at 150 rpm for 18-20 h until the logarithmic growth phase was reached, and the OD was adjusted. 600 The value is 1.0 (viable bacteria count is 1.0 × 10⁻⁶). 9 The P10 activated bacterial suspension was obtained by measuring cfu / mL. Adding biochar: Add the pretreated biochar to fresh LB liquid medium at a ratio of 1g:50mL. After autoclaving at 121℃ for 20min, inoculate with 1mL of P10 activated bacterial suspension. Fix at 30℃ and 150rpm for 20h by shaking. Centrifuge at 6000rpm for 15min, discard the supernatant, and wash the precipitate twice with 10mL of deionized water each time. After natural drying, obtain the cadmium-resistant and growth-promoting biochar agent.
[0022] Example 2: Preparation method of cadmium-resistant and growth-promoting biochar agent (1) Biochar pretreatment: After passing coconut shell biochar through a 50-mesh sieve, wash it 4 times with deionized water, each time using 250 mL of deionized water. Then soak it in 0.1 mol / L HCl solution and stir continuously for 1.5 h. Wash it with deionized water until the pH is 7.0-7.2. Dry it at 45℃ to constant weight to obtain pretreated biochar for later use. (2) Activation of the strain: 55 μL of Burkholderia cepacia P10 bacterial culture preserved with glycerol was inoculated into 45 mL of LB liquid medium and activated overnight at 32°C; 0.9 mL of the activated bacterial culture was transferred into 55 mL of fresh LB liquid medium and cultured at 32°C with shaking at 160 rpm for 18 h until the logarithmic growth phase was reached, and the OD was adjusted.600 A value of 0.9 indicates the presence of P10 activated bacterial suspension. (3) Adding biochar: Add the pretreated biochar to fresh LB liquid medium at a ratio of 2g:50mL. After autoclaving at 121℃ for 20min, add 0.8mL of P10 activated bacterial suspension. Shake and fix at 32℃ and 160rpm for 18h. Centrifuge at 6000rpm for 16min, discard the supernatant, wash the precipitate three times with 12mL of deionized water each time, and air dry to obtain cadmium-resistant growth-promoting biochar agent.
[0023] Example 3: Preparation method of cadmium-resistant and growth-promoting biochar agent (1) Biochar pretreatment: After passing coconut shell biochar through a 30-mesh sieve, wash it twice with deionized water, each time using 150 mL of deionized water. Then soak it in 0.1 mol / L HCl solution and stir continuously for 0.8 h. Wash it again with deionized water until the pH is 6.8-7.0. Dry it at 35℃ to constant weight to obtain pretreated biochar for later use. (2) Activation of the strain: 45 μL of Burkholderia cepacia P10 bacterial culture preserved with glycerol was inoculated into 55 mL of LB liquid medium and activated overnight at 28°C; 1.1 mL of the activated bacterial culture was transferred into 45 mL of fresh LB liquid medium and cultured at 28°C and 140 rpm for 20 h with shaking until the logarithmic growth phase was reached, and the OD was adjusted. 600 A value of 1.1 indicates the presence of P10 activated bacterial suspension. (3) Adding biochar: Add the pretreated biochar to fresh LB liquid medium at a ratio of 0.5g:50mL. After autoclaving at 121℃ for 20min, add 1.2mL of P10 activated bacterial suspension. Shake and fix at 28℃ and 140rpm for 22h. Centrifuge at 6000rpm for 14min, discard the supernatant, wash the precipitate twice with deionized water (8mL each time), and air dry to obtain cadmium-resistant growth-promoting biochar agent.
[0024] This invention has conducted extensive experiments to demonstrate the preparation process of the cadmium-resistant and growth-promoting biochar agent and to determine its performance, as detailed below: 1. Study on cadmium resistance of Burkholderia cepacia P10 strain The following method for preparing LB liquid culture medium used in the experiment is as follows: Take 10.0 g of tryptone, 5.0 g of yeast extract and 10.0 g of sodium chloride, add 1000 mL of distilled water, adjust the pH to 7.0-7.2, and sterilize at 121℃ for 20 min; The following method for preparing LB solid culture medium used in experiments is as follows: Add 1.5-2.0% agar to LB liquid culture medium and sterilize at 121℃ for 20 min.
[0025] 1.1 Growth characteristics of strain P10 under cadmium stress Activation: Inoculate 50 μL of *Burkholderia cepacia* P10 culture preserved with glycerol into 50 mL of LB broth and incubate overnight at 30°C to activate the strain. Transfer 1 mL of the activated culture to 50 mL of fresh LB broth and incubate at 30°C with shaking at 150 rpm for 18-20 h until the logarithmic growth phase, adjusting the OD... 600 The value is 1.0 (viable bacteria count is 1.0 × 10⁻⁶). 9 The P10 activated bacterial suspension was obtained by measuring cfu / mL. When determining the growth curve, the activated P10 strain culture was inoculated at a rate of 2% into LB liquid medium containing different concentrations of CdCl2 (0, 200, 400, 600, 800 mg / L), and cultured at 30°C with shaking at 150 rpm. OD was measured every 6 hours. 600 value.
[0026] The results are as follows Figure 1 As shown, the growth curves of strain P10 under different concentrations of cadmium added indicate that the bacterial growth is still relatively good under cadmium stress below 600 mg / L; cadmium stress of 800 mg / L significantly prolongs the lag phase of the strain, but it still has a certain amount of growth afterward.
[0027] 1.2 Growth-promoting characteristics of strain P10 under cadmium stress Under the same cadmium stress conditions as under section “1.1”, the supernatant of the culture medium was used to determine the ability to produce siderophores by the CAS method and the relative content of siderophores was calculated. The extracellular polysaccharide content of the strain was determined by the sulfuric acid phenol method.
[0028] The results are as follows Figure 2 and Figure 3 As shown, under 400 mg / L CdCl2 stress, the ability of strain P10 to produce siderophores decreased; however, even under 600 mg / L CdCl2 stress, the relative content of siderophores secreted by strain P10 could still reach 25.3%. With increasing cadmium stress, the ability of P10 to secrete EPS also decreased slightly, but under 600 mg / L stress, its EPS production was 21.29 μg / mL. This indicates that strain P10 still has a strong growth-promoting ability under high cadmium stress.
[0029] 1.3 Cadmium adsorption capacity and adsorption mechanism of strain P10 P10 cells in the logarithmic growth phase were inoculated into LB medium containing different concentrations of CdCl2 and cultured at 30°C and 150 rpm using a shaker. After culture, the cells were collected, eluted with EDTA, digested with HNO3 / HClO4, and the Cd concentrations in the culture medium, eluent, and cells were determined by ICP-AES. 2+ Content. Meanwhile, the remaining bacterial cells were fixed with glutaraldehyde, and the morphology of the fixed cells was observed using scanning electron microscopy, while the intracellular cadmium distribution was observed using transmission electron microscopy.
[0030] Cadmium content in different parts such as Figure 4 As shown, Cd adsorbed extracellularly by the bacteria 2+ The Cd content was significantly higher than that inside the bacterial cell, and the supernatant contained Cd... 2+ The content was significantly lower than that of intracellular and extracellular Cd. 2+ The content initially indicated that P10 chelates Cd. 2+ The adsorption of Cd mainly occurs on the cell surface; even in a culture medium supplemented with 600 mg / L CdCl2, the extracellular adsorption of strain P10 still accounts for 46.19%. Secondly, a certain concentration of Cd can be deposited or polymerized intracellularly. 2++ .
[0031] Scanning electron microscopy and transmission electron microscopy observations, such as Figure 5 As shown, the control group cells had a regular morphology, appearing as short rods with blunt ends. When the CdCl2 concentration in the culture medium was 200 mg / L, the cells exhibited a regular morphology and were actively dividing, with a small amount of black substance (cadmium) adhering to the cell surface and a small amount of black clumps (cadmium) accumulating inside the cells. Even at CdCl2 concentrations of 600 mg / L and 800 mg / L, the cells remained mostly intact, and binary fission was observed. Overall, with increasing cadmium concentration, the amount of black substance on the cell surface and inside the cells increased, indicating that strain P10 had extremely strong adsorption, uptake, and tolerance to cadmium.
[0032] 2. Research on the preparation process of cadmium-resistant and growth-promoting biochar inoculant 2.1 Determining the addition ratio of cadmium-resistant and growth-promoting biochar inoculant A growth-promoting bacterial inoculum was prepared by combining the highly cadmium-tolerant growth-promoting strain P10 with highly efficient cadmium-resistant biochar. First, experiments were conducted to investigate the effect of different biochar addition amounts on the growth of strain P10, in order to determine the optimal biochar addition amount.
[0033] 1) Biochar pretreatment: The biochar is coconut shell biochar. After passing the biochar through a 40-mesh sieve, it is washed three times with 200 mL of deionized water each time. Then it is soaked in 0.1 mol / L HCl solution and stirred continuously for 1 hour. After washing with deionized water until the pH is 7.0, it is dried at 40℃ to constant weight to obtain pretreated biochar for later use. 2) Activation of the strain: 50 μL of *Burkholderia cepacia* P10 culture preserved with glycerol was inoculated into 50 mL of LB broth and incubated overnight at 30°C to activate the bacterial culture. 1 mL of the activated bacterial culture was then transferred to 50 mL of fresh LB broth and cultured at 30°C with shaking at 150 rpm for 18-20 h until the logarithmic growth phase. The OD was then adjusted. 600 The value is 1.0 (viable bacteria count is 1.0 × 10⁻⁶). 9 The P10 activated bacterial suspension was obtained by measuring cfu / mL. 3) Effect of different biochar addition amounts: 0.5g, 1g, 1.5g, 2g, and 2.5g of biochar were added to 50mL of culture medium to achieve final concentrations of 0.01g / mL, 0.02g / mL, 0.03g / mL, 0.04g / mL, and 0.05g / mL, respectively. After sterilization, 1mL of activated P10 bacterial suspension was inoculated and co-cultured for 20h. 1mL of the culture solution was serially diluted and evenly spread onto solid culture medium. After incubation at constant temperature, the colony counts of different treatment groups were recorded. The colony count of the culture medium without biochar served as a control. Each treatment was performed in triplicate. The results are as follows: Table 1. Effects of different biochar addition amounts on the growth of strain P10 The results are shown in Table 1. The biochar agent for promoting growth prepared by adding 0.02 g of biochar per mL of LB liquid medium had the highest number of viable bacteria, thus determining the biochar content added to the agent.
[0034] 2.2 Preparation method of cadmium-resistant and growth-promoting biochar inoculant The composition of the cadmium-resistant growth-promoting biochar inoculum: It is prepared by compounding Burkholderia cepacia P10 with biochar. The preparation method includes the following steps: biochar pretreatment, strain activation, and expansion culture are the same as in section "2.1". When preparing the biochar inoculum, 1g of pretreated biochar is added to 50mL of LB liquid medium for sterilization, and then transferred to 1mL of activated P10 strain culture (OD200). 600 =1.0, viable bacteria count is 1.0×10 9 The biochar agent was obtained by centrifuging at 6000 rpm for 15 min at 30℃ for 20 h, discarding the supernatant, washing the precipitate with deionized water and drying it naturally. The number of viable bacteria in the biochar agent was determined by dilution and coating method, and the micromorphology of the growth-promoting biochar agent was observed by scanning electron microscopy.
[0035] The viable cell count results showed that the viable count of Burkholderia cepacia P10 in this cadmium-resistant growth-promoting biochar agent was 1.03 × 10⁻⁶. 15 cfu / g. Scanning electron microscopy (SEM) results are shown below. Figure 6As shown, a large number of rod-shaped P10 strain bacteria cells are adsorbed inside and on the surface of the bacterial agent.
[0036] 3. Effects of growth-promoting biochar inoculant on the growth and development of potted peppers Pot experiments were conducted to verify the effects of the growth-promoting biochar inoculant prepared under section "2.2" on the growth and development of chili peppers, fruit yield and quality, and cadmium accumulation in soil, plants and fruits.
[0037] 3.1 Experimental crop: Chili pepper 3.2 Test Variety: Zhuojiaoxin 52 3.3 Test period: March 28, 2025 - August 3, 2025 3.4 Grouping: This experiment was conducted using a pot method. Each pot contained 2000g of sterile soil. A certain amount of CdCl2 solution was first added to the soil to achieve final concentrations of 10, 30, 50, and 70 mg / kg. After equilibration for one month, chili seedlings with uniform growth were transplanted into plastic pots and allowed to recover for 15 days before treatment. The experiment included different cadmium stress groups (10, 30, 50, 70 mg / kg), different cadmium stress + biochar treatment groups (10T, 30T, 50T, 70T), different cadmium stress + liquid inoculant treatment groups (10P, 30P, 50P, 70P), and different cadmium stress + growth-promoting biochar inoculant treatment groups (10PT, 30PT, 50PT, 70PT). Control groups, biochar treatment groups (Tck), liquid inoculant treatment groups (Pck), and growth-promoting biochar inoculant treatment groups (PTck) were also included. Each treatment had six replicates. For each treatment, 0.01 g of biochar and growth-promoting bacteria biochar inoculant were weighed and diluted in 50 mL of sterile water. The liquid inoculant was prepared by centrifuging a 18-20 h culture broth of P10 strain, discarding the supernatant, and resuspending the precipitate in 50 mL of sterile water. For the control group, 50 mL of water was added and applied to the roots of the pepper plants. Treatment was repeated every 15 days. After the peppers reached the seedling, flowering, and peak fruiting stages, the growth and physiological indicators of the pepper plants were measured, the physicochemical properties of the soil were measured, and the cadmium accumulation in the plants, fruits, and soil was determined. The experimental results and analysis are as follows: Table 2. Effects of different treatments under different concentrations of cadmium stress on growth indicators of pepper seedlings. Note: P represents liquid inoculant of strain P10, T represents biochar addition, PT represents growth-promoting biochar inoculant, CK is the control, and 10, 30, 50, and 70 represent cadmium stress concentrations of 10, 30, 50, and 70 mg / kg, respectively; values are expressed as mean ± standard deviation, and different lowercase letters in the same column indicate significant differences (P < 0.05). The same applies to the following tables. Table 2 shows that, during the seedling stage of peppers, without cadmium stress, the growth-promoting effect of the biochar inoculant treatment (PT) was superior to that of the single inoculant treatment (P). Under cadmium stress of 10 mg / kg, the stem diameter, plant height, root weight, total root surface area, and chlorophyll content of peppers treated with PT were significantly higher than those of other treatments. The inhibitory effect of cadmium stress on plants increased with increasing stress concentration, but the chlorophyll content of plants treated with PT was significantly higher than that of the corresponding stress groups. Most growth indicators were higher than those of individual stress treatments to varying degrees, and the plants exhibited lush foliage and robust root systems. For example, at 70 mg / kg, the stem diameter, fresh weight, root weight, and total root surface area of the PT treatment group increased by 27.78%, 14.66%, 2.67%, and 39.01%, respectively, compared with the stress treatment.
[0038] Table 3. Available cadmium in soil, cadmium content in plant roots and stems during the seedling stage under different treatments. Table 3 shows that during the seedling stage of chili peppers, PT treatment significantly reduced the content of available cadmium in the soil and inhibited the translocation of cadmium to the aboveground parts of the plant, and was superior to P or T treatments to varying degrees. In cadmium-contaminated soils at concentrations of 10, 30, 50, and 70 mg / kg, compared with the control (CK), PT treatment reduced cadmium accumulation in chili pepper roots by 66.11%, 18.43%, 24.72%, and 33.14%, respectively, and cadmium accumulation in stems by 79.01%, 124.72%, 50.40%, and 17.15%, respectively.
[0039] Table 4. Effects of different treatments under different concentrations of cadmium stress on growth indicators of pepper plants during flowering. As shown in Table 4, as the plants entered the flowering stage, the growth indicators of the PT treatment group were still significantly higher than those of the CK group and superior to those of the P and T treatment groups. The inhibitory effect of cadmium stress on plants continued to increase with increasing stress concentration, and the growth indicators of the PT treatment plants were mostly significantly higher than those of the corresponding single stress treatments and superior to the P and T treatments to varying degrees. Under 10 mg / kg cadmium stress, the fresh weight and root weight of the PT treatment were significantly increased by 49.6% and 109.0% compared with the single cadmium stress group, respectively; while under 70 mg / kg cadmium stress, the stem diameter, plant height, fresh weight, dry weight, root weight, total root surface area, and chlorophyll content of the PT treatment plants were significantly increased by 53.56%, 46.05%, 48.80%, 70.83%, 14.67%, and 67.53%, respectively. Overall, the higher the cadmium stress concentration, the stronger the effect of PT treatment in alleviating cadmium stress on plants.
[0040] Table 5. Effects of four treatments under different concentrations of cadmium stress on growth indicators of pepper plants and fruits in the fruiting stage. As shown in Table 5, during the peak fruiting period, under cadmium-free conditions, PT treatment significantly promoted the growth of peppers, with the number of fruits per plant (22.3) increasing significantly by 62.8% compared to the control (13.7). Stem diameter, root surface area, and root length were also significantly higher in PT treatment than in the control. Under cadmium stress of 10 mg / kg, PT treatment significantly increased plant height, root weight, root surface area, and root length. When the cadmium stress concentration increased to 70 mg / kg, stem diameter, plant height, fresh weight, root weight, and root length were all significantly higher than under cadmium stress alone. At cadmium concentrations of 10–70 mg / kg, the number of fruits per plant decreased significantly with increasing cadmium stress, but the number of fruits per plant increased by 9.11%, 36.08%, 400%, and 254.04% respectively under PT treatment. Biochar inoculants showed a more significant yield-promoting effect on plants under high cadmium stress.
[0041] Table 6. Cadmium content in soil, plant roots, stems, and fruits during the peak fruiting period under different treatments. As shown in Table 6, during the peak fruiting period of chili peppers, except for the 10 mg / kg cadmium stress group, the cadmium content in the roots, stems, and soil of other cadmium-stressed plants was significantly reduced in the PT treatment groups, and the reduction was greater with increasing stress intensity. Furthermore, under cadmium stress of 10-70 mg / kg, the cadmium content in the fruits of the PT treatment groups was significantly lower than that of the cadmium stress treatment alone, and this effect was more pronounced with increasing cadmium stress concentration; compared with the corresponding cadmium stress groups alone, the cadmium content in the fruits treated by PT decreased by 28.03%, 33.50%, 43.69%, and 45.45%, respectively. P or T treatments also reduced the cadmium content in the soil, plant roots and stems, and fruits, but their effects were weaker than the combined PT treatment. Taking a cadmium stress concentration of 70 mg / kg as an example, the available cadmium in the soil was reduced by 21.00% under PT treatment, while the cadmium content in the roots, stems and fruits of the plants was reduced by 54.35%, 44.71% and 45.45% respectively. This indicates that PT treatment significantly inhibited the transport and accumulation of cadmium in the soil to the roots and stems, thereby reducing cadmium accumulation in the fruits.
Claims
1. A method for preparing a cadmium-resistant, growth-promoting biochar agent, characterized in that: The preparation method is carried out according to the following steps: (1) Biochar pretreatment: After passing the biochar through a 30-50 mesh sieve, wash it with deionized water 2-4 times, each time using 150-250 mL of deionized water. Then soak it in 0.1 mol / L HCl solution and stir continuously for 0.8-1.5 h. Wash it with deionized water until the pH is 6.8-7.
2. Dry it at 35-45℃ to constant weight to obtain pretreated biochar for later use. (2) Activation of bacterial strain: 45-55 μL of Burkholderia cepacia P10 bacterial suspension preserved in glycerol was inoculated into 45-55 mL of LB liquid medium and activated overnight at 28-32℃; 0.9-1.1 mL of activated bacterial suspension was transferred into 45-55 mL of fresh LB liquid medium and cultured at 28-32℃ with shaking at 140-160 rpm for 18-20 h until the logarithmic growth phase was reached, and the OD was adjusted. 600 A value of 0.9-1.1 indicates the presence of P10 activated bacterial suspension. (3) Add biochar: Add the pretreated biochar to fresh LB liquid medium. The ratio of biochar to LB liquid medium is 0.5-2g:50mL. After autoclaving, add 0.8-1.2mL of P10 activated bacterial suspension. Shake and fix at 28-32℃ and 140-160rpm for 18-22h. Then centrifuge at 6000rpm for 14-16min. After discarding the supernatant, wash the precipitate with deionized water 2-3 times. The amount of deionized water used each time is 8-12mL. After natural drying, the cadmium-resistant growth-promoting bacterial biochar agent is obtained.
2. The method for preparing the cadmium-resistant and growth-promoting biochar agent according to claim 1, characterized in that: In step (1), the biochar pretreatment is as follows: after passing the biochar through a 40-mesh sieve, it is washed three times with deionized water, with 200 mL of deionized water used each time. Then, it is soaked in 0.1 mol / L HCl solution and stirred continuously for 1 hour. After washing with deionized water until the pH is 7.0, it is dried at 40°C to constant weight to obtain pretreated biochar for later use.
3. The method for preparing the cadmium-resistant and growth-promoting biochar agent according to claim 1 or 2, characterized in that: In step (1), the biochar is coconut shell biochar.
4. The method for preparing the cadmium-resistant and growth-promoting biochar agent according to claim 1, characterized in that: In step (2), the strain is activated as follows: 50 μL of Burkholderia cepacia P10 bacterial culture preserved with glycerol is inoculated into 50 mL of LB liquid medium and activated overnight at 30°C; 1 mL of the activated bacterial culture is transferred to 50 mL of fresh LB liquid medium and cultured at 30°C and 150 rpm for 18-20 h until the logarithmic growth phase is reached, and the OD is adjusted. 600 A value of 1.0 indicates the presence of P10 activated bacterial suspension.
5. The method for preparing the cadmium-resistant and growth-promoting biochar agent according to claim 1 or 4, characterized in that: In step (2), the LB liquid culture medium is prepared by adding 10.0g of tryptone, 5.0g of yeast extract and 10.0g of sodium chloride to 1000mL of distilled water, adjusting the pH to 7.0-7.2, and sterilizing at 121℃ for 20min.
6. The method for preparing the cadmium-resistant and growth-promoting biochar agent according to claim 1, characterized in that: In step (3), biochar is added: pretreated biochar is added to fresh LB liquid culture medium at a ratio of 1g:50mL. After autoclaving at 121℃ for 20min, 1mL of P10 activated bacterial suspension is added. The mixture is then fixed by shaking at 30℃ and 150rpm for 20h, followed by centrifugation at 6000rpm for 15min. The supernatant is discarded, and the precipitate is washed twice with 10mL of deionized water each time. After natural drying, the cadmium-resistant growth-promoting bacterial biochar agent is obtained. The viable count of Burkholderia cepacia P10 strain in the cadmium-resistant growth-promoting bacterial biochar agent is 0.98×10⁻⁶. 15 -1.08×10 15 cfu / g.
7. The method for preparing the cadmium-resistant and growth-promoting biochar agent according to claim 1 or 6, characterized in that: In step (3), the LB liquid culture medium is prepared by adding 10.0g of tryptone, 5.0g of yeast extract and 10.0g of sodium chloride to 1000mL of distilled water, adjusting the pH to 7.0-7.2, and sterilizing at 121℃ for 20min.
8. The method for preparing the cadmium-resistant and growth-promoting biochar agent according to claim 1 or 6, characterized in that: In step (3), the viable count of Burkholderia cepacia P10 strain in the prepared cadmium-resistant growth-promoting biochar agent is 1.03 × 10⁻⁶. 15 cfu / g.
9. The application of the cadmium-resistant growth-promoting biochar agent according to claim 1 in the preparation of a chili pepper growth promoter, characterized in that: Cadmium-resistant and growth-promoting biochar inoculants can be used in areas with high soil cadmium background values. They can promote the growth and development of chili peppers, improve fruit yield and quality, and reduce the accumulation of cadmium in soil, plants and fruits.
10. The application according to claim 8, characterized in that: The method of applying the cadmium-resistant and growth-promoting biochar agent is to dilute it with water and then drench it on the roots of the chili pepper plant at a rate of 0.01g per plant, once every 15 days.