Microorganism-based nano-enzyme as well as preparation method and application thereof
By preparing microbial-based nanozymes and using teliospores of Ustilago maydis as templates for manganese ion deposition and slow-release coating, the problem of tobacco bacterial wilt control was solved, achieving an environmentally friendly and highly effective control effect.
Patent Information
- Application Number
- CN202510950204.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-10
- Publication Date
- 2025-10-28
AI Technical Summary
Current technologies lack research on the use of microbial-based nanozymes to control tobacco bacterial wilt, and there are problems such as environmental pollution, soil degradation, and increased bacterial resistance.
By pretreating teliospores of Ustilago maydis with low-temperature plasma and then carbonizing them with high temperature to generate biomass carbon microparticles, which are used as templates for manganese ion deposition, combined with slow-release coating technology, microbial-based nanozymes are prepared for the prevention and control of tobacco bacterial wilt.
It provides a safe and efficient biological control method, reduces preparation costs, reduces environmental pollution, and improves control effects, exhibiting targeted and highly effective inhibition of tobacco bacterial wilt.
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Figure CN120838404A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant disease control technology, specifically relating to a microbial-based nanozyme, its preparation method, and its application. Background Technology
[0002] Tobacco bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum, and it is a serious disease affecting tobacco yield and quality. Once it occurs, it causes poor growth and wilting of tobacco plants, resulting in huge economic losses to the tobacco industry. Currently, the control of tobacco bacterial wilt mainly relies on chemical pesticides, but long-term use of chemical pesticides leads to environmental pollution, soil degradation, and increased bacterial resistance. Integrated management of tobacco bacterial wilt has become a research hotspot, including the breeding of disease-resistant varieties, optimization of agricultural management practices, and the development of biological control technologies. Among these, biological control technologies have gained attention due to their green, environmentally friendly, and sustainable nature, and the natural source of microorganisms makes them play an important role in ecological balance and disease management.
[0003] Nanomaterials have demonstrated broad application potential in multiple fields due to their unique physicochemical properties. In recent years, novel strategies combining nanotechnology with traditional biological control have emerged. For example, by synthesizing specific nanozymes, the cell walls of pathogens can be disrupted, thereby inhibiting their growth and development; applying microorganisms to the synthesis of nanozymes can not only provide biological templates but also enhance the biocompatibility and environmental friendliness of the nanozymes.
[0004] Currently, there is a lack of research and application of microbial-based nanozymes in the control of tobacco bacterial wilt, and the stability, biosafety, and application effects of microbial-based nanozymes in different soil environments require further investigation. Therefore, those skilled in the art are eager to develop a strategy for the biocontrol of tobacco bacterial wilt based on microbial-based nanozymes, aiming to combine nanotechnology with the growth-promoting properties of microorganisms to leverage the potential of microbial nanozymes in inhibiting bacterial growth, thus providing a safe and efficient new control method for tobacco bacterial wilt. Summary of the Invention
[0005] This invention addresses the technical problems of existing chemical control methods for tobacco bacterial wilt, which include environmental pollution, soil degradation, and increased bacterial resistance. It provides a microbial-based nanozyme, its preparation method, and its application.
[0006] One objective of this invention is to provide a method for preparing microbial-based nanozymes, the method comprising the following steps: S1: High-temperature carbonization treatment of pretreated Ustilago esculent teliospores to obtain biomass carbon microparticles; S2: Using the biomass carbon microparticles obtained in S1 as templates, manganese ion deposition was performed in a manganese source solution, followed by washing and drying to obtain a Ustilago esculent teliospore-manganese-based nanozyme complex. S3: The corn smut spore-manganese-based nanozyme complex obtained in S2 was encapsulated with a biodegradable material using a slow-release coating technology to obtain a microbial-based nanozyme.
[0007] In a preferred embodiment of the present invention, the teliospores of the genus *Ustilago maydis* mentioned in S1 include, but are not limited to, *Ustilago maydis* of corn nodules and *Ustilago maydis* of sorghum spikes.
[0008] In a preferred embodiment of the present invention, the pretreatment step in S1 is: low-temperature plasma pretreatment of teliospores of the genus Ustilago maydis.
[0009] In a preferred embodiment of the present invention, the high-temperature carbonization treatment in S1 is carried out at a temperature of 300-500°C for 1-2 hours.
[0010] In a preferred embodiment of the present invention, the manganese source solution in S2 includes, but is not limited to, manganese nitrate solution and manganese chloride solution; the pH value of the manganese source solution is 6-7.
[0011] In a preferred embodiment of the present invention, the manganese ion deposition treatment step in S2 is as follows: the biomass carbon microparticles obtained in S1 are mixed with the manganese source solution at a mass-volume ratio of 1:1, and stirred at room temperature for 4-6 hours. The pH of the above mixed solution is adjusted to 10 using a NaOH solution of ≤1 mmol / L, and then the temperature is raised to 60-80°C and the reaction continues for 6-12 hours.
[0012] In a preferred embodiment of the present invention, the biodegradable material described in S3 includes, but is not limited to, polylactic acid.
[0013] A second objective of this invention is to provide a microbial-based nanozyme, which is obtained using the above-described preparation method.
[0014] The third objective of this invention is to provide the application of the above-mentioned microbial-based nanozymes in the prevention and control of tobacco bacterial wilt.
[0015] In a preferred embodiment of the present invention, the application involves spreading microbial-based nanozymes on tobacco planting soil for prevention and control, wherein the amount of microbial-based nanozymes applied is 5-10 grams per square meter.
[0016] The beneficial effects of this invention are as follows: This invention provides a microbial-based nanozyme. Low-temperature plasma pretreatment of *Ustilago maydis* teliospores removes impurities from the spore surface and increases their specific surface area. The pretreated teliospores are then subjected to high-temperature carbonization. Using the generated biomass carbon microparticles as templates, manganese-based nanozymes are obtained through manganese ion deposition. This invention introduces auxiliary materials such as selenides to adjust the nanozyme structure and catalytic activity. Utilizing the catalytic activity and bacterial wall-destructive ability of this manganese-based nanozyme, it specifically inhibits bacteria that cause tobacco bacterial wilt, thereby achieving the purpose of treating and preventing tobacco bacterial wilt. This invention employs a slow-release coating technology to encapsulate the *Ustilago maydis* teliospore-manganese-based nanozyme complex with biodegradable materials to prolong its action time in the soil, while also helping to reduce potential impacts on non-target organisms and improve the control effect.
[0017] The microbial-based nanozyme preparation method provided by this invention utilizes the host pathogens of crops as material sources, thereby reducing preparation costs. The provided microbial-based nanozyme reduces the use of chemical pesticides, lessens the burden on the environment, is more environmentally friendly, has no secondary pollution, and is highly safe. The provided microbial-based nanozyme is more targeted and efficient in inhibiting the bacterial wilt pathogen of tobacco, improving the control effect of tobacco bacterial wilt and providing a new approach for its application in plant disease control. Attached Figure Description
[0018] Figure 1 This is a transmission electron microscope image of corn smut spores after high-temperature carbonization treatment in Example 1; Figure 2 This is a transmission electron microscope image of the corn nodule black spore-manganese-based nanozyme complex in Example 1; Figure 3 This is a transmission electron microscope image of the corn spore-manganese nanozyme complex after low-temperature plasma pretreatment in Example 2. Detailed Implementation
[0019] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.
[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.
[0021] Example 1: S1: High-temperature carbonization treatment of corn smut spores is performed at 400°C for 1 hour to obtain biomass carbon microparticles. The transmission electron microscopy image of the corn smut spores after the high-temperature carbonization treatment is shown below. Figure 1 As shown; S2: Using the biomass carbon microparticles obtained in S1 as a template, manganese ion deposition treatment was performed in a manganese nitrate solution (pH 6-7). The manganese ion deposition treatment steps were as follows: 10 mg of biomass carbon microparticles were added to 10 mL of manganese nitrate solution (50 mmol / L), and the mixture was stirred at room temperature for 4 hours. The pH of the mixture was adjusted to 10 using NaOH solution (≤1 mmol / L), and the temperature was raised to 60℃ for another 10 hours. The mixture was washed with water and dried to obtain a corn spore-manganese nanozyme complex. The transmission electron microscopy image of the corn spore-manganese nanozyme complex is shown below. Figure 2 As shown.
[0022] Example 2: S1: The corn nodule spores pretreated with low-temperature plasma are subjected to high-temperature carbonization treatment at a temperature of 400°C for 1 hour to obtain biomass carbon microparticles. S2: Using the biomass carbon microparticles obtained in S1 as a template, manganese ion deposition treatment was performed in a manganese nitrate solution (pH 6-7). The manganese ion deposition treatment steps were as follows: 10 mg of biomass carbon microparticles were added to 10 mL of manganese nitrate solution (50 mmol / L), and the mixture was stirred at room temperature for 4 hours. The pH of the mixture was adjusted to 10 using NaOH solution (≤1 mmol / L), and the temperature was raised to 60℃ for another 10 hours. The mixture was washed with water and dried to obtain the corn spore-manganese nanozyme complex. The transmission electron microscope image of the corn spore-manganese nanozyme complex after low-temperature plasma pretreatment is shown below. Figure 3 As shown; S3: The corn spore-manganese-based nanozyme complex obtained in S2 was encapsulated with polylactic acid biodegradable material using a slow-release coating technology to obtain microbial-based nanozymes.
[0023] Comparative Example 1: This comparative example involves pretreating corn nodule spores with low-temperature plasma and then subjecting them to high-temperature carbonization treatment at 400°C for 1 hour to obtain biomass carbon microparticles.
[0024] Comparative Example 2: This comparative example is a template-free synthesized manganese-based nanozyme, which undergoes manganese ion deposition treatment in a manganese chloride solution (pH 6-7). The manganese ion deposition treatment steps are as follows: 10 mL of manganese chloride solution (50 mmol / L) is stirred at room temperature for 4 hours, the pH of the above mixed solution is adjusted to 10 using NaOH solution (≤1 mmol / L), and the temperature is raised to 60℃ and the reaction is continued for 10 hours; after washing with water and drying, the manganese-based nanozyme is obtained.
[0025] Effect Experiment: 1. The antibacterial effects of the corn nodule smut spore-manganese nanozyme complex prepared in Example 1, the biomass carbon microparticles prepared in Comparative Example 1, and the manganese nanozyme prepared in Comparative Example 2 were detected using the Oxford cup method. The specific steps are as follows: Take 500 μL 1×10 8 CFU / mL of Ralstonia solanacearum bacterial suspension was evenly spread on solid LB agar plates (solid LB composition: 1% tryptone, 0.5% yeast extract, 1% sodium chloride) and incubated at 37℃ for 1 h. Two Oxford cups (6 mm inner diameter) were placed on the solid medium, and 50 μL of the corn nodule spore-manganese nanozyme complex prepared in Example 1 with concentrations of 10 mg / mL, 50 mg / mL, and 100 mg / mL were added respectively; as well as biomass carbon microparticles prepared in Comparative Example 1 with a concentration of 100 mg / mL and manganese nanozyme prepared in Comparative Example 2 with a concentration of 100 mg / mL. Sterile water was selected as a blank control group. After incubation at 37℃ for 24 h, the antibacterial effect was observed. Each treatment was set up in triplicate, and the antibacterial rate was counted.
[0026] Antibacterial rate = (diameter of inhibition zone - diameter of Oxford cup) / diameter of inhibition zone × 100%.
[0027] The results are shown in Table 1. The corn spore-manganese nanozyme complex prepared in Example 1 has a good antibacterial effect against Ralstonia solanacearum, and the antibacterial effect increases with the increase of the concentration of the corn spore-manganese nanozyme complex. However, the biomass carbon microparticles in Comparative Example 1 have no inhibitory effect on Ralstonia solanacearum. The manganese nanozyme in Comparative Example 2 has a certain antibacterial effect on Ralstonia solanacearum, but its antibacterial rate is significantly lower than that of the corn spore-manganese nanozyme complex at the same concentration.
[0028] Table 1
[0029] 2. Application of corn smut spore-manganese-based nanozyme complex in the control of tobacco bacterial wilt The corn smut spore-manganese nanozyme complex prepared in Example 1 was evenly spread on the tobacco planting soil. The sample area was 10 square meters. It was applied according to the standard of 5-10 grams per square meter. The application rates for the sample plots were as follows: sample plot 1: 5 g / m², sample plot 2: 7.5 g / m², sample plot 3: 10 g / m², and sample plot 4: The sample plot without corn smut spore-manganese nanozyme complex was used as a blank control group. The control effect of the above sample plots on tobacco bacterial wilt was tested.
[0030] Prevention efficacy (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.
[0031] The results are shown in Table 2. The disease index of tobacco bacterial wilt was low in the plots where the corn smut spore-manganese nanozyme complex prepared in Example 1 was applied. The control efficacy of tobacco bacterial wilt in the plots with application rates of 5 g, 7.5 g and 10 g / m² was 39.99%, 40.25% and 41.48%, respectively.
[0032] Table 2
[0033] 3. The antibacterial effect of the microbial-based nanozyme prepared in Example 2 was detected using the Oxford cup method. The specific steps are as follows: Take 500 μL 1×10 8 CFU / mL of Ralstonia solanacearum bacterial suspension was evenly spread on solid LB agar plates (solid LB composition: 1% tryptone, 0.5% yeast extract, 1% sodium chloride) and incubated at 37℃ for 1 h. Two Oxford cups (6 mm inner diameter) were placed on the solid culture medium, and 50 μL of the microbial-based nanozyme prepared in Example 1 at concentrations of 10 mg / mL, 50 mg / mL, and 100 mg / mL were added respectively, along with sterile water as a blank control. After incubation at 37℃ for 24 h, the antibacterial effect was observed. Each treatment was set up in triplicate, and the antibacterial rate was calculated.
[0034] Antibacterial rate = (diameter of inhibition zone - diameter of Oxford cup) / diameter of inhibition zone × 100%.
[0035] The results are shown in Table 3. The microbial-based nanozyme prepared in Example 2 has a good antibacterial effect against Ralstonia solanacearum. Compared with the antibacterial effect of the corn spore-manganese-based nanozyme complex prepared in Example 1, the antibacterial rate against Ralstonia solanacearum was increased from 67.66%-81.52% to 70.54%-86.32%.
[0036] Table 3
[0037] 4. Application of microbial-based nanozymes in the control of tobacco bacterial wilt The microbial-based nanozyme prepared in Example 2 was evenly spread on the tobacco planting soil. The sample area was 10 square meters. It was applied according to the standard of 5-10 grams per square meter. The application rates for the sample plots were as follows: sample plot 5: 5 g / m², sample plot 6: 7.5 g / m², sample plot 4: 10 g / m², and sample plot 8: The sample plot without microbial-based nanozyme application was used as a blank control group. The efficacy of the above sample plots was tested.
[0038] Prevention efficacy (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.
[0039] The results are shown in Table 4. The disease index of tobacco bacterial wilt was lower in the plots treated with the microbial nanozyme prepared in Example 2. The control efficacy of the plots treated with 5 g, 7.5 g, and 10 g / m² was 42.31%, 45.35%, and 47.84%, respectively. Compared with the antibacterial effect of the corn smut spore-manganese nanozyme complex prepared in Example 1, the antibacterial effect was significantly improved, from 39.99%-41.48% to 42.31%-47.84%.
[0040] Table 4
[0041] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A method for preparing a microbial-based nanozyme, characterized in that, The preparation method includes the following steps: S1: High-temperature carbonization treatment of pretreated Ustilago esculent teliospores to obtain biomass carbon microparticles; S2: Using the biomass carbon microparticles obtained in S1 as templates, manganese ion deposition was performed in a manganese source solution, followed by washing and drying to obtain a Ustilago esculent teliospore-manganese-based nanozyme complex. S3: The corn smut spore-manganese-based nanozyme complex obtained in S2 was encapsulated with a biodegradable material using a slow-release coating technology to obtain a microbial-based nanozyme.
2. The preparation method according to claim 1, characterized in that, The teliospores of the genus *Ustilago maydis* mentioned in S1 include, but are not limited to, *Ustilago maydis* of corn nodules and *Ustilago maydis* of sorghum spikes.
3. The preparation method according to claim 1, characterized in that, The pretreatment step described in S1 is: low-temperature plasma pretreatment of teliospores of the genus Ustilago maydis.
4. The preparation method according to claim 1, characterized in that, The high-temperature carbonization process described in S1 is carried out at a temperature of 300-500℃ for 1-2 hours.
5. The preparation method according to claim 1, characterized in that, The manganese source solution mentioned in S2 includes, but is not limited to, manganese nitrate solution and manganese chloride solution; the pH value of the manganese source solution is 6-7.
6. The preparation method according to claim 1, characterized in that, The manganese ion deposition treatment step described in S2 is as follows: the biomass carbon microparticles obtained in S1 are mixed with the manganese source solution at a mass-volume ratio of 1:1, and the mixture is stirred at room temperature for 4-6 hours. The pH of the mixture is adjusted to 10 using a NaOH solution of ≤1 mmol / L, and the mixture is then heated to 60-80℃ and the reaction continues for 6-12 hours.
7. The preparation method according to claim 1, characterized in that, The biodegradable materials described in S3 include, but are not limited to, polylactic acid.
8. A microbial-based nanozyme, characterized in that, The microbial-based nanozyme is obtained by the preparation method described in any one of claims 1 to 7.
9. The application of the microbial-based nanozyme according to claim 8 in the prevention and control of tobacco bacterial wilt.
10. The application according to claim 9, characterized in that, The application involves spreading microbial-based nanozymes in the soil of tobacco plantations for prevention and control, with an application rate of 5-10 grams per square meter.