A method for improving the colonization efficiency of rhizosphere growth promoting bacteria by using nanomaterials and magnetic interaction system

Abstract

This disclosure relates to a method for improving the colonization efficiency of rhizosphere growth-promoting bacteria using nanomaterials and a magnetic interaction system. The method includes: S1, mixing modified iron oxide nanoparticles with a suspension of rhizosphere growth-promoting bacteria and then conducting a first culture to obtain modified rhizosphere growth-promoting bacteria; S2, applying a dispersion of magnetic iron oxide nanoparticles to the roots of a plant for a second culture to obtain modified plants; S3, applying the suspension of the modified rhizosphere growth-promoting bacteria to the roots of the modified plants for a third culture; wherein the modified iron oxide nanoparticles are cationic polymer-modified iron oxide nanoparticles. This method constructs a plant-microorganism magnetic interaction system, improving the colonization ability of rhizosphere growth-promoting bacteria in plant roots, and also enhancing the nitrogen-fixing ability of the rhizosphere growth-promoting bacteria, effectively promoting plant growth.

Description

Technical Field

[0001] This disclosure relates to the field of biological nitrogen fixation, and more specifically, to a method for improving the colonization efficiency of rhizosphere-promoting bacteria using nanomaterials and magnetic interaction systems. Background Technology

[0002] In agriculture, nitrogen is an indispensable nutrient for crop growth. As a foliar fertilizer, it directly participates in chlorophyll synthesis and protein accumulation, playing a crucial role in improving crop yield and quality. However, current nitrogen fertilizer application faces severe challenges: low nitrogen fertilizer utilization rate and excessive application not only lead to resource waste but also trigger a series of environmental problems, including soil acidification and compaction, water eutrophication, and imbalance of soil microbial communities. Therefore, developing green alternative technologies to reduce reliance on nitrogen fertilizer and improve nitrogen use efficiency has become an urgent need for sustainable agricultural development.

[0003] Nitrogen-fixing microorganisms play a crucial role in sustainable agricultural development. Through biological nitrogen fixation at normal temperature and pressure, they convert atmospheric nitrogen into ammonia, which plants can absorb and utilize, effectively replenishing the soil nitrogen pool. Studies have shown that an efficient nitrogen-fixing system can fix 50 to 100 kg of nitrogen per hectare per year, sufficient to meet up to 90% of the nitrogen requirements of host plants, demonstrating its enormous potential to replace chemical nitrogen fertilizers. Field practice has proven that inoculating crops such as *Azologia azotocinus* can reduce the application of chemical nitrogen fertilizers to crops like cereals and corn by 25% to 50%. Although some nitrogen-fixing microbial strains have been commercially produced and applied in the field, their large-scale application still faces significant technical bottlenecks. The core challenges are: low colonization efficiency of inoculated microorganisms in the crop rhizosphere, unstable efficacy in field conditions, and insufficient survival rates under adverse abiotic stresses such as drought and salinity. These factors severely restrict the stable realization of their practical effects and their large-scale promotion.

[0004] To overcome the aforementioned technical bottlenecks, it is urgent to develop novel efficiency-enhancing strategies that can actively guide and strengthen microbial root colonization. Summary of the Invention

[0005] The purpose of this disclosure is to provide a method for improving the colonization efficiency of rhizosphere growth-promoting bacteria using nanomaterials and magnetic interaction systems. This method constructs a plant-microbe magnetic interaction system, which improves the colonization ability of rhizosphere growth-promoting bacteria in plant roots, and also enhances the nitrogen fixation ability of rhizosphere growth-promoting bacteria, effectively promoting plant growth.

[0006] To achieve the above objectives, this disclosure provides a method for improving the colonization efficiency of rhizosphere growth-promoting bacteria using nanomaterials and magnetic interaction systems, the method comprising:

[0007] S1. After mixing the modified iron oxide nanoparticles with the bacterial suspension of rhizosphere growth-promoting bacteria, a first culture was carried out to obtain the modified rhizosphere growth-promoting bacteria.

[0008] S2. Apply the dispersion of magnetic iron oxide nanoparticles to the roots of plants for a second culture to obtain modified plants.

[0009] S3. Apply the bacterial suspension of the modified rhizosphere growth-promoting bacteria to the roots of the modified plant for a third culture.

[0010] The modified iron oxide nanoparticles are iron oxide nanoparticles modified with cationic polymers.

[0011] Optionally, in step S1, the viable concentration of the rhizosphere growth-promoting bacteria in the bacterial suspension is 10. 5 -10 10 CFU / mL;

[0012] The amount of modified iron oxide nanoparticles added is 0.1-5.0 g relative to each 1 L of the rhizosphere growth-promoting bacteria suspension;

[0013] The rhizosphere growth-promoting bacteria are nitrogen-fixing microorganisms.

[0014] Optionally, the nitrogen-fixing microorganism is selected from one or more of Pseudomonas schrenckii, Burkholderia spp., Azotobacter halophyte, and Azotobacter brasiliensis.

[0015] Optionally, the conditions for the first culture include: a temperature of 25-35℃, a rotation speed of 50-1000 rpm, and a time of 30-120 min.

[0016] Optionally, in step S1, the method for preparing the modified iron oxide nanoparticles includes: mixing an iron source, a cationic polymer, and a solvent under an inert atmosphere to obtain a first mixture; mixing the first mixture with a pH adjuster to obtain a second mixture and reacting it to obtain a third mixture; and subjecting the third mixture to magnetic separation, washing, and drying in sequence.

[0017] The reaction is selected from any of the following pathways: (1) hydrothermal reaction; (2) coprecipitation reaction and hydrothermal reaction; (3) hydrothermal reaction and isothermal oscillation reaction;

[0018] The modified iron oxide nanoparticles have an average particle size of 20-100 nm.

[0019] The iron source is selected from one or more of FeCl·6H2O, FeSO4·7H2O, FeCl2·4H2O, Fe(NO3)3·9H2O and Fe(Ac)3·4H2O;

[0020] The cationic polymer is selected from one or more of polyethyleneimine, triphenylphosphine chitosan and its derivatives, polylysine, polydimethyldiallylammonium chloride, chitosan quaternary ammonium salt and polymethacryloyloxyethyltrimethylammonium chloride;

[0021] The solvent is selected from one or more of sulfuric acid, water, ethanol, ethylene glycol and N,N-dimethylformamide;

[0022] The pH adjuster is selected from one or more of potassium nitrate, sodium hydroxide, ammonia, sodium bicarbonate, hydrochloric acid, and acetic acid.

[0023] Optionally, the content of the iron source, calculated as iron, is 1-1000 mg per 1-1000 mL of solvent, and the content of the cationic polymer is 1-1000 mg.

[0024] The pH value of the second mixture is 9-11;

[0025] The conditions for the hydrothermal reaction include: a temperature of 50-100℃, a stirring rate of 100-500 rpm, and a time of 0.5-24 h.

[0026] The conditions for the coprecipitation reaction include: a temperature of 25-60℃ and a time of 5-60 min;

[0027] The conditions for the isothermal oscillation reaction include: a temperature of 20-37℃, an oscillation rate of 150-200 rpm, and a time of 12-24 h.

[0028] Optionally, in step S2, the content of the magnetic iron oxide nanoparticles is 20-2000 mg relative to each 1 L of the dispersion of the magnetic iron oxide nanoparticles; the solvent of the dispersion of the magnetic iron oxide nanoparticles is water and / or Hoagland nutrient solution; and the average particle size of the magnetic iron oxide nanoparticles is 20-500 nm.

[0029] The amount of the dispersion of the magnetic iron oxide nanoparticles applied is 50-100 mL / plant.

[0030] The plants mentioned include rice, corn, and tomatoes.

[0031] Optionally, the conditions for the second culture include: a temperature of 25-30℃, a light condition of 16h light / 8h darkness and root protection from light, and a duration of 5-7 days.

[0032] Optionally, in the bacterial suspension of the modified rhizosphere growth-promoting bacteria, the viable bacterial concentration of the modified rhizosphere growth-promoting bacteria is 10. 5 -10 10CFU / mL;

[0033] The application rate of the modified rhizosphere growth-promoting bacteria suspension is 10-100 mL / plant.

[0034] Optionally, the conditions for the third culture include: a temperature of 25-30°C and a time of 1-7 days.

[0035] Through the above technical solution, this disclosure constructs a plant-microorganism magnetic interaction system. Due to the inherent tendency of modified iron oxide nanoparticles to adsorb onto surfaces, these nanoparticles attach to the surface of rhizosphere growth-promoting bacteria. Simultaneously, the magnetic interaction between the magnetic iron oxide nanoparticles on the plant roots and the modified iron oxide nanoparticles on the surface of the rhizosphere growth-promoting bacteria guides the bacteria to accumulate in the plant roots, thereby improving the colonization efficiency of the bacteria on the plant root surface. Furthermore, the modified iron oxide nanoparticles can undergo slow-release degradation in the root microenvironment, continuously providing iron ions and offering in-situ replenishment of key metal cofactors for nitrogenase. This effectively ensures the efficiency of its biosynthesis and electron transfer, significantly enhancing the catalytic activity of nitrogen-fixing microorganisms and thus strengthening their nitrogen-fixing capacity, thereby better promoting plant growth.

[0036] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0037] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0038] Figure 1 It is the process of constructing a magnetic interaction system.

[0039] Figure 2 These are scanning electron microscope (SEM) images of A1501 modified with three different types of iron oxide nanoparticles; where a is unmodified A1501, b is A1501 modified with adsorbed polyethyleneimine, c is A1501 modified with adsorbed triphenylphosphine-chitosan, and d is A1501 modified with adsorbed polylysine (PLL).

[0040] Figure 3 This describes the colonization of A1501 in rice within a magnetic interaction system; where a represents unmodified A1501, b represents A1501 adsorbed with polyethyleneimine-modified nano-Fe3O4, c represents A1501 adsorbed with triphenylphosphine-chitosan-modified nano-Fe3O4, and d represents A1501 adsorbed with polylysine (PLL)-modified nano-Fe3O4.

[0041] Figure 4 The effect of modified iron oxide nanomaterials on the activity of A1501 nitrogenase.

[0042] Figure 5 The effect of modified iron tetroxide nanomaterials on the activity of A1501 nitrogenase colonized in rice roots.

[0043] Figure 6 It refers to the root-modified and root-unmodified A1501 nitrogenase activities. Detailed Implementation

[0044] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0045] This disclosure provides a method for improving the colonization efficiency of rhizosphere growth-promoting bacteria using nanomaterials and magnetic interaction systems. The method includes:

[0046] S1. After mixing the modified iron oxide nanoparticles with the bacterial suspension of rhizosphere growth-promoting bacteria, a first culture was carried out to obtain the modified rhizosphere growth-promoting bacteria.

[0047] S2. Apply the dispersion of magnetic iron oxide nanoparticles to the roots of plants for a second culture to obtain modified plants.

[0048] S3. Apply the bacterial suspension of the modified rhizosphere growth-promoting bacteria to the roots of the modified plant for a third culture.

[0049] The modified iron oxide nanoparticles are iron oxide nanoparticles modified with cationic polymers.

[0050] like Figure 1As shown, this disclosure constructs a plant-microbe magnetic interaction system. Due to the inherent tendency of modified iron oxide nanoparticles to adsorb onto surfaces, the modified iron oxide nanoparticles are co-cultured with rhizosphere growth-promoting bacteria, causing the modified iron oxide nanoparticles to attach to the surface of the bacteria. Applying magnetic iron oxide nanoparticles to the substrate around plant roots induces the formation of a magnetic iron film on the roots. Simultaneously, the magnetic interaction between the magnetic iron oxide nanoparticles on the plant roots and the modified iron oxide nanoparticles on the surface of the rhizosphere growth-promoting bacteria guides the bacteria to accumulate in the plant roots, thereby improving the colonization efficiency of the bacteria on the plant root surface. Furthermore, the modified iron oxide nanoparticles can undergo slow-release degradation in the root microenvironment, continuously providing iron ions. Iron is a core component of nitrogenase ferritin clusters and the active centers of molybdenum-iron protein P-cluster and FeMo-co. This process provides in-situ replenishment of key metal cofactors for nitrogenase, effectively ensuring its biosynthesis and electron transfer efficiency, thereby significantly enhancing the catalytic activity of nitrogen-fixing microorganisms and thus enhancing their nitrogen-fixing capacity, which in turn can better promote plant growth.

[0051] To ensure sufficient modified iron oxide nanoparticles coat the surface of the rhizosphere growth-promoting bacteria, in one embodiment, in step S1, the viable bacterial concentration of the rhizosphere growth-promoting bacteria in the bacterial suspension is 10. 5 -10 10 CFU / mL; the amount of modified iron oxide nanoparticles added is 0.1-5.0 g relative to each 1 L of the bacterial suspension of the rhizosphere growth-promoting bacteria;

[0052] The rhizosphere growth-promoting bacteria are nitrogen-fixing microorganisms; preferably, the nitrogen-fixing microorganisms are selected from one or more of Pseudomonas schrenckii, Burkholderia spp., Azotobacter halophyte, and Azotobacter brasiliensis.

[0053] In order to further coat the surface of the rhizosphere growth-promoting bacteria with sufficient modified iron oxide nanoparticles, in one embodiment, the conditions for the first culture include: a temperature of 25-35°C, a rotation speed of 50-500 rpm, and a time of 30-120 min.

[0054] In one embodiment, the method for preparing the modified iron oxide nanoparticles includes: mixing an iron source, a cationic polymer, and a solvent under an inert atmosphere to obtain a first mixture; mixing the first mixture with a pH adjuster to obtain a second mixture and reacting it to obtain a third mixture; and subjecting the third mixture to magnetic separation, washing, and drying treatments in sequence.

[0055] The reaction is selected from any of the following pathways: (1) hydrothermal reaction; (2) coprecipitation reaction and hydrothermal reaction; (3) hydrothermal reaction and isothermal oscillation reaction.

[0056] In the above embodiments, the reaction may be solely a hydrothermal reaction; it may also be a combination of a coprecipitation reaction and a hydrothermal reaction, in which case the reaction includes a sequential coprecipitation reaction and a hydrothermal reaction; or it may be a combination of a hydrothermal reaction and a isothermal oscillation reaction, in which case the reaction includes a sequential hydrothermal reaction and a isothermal oscillation reaction. After the reaction is completed, the solid produced by the reaction is subjected to sequential magnetic separation, washing, and drying treatments. The magnetic separation, washing, and drying treatments can be performed using methods conventionally employed by those skilled in the art, and will not be described in detail here.

[0057] In this disclosure, the modified iron oxide nanoparticles used in the preparation of the modified rhizosphere growth-promoting bacteria are iron oxide nanoparticles modified with cationic polymers. These modified iron oxide nanoparticles can modify the rhizosphere growth-promoting bacteria through electrostatic interactions, which facilitates the coating of the modified iron oxide nanoparticles on the surface of the rhizosphere growth-promoting bacteria and also facilitates the adsorption of the modified rhizosphere growth-promoting bacteria on the surface of plant roots. The average particle size of the modified iron oxide nanoparticles is 20-100 nm.

[0058] In the above embodiments, the first mixture obtained by mixing the iron source, cationic polymer, and solvent can be specifically achieved by dissolving the iron source and cationic polymer together in the solvent; or by dissolving the cationic polymer in the solvent first, and then adding the iron source. The second mixture obtained by mixing the first mixture with a pH adjuster can be achieved by adding the pH adjuster dropwise to the first mixture, or by adding the first mixture dropwise to the pH adjuster, adjusting the pH of the second mixture to 9-11. An inert atmosphere is used to remove oxygen from the solution; the inert atmosphere can be nitrogen or a rare gas.

[0059] In the above embodiments, the iron source can be selected from one or more of FeCl·6H2O, FeSO4·7H2O, FeCl2·4H2O, Fe(NO3)3·9H2O and Fe(Ac)3·4H2O;

[0060] The cationic polymer may be selected from one or more of polyethyleneimine, triphenylphosphine chitosan and its derivatives, polylysine, polydimethyldiallylammonium chloride (PDADMAC), chitosan quaternary ammonium salt (HTCC), and polymethacryloyloxyethyltrimethylammonium chloride (PMETAC);

[0061] The solvent may be selected from one or more of sulfuric acid, water, ethanol, ethylene glycol and N,N-dimethylformamide;

[0062] The pH adjuster may be selected from one or more of potassium nitrate, sodium hydroxide, ammonia, sodium bicarbonate, hydrochloric acid, and acetic acid.

[0063] In one embodiment, the content of the iron source, calculated as iron element, is 1-1000 mg per 1-1000 mL of solvent, and the content of the cationic polymer is 1-1000 mg; the pH value of the second mixture is 9-11.

[0064] The conditions for the hydrothermal reaction include: a temperature of 50-100℃, a stirring rate of 100-500 rpm, and a time of 0.5-24 h.

[0065] The conditions for the coprecipitation reaction include: a temperature of 25-60℃ and a time of 5-60 min;

[0066] The conditions for the isothermal oscillation reaction include: a temperature of 20-37℃, an oscillation rate of 150-200 rpm, and a time of 12-24 h.

[0067] In one embodiment, in step S2, the content of the magnetic iron oxide nanoparticles is 20-2000 mg relative to each 1 L of the dispersion of the magnetic iron oxide nanoparticles; in this disclosure, the magnetic iron oxide nanoparticles are magnetized iron oxide nanoparticles, and preferably, the average particle size of the magnetic iron oxide nanoparticles is 20-500 nm.

[0068] In the above embodiments, the solvent for the dispersion of magnetic iron oxide nanoparticles can be water or Hoagland nutrient solution, preferably Hoagland nutrient solution.

[0069] In one embodiment, the amount of the dispersion of the magnetic iron oxide nanoparticles applied is 50-100 mL / plant; wherein the plant includes rice, corn and tomato.

[0070] In one embodiment, the conditions for the second culture include: a temperature of 25-30°C, a light condition of 16 hours of light / 8 hours of darkness and root protection from light, and a duration of 5-7 days.

[0071] In one embodiment, the viable bacterial concentration of the modified rhizosphere growth-promoting bacteria in the bacterial suspension is 10. 5 -10 10 CFU / mL;

[0072] The amount of bacterial suspension of the modified microorganism applied is 10-100 mL / strain;

[0073] In one embodiment, the conditions for the third culture include: a temperature of 25-30°C and a time of 1-7 days.

[0074] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0075] All raw materials used in the embodiments can be obtained through commercial purchase.

[0076] The magnetic nano-iron oxide was purchased from Ningbo Luofei Nanotechnology Co., Ltd., with an average particle size of 100 nm.

[0077] Example 1

[0078] This example illustrates the preparation of polyethyleneimine (PEI) modified iron oxide nanoparticles:

[0079] (1) In a three-necked round-bottom flask continuously purged with nitrogen, 0.616 g of ferrous sulfate heptahydrate and 0.7 g of 50% polyethyleneimine solution (molecular weight 70,000) were dissolved together in 30 mL of 0.01 M sulfuric acid solution. The mixture was stirred continuously under a nitrogen atmosphere for 2 hours and is denoted as solution A.

[0080] (2) Dissolve 2.728g of potassium nitrate and 0.972g of sodium hydroxide in 270mL of distilled water to prepare solution B.

[0081] (3) Nitrogen gas was introduced into solution B for 5 minutes to remove dissolved oxygen. Then, under the condition of maintaining a nitrogen atmosphere and stirring, solution A obtained in step (1) was slowly and evenly added to solution B using a constant pressure dropping funnel. During the addition process, a black precipitate gradually formed in the system. The pH of the reaction system was adjusted to 9. After solution A was added, the coprecipitation reaction was continued for 5 minutes at room temperature (25°C).

[0082] (4) Transfer all the co-precipitated reaction solution to a constant temperature water bath preheated to 90°C. Under nitrogen protection and stirring at 500 rpm, carry out a hydrothermal reaction for 24 hours to allow the iron oxide nanoparticles to grow fully and complete the PEI coating.

[0083] (5) After the hydrothermal reaction was completed, the reaction system was cooled in an ice-water bath. Subsequently, the generated black magnetic precipitate of polyethyleneimine-modified iron oxide nanoparticles was magnetically separated and collected using a permanent magnet. The precipitate was washed five times alternately with deionized water and anhydrous ethanol to thoroughly remove impurities.

[0084] (6) The washed product was placed in a drying oven at 60°C and dried for 12 hours to obtain polyethyleneimine modified iron oxide nanoparticles with an average particle size of 50 nm.

[0085] Example 2

[0086] This example illustrates the preparation of triphenylphosphine-chitosan (ChiP) modified iron oxide nanoparticles:

[0087] (1) Accurately weigh 100 mg of chitosan powder and place it in a 50 mL Erlenmeyer flask. Add 18 mL of deionized water. Then slowly add 10 μL of glacial acetic acid and stir at room temperature with a magnetic stirrer until the chitosan is completely dissolved to form a transparent colloidal solution. Add 2 mL of 500 mM MES buffer solution with a pH of 6.0 to this solution and mix well.

[0088] (2) Accurately weigh 70 mg of carboxyl-functionalized phosphate compound, 150 mg of carbodiimide (EDC), and 100 mg of N-hydroxysuccinimide ester (NHSS), and dissolve them together in 5 mL of 50 mM MES buffer solution with a pH of 6.0. Stir the mixture at 500 rpm for 30 minutes at room temperature to fully activate the carboxyl groups.

[0089] (3) The activating reagent solution obtained in step (2) was slowly added dropwise to the chitosan solution, and the reaction was carried out under nitrogen protection at room temperature with continuous stirring for 24 hours. After the reaction was completed, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 6000 Da, and purified by dialysis with deionized water for 5 days, changing the dialysis solution 3 times a day to completely remove unreacted byproducts and salts. Finally, the purified product was dried to constant weight in a vacuum desiccator to obtain a white flocculent solid product, triphenylphosphine chitosan derivative (ChiP).

[0090] (4) Weigh an appropriate amount of the triphenylphosphine chitosan derivative (ChiP) obtained in the above steps, dissolve it in an appropriate amount of deionized water, and prepare a transparent solution with a concentration of 5 mg / mL. Then, according to the molar ratio of Fe... 3+ Fe 2+ Weigh out 0.5417 g of FeCl3·6H2O and 0.2780 g of FeSO4·7H2O in a 2:1 ratio and add them to 100 mL of ChiP solution. Stir at room temperature until the iron salts are completely dissolved to form a homogeneous mixed solution.

[0091] (5) Under nitrogen protection and continuous stirring, ammonia solution was slowly added dropwise to the above mixed solution using a constant pressure dropping funnel to adjust the pH of the reaction system to 9. As the alkaline solution was added, the solution gradually turned black, indicating that Fe3O4 nanoparticles began to form. The reaction was carried out at 80°C and 500 rpm for 60 minutes with continuous stirring to ensure the reaction proceeded fully.

[0092] (6) After the hydrothermal reaction was completed, the reaction system was cooled in an ice-water bath. Subsequently, the generated black magnetic precipitate of ChiP-modified iron oxide nanoparticles was magnetically separated and collected using a permanent magnet. The precipitate was washed five times alternately with deionized water and anhydrous ethanol to thoroughly remove impurities.

[0093] (7) The washed product was placed in a drying oven at 60°C and dried for 12 hours to obtain ChiP modified iron oxide nanoparticles with an average particle size of 50 nm.

[0094] Example 3

[0095] This example illustrates the preparation of polylysine (PLL) modified iron oxide nanoparticles:

[0096] (1) Weigh polylysine (PLL, molecular weight 15-30 kDa), dissolve it in MES buffer solution with the same pH 6.0, and prepare a 6 mg / mL PLL solution. Then, according to the molar ratio of Fe... 3+ Fe 2+ Accurately weigh 0.5417 g FeCl3·6H2O and 0.2780 g FeSO4·7H2O in a 2:1 ratio and add them to 100 mL of PLL solution. Stir at room temperature until the iron salts are completely dissolved to form a homogeneous mixed solution.

[0097] (2) Under nitrogen protection and continuous stirring, ammonia solution was slowly added dropwise to the above mixed solution using a constant pressure dropping funnel to adjust the pH of the reaction system to 9. As the alkaline solution was added, the solution gradually turned black, indicating that Fe3O4 nanoparticles began to form. The reaction was continuously stirred at 80°C and 500 rpm for 60 minutes to ensure the reaction proceeded fully. The mixed system was then placed in a shaker at 25°C and 200 rpm for 18 hours at room temperature.

[0098] (3) After the reaction is complete, magnetic separation is performed using a strong permanent magnet, and the supernatant is discarded. The precipitate is washed five times with deionized water to remove unreacted PLL and byproducts. The final product, PLL-modified iron oxide nanoparticles, is washed three times with deionized water or PBS buffer.

[0099] (4) The washed product was placed in a drying oven at 60°C and dried for 12 hours to obtain PLL-modified iron oxide nanoparticles with an average particle size of 50 nm.

[0100] Example 4

[0101] This example illustrates the determination of the modification efficiency of modified iron oxide nanoparticles on bacterial strains:

[0102] (1) Using a sterile inoculation loop, pick a single, morphologically typical fresh and activated colony of Pseudomonas stearothermiae A1501 from the plate and inoculate it into a 15 mL sterile centrifuge tube containing 5 mL of LB liquid medium. Fix the centrifuge tube at an angle in a constant temperature shaker and incubate overnight at 30 °C and 220 rpm to obtain a seed culture in the logarithmic growth phase.

[0103] (2) Transfer the primary seed culture at a volume ratio of 1:100 to an Erlenmeyer flask containing 300 mL of LB liquid medium. Place the flask in a constant temperature shaker and culture overnight at 30°C and 220 rpm to allow the bacteria to proliferate fully.

[0104] (3) Centrifuge the expanded bacterial culture at 4°C and 6000×g for 15 minutes and discard the supernatant. Resuspend the obtained bacterial pellet in pre-cooled phosphate buffer (PBS, pH 7.4) and gently wash by pipetting. Repeat this process three times to thoroughly remove residual components of the culture medium.

[0105] (4) Resuspend the washed bacterial body in an appropriate amount of PBS buffer and measure its OD. 600 Adjust it to around 1, and use a UV-Vis spectrophotometer to measure its optical density value at a wavelength of 600 nm, which is recorded as the initial OD value.

[0106] (5) Take a certain mass of modified iron oxide nanoparticles and the above bacterial suspension (live bacteria concentration of 10). 10 Mix (CFU / mL) in a 50mL centrifuge tube; following the same procedure, prepare bacterial suspensions containing 0.5, 2.5, and 5.0 g / L polyeneimide-modified iron tetroxide nanoparticles (denoted as PEI-modified), 0.5, 2.5, and 5.0 g / L triphenylphosphine-chitosan (ChiP)-modified iron tetroxide nanoparticles (denoted as ChiP-modified), and 0.5, 2.5, and 5.0 g / L polylysine-modified iron tetroxide nanoparticles (denoted as PLL-modified). Place the centrifuge tubes on a constant temperature mixer and incubate with gentle shaking at 30℃ and 1000 rpm for 60 minutes.

[0107] (6) After incubation, take 1 mL of the bacterial suspension from step (5) into a 2 mL centrifuge tube, place the centrifuge tube in the magnetic field generated by the permanent magnet, and let it stand so that the nanoparticle-bacterial complex is fully adsorbed onto the tube wall. Carefully aspirate the supernatant. To improve the separation purity, PBS buffer can be added back to the precipitate, and the magnetic separation operation can be performed again after gentle resuspending. This washing step can be repeated several times until the supernatant becomes clear and transparent.

[0108] (7) After magnetic separation, the final supernatant was aspirated and its optical density was measured at a wavelength of 600 nm using a UV-Vis spectrophotometer. This value was recorded as the OD value after capture.

[0109] (8) The magnetic capture efficiency (MCE) of bacteria is calculated using the following formula:

[0110] MCE (%) = [(Initial OD value - OD value after capture) / Original OD value] × 100%.

[0111] (9) After fixing the A1501 adsorbed modified iron oxide nanoparticles with 2.5% glutaraldehyde for 1 hour, it was observed by scanning electron microscopy.

[0112] Experimental results are as follows Figure 2 As shown in Table 1:

[0113] Table 1

[0114]

[0115] As shown in Table 1, when the OD of A1501 bacterial culture... 600 When the value is 1, the magnetic capture efficiency of 5.0 g / L modified iron oxide nanoparticles for the strain can reach over 90%; Figure 2 As can be seen from the scanning electron microscopy results, the nanoparticles successfully adhered to the surface of the bacteria.

[0116] Example 5

[0117] This example illustrates the determination of the effect of magnetic interaction systems on the colonization efficiency of bacterial strains.

[0118] (1) The rice variety used in the experiment was Zhonghua 11. The seeds were dehulled using a dehulling machine, and seeds with intact, plump grains and no disease spots were manually selected for subsequent experiments.

[0119] (2) Completely immerse the selected seeds in a 75% (v / v) ethanol solution and shake on a shaker for 5 minutes to remove surface grease. After discarding the ethanol, add a 5% (v / v) sodium hypochlorite solution and shake for 10 minutes. Repeat this step once to ensure thorough elimination of microorganisms on the surface. After disinfection, rinse the seeds three times with sterile distilled water, shaking on a shaker for 5 minutes each time to thoroughly remove any residual disinfectant. Finally, air dry the seeds in a laminar flow hood for about 30 minutes until there is no obvious moisture on the seed surface.

[0120] (3) The surface-dried and sterilized seeds were evenly sown in petri dishes with 1 / 2 MS semi-solid medium (with 0.8% agar powder added and pH adjusted to 5.8) as the substrate. The petri dishes were placed in a controlled artificial climate chamber for dark germination. The culture conditions were set as follows: day and night temperature 30℃ / 25℃, photoperiod 16 hours light / 8 hours dark, and relative humidity period 90% / 50%. Under these conditions, the seedlings were cultured for 8 days to obtain sterile rice seedlings with uniform growth.

[0121] (4) After 8 days of cultivation, carefully remove the seedlings from the culture medium and gently rinse the roots with distilled water to remove the culture medium. Select seedlings with uniform growth and consistent health for hydroponic experiments. The hydroponic substrate is 1 / 2 Hoagland nutrient solution (pH=5.8).

[0122] (5) Disperse the magnetic nano-iron oxide powder in Hoagland nutrient solution and sonicate it for 30 minutes to ensure that it is fully dispersed and forms a uniform suspension (also known as dispersion), so that the concentration of magnetic nano-iron oxide reaches 100 mg / L. Place the hydroponic seedlings in an artificial climate chamber, and apply 50 mL of magnetic nano-iron oxide nanoparticle dispersion per plant. The culture conditions are: day and night temperature 30℃ / 25℃, 16h light / 8h darkness and root protection from light, and continue to culture for 5 days.

[0123] (6) Using a sterile inoculation loop, pick a single, morphologically typical fresh and activated colony of *Pseudomonas stearothermii* A1501 from the plate and inoculate it into a 15 mL sterile centrifuge tube containing 5 mL of LB liquid medium. Fix the centrifuge tube at an angle in a constant temperature shaker and incubate overnight at 30°C and 220 rpm to obtain a seed culture in the logarithmic growth phase.

[0124] (7) Transfer the primary seed culture at a volume ratio of 1:100 to an Erlenmeyer flask containing 300 mL of LB liquid medium. Place it in a constant temperature shaker and culture overnight at 30°C and 220 rpm to allow the bacteria to proliferate fully.

[0125] (8) Centrifuge the expanded bacterial culture at 4°C and 6000×g for 15 minutes and discard the supernatant. Resuspend the obtained bacterial pellet in pre-cooled phosphate buffer (PBS, pH 7.4) and gently wash by pipetting. Repeat this process three times to thoroughly remove residual components of the culture medium.

[0126] (9) Resuspend the washed bacterial body in an appropriate amount of PBS buffer and measure its OD. 600 Adjust to approximately 1, then take a certain mass of iron oxide nanoparticles and mix them with the above bacterial suspension (live bacteria concentration of 10). 10Mix (CFU / mL) in a 50mL centrifuge tube to achieve a concentration of ferric oxide of 5.0 g / L; take a certain mass of polyeneimide-modified ferric oxide nanoparticles and mix with the above bacterial suspension (live bacteria concentration of 10 CFU / mL). 10 Mix (CFU / mL) in 50mL centrifuge tubes to achieve a polyeneimide-modified iron oxide concentration of 5.0 g / L. Prepare 5.0 g / L triphenylphosphine-chitosan (ChiP)-modified iron oxide nanoparticle bacterial suspensions (denoted as ChiP-modified) and 5.0 g / L polylysine-modified iron oxide nanoparticle bacterial suspensions (denoted as PLL-modified) using the same method. Place the centrifuge tubes on a constant-temperature mixer and incubate with gentle shaking at 30℃ and 1000 rpm for 60 minutes.

[0127] (10) Centrifuge the modified strain obtained in step (9) at 6000×g for 15 minutes, discard the supernatant, and gently resuspend it in a certain volume of Hoagland nutrient solution to allow its OD to adjust. 600 It is 0.1.

[0128] (11) Inoculate the Hoagland bacteria suspension obtained in step (10) into the rice seedlings obtained in step (5) (10 mL / seedling). After culturing at 25°C for 1 day, cut off the rice roots with sterile scissors, place them in a 50 mL sterile centrifuge tube, add 10 mL of PBS, vortex for 15 minutes, and determine the colony count using the dilution plating method.

[0129] (12) Wipe the cut rice roots dry with filter paper, weigh them, and use the plate count method to calculate the number of nitrogen-fixing microorganisms colonized per gram of root system.

[0130] The formula is: Colony number (CFU / g) = (Dilution factor × Corresponding gradient colony number) / Root weight.

[0131] The experimental results are shown in Table 2 and Figure 3 As shown:

[0132] Table 2

[0133]

[0134] Table 2 shows that the colony count in the A1501 treatment group, which absorbed the nanomaterials, was approximately 10 times higher than that in the control group. Figure 3 As can be seen from the scanning electron microscope, the modified A1501 was successfully colonized in the rice root system.

[0135] Example 6

[0136] This example illustrates the determination of the following effects of magnetic interactions on plant growth promotion:

[0137] (1) The rice variety used in the experiment was Zhonghua 11. The seeds were dehulled using a dehulling machine, and seeds with intact, plump grains and no disease spots were manually selected for subsequent experiments.

[0138] (2) Completely immerse the selected seeds in a 75% (v / v) ethanol solution and shake on a shaker for 5 minutes to remove surface grease. After discarding the ethanol, add a 5% (v / v) sodium hypochlorite solution and shake for 10 minutes. Repeat this step once to ensure thorough elimination of microorganisms on the surface. After disinfection, rinse the seeds three times with sterile distilled water, shaking on a shaker for 5 minutes each time to thoroughly remove any residual disinfectant. Finally, air dry the seeds in a laminar flow hood for about 30 minutes until there is no obvious moisture on the seed surface.

[0139] (3) The surface-dried sterilized seeds were evenly sown in petri dishes with 1 / 2 MS semi-solid medium (with 0.8% agar powder added and pH adjusted to 5.8) as the substrate. The petri dishes were placed in a controlled artificial climate chamber for dark germination. The culture conditions were set as follows: day and night temperature 30℃ / 25℃, photoperiod 16 hours light / 8 hours dark, and relative humidity period 90% / 50%. Under these conditions, the seedlings were cultured for 8 days to obtain sterile rice seedlings with uniform growth.

[0140] (4) After 8 days of cultivation, carefully remove the seedlings from the culture medium and gently rinse the roots with distilled water to remove the culture medium. Select seedlings with uniform growth and consistent health for hydroponic experiments. The hydroponic substrate is 1 / 2 Hoagland nutrient solution (pH=5.8).

[0141] (5) First, the magnetic nano-iron oxide powder was dispersed in a small amount of distilled water and ultrasonically treated for 30 minutes to ensure that it was fully dispersed and formed a uniform suspension (also known as dispersion). Then, it was added to the nutrient solution to make the concentration reach 0.1 g / L. Ten seedlings were cultured in each large test tube. The hydroponic seedlings were placed in an artificial climate chamber. The amount of magnetic nano-iron oxide nanoparticle dispersion applied was 50 mL / plant. The culture conditions were: day and night temperature 30℃ / 25℃, 16h light / 8h darkness and root protection from light, and cultured for 5 days.

[0142] (6) Using a sterile inoculation loop, pick a single, morphologically typical fresh and activated colony of *Pseudomonas stearothermiae* A1501 from the plate and inoculate it into a 15 mL sterile centrifuge tube containing 5 mL of LB liquid medium. Fix the centrifuge tube at an angle in a constant temperature shaker and incubate overnight at 30°C and 220 rpm to obtain a seed culture in the logarithmic growth phase.

[0143] (7) Transfer the primary seed culture at a volume ratio of 1:100 to an Erlenmeyer flask containing 300 mL of LB liquid medium. Place it in a constant temperature shaker and culture overnight at 30°C and 220 rpm to allow the bacteria to proliferate fully.

[0144] (8) Centrifuge the expanded bacterial culture at 4°C and 6000×g for 15 minutes and discard the supernatant. Resuspend the obtained bacterial pellet in pre-cooled phosphate buffer (PBS, pH 7.4) and gently wash by pipetting. Repeat this process three times to thoroughly remove residual components of the culture medium.

[0145] (9) Resuspend the washed bacterial body in an appropriate amount of PBS buffer and measure its OD. 600 Adjust to approximately 1, then take a certain mass of iron oxide nanoparticles and mix them with the above bacterial suspension (live bacteria concentration of 10). 10 Mix (CFU / mL) in a 50mL centrifuge tube to achieve a concentration of 5g / L for ferric oxide; take a certain mass of polyeneimide-modified ferric oxide nanoparticles and mix with the above bacterial suspension (live bacteria concentration of 10). 10 Mix (CFU / mL) in 50mL centrifuge tubes to achieve a polyeneimide-modified iron oxide concentration of 5.0 g / L. Prepare 5.0 g / L triphenylphosphine-chitosan (ChiP)-modified iron oxide nanoparticle bacterial suspensions (denoted as ChiP-modified) and 5.0 g / L polylysine-modified iron oxide nanoparticle bacterial suspensions (denoted as PLL-modified) using the same method. Place the centrifuge tubes on a constant-temperature mixer and incubate with gentle shaking at 30℃ and 1000 rpm for 60 minutes.

[0146] (10) Centrifuge the modified strain obtained in step (9) at 6000×g for 15 minutes, discard the supernatant, and gently resuspend it in a certain volume of Hoagland nutrient solution to make its OD600 0.1.

[0147] (11) The Hoagland bacteria suspension obtained in step (10) was inoculated into the rice seedlings obtained in step (5) (10 mL / seedling), and cultured at 25℃ for 7 days. The physiological indicators such as rice plant height and root length were measured.

[0148] The experimental results are shown in Table 3:

[0149] Table 3

[0150]

[0151] As shown in Table 3, the magnetic interaction system constructed in this disclosure has a significant promoting effect on rice growth.

[0152] Example 7

[0153] This example illustrates the effect of magnetic interaction systems on the nitrogenase activity of bacterial strains.

[0154] (1) Using a sterile inoculation loop, pick a single, morphologically typical fresh and activated colony of Pseudomonas schlegelii A1501, a nitrogen-fixing microorganism, from the plate and inoculate it into a 15 mL sterile centrifuge tube containing 5 mL of LB liquid medium. Fix the centrifuge tube at an angle in a constant temperature shaker and incubate overnight at 30 °C and 220 rpm to obtain a seed culture in the logarithmic growth phase.

[0155] (2) Transfer the primary seed culture at a volume ratio of 1:100 to an Erlenmeyer flask containing 300 mL of LB liquid medium. Place the flask in a constant temperature shaker and culture overnight at 30°C and 220 rpm to allow the bacteria to proliferate fully.

[0156] (3) Centrifuge the expanded bacterial culture at 4°C and 6000×g for 15 minutes and discard the supernatant. Resuspend the obtained bacterial pellet in pre-cooled phosphate buffer (PBS, pH 7.4) and gently wash by pipetting. Repeat this process three times to thoroughly remove residual components of the culture medium.

[0157] (4) Resuspend the washed bacterial body in an appropriate amount of PBS buffer and measure its OD. 600 Adjust to approximately 1, then take a certain mass of iron oxide nanoparticles and mix them with the above bacterial suspension (live bacteria concentration of 10). 10 Mix (CFU / mL) in a 50mL centrifuge tube to achieve a concentration of 5g / L for ferric oxide; take a certain mass of polyeneimide-modified ferric oxide nanoparticles and mix with the above bacterial suspension (live bacteria concentration of 10). 10 Mix (CFU / mL) in 50mL centrifuge tubes to achieve a polyeneimide-modified iron oxide concentration of 5.0 g / L. Prepare 5.0 g / L triphenylphosphine-chitosan (ChiP)-modified iron oxide nanoparticle bacterial suspensions (denoted as ChiP-modified) and 5.0 g / L polylysine-modified iron oxide nanoparticle bacterial suspensions (denoted as PLL-modified) using the same method. Place the centrifuge tubes on a constant-temperature mixer and incubate with gentle shaking at 30℃ and 1000 rpm for 60 minutes.

[0158] (5) Take 10 mL of the bacterial cells obtained in step (4), place them in a sterile centrifuge tube, centrifuge at 4°C and 6000×g for 15 minutes, discard the supernatant, and collect the bacterial cell precipitate. Then, resuspend the bacterial cells in an appropriate amount of nitrogen-free K medium to adjust their OD. 600 It is 1.0.

[0159] (6) Use 120mL saline bottles as reaction vessels. Accurately add 18mL of nitrogen-free K medium and 2mL of OD prepared in step (5) to each bottle. 600A bacterial suspension with a concentration of 1.0 μL was used to make a total reaction volume of 20 mL, which was then used to determine the activity of A1501 nitrogenase modified with nanomaterials. Five replicates were required for each experimental group.

[0160] (7) The bacterial suspension obtained in step (5) of this embodiment was inoculated into the rice seedlings obtained in step (5) of this embodiment (10 mL / seedling) and cultured at 25°C. One day after inoculation, the rice roots were cut off and placed in a reaction container (120 mL saline bottle). 20 mL of nitrogen-free K medium was precisely added to each bottle to determine the activity of A1501 nitrogenase under root colonization conditions. Five parallel replicates were set up for each experimental group.

[0161] (8) Use butyl rubber stoppers to seal the mouths of the saline bottles from steps (6) and (7) respectively, and use aluminum caps with capping devices to seal them to ensure the airtightness of the reaction system.

[0162] (9) An anaerobic environment was created using an inert gas displacement method. A gas-filling needle was inserted into the bottle through a rubber stopper, and high-purity argon gas was continuously introduced into the bottle for 4 minutes to effectively expel the air inside. Subsequently, a pre-mixed gas mixture was injected into the bottle, resulting in a final concentration containing 0.5% (v / v) oxygen and 10% (v / v) acetylene. Acetylene serves as a specific substrate for nitrogenase.

[0163] (10) Place the prepared reaction flask in a constant temperature shaker and incubate the reaction at 30°C and 200 rpm. Collect gas samples every 2 hours after the start of incubation. When sampling, use a gas-tight micro-syringe to pass through the rubber stopper and extract 0.25 mL of gas sample from the gas phase portion of the flask for subsequent gas chromatography analysis.

[0164] (11) The collected gas samples were analyzed using a gas chromatograph equipped with a flame ionization detector (FID) to detect the peak area of ​​ethylene. Nitrogenase activity was calculated using the following formula:

[0165] Nitrogenase activity [nmol C2H4 (mg·protein·h)] -1 = [Ethylene peak area on recorder × gas phase volume in bottle / gas sample injection volume] / [protein concentration × peak area of ​​1 nmol standard ethylene × reaction time].

[0166] Experimental results are as follows Figure 4 and 5 As shown, by Figure 4 It can be seen that the nitrogenase activity of the modified A1501 was significantly increased compared with that of the control nitrogenase. Figure 5 It can be seen that the activity of A1501 nitrogenase colonized in the root system also increased significantly.

[0167] Comparative Example 1

[0168] The method described in Example 6 was used, except that step (5) was not performed, that is, the magnetic iron oxide nanoparticles were not used to treat the plant roots. The A1501 nitrogenase activity under root colonization conditions was tested using the method in Example 7.

[0169] The results are as follows Figure 6 As shown, in Figure 6 In the study, unmodified roots represent plant roots not treated with magnetic iron oxide nanoparticles; modified roots represent plant roots treated with magnetic iron oxide nanoparticles; and the nitrogenase activity of A1501 in modified roots was significantly higher than that in unmodified roots.

[0170] The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings. However, the present disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0171] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0172] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A method for improving the colonization efficiency of rhizosphere growth-promoting bacteria using nanomaterials and magnetic interaction systems, characterized in that, The method includes: S1. After mixing the modified iron oxide nanoparticles with the bacterial suspension of rhizosphere growth-promoting bacteria, a first culture was carried out to obtain the modified rhizosphere growth-promoting bacteria. S2. Apply the dispersion of magnetic iron oxide nanoparticles to the roots of plants for a second culture to obtain modified plants. S3. Apply the bacterial suspension of the modified rhizosphere growth-promoting bacteria to the roots of the modified plant for a third culture. The modified iron oxide nanoparticles are cationic polymer-modified iron oxide nanoparticles. The preparation method of the modified iron oxide nanoparticles includes: mixing an iron source, a cationic polymer, and a solvent under an inert atmosphere to obtain a first mixture; mixing the first mixture with a pH adjuster to obtain a second mixture and reacting it to obtain a third mixture; and subjecting the third mixture to magnetic separation, washing, and drying in sequence. The reaction is selected from any of the following pathways: (1) hydrothermal reaction; (2) coprecipitation reaction and hydrothermal reaction; (3) hydrothermal reaction and isothermal oscillation reaction; The modified iron oxide nanoparticles have an average particle size of 20-100 nm. The iron source is selected from one or more of FeCl3·6H2O, FeSO4·7H2O, FeCl2·4H2O, Fe(NO3)3·9H2O and Fe(Ac)3·4H2O; The cationic polymer is selected from one or more of polyethyleneimine, triphenylphosphine chitosan and its derivatives, polylysine, polydimethyldiallylammonium chloride, chitosan quaternary ammonium salt and polymethacryloyloxyethyltrimethylammonium chloride; The solvent is selected from one or more of sulfuric acid, water, ethanol, ethylene glycol and N,N-dimethylformamide; The pH adjuster is selected from one or more of potassium nitrate, sodium hydroxide, ammonia, sodium bicarbonate, hydrochloric acid, and acetic acid; The plants include rice, corn, and tomatoes; The rhizosphere growth-promoting bacteria are nitrogen-fixing microorganisms.

2. The method according to claim 1, wherein, In step S1, the viable bacterial concentration of the rhizosphere growth-promoting bacteria in the bacterial suspension is 10. 5 -10 10 CFU / mL; The amount of modified iron oxide nanoparticles added is 0.1-5.0g per 1L of the rhizosphere growth-promoting bacteria suspension.

3. The method according to claim 2, wherein, The nitrogen-fixing microorganisms are selected from one or more of Pseudomonas schrenckii, Burkholderia spp., Azotobacter halojicus, and Azotobacter brasiliensis.

4. The method according to claim 3, wherein, The conditions for the first culture include: a temperature of 25-35℃, a rotation speed of 50-1000 rpm, and a time of 30-120 min.

5. The method according to claim 1, wherein, The content of the iron source, calculated as iron element, is 1-1000 mg per 1-1000 mL of solvent, and the content of the cationic polymer is 1-1000 mg. The pH value of the second mixture is 9-11; The conditions for the hydrothermal reaction include: a temperature of 50-100℃, a stirring rate of 100-500 rpm, and a time of 0.5-24 h. The conditions for the coprecipitation reaction include: a temperature of 25-60℃ and a time of 5-60 min; The conditions for the isothermal oscillation reaction include: a temperature of 20-37℃, an oscillation rate of 150-200 rpm, and a time of 12-24 h.

6. The method according to claim 1, wherein, In step S2, the content of magnetic iron oxide nanoparticles is 20-2000 mg relative to each 1 L of the dispersion of magnetic iron oxide nanoparticles; the solvent of the dispersion of magnetic iron oxide nanoparticles is water and / or Hoagland nutrient solution; and the average particle size of the magnetic iron oxide nanoparticles is 20-500 nm. The amount of the dispersion of the magnetic iron oxide nanoparticles applied is 50-100 mL / plant.

7. The method according to claim 6, wherein, The second cultivation conditions include: a temperature of 25-30℃, a light condition of 16 hours of light / 8 hours of darkness and root protection from light, and a duration of 5-7 days.

8. The method according to claim 1, wherein, In the bacterial suspension of the modified rhizosphere growth-promoting bacteria, the viable bacterial concentration of the modified rhizosphere growth-promoting bacteria is 10. 5 -10 10 CFU / mL; The application rate of the modified rhizosphere growth-promoting bacteria suspension is 10-100 mL / plant.

9. The method according to claim 8, wherein, The conditions for the third culture include: a temperature of 25-30℃ and a time of 1-7 days.

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