Synthesis of nano-isoflavones and its application in soybean root rot

Abstract

The application provides a synthesis method of nano-isoflavones and application of the nano-isoflavones in soybean root rot, and the method comprises the following steps: step 1, preparing A liquid, B liquid and C liquid, three solutions for reaction. The A liquid is a 0.3% chitosan (w / v) solution, the B liquid is a 0.6% genistein (w / v) solution, and the C liquid is a 0.3% sodium tripolyphosphate (w / v) solution. Step 2, mixing the three solutions A, B and C in step 1 according to a volume ratio of 4:1:2, and preparing under the condition of 45 DEG C and magnetic stirring and ultrasonic. The application not only helps to reduce the use of chemical pesticides and reduce environmental pollution, but also optimizes the soil structure and improves the sustainability of the agricultural ecosystem, and provides theoretical support and feasible path for the development of environment-friendly nano materials and the green prevention and control of soybean root rot.

Description

Technical Field

[0001] This invention belongs to the field of crop disease resistance technology, and in particular relates to the synthesis of nano-isoflavones and their application in soybean root rot. Background Technology

[0002] Soybeans, as a globally important grain and oil crop, are a key source of high-quality protein and lipids for humankind. For a long time, limited soybean planting area, low yields, and poor quality have led to reliance on imports, which not only restricts the development of the soybean industry but also threatens food security. Besides the limitation of arable land, root rot is a significant factor restricting the improvement of soybean yield and quality. Currently, the control of this disease mainly relies on chemical pesticides, but due to the complexity of the pathogen, the lack of resistant varieties, the slow effectiveness of bio-fertilizers, and problems such as pesticide residues and environmental pollution, existing control strategies are insufficient to meet the needs of efficient, safe, and sustainable agriculture. Therefore, there is an urgent need to develop efficient and environmentally friendly new control technologies to achieve synergistic development between disease management and agricultural ecological security.

[0003] Nanotechnology, as an emerging method for disease control, can effectively increase the specific surface area and bioactivity of nanomaterials, and has been proven to improve crop disease resistance. For example, Fe-NPs, Cu-CNFs, CuO-NPs, and MgO-NPs have been reported to effectively increase the biomass of crops such as soybeans, rice, corn, cucumbers, tomatoes, and lettuce, thus enhancing their disease resistance. However, current research mainly focuses on inorganic nanomaterials (such as metals and their oxides). Although these materials exhibit strong antibacterial activity, their long-term environmental fate, bioaccumulation effects, and potential impacts on plant-microbe interactions remain unclear, posing ecological safety risks. In contrast, isoflavones, as natural antibacterial metabolites of leguminous plants, possess both the function of regulating plant immunity and optimizing rhizosphere microecology, making them potential candidates for green disease control in agriculture. However, isoflavones are limited by their poor solubility, low stability, and easy degradation, hindering their full development in agricultural applications. Nanoparticle modification of isoflavones not only improves their stability and solubility but also enhances their targeted antibacterial properties, activates plant immunity, and regulates the microecological system, significantly increasing their application potential and possibilities in plant disease control. Currently, research on nano-isoflavones mainly focuses on the pharmaceutical and food fields, with almost no application in agricultural disease management. How nano-isoflavones regulate soybean physiological and biochemical indicators and rhizosphere microbial composition, thereby enhancing crop disease resistance, disrupting pathogen cell structure and physiological functions to limit pathogen invasion, and their underlying mechanisms still lack systematic research. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for synthesizing nano-isoflavones and their application in soybean root rot. This method combines nanotechnology with plant antigen antibacterial strategies to create novel nano-isoflavone materials that are highly effective in disease resistance, environmentally friendly, and easily metabolized. This breaks through the bottlenecks of traditional isoflavone applications and systematically analyzes the mechanism of soybean root rot control. This not only helps reduce the use of chemical pesticides and lower environmental pollution but also optimizes soil health and enhances the sustainability of agricultural ecosystems, providing theoretical support and a feasible path for the development of green nanomaterials and the control of soybean root rot.

[0005] The present invention adopts the following technical solution: Methods for synthesizing nano-isoflavones include: Step 1. Prepare solutions A, B, and C, three solutions for the reaction; Among them, solution A is a 0.3% chitosan (w / v) solution, solution B is a 0.6% genistein (w / v) solution, and solution C is a 0.3% sodium tripolyphosphate (w / v) solution; Step 2. Mix the three solutions A, B, and C from Step 1 in a volume ratio of 4:1:2 and stir magnetically at 45°C to obtain the final product.

[0006] Furthermore, the preparation of solution A includes: dissolving chitosan in a 0.8% glacial acetic acid (v / v) solution, stirring magnetically at 60°C for 30 min to obtain a chitosan (w / v) solution with a concentration of 0.3%.

[0007] Furthermore, the preparation of solution B includes: dissolving isoflavones in a 60% ethanol (v / v) solution, stirring magnetically at 60°C for 30 min to obtain a 0.6% genistein (w / v) solution.

[0008] Furthermore, the preparation of solution C includes: dissolving sodium tripolyphosphate in deionized water to obtain a 0.3% sodium tripolyphosphate (w / v) solution.

[0009] Further, step 2 includes: taking 4 portions of solution A and heating them to 60°C, slowly adding 1 portion of solution B under magnetic stirring, using a magnetic stirrer (800 rpm / min) and ultrasonic probe (300W, 3s operation / 6s interval), adjusting the temperature to 45°C, and stirring thoroughly for 20 minutes until solutions A and B are evenly dispersed. Then, adding 2 portions of solution C dropwise using a burette, expected to take 100 minutes. Subsequently, the precipitate is centrifuged at 5000 rpm / min and resuspended in deionized water, repeatedly washed until the pH of the washing solution is neutral to remove ethanol and glacial acetic acid. Finally, the precipitate is collected and frozen at -80°C for 12 hours, followed by vacuum freeze-drying for 72 hours to obtain nano-isoflavone powder.

[0010] Furthermore, the nano-isoflavones are spherical with a particle size of 44.46±6.31 nm.

[0011] Furthermore, nano-isoflavones exhibit good biocompatibility, show no significant toxicity to soybean growth, and promote plant growth and development.

[0012] Furthermore, nano-isoflavones significantly inhibited the spore germination rate of *Fusarium oxysporum*, the main pathogen of soybean root rot, under both acidic and alkaline liquid culture conditions, with an inhibition rate of 100% at medium to high concentrations. In solid culture media, it significantly inhibited the radial growth of *Fusarium oxysporum* hyphae, with the inhibitory effect showing a dose-dependent relationship. Nano-isoflavones exhibit a trace-level, highly effective, and stable inhibitory effect on the growth and development of *Fusarium oxysporum*.

[0013] Furthermore, when controlling soybean root rot, nano-isoflavones have a considerable control effect compared to other materials.

[0014] Furthermore, the optimal working concentration range for nano-isoflavones in controlling soybean root rot is 10-50 mg / kg.

[0015] The beneficial effects of this invention are as follows:

[0016] 1. Component advantages: The components of nano isoflavones are mainly composed of chitosan, sodium tripolyphosphate and isoflavones. Its elemental composition is mainly C, H, O and Na. It is easily metabolized and transformed, and will not cause cumulative effects in the environment. It has environmentally friendly and sustainable characteristics.

[0017] 2. Advantages in biocompatibility: Since the main components of nano-isoflavones, chitosan and isoflavones, are derived from plants and animals respectively, this nanomaterial has good biocompatibility. It can be observed that it has good biocompatibility in a relatively simple hydroponic environment, and it is free from plant toxicity while helping soybean growth and development.

[0018] 3. Antibacterial Advantages: Nano-isoflavones possess superior antibacterial effects. Even at low concentrations, they exhibit significant inhibitory effects against root rot pathogens, and at medium to high concentrations, they can achieve 100% inhibition. Compared with other nanomaterials on the market used to control soybean root rot, such as nano-copper oxide, nano-iron oxide, and nano-silver, their inhibitory effect is significantly superior.

[0019] 4. Advantages in controlling root rot: Nano-isoflavones exhibit a trace amount and high efficiency in controlling soybean root rot. Compared with the optimal working concentrations of other nanomaterials for root rot, such as 100 mg / kg nano-copper oxide and 50 mg / kg nano-iron oxide, nano-isoflavones have a lower working concentration and better control effect. Attached Figure Description

[0020] Figure 1This is a macroscopic diagram showing nano-isoflavones and other materials. A represents chitosan; B represents isoflavones (category: genistein); C represents sodium tripolyphosphate; L represents a simple mixture of chitosan, isoflavones, and sodium tripolyphosphate in a 2:1:1 mass ratio; N represents nano-isoflavones.

[0021] Figure 2 Material characterization of nano-isoflavones. A shows the macroscopic phenotype of the materials required for the synthesis of nano-isoflavones and their bulk composition; B shows the scanning electron microscope (SEM); C shows the transmission electron microscope (TEM).

[0022] Figure 3 The effect of nano-isoflavones on soybean growth. A is a comparison of soybean growth phenotypes under different treatments after 14 days of hydroponics; B is soybean plant height and root length; C is stem diameter; D is leaf area; E is aboveground / belowground dry weight.

[0023] Figure 4 The inhibitory effect of nano-isoflavones on the germination of *F. oxysporum* spores under alkaline conditions is shown in Figure A. Phenotype of *F. oxysporum* on mung bean soup medium 3 days after inoculation; Figure B shows the inhibitory effect of different treatments on the germination of *F. oxysporum* spores; Figure C shows the inhibition rate of different treatments on the germination of *F. oxysporum* spores. Different lowercase letters represent significant differences (P < 0.05). A1, A2, A3, A4, and A5 represent 20, 100, 200, 1000, and 2000 mg / L chitosan, respectively. B1, B2, B3, B4, and B5 represent 10, 50, 100, 500, and 1000 mg / L isoflavones, respectively. C1, C2, C3, C4, and C5 represent 10, 50, 100, 500, and 1000 mg / L sodium tripolyphosphate (STPP), respectively. L1, L2, L3, L4, and L5 represent 40, 200, 400, 2000, and 4000 mg / L of bulk ABC mixtures, respectively. N1, N2, N3, N4, and N5 represent 40, 200, 400, 2000, and 4000 mg / L of nano-isoflavones, respectively, and the same applies below.

[0024] Figure 5 The inhibitory effect of nano-isoflavones on the germination of F. oxysporum spores under acidic conditions is shown in Figure A. phenotype of F. oxysporum on PDB medium 3 days after inoculation; Figure B shows the inhibitory effect of different treatments on the germination of F. oxysporum spores; Figure C shows the inhibition rate of different treatments on the germination of F. oxysporum spores.

[0025] Figure 6 The inhibitory effect of nano-isoflavones on the radial growth of F. oxysporum hyphae. A shows the inhibitory effect of different treatments on the radial growth of F. oxysporum hyphae; B shows the inhibition rate of different treatments on the radial growth of F. oxysporum spore hyphae.

[0026] Figure 7 The study investigated the control effects of different materials on soybean root rot. A represents soybean growth phenotype; B represents soybean plant height and stem diameter; C represents soybean leaf area and root volume; D represents soybean aboveground and underground dry weight; and E represents disease severity. Significance analysis results are comparisons between each treatment and the control group (CK-F). * represents P < 0.05, and ** represents P < 0.01.

[0027] Figure 8 The study investigated the control effect of nano-isoflavones on soybean root rot. A represents soybean growth phenotype; B represents soybean plant height and stem diameter; C represents soybean leaf area and root volume; D represents soybean aboveground and underground dry weight; E represents chlorophyll a, chlorophyll b, and xanthophyll content; F represents growth and development status (V1 shows the emergence of the first compound leaf, V2 shows the emergence of the second compound leaf); G represents disease severity. N5, N10, N20, N50, and N100 represent 5, 10, 20, 50, and 100 mg / L of nano-isoflavones, respectively. Different lowercase letters indicate statistically significant differences (P < 0.05).

[0028] Figure 9 This is a flowchart illustrating the preparation process of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0030] Example 1. Synthesis of nano-isoflavones: 1.1 Experimental Materials The reagents required for the synthesis of nano-isoflavones are shown in Table 1: Table 1. Raw materials required for the synthesis of nano-isoflavones 1.2 Synthesis steps of nano-isoflavones The experiment first requires the preparation of three reaction solutions, A, B, and C, as follows: Solution A: Dissolve chitosan in 0.8% glacial acetic acid (v / v) solution and stir magnetically at 60℃ for 30 min to obtain a chitosan (w / v) solution with a concentration of 0.3%; Solution B: Dissolve isoflavones in 60% ethanol (v / v) solution and stir magnetically at 60°C for 30 min to obtain a 0.6% genistein (w / v) solution; Solution C: Dissolve sodium tripolyphosphate (STPP) in deionized water to obtain a 0.3% (w / v) STPP solution.

[0031] In the synthesis of nano-isoflavones, the final volume ratio of solutions A, B, and C was 4:1:2 (mass ratio = 2:1:1), and the reaction needed to be carried out at 45℃ using a magnetic stirrer and ultrasonic probe. Specifically, first, four portions of solution A were heated to 60℃, and the temperature was adjusted to 45℃ using a magnetic stirrer (800 rpm / min) and ultrasonic probe (300W, 3s on / 6s off). The mixture was stirred thoroughly for 20 minutes until solutions A and B were uniformly dispersed. Then, two portions of solution C were added dropwise using a burette, with an estimated time of 100 minutes. After titration, the mixture was allowed to stand for 1 minute; the separation of the colloid from the solution indicated successful synthesis. Subsequently, the precipitate was centrifuged at 5000 rpm / min and resuspended in deionized water. The mixture was repeatedly washed until the pH of the washing solution was neutral to remove ethanol and glacial acetic acid. Finally, the precipitate was collected and frozen at -80℃ for 12 hours, followed by vacuum freeze-drying for 72 hours to obtain nano-isoflavone powder, as shown below. Figure 9 As shown.

[0032] 2. Plant safety of nano-isoflavones: The biosafety of nano-isoflavones on soybeans was evaluated using a hydroponic experiment. The hydroponic system was 1 / 4 Hoagland nutrient solution (calcium nitrate 0.945g, potassium sulfate 0.607g, ammonium dihydrogen phosphate 0.115g, magnesium sulfate 0.493g, EDTA iron sodium salt 0.02g, ferrous sulfate 0.15g, boric acid 2.86mg, manganese sulfate 4.5mg, copper sulfate 0.05mg, zinc sulfate 0.22mg, ammonium sulfate 0.02mg, deionized water 1000mL); the soybean variety was Nan Dou 12. The experiment was the same as the antibacterial experiment, including 26 treatments, as shown in Table 2.

[0033] Specifically, healthy soybean seeds of uniform size were selected, sterilized with 30% H2O2 for 10 min, rinsed three times with sterile water, and then placed in sterile vermiculite. They were cultured at 28°C for 3 days under alternating light and dark conditions (8h / 16h). Healthy seedlings of uniform growth were then transplanted into 50 mL centrifuge tubes (containing 45 mL of a modified 1 / 4 Hoagland nutrient solution for each material). Each treatment consisted of five centrifuge tubes, with one soybean seedling in each tube. The tubes were cultured at 28°C for 8h / 16h under alternating light and dark conditions, with an appropriate amount of 1 / 4 Hoagland nutrient solution added daily until 14 days, at which point plant samples were collected. Agronomic traits, including plant height, root length and stem diameter, leaf area, and aboveground and belowground dry weight, were measured to assess phytotoxicity. The experiment was independently repeated three times.

[0034] 3. Antibacterial experiment of nano-isoflavones: The inhibitory effects of nano-isoflavones on spore germination and mycelial growth of *Fusarium oxysporum* (strain B3S1, GenBank: MN892311.1), the main pathogen of soybean root rot, were determined using liquid shaking culture and plate inoculation methods. Overall, the antibacterial experiments included two main categories, each containing 26 treatments: one control (CK) and five different materials with five concentration gradients, totaling 25 treatment groups. The specific settings are shown in Table 2.

[0035] Table 2 Antibacterial Experimental Treatment Settings Note: A, B, C, L, and N represent chitosan, isoflavones, sodium tripolyphosphate, a bulk mixture of ABC, and nano-isoflavones, respectively. 1, 2, 3, 4, and 5 represent five concentrations in mg / L. The mass ratio of substances A, B, and C required for the synthesis of nano-isoflavones is 2:1:1, and the concentrations for each treatment are adjusted proportionally based on the isoflavone concentration.

[0036] First, the pathogenic fungus was inoculated into a 90 mm diameter potato glucose agar (PDA, 6 g potato extract, 20 g glucose, 20 g agar, 1000 mL deionized water) medium and activated for 5 days. Then, 5 mm diameter mycelial cakes were prepared along the outer ring using a punch for later use.

[0037] Spore germination assay: The spore germination assay was conducted under two pH conditions, involving two types of liquid culture media: one was a weakly acidic potato glucose broth (PDB, 5g potato extract, 10g peptone, 15g glucose, and 5g sodium chloride, 1000mL deionized water); the other was a weakly alkaline mung bean soup (15g mung beans, 1000mL deionized water). Specifically, three 5mm diameter fungal discs were inoculated into either PDB or mung bean soup medium (v medium: Erlenmeyer flask = 100 / 150mL), and cultured at 28℃ with shaking at 150rpm / min for 3 days. The culture solution was then serially diluted with sterile water to 10⁻⁶. -4 100 μL of the diluted solution was plated on PDA medium and incubated in the dark at 25°C for 2 days. The spore germination inhibition rate (GI) of each treatment was calculated using the following formula:

[0038] GI (%) = (G1 - G2) / G1 × 100 Wherein, G1 represents the number of spores that germinated in the pathogen control CK2, and G2 represents the number of spores that germinated in the mixed culture of the material and the fungus. Each treatment contains 3 replicates, and the experiment is independently repeated three times.

[0039] Mycelial growth inhibition test: 5 mm fungal discs were inoculated into the center of a 90 mm diameter modified PDA medium and incubated in the dark at 25°C for 5 days. The mycelial growth diameter of the pathogen in each treatment group was measured using calipers, and the mycelial growth inhibition rate (PI) was calculated according to the following formula: PI(%)=(R1-R2) / (R1-5)×100 Where R1 is the mycelial diameter of the pathogen control CK2, R2 is the mycelial diameter of the mixed culture of material and fungus, and 5 represents the size of the mycelial cake (mm). Each treatment contains 3 replicates, and the experiment is independently repeated three times.

[0040] 4. The effect of nano-isoflavones on the control of soybean root rot: The pathogens required for soil improvement were propagated using sorghum grain inoculation. Specifically, *Fusarium oxysporum* (Fo) was cultured in PDA for 5 days. Then, 8mm diameter mycelial cakes were prepared along the outer ring of the hyphae using a punch. Six mycelial cakes were inoculated into sorghum seeds that had undergone double autoclaving and cultured in the dark at 25°C for 7-10 days, gently shaking daily to ensure uniform colonization. The propagated culture was then dried in a 25°C oven for 12 hours and pulverized into powder. The powdered propagated culture was thoroughly mixed with sterile nutrient soil to prepare disease-infected soil. A mixture of Fo-free sorghum grain powder and sterile soil served as a control. The bacterial load per gram of propagated culture was calculated by dilution and plating, and the number of spores germinating was recorded. In summary, the final inoculation concentration of Fo was ensured to be 1×10⁻⁶. 6 ppg per gram of soil.

[0041] 4.1 Assessment of Disease Prevention and Control for Different Materials: Based on the results of antibacterial and plant safety tests, three concentrations corresponding to five materials were further screened to conduct further disease control experiments. In addition, sterile controls and pathogen controls were set up respectively, for a total of 17 treatments to jointly evaluate the disease control effect of nano-isoflavones. The specific treatment settings are shown in Table 3.

[0042] Table 3 Experimental treatment settings for soybean root rot control Note: The concentration units for each material are milligrams per kilogram of soil (mg / kg). The pathogen concentration in the diseased soil is 1×10⁻⁶. 6 ppg per gram of soil, the same below.

[0043] 4.2 Screening of the optimal disease control concentration for nano-isoflavones: The concentration of nano-isoflavones for disease control was further refined. A total of seven treatments were set up in the experiment, as detailed in Table 4:

[0044] Table 4 Experimental treatment settings for the control of soybean root rot by nano-isoflavones The two disease control experiments were similar to plant safety experiments. Soybean seeds were sterilized with 30% H2O2 and germinated in sterile vermiculite for 3 days. Seedlings with uniform growth were transplanted into pots (70mm × 70mm) modified with improved materials and pathogens. Each pot contained 50g of soil and planted 2 seedlings. Each treatment contained 5 pots, for a total of 10 seedlings. All potted plants were arranged in a greenhouse according to a completely randomized block design and cultured for 14 days under 28-35℃, 14 light / 10h dark conditions. The seedlings were then carefully removed, the soil was washed off the roots, and phenotypic data were measured. Symptoms of soybean root rot were observed, and the disease index was calculated based on mortality and a disease grade of 0-4 to evaluate the disease control effect of each treatment. The disease index (DI) formula is as follows:

[0045] DI (%) = ( )×100 result: 5. Material characterization of nano-isoflavones like Figure 1 As shown, at a macroscopic level, chitosan (A) is pale yellow, isoflavone (B) is off-white with a slippery feel, sodium tripolyphosphate (C) is white with a rough, gritty feel, bulk mixture (L) is off-white with an uneven texture, and nano-isoflavone (N) is off-white with a uniform and fluffy texture. Figure 2 As shown, further material characterization of the nano-isoflavones was performed. Scanning electron microscopy (SEM) revealed that the nano-isoflavone powder had a uniform texture and consisted of irregularly shaped spheres. Figure 2 Similarly, transmission electron microscopy (TEM) revealed that the nano-isoflavones exhibited irregular spherical shapes in ethanol solvent. Figure 2 (B); Particle size distribution analysis revealed that the diameter of the nano-isoflavones was approximately 44.46 ± 6.31 nm. Figure 2 (C)

[0046] 6. Nano-isoflavones exhibit excellent biocompatibility and promote soybean growth: The plant safety of each material, such as Figure 3 As shown in Figures A, B, C, D, and E, specifically, under hydroponic conditions, the soybean plant height, root length, stem diameter, leaf area, and above-ground and underground dry weight were 10.3 cm, 11.82 cm, 2.38 cm, and 69.76 cm, respectively. 2The concentrations of chitosan (A) and isoflavone (B) were 23.38 mg / plant and 10.07 mg / plant, respectively. Compared with the control, chitosan (A) treatment increased plant height, root length, stem diameter, and underground dry weight by 12.23–32.62%, -4.9–12.69%, -3.19–7.11%, and 1.79–6.82%, respectively. Isoflavone (B) treatment increased plant height, stem diameter, leaf area, aboveground and underground dry weight by 18.45–37.86%, -6.48–8.12%, -1.57–11.84%, -0.64–15.30%, and -1.71–10.25%, respectively. After treatment with nano-isoflavones (N), soybean plant height, root length, stem diameter, leaf area, and aboveground and underground dry weight increased by 27.18-38.83, -1.69-14.38, 0.63-15.15, 0.65-19.26, 4.19-16.90, and 3.57-39.32, respectively.

[0047] In contrast to the control group, treatment with sodium tripolyphosphate (C) resulted in reductions of stem diameter (0.50-19.44%), leaf area (-1.6-41.04%), aboveground dry weight (0.35-27.00%), and underground dry weight (-1.7-46.83%), respectively. Treatment with ion-dispersed bulk (L) compounds resulted in reductions of soybean root length (2.53-20.14%), leaf area (8.38-50.83%), aboveground dry weight (-1.57-40.06%), and underground dry weight (-10.11-53.9%), respectively.

[0048] Overall, different concentrations of chitosan, isoflavones, and nano-isoflavones all promoted soybean growth and development to some extent, with no significant phytotoxicity and good biocompatibility. However, the mixed treatment with sodium tripolyphosphate and ion-soluble bulk compounds had a certain negative impact on soybean growth, with high concentrations showing a particularly prominent inhibitory effect and exhibiting certain phytotoxicity.

[0049] 7. Nano-isoflavones effectively inhibit the germination and mycelial growth of soybean root rot fungus (F. oxysporum): 7.1 nanometers of isoflavones effectively inhibit the germination of Fusarium oxysporum: 7.1.1 Under alkaline conditions: Under alkaline conditions (mung bean soup culture medium), nano-isoflavones can inhibit the proliferation of pathogens by encapsulating them.

[0050] from Figure 4In medium A, the addition of nano-isoflavones (N) changed the medium from reddish-brown to transparent. Furthermore, in N3, pathogens added at the outer edge were observed to be encapsulated by nano-isoflavones, which may be an important mechanism for inhibiting pathogen reproduction. Additionally, ion mixing (L) and chitosan (A) treatments also made the medium transparent, but only at high concentrations. Isoflavones (B) and sodium tripolyphosphate (C) affected the medium color to some extent, but did not affect the overall condition.

[0051] from Figure 4 As shown in Figures B and C, different concentrations of nano-isoflavones exhibited inhibition rates of 44.30-100% on F. oxysporum spore germination. Low concentrations (such as N1 and N2) showed significant antibacterial activity, while medium-to-high concentrations (such as N3, N4, and N5) could inhibit spore germination by 100%. Bulk mixed treatment showed inhibition rates of 1.26-75.95% on pathogen spore germination, with the highest concentration, L5, failing to achieve 100% inhibition, indicating its antibacterial ability was far inferior to that of nano-treatment. As important components for synthesizing nano-isoflavones, chitosan, isoflavones, and sodium tripolyphosphate (STPP) (C) showed inhibition ranges of 13.92-63.29%, 34.17-86.08%, and -143.04-55.70% on F. oxysporum spore germination, respectively. Chitosan and isoflavones at different concentrations all showed good antibacterial activity, while sodium tripolyphosphate showed a trend of inhibition at low concentrations and promotion of germination at high concentrations.

[0052] 7.1.2 Under acidic conditions pass Figure 5 As shown, in PDB acidic medium, nano-isoflavones (N) can make the medium clear and, similar to alkaline conditions, prevent the proliferation and spread of pathogens by encapsulation. However, ionic mixtures (L), chitosan (A), isoflavones (B), and STPP (D) did not significantly improve the transparency of the medium, nor did they encapsulate pathogens to prevent their proliferation.

[0053] The inhibitory effect on spore germination under acidic conditions is as follows: Figure 5 As shown in Figures B and C, nano-isoflavones maintained stable antibacterial activity. The inhibition rates of N1, N2, and N3 on F. oxysporum germination were 51.43%, 49.52%, and 92.29%, respectively, while the inhibition rates of high concentrations of N4 and N5 were both 100%, highly consistent with the results under alkaline conditions. The antibacterial rate of the ion mixture was enhanced compared to alkaline conditions, with an inhibition range of 26.67-92.38%. The inhibition rates of chitosan, isoflavones, and STPP on F. oxysporum spore germination were 0.95-98.10%, 27.62-91.43%, and -16.19-34.28%, respectively, showing an overall trend consistent with alkaline conditions.

[0054] 7.2 nm isoflavones effectively inhibit the mycelial growth of Fusarium oxysporum: like Figure 6 As shown, different concentrations of nano-isoflavones (N) significantly inhibited the radial growth of *F. oxysporum* hyphae in PDA medium, and the antibacterial ability gradually increased with increasing concentration, ranging from 22.21% to 85.47%. The inhibition rate of the ion mixture (L) ranged from -7.26% to 13.01%, showing a trend of low concentration inhibiting growth while high concentration promoting growth. Chitosan (A) inhibited the radial growth of *F. oxysporum* by -6.56% to 1.35%, primarily promoting mycelial growth. Different concentrations of isoflavones (B) all exhibited certain antibacterial effects, ranging from 11.98% to 26.42%. STPP (C), similar to chitosan, showed a good growth-promoting effect, with an inhibition rate of -11.31% to 0.72%, and the growth-promoting ability increased with increasing concentration.

[0055] 8. Nano-isoflavones can effectively control soybean root rot disease: 8.1 Regulatory Effects of Different Materials on Soybean Root Rot Disease Based on the antibacterial and hydroponic safety test results of each material, three low-concentration treatments were selected to evaluate the control effect of each material on root rot disease, such as... Figure 7 As shown in the figure. The results indicated that chitosan, isoflavones, and nano-isoflavones had certain preventive and control effects on soybean root rot, especially low concentrations of nano-isoflavones. Specifically, compared with healthy controls, infected soybeans showed significant reductions in stem diameter, leaf area, root area, aboveground and underground dry weight by 18.14%, 33.76%, 82.24%, 21.13%, and 59.72%, respectively, with a disease index reduction of 0-23.81%. Different concentrations of isoflavones also improved soybean growth to some extent. Compared with infected controls, plant height, stem diameter, leaf area, root area, aboveground and underground dry weight increased by 9.75-17.21%, 4.28-10.83%, 11.63-25.62%, 29.14-69.29%, 24.60-28.09%, and 7.89-12.12%, respectively, with a disease index reduction of 14.29-33.33%. Similar to chitosan and isoflavones, nano-isoflavones of different concentrations have a strong control effect on root rot disease, and their control effect is negatively correlated with the concentration used. Among them, N1 and N2 have significant effects on soybean disease regulation, especially the low concentration N1 treatment, which significantly increased plant height, stem diameter and leaf area, root area, and aboveground and underground dry weight by 25.98%, 23.79%, 70.80%, 236.45%, 39.01%, and 52.51%, respectively, and significantly reduced the disease index by 52.38%.

[0056] Meanwhile, the other two types of materials, sodium tripolyphosphate and ion-mixed assembly treatment, exhibited completely opposite disease-regulating effects compared to the three materials mentioned above. They provided almost no assistance to soybeans in resisting root rot, and even promoted the occurrence of the disease. Specifically, compared with the susceptible control, the three concentrations of sodium tripolyphosphate reduced soybean plant height, stem diameter, leaf area, root area, and aboveground and underground dry weight by 7.04-9.41%, 1.67-7.96%, 7.70-30.97%, -21.67-36.85%, 9.26-16.40%, and 16.38-22.43%, respectively, while increasing the disease index by 0-14.29%. Compared with the diseased control, the three concentrations of bulk ion-mixed materials increased the height of soybean plants, stem diameter, leaf area, root area, and above-ground and underground dry weight by -0.96-12.38%, -0.56-1.28%, 14.06-45.84%, 19.21-30.86%, 18.32-23.71%, and 17.70-34.00%, respectively, and the disease index increased by 33.33-47.62%.

[0057] Since low concentrations of nano-isoflavones have shown excellent disease control capabilities, further refined experiments were conducted on nano-isoflavones to determine their minimum effective concentration.

[0058] 8.2 The regulatory effects of different concentrations of nano-isoflavones on soybean root rot: like Figure 8 As shown, compared with healthy controls, diseased soybeans exhibited significantly reduced plant height, stem diameter, leaf area, root area, aboveground / belowground dry weight, chlorophyll a, chlorophyll b, and carotenoid content by 37.09%, 13.25%, 77.22%, 77.99%, 59.89%, 78.55%, 44.41%, 36.33%, and 46.78%, respectively, with a disease index reaching 75. Furthermore, after 14 days of growth, the plants only reached stage V1, while 90% of healthy controls entered stage V2. Compared with diseased controls, application of nano-isoflavones at concentrations of 5, 10, 20, 50, and 100 all showed varying degrees of positive regulatory effects on root rot.

[0059] Specifically, 5 mg / kg nano-isoflavones significantly increased soybean stem diameter, leaf area, root volume, and aboveground dry weight by 13.25%, 45.12%, 107.78%, 16.44%, and 146.18%, respectively, while significantly reducing the disease index by 23.33, resulting in 20% of soybeans entering the v2 stage. 10 mg / kg nano-isoflavones significantly increased soybean plant height, stem diameter, leaf area, root volume, aboveground dry weight, chlorophyll a, chlorophyll b, and carotenoids by 53.16%, 9.41%, 196.07%, 271.37%, 93.15%, 88.90%, 76.92%, and 66.54%, respectively, while significantly reducing the disease index by 40%, resulting in 60% of soybeans entering the v2 stage. 20 mg / kg nano-isoflavones significantly increased soybean stem diameter, leaf area, aboveground dry weight, chlorophyll a, chlorophyll b, and carotenoids by 12.38%, 134.25%, 67.91%, 102.03%, 92.97%, 78.09%, and 81.46%, respectively, while significantly reducing the disease index by 30%, resulting in 50% of soybeans entering the V2 stage. 50 mg / kg nano-isoflavones significantly increased soybean plant height, stem diameter, leaf area, aboveground and underground dry weight, chlorophyll a, chlorophyll b, and carotenoids by 33.64%, 15.68%, 213.24%, 216.75%, 94.17%, 92.62%, 76.77%, and 78.24%, respectively, while significantly reducing the disease index by 43.33%, resulting in 70% of soybeans entering the V2 stage. 100 mg / kg nano-isoflavones significantly increased soybean leaf area, aboveground and underground dry weight by 54.70%, 21.12%, 50.42%, 42.87%, and 40.23%, respectively, while significantly reducing the disease index by 20%, and causing 30% of soybeans to enter the v2 stage.

[0060] Overall, all five concentrations of nano-isoflavones showed considerable control effects on root rot, with 10-50 mg / kg showing the best control effect.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for synthesizing nano-isoflavones, characterized in that, include: Step 1. Prepare solutions A, B, and C, three solutions for the reaction; Among them, solution A is a 0.3% chitosan w / v solution, solution B is a 0.6% genistein w / v solution, and solution C is a 0.3% sodium tripolyphosphate w / v solution; Step 2. Mix the three solutions A, B, and C from Step 1 in a volume ratio of 4:1:2 and stir magnetically at 45°C to obtain the final product.

2. The method according to claim 1, characterized in that, The preparation of solution A includes: dissolving chitosan in a 0.8% glacial acetic acid v / v solution, stirring magnetically at 60°C for 30 min to obtain a chitosan w / v solution with a concentration of 0.3%.

3. The method according to claim 1, characterized in that, The preparation of solution B includes: dissolving isoflavones in a 60% ethanol v / v solution, stirring magnetically at 60°C for 30 min to obtain a 0.6% genistein w / v solution.

4. The method according to claim 1, characterized in that, The preparation of solution C includes: dissolving sodium tripolyphosphate in deionized water to obtain a 0.3% sodium tripolyphosphate w / v solution.

5. The method according to claim 1, characterized in that, Step 2 includes: taking 4 portions of solution A and heating them to 60°C, slowly adding 1 portion of solution B under magnetic stirring and ultrasonic probe conditions, adjusting the temperature to 45°C, and stirring thoroughly for 20 minutes until solutions A and B are evenly dispersed. Then, adding 2 portions of solution C dropwise using a burette, with an estimated time of 100 minutes. Subsequently, the precipitate is centrifuged at 5000 rpm / min and resuspended in deionized water, repeatedly washed until the pH of the washing solution is neutral to remove ethanol and glacial acetic acid. Finally, the precipitate is collected and frozen at -80°C for 12 hours, followed by vacuum freeze-drying for 72 hours to obtain nano-isoflavone powder.

6. The application of the nano-isoflavone synthesis method according to any one of claims 1-5 in the treatment of soybean root rot, characterized in that, The nano-isoflavones are spherical with a particle size of 44.46±6.31 nm.

7. The application of the nano-isoflavone synthesis method according to any one of claims 1-5 in the treatment of soybean root rot, characterized in that, Nano-isoflavones can be used to inhibit the growth of Fusarium oxysporum.

8. The application of the nano-isoflavone synthesis method according to any one of claims 1-5 in soybean root rot, characterized in that, When controlling soybean root rot, the working concentration is 10-50 mg / kg.

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