Drought and high temperature resistant slow-release fertilizer containing nano selenium and preparation method thereof

Abstract

The application relates to the field of slow-release fertilizers, and particularly discloses a nano-selenium drought-resistant and high-temperature-resistant slow-release fertilizer and a preparation method thereof. 0 The method comprises the following steps: fixing glucose oxidase on the surface of TiO2 nanoparticles, and performing cascade reaction of the glucose oxidase, glucose and sodium selenite under mild conditions to realize synchronous synthesis of Se The method is green, mild and controllable, the obtained selenium fertilizer has excellent stability and slow-release performance, can significantly improve the selenium content and yield of crops, and is suitable for selenium-rich functional agriculture.

Description

Technical Field

[0001] This invention belongs to the field of slow-release fertilizer preparation technology, specifically relating to a nano-selenium drought-resistant and high-temperature-resistant slow-release fertilizer and its preparation method. Background Technology

[0002] Global warming and abnormal climate, with frequent high temperatures and droughts, have severely impacted plant growth, especially the production of food crops.

[0003] Selenium is a recognized element for drought and heat resistance. It exerts its antioxidant effect by increasing the activity of glutathione peroxidase (GSH-Px), acting as a direct scavenger of excess free radicals. Simultaneously, selenium can alter the activity of other reactive oxygen species defense enzymes, such as superoxide dismutase (SOD) and peroxidase (POD), maintaining a dynamic balance among various antioxidant systems in the body. This protects the integrity of plant cell membrane structure and function under drought and heat stress, reduces electrolyte leakage, and enhances plant resistance to high temperatures and drought. Selenium significantly improves root vigor, increases proline accumulation, and leads to significant potassium accumulation in leaves. The synergistic effect of these drought- and heat-resistant factors further enhances the crop's ability to resist drought and high temperatures.

[0004] However, in practical applications, selenium exhibits a characteristic of low-promotion and high-inhibition (low dosage promotes plant growth; high dosage inhibits it). The concentration and range of selenium application are extremely narrow; for example, in rice seedlings, 0.01 ppm-1 ppm promotes root growth, while concentrations exceeding 3 ppm significantly inhibit root growth. Without slow-release and controlled-release technologies and materials, applying selenium-containing fertilizers to the soil at dosages beneficial to plant growth will result in selenium being fixed in near-neutral to acidic soils by forming poorly water-soluble oxides or hydrated oxides with iron, or it may be lost with water. Excessive application can immediately cause toxicity to plants. When its effects are truly needed, it is difficult to replenish and adjust it as readily as under experimental conditions or with sufficient labor. During dry and hot seasons, manpower and water resources are limited. Nano-selenium has become a research hotspot for new selenium fertilizers due to its high biological activity and low toxicity. However, nano-selenium is prone to aggregation and oxidation during preparation, storage, and application, severely affecting its fertilizer efficiency. In existing technologies, the preparation of nano-selenium often employs chemical reduction methods (using ascorbic acid, sodium borohydride, etc.) or natural extract reduction methods (such as raisin extract), but these methods suffer from problems such as reducing agent toxicity, poor product stability, and low loading efficiency. Furthermore, the slow-release performance of nano-selenium still needs improvement. Therefore, researching a new generation of slow-release selenium fertilizers that can maintain the chemical activity of selenium in drought-resistant and high-temperature-resistant materials while effectively leveraging its synergistic effects is of paramount importance. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a nano-selenium drought-resistant and high-temperature-resistant slow-release fertilizer, which achieves Selenium loading through a glucose oxidase (GOx) cascade reaction and TiO2 carrier loading. 0 By simultaneously synthesizing and depositing the nano-selenium and combining it with a chitosan-calcium alginate multi-level encapsulation structure, a highly stable and bioactive nano-selenium slow-release fertilizer was prepared. This solved the technical problems of easy agglomeration and oxidation of nano-selenium during preparation, storage and application. At the same time, it achieved the controlled slow release of selenium, extended its action time, improved selenium utilization, overcame the application difficulties caused by the "low-promoting and high-inhibiting" characteristics of selenium, and effectively exerted the antioxidant synergistic effect of selenium under drought and high temperature stress conditions, thereby enhancing the crop's ability to resist adversity.

[0006] Technical solution

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a method for preparing a nano-selenium drought-resistant and high-temperature-resistant slow-release fertilizer, comprising the following steps:

[0009] Step 1: Immobilize glucose oxidase (GOx) on the surface of anatase TiO2 nanoparticles to obtain immobilized GOx@TiO2;

[0010] Step 2: The immobilized GOx@TiO2 is mixed with D-glucose and sodium selenite and reacted. Glucose oxidase catalyzes the generation of H2O2 from D-glucose, and H2O2 reduces Se in situ. 4+ For Se 0 And the generated Se 0 Simultaneous deposition on the TiO2 surface to form a Se@TiO2 complex;

[0011] Step 3: Perform multi-stage stabilization treatment on the Se@TiO2 complex to obtain nano-selenium slow-release fertilizer.

[0012] In step 1, TiO2 nanoparticles are dispersed in PBS buffer (pH 6.5-7.0, 20-50 mM) at a concentration of 10-50 mg / mL, and GOx lyophilized powder is added. The mass ratio of GOx to TiO2 is 1:10-1:50. Adsorption is carried out at 4℃ and 50-150 rpm for 1-4 hours. Then, the mixture is centrifuged at 8000-12000 rpm for 5-15 minutes. The precipitate is washed with PBS 1-3 times to remove unadsorbed enzyme, thus obtaining immobilized GOx@TiO2. The TiO2 surface is rich in Ti-OH hydroxyl groups, which can stably immobilize GOx through multi-site hydrogen bonding and coordination. After immobilization, the enzyme conformation changes little, and the activity retention rate is high, providing a stable catalyst for subsequent cascade reactions. At the same time, the catalytic activity of the enzyme is confined to the carrier surface, which is conducive to the realization of Se... 0 This laid the foundation for synchronous deposition.

[0013] Preferably, in step 1, catalase (CAT) and GOx can be further introduced and co-immobilized on the TiO2 surface, with the mass ratio of CAT to GOx being 1:5 to 5:1, forming a dual-enzyme cascade amplification reduction system. In a single GOx system, the generation rate of H2O2 is greater than the consumption rate, leading to the accumulation of H2O2. The excess H2O2 will, in turn, oxidize the newly formed Se. 0 It also inhibits GOx activity. After the introduction of CAT, CAT decomposes excess H2O2 into H2O and O2, maintaining the H2O2 concentration at an appropriate level. Because H2O2 reduces Se... 4 The rate of ⁺ decomposition is much faster than that of CAT decomposition of H₂O₂ in the low concentration range. CAT does not preempt the H₂O₂ required for normal reduction, but rather precisely removes those that cannot be decomposed by Se. 4+ Excess H2O2 is consumed in a timely manner, thereby achieving a higher selenium yield and a more stable product.

[0014] In step 2, the mixing reaction is carried out at pH 6.5-7.0 and 30-40℃. Based on the final volume of the reaction solution, the amount of sodium selenite added is 0.9-3.5 mg / mL, the amount of D-glucose added is 9-36 mg / mL, and the amount of immobilized GOx@TiO2 added is 5-25 μg / mL based on the mass of GOx. The reaction time is 4-6 hours, carried out under light-protected conditions. GOx specifically catalyzes the reaction of D-glucose with O2 to generate H2O2. This reaction can be carried out efficiently at near-neutral and mild temperatures, avoiding the use of high temperatures, strong alkalis, or chemical reducing agents. The H2O2 generation rate can be precisely controlled by adjusting the glucose concentration and the amount of GOx. The generated H2O2 immediately decomposes the selenite. 4+ In-situ restoration to Se 0 Because GOx is fixed on the TiO2 surface, H2O2 is locally generated on the support surface, forming a concentration gradient that forces the reduction reaction to be strictly confined to the surface microregions. 0 Preferential heterogeneous nucleation and growth on the TiO2 surface, rather than homogeneous nucleation in solution, thus achieving Se... 0 The synthesis and loading are integrated, and the product particle size can be controlled between 20-40nm, with uniform average particle size distribution and strong loading.

[0015] Step 3, the multi-level stabilization process includes the following steps:

[0016] Step 3.1: The Se@TiO2 complex is dispersed in a chitosan solution for surface modification to obtain a chitosan-coated Se@TiO2 dispersion. Specifically, the Se@TiO2 complex is dispersed in a chitosan solution at a concentration of 10-50 mg / mL, where the chitosan solution concentration is 2-5 mg / mL and dissolved in a 10 mg / mL acetic acid aqueous solution. The modification time is 30-60 minutes. Under acidic conditions, the amino groups on the chitosan molecular chains are protonated and become positively charged, thus encapsulating the Se@TiO2 surface through electrostatic adsorption to form a positively charged layer. Simultaneously, the long chitosan chains generate steric hindrance, preventing particle approach and aggregation, ensuring uniform dispersion of Se@TiO2 in the solution and providing a well-dispersed precursor for subsequent encapsulation processes.

[0017] Preferably, the chitosan solution in step 3.1 can be replaced with a chitosan-dextran copolymer solution instead of a pure chitosan solution. The mass ratio of chitosan to dextran in the copolymer is 3:1 to 1:1. The chitosan portion provides surface modification and positive charge functionality for Se@TiO2, while the dextran segments impart enzyme-responsive properties. Specifically, the dextran is selected from dextrans containing β-1,4-glycosidic bonds, including at least one of cellulosic oligosaccharides, lichen polysaccharides, or mixed-bond β-glucans. Rhizosphere microorganisms (such as Pseudomonas, Bacillus, etc.) proliferate in large numbers in the crop rhizosphere, continuously secreting extracellular enzymes such as β-glucosidase, which can specifically cleave the β-1,4-glycosidic bonds of the dextran segments, resulting in a loose copolymer layer structure. Se@TiO2 is preferentially released in the rhizosphere region, achieving enzyme-responsive targeted release. This preferred scheme, while retaining the original dispersing and stabilizing function of chitosan, additionally adds an enzyme-responsive intelligent release function, further improving the utilization efficiency of selenium.

[0018] Step 3.2: Add sodium alginate to the chitosan-modified Se@TiO2 dispersion obtained in Step 3.1 to a final concentration of 15-30 mg / mL. After mixing thoroughly, add the mixture dropwise into a CaCl2 solution for cross-linking. The volume ratio of the total mixture of the chitosan-modified Se@TiO2 dispersion and sodium alginate to the CaCl2 solution is 1:5-1:20. The concentration of the CaCl2 solution is 10-50 mg / mL. The cross-linking time is 20-60 minutes, forming calcium alginate gel microspheres embedded with Se@TiO2. The G block in the sodium alginate molecular chain interacts with the CaCl2... 2+ Specific ionic cross-linking occurs, forming a three-dimensional network gel. These gel microspheres can physically isolate oxygen and delay the degradation of Se. 0 It oxidizes and gradually absorbs water, swells, and degrades in soil or water, causing Se@TiO2 to be released slowly, thus achieving a continuous supply of selenium.

[0019] Step 3.3: After drying the microspheres, add citric acid crystals and mix thoroughly. Seal and store. Specifically, add citric acid crystals at a ratio of 0.5-2% of the microsphere mass, and shake or stir in a sealed container for 5-15 minutes until thoroughly mixed. Citric acid is a natural organic acid with reducing and free radical scavenging capabilities. During microsphere storage, it can remove trace amounts of reactive oxygen species that have penetrated the microspheres and inhibit selenium formation. 0 It undergoes chemical oxidation, slowly dissolves and releases antioxidant activity when exposed to moisture, thus extending the product's shelf life.

[0020] The nano-selenium slow-release fertilizer prepared by the above method has a core of Se. 0 A Se@TiO2 composite is formed by loading nanoparticles onto the surface of anatase TiO2 nanoparticles. A chitosan modification layer is coated on the surface of the composite, and calcium alginate gel microspheres are embedded inside it. Citric acid is dispersed in the microspheres or on the surface of the microspheres as a solid antioxidant. The Se... 0 The average particle size of the nanoparticles is 20-40 nm, and the diameter of the calcium alginate microspheres is 1-5 mm.

[0021] Compared with the prior art, the present invention has the following significant advantages:

[0022] ① Solving the stability problem of nano-selenium: This invention fixes GOx on the TiO2 surface and utilizes an enzymatic cascade reaction to generate H2O2 in situ under mild conditions and reduce Se. 4+ For Se 0 , achieved Se 0 The simultaneous synthesis and loading of the product avoided the aggregation problem of free nano-selenium. Simultaneously, a multi-level stabilization strategy involving chitosan dispersion, calcium alginate microsphere encapsulation, and citric acid antioxidation enabled the product to achieve Se2O2 stabilization after 30 days of accelerated aging at 40℃ and 75%RH. 0 The retention rate is still 94–98%, which is significantly better than existing technologies (compared to only 33–61%).

[0023] ② Achieving controlled and slow-release of selenium, solving the problem of "low-promoting and high-inhibiting": This invention achieves slow release of selenium through the gel network structure of calcium alginate microspheres. The cumulative release rate after 240 hours is only 52-68%, while the comparative example releases 60-85% within 24 hours. The slow-release characteristic ensures a continuous supply of selenium in the soil, avoiding the high-concentration toxicity caused by concentrated release, while reducing leaching losses and improving selenium utilization, allowing crops to obtain an adequate supply of selenium throughout their growth period.

[0024] ③ Green and mild preparation process: The present invention uses an enzymatic cascade reaction to carry out the reaction under near-neutral (pH 6.5-7.0) and mild temperature (30-40℃) conditions, avoiding the use of high temperature, strong alkali and toxic chemical reducing agents. The by-product is biodegradable gluconic acid, which meets the requirements of green chemistry and is suitable for large-scale production.

[0025] ④ Significantly increases crop selenium content and yield: Pot experiments showed that after applying the product of this invention, the selenium content of tomato fruits reached 0.48-0.52 μg / g, which is 1.5-1.7 times higher than the existing technology (CN121318555A method); the yield per plant increased by 29-32% compared with the blank control and by 15-18% compared with the existing technology. This indicates that the product can significantly increase crop yield and selenium content under normal conditions, and can effectively alleviate oxidative damage and maintain photosynthetic efficiency under drought and high temperature stress conditions, showing significant drought and high temperature resistance. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the preparation method of the nano-selenium slow-release fertilizer of the present invention. Detailed Implementation

[0027] Example 1: Basic Scheme (TiO2 Loading GOx + Multi-stage Stabilization)

[0028] Step 1: Immobilization of GOx

[0029] 500 mg of anatase TiO2 nanoparticles (average particle size 25 nm) were dispersed in 50 mL of PBS buffer (pH 6.8, 50 mM) and ultrasonically dispersed for 10 min to obtain a TiO2 suspension with a concentration of 10 mg / mL. 25 mg of GOx lyophilized powder (GOx to TiO2 mass ratio 1:20) was added, and adsorption was performed at 4 °C and 100 rpm for 2 h. The mixture was then centrifuged at 10,000 rpm for 10 min, and the precipitate was washed twice with PBS and redispersed in 25 mL of PBS to obtain an immobilized GOx@TiO2 suspension (containing approximately 1 mg / mL of GOx).

[0030] Step 2: Cascade Reaction and Synchronous Loading

[0031] Take 5 mL of the above immobilized GOx@TiO2 suspension (containing 100 mg TiO2 and 5 mg GOx) and add:

[0032] 3.6g D-glucose (final concentration approximately 18 mg / mL), 350mg sodium selenite (final concentration approximately 1.75mg / mL), and PBS to bring the total volume to 200mL.

[0033] The reaction system was placed in a constant temperature shaker at 30℃ (100 rpm) and reacted in the dark for 5 hours. After the reaction was completed, the solution turned orange-red. The red precipitate Se@TiO2 was collected by centrifugation at 10000 rpm for 10 minutes, washed twice with deionized water, and then dried.

[0034] Step 3: Multi-stage stabilization processing

[0035] Step 3.1 (Chitosan Modification): The above Se@TiO2 precipitate was redispersed at a concentration of about 20 mg / mL in 12.5 mL of chitosan solution (concentration of 5 mg / mL, dissolved in 10 mg / mL acetic acid aqueous solution, pH 4.5), and stirred for 30 min to obtain a chitosan-coated Se@TiO2 dispersion.

[0036] Step 3.2 (Calcium alginate microsphere encapsulation): Add 250 mg of sodium alginate powder to the above dispersion (to achieve a final sodium alginate concentration of 20 mg / mL), and stir until homogeneous. Using a syringe, dropwise add the mixture to 125 mL of CaCl2 solution (concentration 20 mg / mL), with a volume ratio of dispersion to CaCl2 solution of 1:10, forming calcium alginate gel microspheres with a diameter of approximately 2-3 mm. After curing for 30 min, wash three times with deionized water.

[0037] Step 3.3 (Citrate Addition and Drying): After freeze-drying the microspheres (-40℃, 10Pa), weigh them. Weigh 5mg of citric acid crystals (1% of the microsphere mass) and place them together with the microspheres in a sealed bag. Manually shake and mix for 10 minutes until homogeneous. Then transfer to a light-proof bottle, seal, and store at 4℃. The final product is designated as S-1.

[0038] Example 2: Two-enzyme cascade amplification system (GOx + catalase)

[0039] The difference from Example 1 is that catalase (CAT) was additionally added during GOx immobilization in step 1, with a GOx to CAT mass ratio of 2:1 (i.e., 25 mg GOx + 12.5 mg CAT), and it was simultaneously immobilized on the TiO2 surface. The reaction conditions remained unchanged. The remaining steps were the same as in Example 1. The final product was designated S-2.

[0040] Example 3: Enzyme-responsive sustained-release microspheres

[0041] The difference from Example 1 is that in step 3.1, the chitosan solution is replaced with a chitosan-cellulose oligosaccharide copolymer solution: the Se@TiO2 complex obtained in step 2 is dispersed at a concentration of 20 mg / mL in 12.5 mL of chitosan-cellulose oligosaccharide copolymer solution and stirred for 40 minutes to obtain a copolymer-coated Se@TiO2 dispersion. The mass ratio of chitosan to cellulose oligosaccharide is 2:1, the degree of polymerization of cellulose oligosaccharide is 2-10, the copolymer concentration is 5 mg / mL, and it is dissolved in a 10 mg / mL aqueous acetic acid solution. The final product is designated as S-3.

[0042] Comparative Example 1 (CN121318555A Method)

[0043] Prepared according to the method of Example 1 of CN121318555A: Take 100 mg of biochar, add 20 mL of sodium selenite solution with a concentration of 100 mg / L, and shake at 25℃ and 150 rpm for 24 h for adsorption. After centrifugation, add raisin extract (liquid-to-solid ratio 1:1), and reduce at 25℃ in the dark for 5 h. Centrifuge, wash, and freeze-dry to obtain biochar-based nano-selenium fertilizer, denoted as D-1.

[0044] Comparative Example 2 (One-step method for free GOx, without immobilization)

[0045] In 200 mL of PBS (pH 6.8, 50 mM), 5 mg of free GOx, 3.6 g of glucose, and 350 mg of sodium selenite were added directly, and the mixture was reacted at 30 °C and 100 rpm in the dark for 5 h. After the reaction, the mixture was centrifuged at 12000 rpm for 15 min, and the red precipitate SeNPs (selenium nanoparticles) was collected and washed twice with deionized water. No further stabilization treatment was performed, and the mixture was directly freeze-dried. This precipitate is designated D-2.

[0046] Performance testing and characterization

[0047] 1. Particle size and dispersibility

[0048] Test methods: The average particle size (Z-Average) and polydispersity index (PDI) were determined by dynamic light scattering (DLS), and the Zeta potential was determined by electrophoretic light scattering. The measurement temperature was 25℃, the scattering angle was 90°, and each sample was measured in triplicate.

[0049] S-1 28±6 0.12 +32.5 S-2 22±4 0.09 +35.1 S-3 35±7 0.15 +28.6 D-1 65±35 0.45 –8.3 D-2 185±120 0.68 +5.1

[0050] Conclusion: All embodiments of the present invention (S-1, S-2, S-3) exhibit small particle size (<40nm) and narrow distribution (PDI<0.15), with absolute Zeta potential values ​​all >28mV, indicating stable particles that are not prone to agglomeration. S-3, due to its larger molecular weight of the chitosan-cellulose oligosaccharide copolymer, has a slightly larger particle size than S-1. The comparative examples (D-1, D-2) have large particle size, wide distribution, low potential, and poor stability.

[0051] 2. Selenium yield

[0052] Test method: Take 5-10 mg of the Se@TiO2 complex obtained from centrifugation and washing in step 2, digest it with nitric acid-hydrogen peroxide using microwave, and then determine the total selenium content using ICP-MS. Selenium yield (%) = (mass of selenium in the product / mass of selenium in the raw material sodium selenite) × 100%.

[0053] S-1 <![CDATA[Se@TiO2 composite]]> 91.5 S-2 <![CDATA[Se@TiO2 composite]]> 94.2 S-3 <![CDATA[Se@TiO2 composite]]> 90.8 D-1 <![CDATA[Composite of biochar and Se 0 > 80 D-2 Free Se nanoparticles (unloaded) 55.6

[0054] Conclusion: The selenium yield of the method of the present invention is >90%, with the highest yield (94.2%) in the two-enzyme system (S-2); the yield of comparative example D-1 is only 80%, and that of comparative example D-2 is as low as 55.6%.

[0055] 3. Storage stability (accelerated aging test)

[0056] Test method: The final microsphere product (obtained in step 3.3) was stored in a constant temperature and humidity chamber at 40±1℃ and 75±2% RH for 30 days in the dark. Samples were taken at 0 days and 30 days, and the total selenium content was determined by ICP-MS. The retention rate (%) was calculated as (selenium content after 30 days / initial selenium content) × 100%.

[0057] S-1 261 247 94.8 S-2 269 259 96.5 S-3 258 253 98.1 D-1 7.3 4.4 60.5 D-2 950 <10 <1.0

[0058] Conclusion: The retention rates of all embodiments of the present invention were >94%, with S-3 exhibiting the highest retention rate (98.1%) due to its more compact and stable chitosan-cellulose oligosaccharide copolymer structure. Comparative Example D-1 was severely oxidized, with a retention rate of only 60.5%; Comparative Example D-2 suffered almost complete loss.

[0059] Additionally, the D1 method resulted in a very low Se content in the product because biochar has limited selenium loading capacity, relying solely on surface adsorption without chemical anchoring; simultaneously, the Se content in the solution... 4+ The total amount is very low, and it is limited by the "ratio of biochar to selenium source solution is 10~100mg:2~20mL". Assuming that the concentration of Se in the solution is forcibly increased... 4+ (The original data in the patent indicated that the concentration of the selenium source solution was 5~100mg / L. Increasing the relative amount of selenium source solution to biochar would likely lead to selenium agglomeration and larger growth particles, so directly increasing this concentration is not feasible.) The objective of this application is to produce a highly concentrated selenium fertilizer that can be stably stored and released in a controlled manner.

[0060] 4. Sustained-release properties

[0061] Test method: Water extraction method was used. A phosphate buffer solution (pH 6.5, 0.05 mol / L) was used as the release medium. An equal amount of selenium (as Se) was weighed out. 0Each final microsphere sample (calculated as 2.0 mg) was placed in an Erlenmeyer flask, 50 mL of release medium was added, and the flask was placed in a constant-temperature shaker at 37 ± 0.5 °C and 50 rpm. At preset time points, 2 mL samples were taken, and an equal volume of fresh release medium was added simultaneously at the same temperature. The samples were filtered through a 0.22 μm filter membrane, and the selenium content was determined by ICP-MS. The cumulative release rate (%) was calculated. Cumulative release rate = (cumulative mass of released selenium / initial total mass of selenium in the sample) × 100%.

[0062] 1h 3.2 2.8 2.5 18.5 45.2 6h 8.7 7.9 6.8 42.3 68.7 24h 18.5 16.3 14.2 65.4 85.6 72h 32.4 29.8 26.5 82.1 92.3 120h 45.6 42.5 38.2 90.5 96.8 240h 68.3 63.1 56.8 95.2 98.5

[0063] Conclusion: All embodiments of the present invention (S-1 to S-3) exhibit good sustained-release performance, with a cumulative release rate of 56–69% over 240 hours. The two-enzyme system (S-2) releases slightly slower than S-1; S-3 has the slowest release (56.8% release over 240 hours) due to limited swelling of the chitosan-cellulose oligosaccharide copolymer layer in neutral pH buffer. The comparative examples release 65–85% within 24 hours and do not possess effective sustained-release capabilities.

[0064] 5. Tomato pot experiment (including drought and high temperature stress)

[0065] Test method: The final microsphere products were uniformly mixed with soil at a ratio of 0.5 mg / kg selenium content (dry soil), and then potted (3 kg soil per pot). Tomato seedlings were transplanted after 7 days of equilibration. Each treatment was replicated four times (i.e., four pots of each treatment, one seedling per pot). A blank control group (CK) was also included, without selenium fertilizer. The experiment consisted of three groups of growth conditions:

[0066] Normal conditions: 25-30℃, normal watering (maintain soil moisture content at 70% of field capacity);

[0067] Drought stress: After the plants have grown normally to the flowering stage, stop watering and maintain the soil moisture content at 30-35% of field capacity for 10 days.

[0068] High temperature stress: After the plants have grown normally to the flowering stage, they are placed in an artificial climate chamber with day / night temperatures set at 42 / 35℃ for 5 consecutive days.

[0069] After the stress ended, the leaves were allowed to recover for 3 days, and the relative water content and malondialdehyde (MDA) content were measured. Fruits were harvested in batches after ripening, and the yield (fresh weight) per plant was calculated. The total selenium content in the fruits was determined using ICP-MS.

[0070] CK normal 85.2 3.2 0.02 285 S-1 normal 86.5 2.8 0.48 368 S-2 normal 87.1 2.5 0.52 375 S-3 normal 87.0 2.6 0.50 372 D-1 normal 83.8 3.5 0.32 320 D-2 normal 81.2 4.1 0.18 298 CK drought 52.3 12.5 0.02 210 S-1 drought 65.8 7.8 0.42 285 S-2 drought 68.2 6.9 0.46 298 S-3 drought 67.5 7.2 0.45 295 D-1 drought 55.6 11.2 0.25 235 D-2 drought 53.1 11.8 0.15 218 CK high temperature 48.5 15.8 0.02 195 S-1 high temperature 62.3 9.5 0.40 272 S-2 high temperature 65.8 8.2 0.44 286 S-3 high temperature 64.5 8.6 0.43 282 D-1 high temperature 51.2 13.6 0.25 218 D-2 high temperature 49.5 14.5 0.14 205

[0071] *Leaf MDA content is expressed on a fresh weight (FW) basis; fruit selenium content is expressed on a dry weight (DW) basis.

[0072] in conclusion:

[0073] (1) Under normal conditions, the selenium content of tomato fruits treated by the various embodiments of the present invention (S-1, S-2, S-3) reached 0.48-0.52 μg / g, which was 24-26 times higher than the blank control (CK) and 1.5-1.7 times higher than the prior art (D-1); the yield per plant was 29-32% higher than the blank control and 15-18% higher than D-1. Among them, S-2 (dual enzyme system) performed the best, and S-3 (enzyme response system) was comparable to S-1.

[0074] (2) Under drought stress, the relative water content of tomato leaves treated with the product of this invention increased by 13.5-15.9 percentage points compared with the blank control, and the MDA content decreased by 37-45%, indicating that the degree of cell membrane lipid peroxidation was significantly reduced; the selenium content of the fruit was maintained at 0.42-0.46 μg / g, and the yield increased by 36-42% compared with the blank control and by 21-27% compared with D-1.

[0075] (3) Under high temperature stress, the product of the present invention also showed significant stress resistance, with leaf MDA content reduced by 40-48%, yield increased by 39-47% compared with blank control, and increased by 25-31% compared with D-1.

[0076] (4) The release characteristics of S-3 (enzyme response system) under the action of rhizosphere microorganisms make it perform well under drought and high temperature stress conditions. It is not significantly different from S-1, but has better targeted release potential compared with the slow release efficiency in non-rhizosphere environments.

[0077] The above results show that the nano-selenium slow-release fertilizer prepared by the present invention significantly improves the bioavailability and utilization efficiency of selenium under the same selenium application conditions through slow-release structure and nano-particle size control, so that the drought resistance and high temperature resistance functions of selenium can be exerted stably, continuously and efficiently, effectively alleviating oxidative damage caused by drought and high temperature stress, and significantly improving the stress resistance of crops.

[0078] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a nano-selenium drought-resistant and high-temperature-resistant slow-release fertilizer, characterized in that, Includes the following steps: Step 1: Immobilize glucose oxidase on the surface of anatase TiO2 nanoparticles to obtain immobilized GOx@TiO2; Step 2: The immobilized GOx@TiO2 is mixed with D-glucose and sodium selenite and reacted. Glucose oxidase catalyzes the generation of H2O2 from D-glucose, and H2O2 reduces Se in situ. 4+ For Se 0 And the generated Se 0 Simultaneous deposition on the TiO2 surface to form a Se@TiO2 complex; Step 3: Perform multi-stage stabilization treatment on the Se@TiO2 complex to obtain nano-selenium slow-release fertilizer.

2. The preparation method according to claim 1, characterized in that, The GOx immobilization method described in step 1 is as follows: TiO2 nanoparticles are dispersed in PBS buffer at a concentration of 10-50 mg / mL, and GOx lyophilized powder is added. The mass ratio of GOx to TiO2 is 1:10-1:

50. Adsorption is carried out for 1-4 hours under stirring at 50-150 rpm. Then, the mixture is centrifuged at 8000-12000 rpm for 5-15 minutes. The precipitate is washed with PBS 1-3 times to remove unadsorbed enzymes, thus obtaining immobilized GOx@TiO2.

3. The preparation method according to claim 1, characterized in that, In step 2, the mixing reaction is carried out at pH 6.5-7.0 and 30-40℃. Based on the final volume of the reaction solution, the amount of sodium selenite added is 0.9-3.5 mg / mL, the amount of D-glucose added is 9-36 mg / mL, and the amount of immobilized GOx@TiO2 added is 5-25 μg / mL based on the mass of GOx.

4. The preparation method according to claim 1, characterized in that, The multi-level stabilization process in step 3 includes the following steps: Step 3.1: The Se@TiO2 complex is dispersed in a chitosan solution and surface modified to obtain a chitosan-coated Se@TiO2 dispersion; Step 3.2: Add sodium alginate to the dispersion, mix well, and then drop it into CaCl2 solution for cross-linking to form calcium alginate gel microspheres embedded with Se@TiO2; Step 3.3: After drying the microspheres, add citric acid crystals, mix evenly, and seal for storage.

5. The preparation method according to claim 4, characterized in that, Step 3.1 specifically involves dispersing the Se@TiO2 composite at a concentration of 10-50 mg / mL in a chitosan solution, wherein the concentration of the chitosan solution is 2-5 mg / mL, and dissolving it in a 10 mg / mL acetic acid aqueous solution for 30-60 minutes. Step 3.2 specifically involves adding sodium alginate to the chitosan-modified Se@TiO2 dispersion obtained in step 3.1, so that the final concentration of sodium alginate is 15-30 mg / mL, and the volume ratio of the total volume of the chitosan-modified Se@TiO2 dispersion and sodium alginate to the volume of the CaCl2 solution is 1:5-1:20; the concentration of the CaCl2 solution is 10-50 mg / mL, and the crosslinking time is 20-60 minutes. Step 3.3 specifically involves mixing citric acid crystals at a ratio of 0.5-2% of the microsphere mass, placing them in a sealed container, shaking or stirring for 5-15 minutes until uniformly mixed, and then storing.

6. The preparation method according to claim 1, characterized in that, In step 1, catalase and GOx are further introduced and co-immobilized on the TiO2 surface. The mass ratio of catalase to GOx is 1:5 to 5:1, forming a dual-enzyme cascade amplification reduction system.

7. The preparation method according to claim 5, characterized in that, Specifically, step 3.1 involves the chitosan solution being a chitosan-dextran copolymer solution, wherein the mass ratio of chitosan to dextran in the copolymer is 3:1 to 1:1, and the dextran segments contain β-1,4-glycosidic bonds.

8. A nano-selenium slow-release fertilizer prepared by the preparation method according to claims 1-7.

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