Nanometer copper hydroxide and preparation method and application thereof

Abstract

The application discloses nano copper hydroxide and a preparation method and application thereof, and relates to the field of nano copper hydroxide. The application is characterized in that: copper chloride is dissolved in deionized water, and then glycol and ethanol are added to obtain a copper chloride solution; sodium hydroxide solution is added dropwise into the copper chloride solution, and after the dropwise addition is completed, the mixture is stirred to obtain a suspension; the suspension is aged, and then filtered to obtain nano copper hydroxide. The nano copper hydroxide can activate plant defense response and reduce the incidence rate, so that the nano copper hydroxide can be used as an effective component of nano pesticide. The nano copper hydroxide can improve the drought resistance of plants and enhance the survival rate of plants in a drought environment.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, specifically to a nano-copper hydroxide, its preparation method, and its application. Background Technology

[0002] my country is a major agricultural country, and grain is a necessity for its people, playing a vital role in the stable growth of the national economy. However, crop diseases are currently severe, with various pathogens causing losses of billions of dollars in crops annually. Currently, these problems are typically addressed through chemical control methods, using pesticides to quickly and comprehensively kill pathogens directly, thus controlling the diseases. However, over-reliance on chemical pesticides can easily lead to crop damage, the development of pesticide resistance in pathogens, and other side effects. It also causes social and ecological problems such as pesticide residues and soil compaction, disrupting the ecological balance, hindering agricultural development, and threatening human health. Therefore, finding new, low-toxicity, highly efficient, and environmentally friendly methods for disease control is urgently needed.

[0003] Similar to animals, plants also possess an immune system, which allows them to resist pathogen infection through their own immunity. Plant immunity does not directly kill pathogens, but rather activates relevant disease-resistance genes by controlling the plant's defense and metabolic systems, thereby enhancing the plant's resistance to pathogens. Once a plant develops an immune response, it can defend against various diseases, extending from local tissues to the entire plant, and its effects are long-lasting, thus exhibiting systemic, broad-spectrum, and stable characteristics.

[0004] Commercially available copper-based pesticides for disease control range in concentration from 0.3 g / L to 1000 g / L. DuPont's Kocide 3000 is the most effective, but its application concentration remains above 0.3 g / L. Although copper ions can enhance plant immunity at low concentrations (0.05 g / L-1 g / L), their field disease control efficacy is poor and unstable. In summary, existing copper-based pesticides still suffer from high application concentrations, poor control efficacy, and poor stability. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a nano-copper hydroxide, its preparation method and application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of this invention provides a method for preparing nano-copper hydroxide, comprising the following steps:

[0008] Copper chloride was dissolved in deionized water, and then ethylene glycol and ethanol were added to obtain a copper chloride solution. Sodium hydroxide solution was added dropwise to the copper chloride solution, and the mixture was stirred after the addition was completed to obtain a suspension. The suspension was aged and filtered to obtain nano copper hydroxide.

[0009] Preferably, the ratio of the amount of copper chloride, ethylene glycol, ethanol, deionized water and sodium hydroxide solution added is (5-8)g:(15-25)mL:(40-60)mL:(40-60)mL:(250-300)mL, and the concentration of sodium hydroxide solution is 0.5mol / L.

[0010] Preferably, the sodium hydroxide solution is added at a rate of 1.5-2.5 mL / min; the stirring time is 1.5-2.5 h, the stirring speed is 300-800 rpm; the aging time is 10-15 h, and the aging temperature is room temperature.

[0011] The second aspect of the present invention provides nano-copper hydroxide obtained by the above preparation method, wherein the nano-copper hydroxide has a long rivet-like structure with a length of 100-500 nm and a diameter of 10-80 nm.

[0012] A third aspect of the present invention provides the application of the above-described nano-copper hydroxide in any of the following:

[0013] (1) Prevention and control of crop diseases;

[0014] (2) Enhance plant immunity and resistance;

[0015] (3) Improve plant drought resistance.

[0016] Preferably, the nano-copper hydroxide is used as a pesticide formulation, and the concentration of the nano-copper hydroxide is 0.001-0.01 g / L.

[0017] More preferably, the pesticide formulation further includes a surfactant, wherein the volume fraction of the surfactant is 0.03-0.05%.

[0018] Preferably, the crop disease is bacterial leaf streak of rice.

[0019] The beneficial effects of this invention are:

[0020] This invention is the first to prepare rivet-shaped nano-copper hydroxide, which can activate plant defense responses and reduce disease incidence. Therefore, nano-copper hydroxide can be used as an effective component of nano-pesticides.

[0021] This invention belongs to a novel nano-pesticide formulation, which has the characteristics of low pollution, high activity and good effect. Its functional active concentration is more than 1 / 100 of that of copper ions and Kocide 3000.

[0022] In this invention, nano-copper hydroxide can improve plant drought resistance and enhance plant survival rate in arid environments.

[0023] This invention is mainly applied by spraying, which is simple and convenient to operate. It can be sprayed in advance during the peak period of plant disease to prevent large-scale disease outbreaks. Attached Figure Description

[0024] Figure 1 Transmission electron microscope and scanning electron microscope images of nano-copper hydroxide;

[0025] Figure 2 Particle size distribution of nano-copper hydroxide;

[0026] Figure 3 The disease control efficacy of nano-copper hydroxide against bacterial leaf streak in rice;

[0027] Figure 4 The effect of nano-copper hydroxide on inducing ROS burst and callosity deposition;

[0028] Figure 5 Nano-copper hydroxide activates the expression of disease-resistant genes;

[0029] Figure 6 Nano-copper hydroxide improves the drought resistance of rice;

[0030] Figure 7 The effect of nano-copper hydroxide on the opening and closing of rice stomata. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] As described in the background section, copper-based pesticides are widely used in the prior art, with copper hydroxide being a particularly prevalent pesticide. Both excessively high and low concentrations of pesticides can affect their control efficacy. Although copper ions can enhance plant immunity at low concentrations (0.05 g / L-1 g / L), their field disease control effect is poor and unstable.

[0033] In response to this situation, this invention conducted in-depth research on copper hydroxide and discovered that different preparation methods can affect the morphology and structure of copper hydroxide, which in turn affects its application effects. This invention, through improvements to the preparation method, yields elongated, rivet-shaped nano-copper hydroxide. Compared to traditional spherical nano-copper hydroxide, it exhibits stronger adhesion to plant leaves. Furthermore, the rivet-shaped nano-copper hydroxide can promote stomatal closure, defend against pathogen invasion, and thus demonstrate more significant application effects.

[0034] The key steps in this invention for generating rivet-shaped nano-copper hydroxide are stirring time, dropping rate, and reaction temperature. A dropping rate of 1.5-2.5 mL / min, a stirring time of 1.5-2.5 h, and aging at room temperature are crucial for ensuring the synthesis of nano-copper hydroxide, preventing its agglomeration, and improving the synthesis rate. Changing the reaction conditions—such as a dropping rate higher than 2.5 mL / min or lower than 1.5 mL / min, a stirring time higher than 2.5 h or lower than 1.5 h, or an aging temperature higher or lower than room temperature—will alter the length and diameter of the nano-copper hydroxide, leading to changes in its morphology, suspension properties, and increased degradability, thus affecting its stability. Using ethylene glycol and ethanol as solvents during preparation provides good suspension properties, effectively preventing the nano-copper hydroxide from agglomerating, and is also low-cost, low-toxicity, and does not affect the subsequent functions of the nano-copper hydroxide.

[0035] This invention also unexpectedly discovered that, at the same concentration, rivet-shaped nano-copper hydroxide can more significantly promote the accumulation of reactive oxygen species and callose deposition in plant leaves, upregulate the expression of disease resistance-related genes, and close plant stomata, while also enhancing plant drought resistance. The disease resistance activity of the nano-copper hydroxide of this invention is significantly stronger than that of traditional copper ions (above 0.5 g / L) and copper hydroxide 3000 (above 0.3 g / L). Nano-copper hydroxide can significantly enhance plant disease resistance at a concentration of 0.005 g / L, and its concentration is 1 / 100th that of copper ions and copper hydroxide 3000, achieving significant application effects at a lower concentration.

[0036] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0037] All experimental materials used in the embodiments of this invention are conventional experimental materials in the art and can be purchased through commercial channels. The rice bacterial leaf streak fungus RS105 used in the following embodiments of this invention is a strain that has been stored in the laboratory for a long time. The copper sulfate was purchased from Sigma, catalog number: C1297, with a purity of ≥99%, and Kocide 3000 was purchased from DuPont, USA.

[0038] Example

[0039] Preparation of nano copper hydroxide:

[0040] Dissolve 6.5 g of copper chloride in 50 mL of deionized water, add 20 mL of ethylene glycol and 50 mL of ethanol to obtain a copper chloride solution. Prepare a 0.5 mol / L sodium hydroxide solution, and pipette 270 mL into a separatory funnel for later use. Add the 0.5 mol / L sodium hydroxide solution dropwise to the copper chloride solution at a rate of 2 mL / min, while stirring at 500 rpm. After the addition is complete, stir continuously for 2 hours. Then age the suspension at room temperature for 12 hours. Wash and collect the product. After filtration, the precipitate obtained is nano-copper hydroxide. Wash the nano-copper hydroxide with distilled water until pH = 7. Finally, collect the nano-copper hydroxide particles in plastic centrifuge tubes and protect them with alcohol.

[0041] The nano-copper hydroxide solution was air-dried on a silicon wafer, then sputtered with gold, and observed under a scanning electron microscope. Alternatively, the nano-copper hydroxide solution was dropped onto a transmission electron microscope slide, dried, and then observed directly under both TEM and SEM.

[0042] The results are as follows Figure 1 As shown, transmission electron microscopy revealed that the nano-copper hydroxide exhibited a rivet-like structure and was relatively uniformly dispersed. Scanning electron microscopy showed that the nano-copper hydroxide exhibited a long, rivet-like structure, consistent with the results of transmission electron microscopy. This structure may make it easier for the nano-copper hydroxide to adhere to plant leaves.

[0043] The particle size of nano-copper hydroxide was determined using "ZetasizerHelix" nanometrics manufactured by Malvern Panalytical, UK, and further measured and verified using ImageJ software in conjunction with scanning electron microscopy and transmission electron microscopy images.

[0044] The results are as follows Figure 2 As shown, the length of the nano copper hydroxide is between 100-500 nm, and the diameter of its long strip is between 10-80 nm.

[0045] Experimental Example

[0046] 1. Disease resistance effect against bacterial leaf streak of rice

[0047] Experimental crop: Rice (specifically, japonica rice Zhonghua 11).

[0048] Cultivation method: Rice is grown in a greenhouse with alternating 16 hours of light and 8 hours of darkness each day. The cultivation temperature is 28℃ and the air humidity is 60-70%. The greenhouse is located in the State Key Laboratory of Crop Biology at Shandong Agricultural University.

[0049] Test method:

[0050] Add 0.05-500 μg / mL of nano-copper hydroxide solution (with 0.03% surfactant Silwet-77) and 0.05-500 μg / mL of copper sulfate (active ingredient Cu). 2+ Add 0.03% surfactant Silwet-77 and 0.05-500 μg / mL of copper hydroxide (active ingredient: copper hydroxide, concentration: 0.05-500 μg / mL, with added 0.03% surfactant Silwet-77) and spray onto rice leaves and stems cultured for 6 weeks. Use a 0.03% Silwet-77 aqueous solution as a control. Two hours later, rinse the pre-cultured rice bacterial leaf streak pathogen RS105 with sterile deionized water and adjust the OD of the bacterial solution. 600 The value was 0.5. Rice leaves were inoculated using a sterile medical syringe. Ten days after inoculation, diseased parts of the rice were cut off, and the phenotype of the lesions was photographed, and the length of the lesions was measured.

[0051] The results are as follows Figure 3 As shown, after spraying nano copper hydroxide, copper sulfate, and copper oxychloride 3000, compared with the control, nano copper hydroxide at a concentration of 5 μg / mL had the best effect on reducing lesion length. Moreover, the ability of 5 μg / mL nano copper hydroxide to reduce lesion length was similar to that of 50-500 μg / mL copper sulfate and copper oxychloride 3000. This indicates that the disease resistance activity of nano copper hydroxide is 10-100 times that of copper sulfate and copper oxychloride 3000.

[0052] The above experimental results show that exogenous spraying of the nano-copper hydroxide (containing 0.03% Silwet-77) of this invention can stimulate rice resistance to bacterial leaf streak and improve rice's disease resistance. This indicates that the nano-pesticide of this invention has the potential to prevent bacterial leaf streak in rice.

[0053] 2. Effects on reactive oxygen species (ROS) bursts and callose deposition in rice

[0054] Experimental crop: Rice (specifically, japonica rice Zhonghua 11).

[0055] Cultivation method: Rice is grown in a greenhouse with alternating 16 hours of light and 8 hours of darkness each day. The cultivation temperature is 28℃ and the air humidity is 60-70%. The greenhouse is located in the State Key Laboratory of Crop Biology at Shandong Agricultural University.

[0056] Test method:

[0057] Add 5 μg / ml of nano copper hydroxide (containing 0.03% Silwet-77) and copper sulfate (the active ingredient is Cu) 2+A solution containing 0.03% surfactant Silwet-77 and 0.05-500 μg / mL of Kocide 3000 (active ingredient Cu(OH)2, with 0.03% surfactant Silwet-77 added) was sprayed onto rice leaves and stems cultured for 6 weeks, with an aqueous solution containing only 0.03% Silwet-77 serving as a control. Two hours later, the leaves were cut and immersed in 0.5 μg / mL DAB solution and 1 mg / mL NaN3 solution under vacuum for 30 min. Leaves immersed in DAB solution were cultured under light for 8 h. Leaves immersed in NaN3 solution were then transferred to 1 mg / mL NBT solution under vacuum for 30 min. Afterward, the DAB and NBT solutions were replaced with 95% ethanol, and the leaves were destained at 95℃. The leaves stained with DAB and NBT were observed under a stereomicroscope. For aniline blue staining, leaves treated for 24 hours were cut and soaked in a lactophenol mixture (20 mL phenol (pre-melted in a 60°C water bath), 20 mL lactic acid, 8 mL pure glycerol, and ddH2O to a final volume of 100 mL). The solution was then placed in a vacuum pump and vacuumed for 30 min. After decolorization, the solution was heated in a 60°C water bath for 30 min, with the mixture changed every 10 min. Finally, the solution was washed three times with ddH2O, followed by the addition of aniline blue solution. The solution was then incubated overnight in the dark, and the leaves were washed three times with water. Callus deposition was observed using a fluorescence microscope. The experimental results are as follows: Figure 4 As shown.

[0058] Figure 4 A showed that NBT staining revealed an increase in superoxide anion accumulation after treatment with nano-copper hydroxide (5 μg / mL), which was evenly distributed in a dotted pattern and was significantly higher than that of copper sulfate and copper hydroxide 3000. Figure 4 B showed that DAB staining revealed an increase in hydrogen peroxide after treatment with nano-copper hydroxide (5 μg / mL), which was significantly higher than that of copper sulfate and copper hydroxide 3000. Figure 4 C showed that callose accumulation was significantly higher after treatment with nano-copper hydroxide (5 μg / mL) than that of copper sulfate and copper hydroxide 3000, and it was distributed in a uniform, dotted pattern. In contrast, copper preparations and copper hydroxide 3000 showed an irregular, uneven, and flaky distribution. These results indicate that the nano-copper preparation has a uniform effect on activating plant immunity.

[0059] 3. Effects on the expression of genes related to disease course

[0060] Experimental crop: Rice (specifically, japonica rice Zhonghua 11).

[0061] Cultivation method: Rice is grown in a greenhouse with alternating 16 hours of light and 8 hours of darkness each day. The cultivation temperature is 26℃ and the air humidity is 60-70%. The greenhouse is located in the State Key Laboratory of Crop Biology at Shandong Agricultural University.

[0062] Experimental methods:

[0063] Add 5 μg / mL nano copper hydroxide (containing 0.03% Silwet-77) and copper sulfate (the active ingredient is Cu) 2+ 0.03% surfactant Silwet-77 and 0.05-500 μg / ml of Kocide 3000 (active ingredient Cu(OH)2, with 0.03% surfactant Silwet-77 added) were sprayed onto rice leaves and stems cultivated for 6 weeks, respectively, with a 0.05% Silwet-77 aqueous solution used as a control. Two hours later, the treated rice leaves were cut, RNA was extracted and reverse transcribed into DNA, and the expression levels of disease-related genes were detected by quantitative real-time PCR. The experimental results are as follows. Figure 5 As shown.

[0064] Figure 5 The results showed that the expression levels of disease-related genes were significantly increased after spraying with nano-copper hydroxide compared to the control. These results indicate that nano-copper hydroxide can increase the expression levels of disease-related genes, which may be related to plant senescence, non-adverse stress, growth and development.

[0065] 4. Impact on the drought resistance of rice

[0066] Experimental crop: Rice (specifically, japonica rice Zhonghua 11).

[0067] Planting method: The rice planting method is the same as in Example 3.

[0068] Experimental methods:

[0069] Add 5 μg / mL nano copper hydroxide (containing 0.03% Silwet-77) and copper sulfate (the active ingredient is Cu) 2+ Adding 0.03% surfactant Silwet-77 and copper hydroxide 3000 (active ingredient Cu(OH)2, with 0.03% surfactant Silwet-77 added) to the leaves of rice plants cultivated for 6 weeks, sprayed on them, with a 0.03% Silwet-77 aqueous solution (CK) as the control. The treated rice was then subjected to drought treatment for 10 days, followed by re-watering. The rice growth status was recorded 7 days later. The experimental results are as follows: Figure 6 As shown.

[0070] Figure 6 The results showed that after 7 days of rehydration following drought treatment, rice treated with nano-copper hydroxide resumed growth, while rice in other groups died. This indicates that nano-copper hydroxide can improve the drought resistance of rice, while traditional copper ions and Cu(OH)2 cannot.

[0071] 5. Effects on stomata of rice leaves

[0072] Experimental crop: Rice (specifically, japonica rice Zhonghua 11).

[0073] Planting method: The rice planting method is the same as in Example 3.

[0074] Experimental methods:

[0075] 5 μg / mL nano-copper hydroxide (containing 0.03% Silwet-77), copper sulfate (active ingredient Cu2+, with 0.03% surfactant Silwet-77 added), and copper sulfate 3000 (active ingredient Cu(OH)2, with 0.03% surfactant Silwet-77 added) were sprayed onto rice leaves cultivated for 6 weeks. A 0.03% Silwet-77 aqueous solution (CK) was used as a control. Four hours after treatment, rice leaves were collected, and the stomatal opening and closing status of the rice was observed using a scanning electron microscope.

[0076] Figure 7 A shows that spraying nano-copper hydroxide promotes stomatal closure in rice, while copper sulfate and copper sulfate 3000 promote stomatal opening. Figure 7 B shows that spraying nano-copper hydroxide promotes stomatal closure in rice, preventing pathogens from invading. In contrast, spraying with copper sulfate and copper hydroxide (such as copper sulfate 3000) opens the stomata, promoting pathogen invasion. These results indicate that nano-copper hydroxide can enhance rice drought resistance and defend against pathogen invasion by closing the stomata.

[0077] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A nano-copper hydroxide, characterized in that, The nano-copper hydroxide has a long, rivet-like structure with a length of 100-500 nm and a diameter of 10-80 nm. The nano-copper hydroxide is prepared by the following method: Copper chloride was dissolved in deionized water, and then ethylene glycol and ethanol were added to obtain a copper chloride solution. Sodium hydroxide solution was added dropwise to the copper chloride solution, and the mixture was stirred after the addition was completed to obtain a suspension. The suspension was aged and filtered to obtain nano copper hydroxide. The ratio of the amount of copper chloride, ethylene glycol, ethanol, deionized water and sodium hydroxide solution added is (5-8)g:(15-25)mL:(40-60)mL:(40-60)mL:(250-300)mL, and the concentration of sodium hydroxide solution is 0.5mol / L. The sodium hydroxide solution was added at a rate of 1.5-2.5 mL / min; the stirring time was 1.5-2.5 h, the stirring speed was 300-800 rpm; the aging time was 10-15 h, and the aging temperature was room temperature.

2. The application of the nano-copper hydroxide described in claim 1 in improving plant drought resistance.

3. The application of the nano copper hydroxide of claim 1 as a pesticide formulation in the prevention and control of crop diseases, wherein the concentration of the nano copper hydroxide in the pesticide formulation is 0.001-0.01 g / L.

4. The application according to claim 3, characterized in that, The pesticide formulation also includes a surfactant, with a volume fraction of 0.03-0.05%.

5. The application according to claim 3, characterized in that, The crop disease mentioned is bacterial leaf streak of rice.

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