Preparation method and application of multifunctional nano growth-promoting bacteria for overcoming successive cropping obstacles

Abstract

The invention relates to a preparation method of a multifunctional nano growth-promoting bacterium for overcoming successive cropping obstacles, which comprises the following steps: converting bacillus subtilis into bacillus subtilis spores under the condition of heating or inoculating bacillus subtilis into a sporulation culture medium containing manganese sulfate, preparing iron nanoparticles by using a chemical reducing agent and a coprecipitation method under the protection of inert gas, and carrying out vacuum drying on the iron nanoparticles to obtain the multifunctional nano growth-promoting bacterium for overcoming successive cropping obstacles. The preparation method is simple, the cost is low, the production process is green and environment-friendly and suitable for large-scale production, the prepared nano growth-promoting bacteria can integrate the biological functions of the iron nanoparticles and the bacillus subtilis, the soil fertility is improved, trace elements are supplemented, and meanwhile the biological functions of the iron nanoparticles and the bacillus subtilis are improved. The fertilizer promotes proliferation of beneficial bacteria, inhibits proliferation of harmful bacteria, improves the utilization rate of nutrients, degrades harmful substances, improves stress resistance of crops, realizes dual promotion of soil improvement and crop growth, and has remarkable social and economic benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of fertilizers, and in particular to a preparation method and application of multifunctional nano growth-promoting bacteria for overcoming continuous cropping obstacles. Background Art

[0002] Intensive cropping, the dominant mode of modern agriculture, has effectively alleviated the conflict between global population growth and limited land resources. However, this model has also led to increasingly serious problems with continuous cropping, primarily manifested in soil degradation, severe soil-borne diseases, stunted plant growth, and reduced crop yields. This problem is particularly acute in the cultivation of traditional Chinese medicinal materials (TCMs). Data show that approximately 70% of TCMs with tuberous roots suffer from severe continuous cropping problems during cultivation. This not only leads to a decline in TCM yield and production, but also causes a continuous reduction in the cultivated area of authentic production areas and migration to other regions, posing a serious challenge to the sustainable development of TCMs. A growing number of studies have confirmed that continuous cropping problems result from the synergistic effects of multiple factors, including deterioration of soil physical and chemical properties, imbalanced rhizosphere microbial community structure, and the secretion and accumulation of autotoxic substances in plant roots. Therefore, the preparation and development of multifunctional fertilizers has become a key strategy for addressing continuous cropping problems.

[0003] Iron nanoparticles have shown broad application potential in the agricultural field due to their unique physical and chemical properties. First, thanks to their nanoscale size, iron nanoparticles can be efficiently absorbed by plants, which helps to improve the utilization rate of iron elements and significantly improve the photosynthetic efficiency and growth rate of plants; secondly, iron nanoparticles have a high specific surface area and reactivity, which helps to inhibit the growth of pathogens in the soil and reduce the occurrence of plant diseases; thirdly, iron nanoparticles can increase soil porosity and aeration, which helps to improve soil physical properties. However, to date, no relevant applications of iron nanoparticles in the field of continuous cropping disorder management have been seen. Not only that, like most nanoparticles applied to farmland, iron nanoparticles may enter the food chain through plants or enter the environment through water circulation, resulting in a reduction in their effective dosage and potential harm to human health. Therefore, optimizing the transport pathway of iron nanoparticles is the key to their successful application.

[0004] As an excellent plant growth-promoting bacterium, Bacillus subtilis is widely used in the agricultural field. It can not only improve soil fertility, improve soil quality, and enhance plant resistance to stress, but also accelerate the degradation of soil organic matter and reduce nitrogen loss in the ecosystem. Research based on the continuous cropping disorder of Panax notoginseng has shown that Bacillus subtilis can not only promote the proliferation of beneficial bacteria in the soil and inhibit the growth of harmful bacteria, but also reshape the imbalance of soil bacterial community structure caused by continuous cropping disorder by changing the physical and chemical properties of the soil such as electrical conductivity and available phosphorus. More importantly, the application of Bacillus subtilis in nanoparticle delivery systems has also shown great potential, which can effectively avoid the loss of nano-ions and the corresponding toxic side effects. Therefore, if iron nanoparticles can be loaded on the surface of Bacillus subtilis, not only can the biological functions of iron nanoparticles and Bacillus subtilis be exerted, but the loss of iron nanoparticles can also be effectively avoided, and the ecological occupation of Bacillus subtilis can be promoted, thereby achieving the purpose of overcoming continuous cropping disorder. However, no relevant public reports have been seen so far. Summary of the Invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a preparation method and application of multifunctional nano-growth-promoting bacteria for overcoming the problem of continuous cropping, which can effectively solve the problem of continuous cropping of plants.

[0006] To achieve the above-mentioned purpose, the technical solution provided by the present invention is a method for preparing a multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles, wherein Bacillus subtilis is converted into Bacillus subtilis spores under the conditions of heating or inoculation into a spore-forming medium containing manganese sulfate, and iron nanoparticles are prepared by a chemical reducing agent and a co-precipitation method under the protection of an inert gas, and loaded onto the Bacillus subtilis spores, wherein the chemical reducing agent is one of KBH4, NaBH4 or N2H4·H2O, the iron nanoparticles are one or more of zero-valent iron nanoparticles, iron oxide nanoparticles, and ferrosoferric oxide nanoparticles, the particle size of the iron nanoparticles is 20-100 nm, the particle size of the Bacillus subtilis spores is 300-1500 nm, and the mass ratio of the iron nanoparticles to the Bacillus subtilis spores is 4-20:100.

[0007] Furthermore, the method prepares multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles.

[0008] The multifunctional nano growth-promoting bacteria for overcoming continuous cropping obstacles are used in the preparation of fertilizers for overcoming continuous cropping obstacles.

[0009] The crops are Chinese medicinal materials.

[0010] The Chinese medicinal materials are Panax notoginseng, ginseng, Rehmannia root, Chinese yam, Coptis root, Salvia miltiorrhiza, Scutellaria baicalensis and Angelica sinensis. The preparation method of the present invention is simple, low-cost, and the production process is green and environmentally friendly, suitable for large-scale production. The prepared nano-growth-promoting bacteria can integrate the biological functions of iron nanoparticles and Bacillus subtilis. While improving soil fertility and supplementing trace elements, it promotes the proliferation of beneficial bacteria, inhibits the proliferation of harmful bacteria, improves nutrient utilization, degrades harmful substances, and improves crop resistance, achieving dual promotion of soil improvement and crop growth. It is a major innovation in solving the problem of continuous cropping obstacles and has significant social and economic benefits. DETAILED DESCRIPTION

[0011] The specific implementation methods of the present invention are described in detail below with reference to examples and specific situations. Example 1

[0012] A method for preparing multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles comprises the following steps: Bacillus subtilis in the exponential growth phase was transferred to a spore-forming medium containing manganese sulfate at a ratio of 5%-10%, and cultured at 37°C and 250 r / min for 48 h until more than 90% of the cells formed spores. The culture medium was filtered through a 5 μm filter membrane, and the filtered spores were washed with deionized water 2-3 times and finally resuspended in sterile water to obtain a Bacillus subtilis spore suspension. Under nitrogen protection, an N2H4·H2O aqueous solution and an FeSO4 aqueous solution were mixed at a molar ratio of 1-1.2:1, and stirred at room temperature until the mixture no longer produced bubbles. Iron nanoparticles were obtained by centrifugation and washing with deionized water. The iron nanoparticles were added to the Bacillus subtilis spore suspension and vigorously stirred for 2-4 h. The reaction solution was heated to 8.0×10 3 Centrifuge at rpm for 10 min, discard the supernatant, wash the precipitate three times with deionized water, and freeze-dry. Example 2

[0013] A method for preparing multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles comprises the following steps: The exponentially growing Bacillus subtilis was transferred to a spore-forming medium containing manganese sulfate at a ratio of 5%-10%, and cultured at 50°C and 200 rpm for 24 h until more than 90% of the cells formed spores. The culture medium was heated at 4°C and 8.0×10 3 The cells were collected by centrifugation at rpm for 10 min, and the supernatant was discarded and resuspended in pre-cooled sterile saline. 3r / min, 4 ℃ centrifugation for 10 min, repeated washing of the precipitate with deionized water for 3 times, centrifugation and washing, filtration to obtain Bacillus subtilis spores, the spores were resuspended in sterile water to obtain a Bacillus subtilis spore suspension; under argon protection, FeCl3·6H2O and FeSO4·7H2O were added to the Bacillus subtilis spore suspension in a molar ratio of 1-1.2:1, stirred at room temperature for 30 min, co-precipitated to synthesize Fe3O4 nanoparticles, slowly added NaOH solution until the solution pH value was 10-12, continued stirring for 60 min, and then allowed to stand for 1-2 h; the reaction solution was heated to 8.0×10 3 Centrifuge at rpm for 10 min, discard the supernatant, wash the precipitate three times with deionized water, and freeze-dry. Example 3

[0014] A method for preparing multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles comprises the following steps: Bacillus subtilis was inoculated into LB medium and cultured at 37°C and 200 r / min with constant temperature shaking until the plateau phase was reached. The Bacillus subtilis solution grown to the plateau phase was heated at 80-90°C for 30-60 min and rapidly cooled to room temperature in an ice bath to obtain a spore solution. FeSO4 and NaBH4 were respectively taken at a molar ratio of 1:0.8-1.0, and 2 / 3 of the moles of FeSO4 and NaBH4 were first added to the above spore solution. After the reaction was continued until no bubbles were generated, 8.0×10 3 rpm for 10 min, wash the precipitate three times with deionized water, stir until the solution turns black-gray, add the remaining 1 / 3 mole of FeSO4 under nitrogen protection, continue stirring at 50-65℃ for 30-45 min, add concentrated ammonia water dropwise until the solution pH is 9-10, continue stirring for 30 min, let it stand for 1-2 h, and add 8.0×10 3 The pellet was centrifuged at 400 rpm for 10 min, the supernatant was discarded, and the pellet was resuspended in ultrapure water and washed three times before freeze-drying.

[0015] The nano-growth-promoting bacteria prepared by the present invention have excellent soil compatibility and targeting, and are mainly used to overcome the problem of continuous cropping of crops, especially the problem of continuous cropping of perennial Chinese medicinal materials. They can not only efficiently degrade the autotoxic substances secreted by the root system of crops and repair the structure of soil microbial communities, but also significantly inhibit the proliferation of soil-borne pathogens, thereby improving the soil environment under the conditions of continuous cropping. By loading the functional strain Bacillus subtilis and growth-promoting substances, the system can accurately deliver the active ingredients to the rhizosphere microdomain of crops and continuously release the active ingredients. It can be used for any of the following: seed coating, seed soaking, root irrigation, and foliar spraying. Taking Panax notoginseng as an example, the relevant experimental data are as follows: Experiment 1: Effects of iron nanoparticles on the proliferation of Bacillus subtilis Whether Bacillus subtilis spores can germinate and proliferate again after being loaded with iron nanoparticles is one of the main factors affecting the activity of nanoprobiotics, so the plate coating method was used to evaluate it. Bacillus subtilis without nanoparticles and nanoprobiotics were added to the culture medium respectively, and the OD 600 The bacterial suspension was diluted to 1.0, and then 10-fold serial dilutions were performed using culture medium. 150 μL of each dilution was plated on a plate and incubated in a 37°C incubator for 12 hours before colony counts were counted to investigate whether the iron nanoparticle loading affected the germination and proliferation of Bacillus subtilis. The experimental results showed no significant difference in cell proliferation between Bacillus subtilis and those without iron nanoparticle loading, indicating that the iron nanoparticle-loaded nanoprobiotics still had good growth activity.

[0016] Experiment 2: Evaluation of enzyme-like activity of iron nanoparticles To determine the peroxidase-like catalytic activity of iron nanoparticles, a colorimetric reaction system using 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) was employed. Specifically, the iron nanoparticles were dispersed in HAc-NaAc buffer (pH 5.5), followed by the addition of 200 μL of a 10 mM TMB solution and 200 μL of a 30% H2O2 solution. The mixture was incubated at room temperature for 10 minutes, and the absorbance at 652 nm was measured. The results showed that the iron nanoparticles exhibited a distinct absorption peak at 652 nm, indicating a certain degree of enzyme-like activity.

[0017] Experiment 3: Degradation of Panax notoginseng autotoxic substances Based on a potted experiment on continuous cropping problems with Panax notoginseng, rhizosphere soil was collected and metabolomics analysis was used to analyze the degradation of Panax notoginseng autotoxic substances. The results showed that iron nanoparticle treatment significantly reduced the production of secondary metabolites such as ginsenoside Rh1, echinocystic acid, ginsenoside Rg3, and ginsenoside F1. Numerous studies have confirmed that these autotoxic substances are one of the main causes of Panax notoginseng root rot and continuous cropping problems. Simultaneously, the secretion of metabolites such as indole and gibberellin was significantly upregulated, which help strengthen the plant's defense mechanisms and promote plant growth.

[0018] Experiment 4: Reconstruction of the Rhizosphere Microecology of Panax notoginseng High-throughput sequencing and microbial diversity analysis were used to evaluate the effects of nano-growth-promoting bacteria on the rhizosphere microbial community of Panax notoginseng. The experimental results confirmed that after treatment with nano-growth-promoting bacteria, the microbial community in the rhizosphere soil of Panax notoginseng underwent significant changes. Specifically, the relative abundance of beneficial bacterial genera such as Sphingomonas increased significantly, while PlectosphaerellaThe relative abundance of harmful bacterial genera, such as Bacillus, decreased. This suggests that nanoparticle treatment may improve the health of the rhizosphere microbiome by increasing the abundance and diversity of beneficial microorganisms and inhibiting the growth of pathogens. Concurrently, the relative abundance of Bacillus increased nearly 52-fold. The results further demonstrate that loading iron nanoparticles onto the surface of growth-promoting bacteria enhances their growth.

[0019] Experiment 5: Panax notoginseng continuous cropping obstacle potting experiment The potted experiment included five groups: a control group without continuous cropping problems, a control group without continuous cropping problems, an iron nanoparticle group, a Bacillus subtilis group, and a group treated with nano-growth-promoting bacteria. Treatments were administered by root irrigation. Results showed that Panax notoginseng plants with continuous cropping problems exhibited significant yellowing of stems and leaves and wilting, while the other treatments alleviated these wilting symptoms to varying degrees. Compared to the iron nanoparticle and Bacillus subtilis groups, the nano-growth-promoting bacteria group exhibited the best growth, showing no noticeable yellowing. Chlorophyll a, chlorophyll b, and total chlorophyll contents were also similar to those in the control group without continuous cropping problems.

[0020] Experiment 6: Field experiment on continuous cropping obstacles of Panax notoginseng A field experiment was conducted at the Panax notoginseng continuous cropping obstacle test station in Wenshan, Yunnan, using foliar spraying of nano-growth-promoting bacteria. During the experiment, the number of fungicide applications in the nano-growth-promoting group was reduced from the standard six times to one. Otherwise, conventional Panax notoginseng field management was followed. Results showed that despite nano-growth-promoting bacteria treatment, the Panax notoginseng plants continued to grow normally, with no noticeable signs of wilting or root rot. Compared to the control group, the root systems of the plants in the nano-growth-promoting group were more developed.

[0021] Experiments show that the present invention has the following outstanding advantages over the prior art: by loading iron-based nanomaterials onto Bacillus subtilis spores, not only the growth-promoting function of Bacillus subtilis is retained, but also the precise release of iron elements is achieved; nano-growth-promoting bacteria can significantly increase the chlorophyll a content, chlorophyll b content, total chlorophyll content and root iron content of continuously cropped Panax notoginseng leaves, and effectively regulate the imbalance of Panax notoginseng flora caused by continuous cropping obstacles. The raw materials used in the present invention are rich in sources, the production process is simple, and it is suitable for large-scale production. The prepared nano-probiotics can integrate the biological functions of iron nanoparticles and Bacillus subtilis, while improving soil fertility and supplementing trace elements, promoting the proliferation of beneficial bacteria, inhibiting the proliferation of harmful bacteria, improving nutrient utilization, degrading harmful substances and improving crop resistance, achieving dual promotion of soil improvement and crop growth, which is a major innovation in solving the problem of continuous cropping obstacles and has significant social and economic benefits.

[0022] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with this profession can make changes or modify the technical content disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention, and all of them fall within the scope of protection of the present invention.

Claims

1. A method for preparing a multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles, characterized in that: Bacillus subtilis is converted into Bacillus subtilis spores under heating or inoculation into a spore-forming medium containing manganese sulfate. Under inert gas protection, iron nanoparticles are prepared using a chemical reducing agent and a co-precipitation method, and loaded onto the Bacillus subtilis spores. The chemical reducing agent is one of KBH4, NaBH4, or N2H4·H2O; the iron nanoparticles are one or more of zero-valent iron nanoparticles, iron oxide nanoparticles, and ferrosoferric oxide nanoparticles; the particle size of the iron nanoparticles is 20-100 nm; the particle size of the Bacillus subtilis spores is 300-1500 nm; and the mass ratio of the iron nanoparticles to the Bacillus subtilis spores is 4-20:

100.

2. The method for preparing the multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles according to claim 1, characterized in that: Bacillus subtilis in the exponential growth phase was transferred to a spore-forming medium containing manganese sulfate at a ratio of 5%-10%, and cultured at 37°C and 250 r / min for 48 h until more than 90% of the cells formed spores. The culture medium was filtered through a 5 μm filter membrane, and the filtered spores were washed with deionized water 2-3 times and finally resuspended in sterile water to obtain a Bacillus subtilis spore suspension. Under nitrogen protection, an N2H4·H2O aqueous solution and an FeSO4 aqueous solution were mixed at a molar ratio of 1-1.2:1, and stirred at room temperature until the mixture no longer produced bubbles. Iron nanoparticles were obtained by centrifugation and washing with deionized water. The iron nanoparticles were added to the Bacillus subtilis spore suspension and vigorously stirred for 2-4 h. The reaction solution was heated to 8.0×10 3 Centrifuge at rpm for 10 min, discard the supernatant, wash the precipitate three times with deionized water, and freeze-dry.

3. The method for preparing the multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles according to claim 1, characterized in that: The exponentially growing Bacillus subtilis was transferred to a spore-forming medium containing manganese sulfate at a ratio of 5%-10%, and cultured at 50°C and 200 rpm for 24 h until more than 90% of the cells formed spores. The culture medium was heated at 4°C and 8.0×10 3 The cells were collected by centrifugation at rpm for 10 min, and the supernatant was discarded and resuspended in pre-cooled sterile saline. 3 r / min, 4 ℃ centrifugation for 10 minutes, repeated washing of the precipitate with deionized water for 3 times, centrifugation and washing, filtering to obtain Bacillus subtilis spores, the spores were resuspended in sterile water to obtain a Bacillus subtilis spore suspension; under argon protection, FeCl3·6H2O and FeSO4·7H2O were added to the Bacillus subtilis spore suspension in a molar ratio of 1-1.2:1, stirred at room temperature for 30 minutes, co-precipitated to synthesize Fe3O4 nanoparticles, slowly added NaOH solution until the solution pH value was 10-12, continued stirring for 60 minutes, and then allowed to stand for 1-2 hours; the reaction solution was heated to 8.0×10 3 Centrifuge at rpm for 10 min, discard the supernatant, wash the precipitate three times with deionized water, and freeze-dry.

4. The method for preparing the multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles according to claim 1, characterized in that: Bacillus subtilis was inoculated into LB medium and cultured at 37°C and 200 r / min with constant temperature shaking until the plateau phase was reached. The Bacillus subtilis solution grown to the plateau phase was heated at 80-90°C for 30-60 min and rapidly cooled to room temperature in an ice bath to obtain a spore solution. FeSO4 and NaBH4 were respectively taken at a molar ratio of 1:0.8-1.0, and 2 / 3 of the moles of FeSO4 and NaBH4 were first added to the above spore solution. After the reaction was continued until no bubbles were generated, 8.0×10 3 rpm for 10 min, wash the precipitate three times with deionized water, stir until the solution turns black-gray, add the remaining 1 / 3 mole of FeSO4 under nitrogen protection, continue stirring at 50-65℃ for 30-45 min, add concentrated ammonia water dropwise until the solution pH is 9-10, continue stirring for 30 min, let it stand for 1-2 h, and add 8.0×10 3 The pellet was centrifuged at 400 rpm for 10 min, the supernatant was discarded, and the pellet was resuspended in ultrapure water and washed three times before freeze-drying.

5. Multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles prepared by the method according to any one of claims 1 to 4.

6. Use of the multifunctional nano-growth-promoting bacteria for overcoming continuous cropping obstacles as claimed in claim 5 in the preparation of fertilizers for overcoming continuous cropping obstacles.

7. The use according to claim 6, characterized in that The crops are Chinese medicinal materials.

8. The use according to claim 7, characterized in that The Chinese medicinal materials are Panax notoginseng, ginseng, rehmannia root, yam, coptis root, salvia miltiorrhiza, scutellaria baicalensis and angelica sinensis.