Preparation method and application of gold nanocluster for preventing and treating pine wood nematodes

Abstract

The invention relates to the field of agricultural protection preparations, in particular to preparation of gold nanoclusters and application of the gold nanoclusters to prevention and treatment of plant parasitic nematodes. The invention provides a preparation method of a gold nanocluster, which comprises the following steps: preparing by using HAuCl4 and L-His to obtain an L-type histidine modified gold nanocluster; the preparation method comprises the steps that a HAuCl4 aqueous solution and an L-His aqueous solution are evenly mixed and then incubated, an obtained reaction solution containing L-His-AuNCs is centrifuged and then subjected to aftertreatment such as ultrafiltration, and the gold nanocluster is obtained. The gold nanocluster can be used for preventing and treating pine wood nematodes.

Description

Technical Field

[0001] The present invention relates to the field of agricultural protection agents, and particularly to the preparation of gold nanoclusters and their application in controlling plant parasitic nematodes. Background Art

[0002] Gold nanoclusters refer to cluster structures with uniform size and morphology obtained through chemical reduction method, photoreduction method and template method (such as thiol ligands, biomacromolecule templates) to achieve atomic precision control; their main application fields include imaging diagnosis, theranostics, and also include applications in optoelectronic devices.

[0003] The invention of CN118421308A, "Method and Application for Ratio Fluorescent Detection of Iodide Ion Based on Composite Nanogold Cluster New Material", discloses a method for synthesizing gold nanoclusters His-AuNCs by water bath method using L-histidine L-His and chloroauric acid as raw materials. Specifically: Mix 7.00 mL of 20 mM chloroauric acid HAuCl4 solution with 42.00 mL of 100 mM histidine L-His solution and react at room temperature for 2 hours; after the reaction is completed, filter the reaction solution and dialyze it with a dialysis bag with a molecular weight cut-off of 1000 Da for 24 hours to obtain a pure His-AuNCs solution; subject the His-AuNCs solution to vacuum freeze-drying to finally obtain a solid powder of His-AuNCs. The invention also provides the application of the above method for ratio fluorescent detection of iodide ion based on composite nanogold cluster new material, that is, for detecting the content of iodide ion in actual samples.

[0004] The invention of CN110016043B, "A Gold Nanocluster with White Light Emission Property and Its Preparation Method", discloses a preparation method of gold nanoclusters with white light emission property, including the following steps: Mix histidine and methionine evenly and then dissolve them in a strong base solution to obtain a mixed solution with a pH of 10-12; then heat the mixed solution to 45°C, stir and add HAuCl4, and stir and react at 45°C for 3.5 h, and then perform freeze-drying to obtain a powder, and the powder is the gold nanoclusters with white light emission property; the molar ratio of histidine, methionine and HAuCl4 is: 6.5:2.1:0.25.

[0005] Pine wilt disease has caused huge losses to the ecology and economy globally. As an extremely destructive invasive pest, Bursaphelenchus xylophilus rapidly causes tree death by destroying the vascular tissue of pine trees. This disease not only affects the growth of pine wood and the quality of forest resources, but also seriously threatens biodiversity and the stability of the ecosystem.

[0006] Although various control methods have been proposed and implemented at present, such as chemical agent treatment, biological control methods, and improved forest management strategies, these methods often have unstable effects, high costs, or may cause secondary impacts on the environment. Therefore, there is an urgent need to develop a new environmentally friendly and efficient control technology to address the damage caused by pine wood nematodes to forestry. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a preparation method and application of gold nanoclusters for controlling pine wood nematodes.

[0008] To solve the above technical problem, the present invention provides a preparation method of gold nanoclusters, which is prepared by using HAuCl4 and L-His to obtain L-histidine (L-His)-modified gold nanoclusters (L-His-AuNCs).

[0009] As an improvement of the preparation method of the gold nanoclusters of the present invention, the following steps are included

[0010] 1), uniformly mix the aqueous solution of HAuCl4 and the aqueous solution of L-His to form a mixed solution; then incubate it under the water bath condition of 25±5°C for 2±0.2 hours to obtain a reaction solution containing L-His-AuNCs (uniform and stable L-His-AuNCs);

[0011] The molar ratio of HAuCl4:L-His = 1:(45±5);

[0012] Note: After mixing the aqueous solution of HAuCl4 (yellow) and the aqueous solution of L-His, the color immediately turns light yellow;

[0013] 2), centrifuge the reaction solution containing L-His-AuNCs obtained in step 1) at 8000-12000 r / min for 1±0.2 h, and absorb the supernatant solution obtained by centrifugation (the purpose of centrifugation is to remove large particles in the reaction solution containing L-His-AuNCs, and the component of the large particles is aggregated L-His-AuNCs particles);

[0014] 3), place the supernatant solution obtained in step 2) in a 1000D ultrafiltration tube and centrifuge it at 3000-5000 r / min for 45±5 min (after centrifugation, L-His-AuNCs still remain in the ultrafiltration tube, and impurities are filtered outside the ultrafiltration tube);

[0015] Add pure water to the ultrafiltration tube and centrifuge, and centrifuge after adding pure water for 2-5 cycles;

[0016] Obtain purified gold nanoclusters (located inside the ultrafiltration tube), which can be taken out from the ultrafiltration tube and stored in a 4°C refrigerator for later use.

[0017] As a further improvement to the method for preparing the gold nanoclusters of the present invention:

[0018] The concentration of the HAuCl4 aqueous solution is 8 - 12 mM (preferably 10 mM),

[0019] The concentration of the L-His aqueous solution is 0.14 - 0.16 M (preferably 0.15 M).

[0020] The present invention also simultaneously provides a use of a kind of gold nanoclusters prepared by the above method: preventing and controlling Bursaphelenchus xylophilus. The gold nanoclusters are dissolved in water (50 ng / ml - 500 ng / ml) to form an anti-nematode agent that is easy to apply. This anti-nematode agent can be directly applied to the branches and trunks of pine trees affected by Bursaphelenchus xylophilus, and the control effect can be achieved through absorption.

[0021] The present invention synthesizes L-histidine (L-His)-modified gold nanoclusters (L-His-AuNCs) with optical activity through a simple one-step synthesis method.

[0022] The present invention ensures the quality and consistency of the obtained gold nanoclusters through filtration and purification steps, providing a basis for further research and application of preventing Bursaphelenchus xylophilus disease.

[0023] The present invention reduces the gold ions in the gold salt solution through a chemical reduction method to prepare gold nanoclusters (using the reducibility of functional groups such as imidazole and amino groups in histidine to reduce the gold ions).

[0024] The main technical advantage of the present invention lies in providing an anti-Bursaphelenchus xylophilus preparation of gold nanocluster materials. By precisely controlling the synthesis and surface modification of gold nanoclusters, it can effectively interfere with reproduction in Bursaphelenchus xylophilus and inhibit its reproduction. Due to the special surface properties and catalytic activity of gold nanoclusters, compared with the control, the research shows that the efficiency of inhibiting the reproduction of Bursaphelenchus xylophilus is significantly improved. With its low lethal toxicity, it achieves an ecological control effect by inhibiting reproduction without directly killing, thereby effectively enhancing the resistance to diseases such as Bursaphelenchus xylophilus.

[0025] In addition, as a substance with low toxicity and high stability, the gold nanocluster material provides a new prevention and control solution without increasing the risk to the environment or the plants themselves. The present invention can not only quickly and efficiently respond to plant diseases, but also has obvious advantages in terms of sustainability and environmental safety.

[0026] The gold nanocluster material of the present invention can more effectively inhibit the reproduction of Bursaphelenchus xylophilus, thereby enhancing the resistance of pine trees to Bursaphelenchus xylophilus. In addition, the application of the gold nanocluster material is relatively simple, providing a completely new solution compared with traditional chemical control methods. The method and product of the present invention not only improve the efficiency and safety of controlling Bursaphelenchus xylophilus, but also may be extended to the control of other plant diseases, demonstrating the broad application prospects of gold nanocluster materials in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following further elaborates on the specific embodiments of the present invention in conjunction with the accompanying drawings.

[0028] Figure 1 It is the test result of the ultraviolet spectrum of gold nanoclusters;

[0029] This Figure 1 shows the absorption spectrum of gold nanoclusters on an ultraviolet spectrometer; it can be seen from the figure that there are absorption peaks at specific wavelengths, indicating the size distribution and preparation consistency of gold nanoclusters.

[0030] Figure 2 It is the test result of the hydrodynamic diameter of gold nanoclusters;

[0031] This Figure 2 reveals the distribution of the hydrodynamic diameter of gold nanoclusters; the results measured by a particle size analyzer show that the nanoparticles are of uniform size and concentrated distribution, suitable for biomedical and agricultural applications.

[0032] Figure 3 It is the test result of the Zeta potential of gold nanoclusters;

[0033] This Figure 3 shows the Zeta potential value of gold nanoclusters, which indicates the surface charge situation and suspension stability of this nanomaterial.

[0034] Figure 4 It is the test result of the transmission electron microscopy (TEM) appearance of gold nanoclusters;

[0035] This Figure 4 shows the morphology and structure of gold nanocluster particles through transmission electron microscopy; it can be clearly seen from the figure the spherical structure and size consistency of the particles, which are very crucial for their application as a disease-resistant agent.

[0036] Figure 5 It is a statistical chart of the growth of Bursaphelenchus xylophilus mixed with different concentrations of gold nanoclusters in a flat plate culture.

[0037] Figure 6 It is a phenotypic photo of Bursaphelenchus xylophilus ingesting mixed gold nanoclusters (shown in red fluorescence) in a flat plate culture;

[0038] The left figure is the green fluorescence image of the mixed gold nanoclusters in the pine wood nematode under the excitation wavelength of 488 nm. The middle figure is the corresponding bright-field image, and the right figure is the merged image after the fluorescence and bright-field are superimposed; the scale bar is 50 μm.

[0039] Figure 7 This is the tissue condition observed by transmission electron microscopy (TEM) after the pine wood nematodes reared on the plate were treated with different concentrations of mixed gold nanoclusters.

[0040] Figure 8 This is the effect of gold nanocluster treatment on the infection of black pine by pine wood nematodes;

[0041] Representative photos of Masson pine seedlings at 0 dpi just after inoculation and 4 weeks and 6 weeks after inoculation; Control is pine wood nematodes treated with water and pine wood nematodes treated with a mixture of gold nanoclusters / L-His-AuNCs at 50 ng / ml.

[0042] Figure 9 This is the effect of gold nanocluster treatment on the stem of black pine;

[0043] Compared with the control, the cross-section of the stem of the untreated black pine showed obvious browning and withering symptoms; while the lesion symptoms of the stem treated with 50 ng / ml of L-His-AuNCs were significantly reduced, and the cross-section was relatively clean, indicating that this treatment has an obvious control effect on pine wilt disease. The scale bar is 1 cm. Specific implementation manners

[0044] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0045] Example 1. A method for synthesizing gold nanoclusters:

[0046] The optically active L-histidine (L-His)-modified gold nanoclusters (L-His-AuNCs) are synthesized by a simple one-step synthesis method. All glassware used in the experiment needs to be soaked and washed with aqua regia (volume ratio of concentrated nitric acid: concentrated hydrochloric acid is 1:3), and dried in an oven for standby.

[0047] The specific steps are as follows:

[0048] (1) Shake the HAuCl4 aqueous solution (1 ml, 10 mM) and the L-His aqueous solution (3 ml, 0.15 M) under the condition of a 25°C water bath to form a uniform mixed solution;

[0049] Keep the mixed solution in a glass bottle. The color of the solution immediately turns light yellow, and incubate the mixture for 2 hours under the condition of a 25°C water bath to form uniform and stable L-His-AuNCs;

[0050] That is, the molar ratio of HAuCl4:L-His is 1:45;

[0051] (2) Aliquot the homogeneous solution obtained in the above step (1) into 2 mL centrifuge tubes, and centrifuge at 10,000 r / min in a high-speed centrifuge for 1 h. Aspirate the supernatant (the purpose of centrifugation is to remove large particles in the reaction solution containing L-His-AuNCs, and the composition of the large particles is aggregated L-His-AuNCs particles);

[0052] (3) Place the supernatant obtained by centrifugation and separation in step (2) into a 1000D ultrafiltration tube, and centrifuge at 4000 r / min for 45 min (after centrifugation, L-His-AuNCs still remain in the ultrafiltration tube, and impurities are filtered outside the ultrafiltration tube). Add pure water to the scale line (that is, add water to 5 ml), and centrifuge again. Such a cycle of adding water and centrifuging is carried out 4 times in total, so as to finally obtain a supernatant containing gold nanoclusters by purification; it can be taken out and stored in a refrigerator at 4 °C for standby.

[0053] (4) Lyophilize the supernatant obtained in step (3) in a freeze dryer until constant weight to obtain the water-soluble gold nanocluster material L-His-AuNCs.

[0054] Characterize the ultraviolet spectrum, particle size, Zeta potential, and TEM images of the water-soluble gold nanocluster material L-His-AuNCs obtained in the above step (4) in a conventional manner. As Figures 1 to 4 described, it can be known that: the ultraviolet absorption peak of L-His-AuNCs is at 250 nm, and under the irradiation of an ultraviolet lamp (365 nm), it will emit blue-green fluorescence ( Figure 1 ), and the detection results by a nanoparticle size analyzer show that the particle size and potential of L-His-AuNCs are 1.2 nm ( Figure 2 ) and -20.2 mV ( Figure 3 ), respectively. In addition, the TEM transmission electron microscope results show that L-His-AuNCs have good dispersibility and no phenomenon of nanoparticle aggregation, and the particle size is consistent with the detection results of the nanoparticle size analyzer ( Figure 4 ).

[0055] Experiment 1: The experiment on the plate growth of pine wood nematodes by gold nanoclusters;

[0056] In this experiment, a PDA plate cultured with Botrytis cinerea was used as the basis, and the test object was pine wood nematodes (Botrytis cinerea can provide a nutrient matrix for pine wood nematodes to support their reproduction).

[0057] First, the pine wood nematodes were cultured on PDA plates for iterative reproduction for one week, and then the following experiments were carried out. In the experiment, three different concentrations of water-soluble gold nanocluster materials were used: 5 ng / ml, 50 ng / ml, and 500 ng / ml. The specific operations are as follows:

[0058] The water-soluble gold nanocluster material obtained in Example 1 was diluted with deionized water to concentrations of 5 ng / ml, 50 ng / ml, and 500 ng / ml respectively; thus, 3 kinds of diluted solutions of gold nanocluster materials were obtained respectively;

[0059] Experimental group: 0.5 ml of the diluted solution of gold nanocluster material was taken and mixed evenly with 1000 pine wood nematodes, and then coated on the PDA plate cultured with Botrytis cinerea. The experiment was continuously carried out for one week under the condition of no light at 28 °C.

[0060] Control group: Deionized water was used to replace the diluted solution of gold nanoparticles, and the rest was the same as the experimental group. That is, a plate growth experiment of pine wood nematodes without treatment with gold nanoclusters was also set up; the purpose was to be used as a control to evaluate the growth of pine wood nematodes without treatment with gold nanoclusters on the Botrytis cinerea PDA plate. The pine wood nematodes were cultured on the PDA plate cultured with Botrytis cinerea, and only water was used to treat the nematodes as a control.

[0061] After one week, all the surviving pine wood nematodes were collected and counted to evaluate the effects of different treatments.

[0062] According to the experimental results (see Figure 5 Statistics), in the control group (nematodes treated only with water), after 7 days, the number of nematodes increased by 1.34 times, showing a significant increase in the number of pine wood nematodes. Under the treatment of the gold nanocluster material at the lowest concentration (5 ng / ml), the number of pine wood nematodes did not increase significantly after 7 days, indicating that this concentration has a certain inhibitory effect on nematode proliferation. While under the treatment of gold nanoclusters at higher concentrations of 50 ng / ml and 500 ng / ml, the number of pine wood nematodes decreased significantly, showing a significant killing effect of this material at higher concentrations. These results indicate that gold nanoclusters have a significant effect on inhibiting the proliferation of pine wood nematodes.

[0063] In addition, the present invention also found that: when the concentration of the L-His aqueous solution in Example 1 was changed to 0.1 M or 0.2 M, and the rest was referred to Example 1, the two kinds of L-His-AuNCs obtained were detected according to Experiment 1 above, and the killing effect at a concentration of 500 ng / ml had no significant difference from the killing effect at a concentration of 50 ng / ml in the present invention; therefore, it is shown that: the gold nanoclusters obtained in Example 1 of the present invention have the best killing effect on pine wood nematodes.

[0064] Experiment 2: Verification experiment on the uptake of gold nanoclusters by pine wood nematodes;

[0065] This experiment aims to verify the uptake ability of pine wood nematodes to gold nanoclusters.

[0066] First, pine wood nematodes were reared on PDA plates cultured with Botrytis cinerea for iterative reproduction for one week. The pine wood nematodes were treated with water-soluble gold nanocluster materials at a concentration of 50 ng / ml. The specific operation was to dilute the nanomaterials and then mix them evenly with 1000 pine wood nematodes and coat them on the PDA plates. The experiment was continuously carried out for one week at 28 °C under dark conditions.

[0067] After one week, all surviving pine wood nematodes were collected and counted by the funnel method and observed using a laser confocal microscope. According to the experimental results (see Figure 6 ), gold nanoclusters showed autofluorescence at an excitation wavelength of 488 nm, and obvious gold nanocluster fluorescence signals could be detected in the reared nematodes, proving that pine wood nematodes can uptake gold nanoclusters.

[0068] In further experiments, the nematodes treated with 50 ng / ml gold nanoclusters were fixed and ultra-thin sections were made, and the tissue localization was observed using a transmission electron microscope. The results showed that gold nanoclusters were significantly observed in the treated nematodes (see Figure 7 ), once again proving that pine wood nematodes can uptake gold nanoclusters.

[0069] Experiment 3. Experiment on the effect of gold nanocluster treatment on the infection of Pinus thunbergii by pine wood nematodes

[0070] This experiment aims to study the effect of gold nanocluster treatment on the infection of Pinus thunbergii by pine wood nematodes. In the experiment, 3000 pine wood nematodes were treated with 50 ng / ml gold nanoclusters.

[0071] An experiment on the effect of pine wood nematodes without gold nanocluster treatment on the infection of Pinus thunbergii was also set up to evaluate the infection effect of pine wood nematodes without gold nanocluster treatment on Pinus thunbergii as a control. 3000 pine wood nematodes were treated with water.

[0072] The specific operation steps are as follows: 1. Hole punching: Punch a hole with a diameter of 0.5 cm in the main trunk of Pinus thunbergii and stuff cotton into it. 2. Nematode injection: Inject the mixed solution of pine wood nematodes treated with gold nanoclusters into the cotton in the hole. 3. Sealing treatment: Wrap and seal the injection hole with breathable tape to ensure that the nematode liquid does not leak. 4. Greenhouse rearing: The treated Pinus thunbergii was placed in a greenhouse at 26 °C for rearing.

[0073] To observe the effect of gold nanocluster treatment, the experimental Pinus thunbergii was photographed and recorded at the time of inoculation (0 dpi), 4 weeks after inoculation (4 wpi), and 6 weeks after inoculation (6 wpi) respectively. It can be seen from the experimental results (see Figure 8), the leaves of the black pine in the control group gradually turned yellow and withered 4 and 6 weeks after inoculation, showing obvious symptoms of infection. However, the black pine inoculated with nematodes treated with gold nanoclusters remained green after 6 weeks, with no obvious death, indicating that the gold nanoclusters treatment has a significant preventive and control effect on the infection of pine wood nematodes.

[0074] In addition, after 7 weeks, the trunk of the black pine was cross-sectioned and observed, and it was found that the black pine in the control group had obvious dark brown spots due to infection by pine wood nematodes (see Figure 9 ); In the experimental group treated with gold nanoclusters, there were basically no black spots or signs of infection on the stems of black pine, further proving the control effect of gold nanoclusters.

[0075] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A preparation method of gold nanoclusters, characterized in that: Prepared using HAuCl4 and L-His to obtain L-histidine modified gold nanoclusters.

2. The preparation method of the gold nanoclusters according to claim 1, wherein Including the following steps: 1), Uniformly mix the HAuCl4 aqueous solution and the L-His aqueous solution to form a mixed solution; then incubate it under the water bath condition of 25±5°C for 2±0.2 hours to obtain a reaction solution containing L-His-AuNCs; The molar ratio of HAuCl4:L-His = 1:(45±5); 2), Centrifuge the reaction solution containing L-His-AuNCs obtained in step 1) at 8000 - 12000 r / min for 1±0.2 h, and suck the supernatant solution obtained by centrifugation; 3), Place the supernatant solution obtained in step 2) in a 1000D ultrafiltration tube and centrifuge it at 3000 - 5000 r / min for 45±5 min; Add pure water to the ultrafiltration tube and then centrifuge, and repeat the process of adding pure water and centrifuging 2 - 5 times; Obtain gold nanoclusters.

3. The preparation method of the gold nanoclusters according to claim 2, characterized in that: The concentration of the HAuCl4 aqueous solution is 8 - 12 mM, The concentration of the L-His aqueous solution is 0.14 - 0.16 M.

4. Use of the gold nanoclusters prepared by any of the methods according to claims 1 to 3, characterized in that: Control pine wood nematodes.

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