Application of chiral gold nanoclusters in improving wheat seedlings resistance to stripe rust

Abstract

The application discloses application of chiral gold nanoclusters in improving wheat resistance to stripe rust and relates to the field of agricultural technology.The application is characterized in that a solution containing L-gold nanoclusters is applied to the leaves of wheat;in the solution, the concentration of the L-gold nanoclusters is 1 g / L.The application finds that the use of L-gold nanoclusters with a specific concentration can significantly reduce the number of spores of the stripe rust fungus on the surface of the leaves of wheat and can play a role in improving the disease resistance of wheat.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, and more specifically to the application of chiral gold nanoclusters in improving the disease resistance of wheat seedlings. Background Technology

[0002] wheat( Triticum aestivum Wheat stripe rust is a staple crop for the global population and the world's third largest food crop, after rice and corn. China is the world's largest wheat producer, and its production is crucial to the national economy. Puccinia striiformis Wheat stripe rust (F. sp. Tritici) is a common plant disease caused by infection. It can occur throughout the entire growth period of wheat and seriously threatens wheat yield. In-depth research on the physiological and ecological response mechanisms of wheat under the stress of wheat stripe rust infection, elucidating the complex interaction between wheat and wheat stripe rust fungus, and improving the disease resistance of plants through modern cultivation techniques are of great strategic significance.

[0003] Currently, over 80% of global wheat production is affected by wheat stripe rust, characterized by high frequency of outbreaks, wide range of infection, severe damage, and the potential for significant losses. It is prevalent in wheat-producing regions worldwide. The stripe rust fungus infects wheat leaves, destroying chlorophyll and severely impacting photosynthesis. Infected wheat plants experience stunted growth and insufficient grain filling, ultimately leading to severe yield reductions, with global annual production losses exceeding 5 million tons.

[0004] Therefore, a deeper understanding of the response mechanism of wheat to wheat stripe rust infection stress and the development of effective strategies to enhance its disease resistance are of great significance for ensuring my country's food security and agricultural production. Summary of the Invention

[0005] Nanomaterials are a class of nanoscale materials with unique structures. Their chiral structures can originate from crystal structures, surface modifications, or external stimuli, and they have attracted considerable attention due to their unique optical, electronic, and magnetic properties. Configurational differences in small chiral compounds (such as amino acids and hormones) can lead to entirely different biological activities. Chiral enantiomer-selective response mechanisms play an irreplaceable regulatory role in molecular recognition, enzymatic catalysis, and cell signal transduction.

[0006] In view of this, the primary objective of the present invention is to provide the application of L-gold nanoclusters in improving the disease resistance of wheat seedlings.

[0007] The second objective of this invention is to provide a method for improving the disease resistance of wheat seedlings under stripe rust infection stress.

[0008] The third objective of this invention is to provide the application of L-gold nanoclusters in improving the antioxidant properties of wheat seedlings under stripe rust infection stress.

[0009] The fourth objective of this invention is to provide the application of L-gold nanoclusters in increasing the reactive oxygen species content in wheat seedlings under stripe rust infection stress.

[0010] The fifth objective of this invention is to provide an application of L-gold nanoclusters in reducing malondialdehyde content in wheat after wheat stripe rust infection.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] Application of chiral gold nanoclusters in improving the resistance of wheat seedlings to stripe rust, wherein the chiral gold nanoclusters are L-gold nanoclusters.

[0013] Furthermore, the L-gold nanoclusters have a diameter of 1.7 nm and a surface charge of -19 mV.

[0014] Furthermore, the solution of the L-gold nanoclusters was applied to the leaves of wheat seedlings, with a concentration of 1 g / L for the L-gold nanoclusters.

[0015] Furthermore, the solution also includes an organosilicon surfactant.

[0016] Furthermore, the volume concentration of the organosilicon surfactant is 0.05%.

[0017] Furthermore, the stripe rust is caused by a strain of *Strombus styracifolius*.

[0018] Furthermore, the preparation method of the L-gold nanoclusters includes the following steps: L-histidine and chloroauric acid are mixed and reacted at 25°C for 2 h to obtain the L-gold nanoclusters.

[0019] A method to improve the disease resistance of wheat seedlings under stripe rust infection stress involves applying a solution containing L-gold nanoclusters to the leaves of wheat seedlings; The solution contained 1 g / L of L-gold nanoclusters.

[0020] Application of L-gold nanoclusters in enhancing the disease resistance of wheat seedlings under stripe rust infection stress.

[0021] Application of L-gold nanoclusters in enhancing the antioxidant properties of wheat seedlings under stripe rust infection stress.

[0022] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention has shown that using L-gold nanoclusters at specific concentrations can significantly improve the growth of wheat under stripe rust infection stress and enhance the disease resistance of wheat seedlings. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 Transmission electron microscopy (TEM) imaging of L-gold nanoclusters; Figure 2 The particle diameter of the L-gold nanoclusters; Figure 3 The zeta potential of L-gold nanoclusters; Figure 4 This is a confocal microscopy image of the colocalization of L / D-gold nanoclusters and wheat mesophyll cells. Green fluorescence is AuNC fluorescence, and red fluorescence is chloroplast autofluorescence. Figure 5 The effect of L / D-gold nanoclusters treatment on the growth of wheat seedlings infected with wheat stripe rust; Figure 6 The effect of L / D-gold nanocluster treatment on the biomass of wheat stripe rust in wheat seedlings infected with wheat stripe rust; Figure 7 The effect of L-gold nanoclusters treatment on the activity of superoxide dismutase (SOD) in wheat leaves under gold nanoclusters; Figure 8 The effect of L-gold nanoclusters treatment on peroxidase (POD) activity in wheat leaves under gold nanoclusters; Figure 9 The effect of L-gold nanocluster treatment on the content of proline, an osmotic regulator, in wheat leaves; Figure 10 The effect of L-gold nanocluster treatment on malondialdehyde content in wheat leaves. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In the following examples, data are presented as Mean ± SE, where n = biological replicates. Least significant difference (LSD) and two-tailed t-tests were used to compare differences between treatments. p<0.05, p<0.01, and p<0.001 are represented, respectively. Different lowercase letters represent p<0.05, and the same lowercase letter represents p≥0.05.

[0027] Example 1 Synthesis of L-gold nanoclusters (levorotatory gold nanoclusters) The synthesis method of L-gold nanoclusters specifically includes the following steps: 1 mL of HAuCl4 aqueous solution (10 mM, Sigma-Aldrich) and 3 mL of L-histidine aqueous solution (150 mM, Sigma-Aldrich) are mixed and placed in a glass bottle. The mixture is stirred at 500 rpm for 2 h at room temperature (25℃). The reaction solution is then filtered using a 1000 Da ultrafiltration tube (molecular weight cutoff 1000 Da, Millipore Inc.) to obtain the retentate, which is the L-gold nanocluster.

[0028] Comparative Example 1 Synthesis of D-gold nanoclusters (dextral gold nanoclusters) It was synthesized using an aqueous solution of D-histidine, with other conditions the same as those for the synthesis of L-gold nanoclusters.

[0029] Characterization of L-gold nanoclusters (1) Test method The size and morphology of the L-gold nanoclusters synthesized in Example 1 were characterized using transmission electron microscopy (TEM) (H-7650, HitAuNCsi); their Zeta potentials were measured using a nanoparticle size analyzer (NanoBrook 173plus). (2) Test results Figure 1 The image is a transmission electron microscope (TEM) image, which shows the morphology of L-gold nanoclusters, which are granular and uniformly dispersed.

[0030] Figure 2 The particle size distribution shows that the diameter of the L-gold nanoclusters is 1.70 nm.

[0031] Figure 3 The results show that the surface charge of the L-gold nanoclusters is -19 mV, based on the zeta potential measurement.

[0032] Example 2 Co-localization of L / D-gold nanoclusters with wheat leaves The L / D-gold nanocluster mixture was prepared as follows: L / D-gold nanocluster + 0.05% Silwet. The control mixture was prepared as follows: ultrapure water + 0.05% Silwet. The mixture was sprayed onto wheat leaves (two-leaf stage) using a spray bottle, following the treatment method in Example 2, and left to stand in low light for 3 hours. Slice preparation: A 5 mm diameter leaf disc was taken from the second true leaf using a perforator. The disc was placed on a glass slide with the leaf surface facing up. One drop of perfluoronaphthylamine (PFD) was added to the leaf, and the leaf was covered with a coverslip, ensuring no air bubbles. The slices were observed under a Leica laser scanning confocal microscope (LASM SP8). The LASM parameters were set as follows: 40x objective lens (add one drop of ultrapure water before observation), 405 nm excitation, laser intensity 30%; PMT1: 450 nm-500 nm (fluorescence of AuNCs); PMT2: 700 nm - 790 nm (chloroplast fluorescence), with 4-6 replicates. The fluorescence intensity of AuNCs was analyzed using LAS (Leica Application Suite) software.

[0033] (2) Experimental results Figure 4 This is a confocal microscope image showing the co-localization of L / D-gold nanoclusters with wheat mesophyll cells. (Example:) Figure 4 In the results of laser confocal microscopy, a significant fluorescence signal was observed in the chloroplasts of wheat leaves treated with L-gold nanoclusters, while no fluorescence signal was found in D-gold nanoclusters and the control group. This result indicates that L-gold nanoclusters have stronger membrane permeability, enabling them to cross the cell membrane barrier more efficiently and enter the cell interior, thereby more effectively performing their biological functions in the cytoplasm or specific organelles.

[0034] Example 3 Effects of different concentrations of L / D-gold nanoclusters on the growth and development of wheat seedlings under stripe rust infection stress (1) Experimental procedure (a) Seedling experiment: The variety used in this experiment was SY, with a growth temperature of 25℃ ± 2℃, humidity of 60%, and light intensity of 200 μmol / m³. 2 s 1 PAR. The light cycle is 14 / 10 h (day / night).

[0035] (b) Seedling experiment: CYR34 stripe rust strain was selected for inoculation and two treatments were set up, namely the control group (Control) and the L / D-gold nanocluster treatment group (AuNCs), with 4 replicates for each treatment.

[0036] (c) Culture method 1) Seed disinfection: Select plump seeds of uniform size, disinfect the surface with 1% sodium hypochlorite for 15 minutes, then rinse with deionized water for 4-6 minutes and air dry at room temperature.

[0037] 2) Soil cultivation: Sow wheat seeds into the soil. Specific method: Mix the substrate (commercial nutrient soil) and vermiculite in a volume ratio of 1:1, add an appropriate amount of water and mix well. Sow the wheat seeds into the soil of the cultivation pot. When they grow to the two-leaf stage, they can be treated.

[0038] (d) Processing methods Seedling Experiment: Treatment was conducted when wheat reached the two-leaf stage. Different treatment solutions were prepared first. The control treatment solution consisted of pure water containing 0.05% Silwet L-77 (purchased from Shanghai Yuanye Biotechnology Co., Ltd.). The L / D-gold nanocluster treatment solution consisted of an L / D-gold nanocluster solution containing 0.05% Silwet L-77. The wheat seedlings were treated by foliar spraying, applying the solution to two leaves. After spraying, excess solution was wiped off with a paper towel. The treated wheat seedlings were placed in low light for 3 hours to acclimate before inoculation with wheat stripe rust and cultured on a culture rack. The specific inoculation method for wheat stripe rust was as follows: stripe rust spores stored at -80℃ were rapidly activated by a 40-45℃ water bath for 5 minutes, then placed in a culture dish lined with moistened filter paper and hydrated at 4℃ in the dark for 10-12 hours to restore spore activity. The activated spores were mixed with a certain amount of mineral oil to form a bacterial solution, which was then sprayed onto wheat leaves. The solution was kept in the dark and moist at 7-10℃ for 48 hours, and then moved to a greenhouse at 15-18℃ for 13-15 days until typical lesions appeared on the leaves.

[0039] (2) Determination of leaf morphology indicators Wheat leaves from different groups were compared to identify the phenotypic characteristics that produced typical lesions.

[0040] (3) Experimental results Figure 5 This study investigated the effects of different concentrations of L / D-gold nanoclusters on the growth of wheat seedlings under stripe rust infection stress. The results showed that, compared to the control group, L-gold nanocluster treatment resulted in a significant immune response in wheat leaves, inhibiting stripe rust infection; while D-gold nanocluster treatment did not produce a significant immune response and failed to inhibit stripe rust infection.

[0041] In summary, wheat treated with 1 g / L L-gold nanoclusters showed the most significant improvement in resistance to wheat stripe rust. Subsequently, the biomass of stripe rust fungus on wheat seedling leaves under the optimal application concentration of 1 g / L L / D-gold nanoclusters was determined.

[0042] Figure 6 The effect of L / D-gold nanoclusters on the biomass of stripe rust fungi in wheat seedlings is shown in the figure. Compared with the control, the biomass of stripe rust fungi in wheat seedlings treated with L-gold nanoclusters was significantly reduced, decreasing by 60.71% compared with the control group, while the biomass of stripe rust fungi in wheat seedlings treated with D-gold nanoclusters was not significantly different from that in the control group.

[0043] The above results indicate that L-gold nanoclusters are the most effective way to enhance wheat resistance to wheat stripe rust through immune induction, significantly better than the control group.

[0044] Example 4 Effects of L-gold nanoclusters on antioxidant enzyme activity in wheat leaves after stripe rust infection (1) Experimental method: The treatment of wheat stripe rust infection was carried out in accordance with the treatment method in Example 2 (d).

[0045] (2) Test method Weigh 0.1 g of wheat sample and add 1 mL of 0.05 mol / L sodium phosphate buffer (PBS). Grind the sample for 180 s (65 Hz) using a grinder, then centrifuge at 12000 r / min and 4℃ for 20 min. The supernatant is the enzyme extract.

[0046] Superoxide dismutase (SOD) assay: In a 5 mL test tube, add 1.5 mL of 0.05 mol / L PBS, 0.3 mL of 0.13 mol / L methionine solution, 0.3 mL of 0.75 mol / L NBT solution, 0.3 mL of EDTA-Na2 solution, and 0.3 mL of 0.02 mol / L riboflavin solution. Add 0.05 mL of enzyme extraction buffer (use buffer solution instead of enzyme solution in the control tube), and finally add 0.25 mL of distilled water to make a total volume of 3 mL. After mixing, place the control tube in the dark, and react the other tubes under 4000 lx sunlight for 20 min. Measure the absorbance of each tube at 560 nm.

[0047] SOD activity can be calculated using the following formula: ; In the formula: ACK is the absorbance of the control tube, AE is the absorbance of the sample tube, V is the total volume of the sample (mL), VT is the amount of sample used during the determination (mL), and m is the fresh weight of the sample (g).

[0048] Peroxidase (POD) assay: Prepare the reaction mixture: Take 200 mL of sodium phosphate buffer (PBS, 0.2 mol / L, pH=6.0), add 0.076 mL of guaiacol stock solution (2-methoxyphenol), heat and stir to dissolve, cool, and then add 0.112 mL of 30% H2O2 solution. Enzyme activity assay: Take 3 mL of the above reaction mixture, add 50 µL of enzyme extraction solution, mix well, and zero the sample using PBS as a control. Measure the absorbance of the mixture at 470 nm. The measurement is performed for 180 s, with readings every 30 s. Calculate the peroxidase activity using the following formula: ; In the formula: △A470 is the average absorbance during the reaction time, m is the fresh sample mass (g), T is the reaction time, V is the enzyme solution volume taken during the determination (mL), and VT is the total volume of the extracted enzyme solution (mL).

[0049] (3) Experimental results Figure 7 and Figure 8 To investigate the effects of L-gold nanocluster treatment on the activity of antioxidant enzymes in wheat leaves under stripe rust infection stress, the activities of two antioxidant enzymes, SOD and POD, in wheat seedling leaves treated with L-gold nanoclusters were measured. Figure 7 The results showed that SOD activity was not significantly different from that of the control group.

[0050] like Figure 8 As shown, the average POD activity of the L-gold nanoclusters treated group was significantly reduced by 20.57% compared with the control group.

[0051] In summary, L-gold nanoclusters significantly reduced POD activity in wheat seedlings infected with wheat stripe rust, while having no significant effect on SOD activity. The activation of the overall antioxidant enzyme system indicates that L-gold nanoclusters provide crucial support for wheat immune induction and resistance to wheat stripe rust by efficiently regulating the ROS pathway.

[0052] Example 5 The Effects of L-Gold Nanoclusters on Osmotic Regulation Substances in Wheat Leaves After Stripe Rust Infection (1) Experimental method: The treatment of wheat stripe rust infection was carried out in accordance with the treatment method in Example 2 (d).

[0053] (2) Test method Proline content determination: Weigh 0.2 g of sample and add 5 mL of 3% sulfosalicylic acid solution. Extract proline by reacting in a boiling water bath for 10 min. Transfer 2 mL of the extract to a 10 mL test tube and add 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin. Mix well and heat the mixture in a boiling water bath for 30 min. After cooling, extract the reaction mixture with 4 mL of toluene. Centrifuge and collect the supernatant of the reaction mixture. Using toluene as a blank control, measure the absorbance at 520 nm.

[0054] Calculate using the following formula: ; In the formula: Vt is the volume of the extract (mL), Vs is the volume of the sample taken during the determination (mL), and m is the sample mass (g).

[0055] (3) Experimental results Figure 9 The effects of L-gold nanoclusters on osmotic regulators in wheat leaves under stripe rust infection stress. Among them... Figure 9 The results showed that the average proline content in the L-gold nanocluster treatment group was significantly lower than that in the control group by 28.87%, indicating that the L-gold nanocluster treatment group effectively alleviated the abnormal accumulation of proline induced by stripe rust infection stress.

[0056] In conclusion, L-gold nanoclusters significantly and effectively inhibited the abnormal accumulation of proline (28.87%), with a significantly better effect than the control group. This indicates that L-gold nanoclusters enhance the resistance of wheat seedlings to wheat stripe rust through multiple pathways of synergistic regulation of osmotic regulators.

[0057] Example 6 Effects of L-gold nanoclusters on malondialdehyde content and electrolyte permeability in wheat leaves infected with wheat stripe rust (1) Experimental method: The treatment of wheat stripe rust infection was carried out in accordance with the treatment method in Example 2 (d).

[0058] (2) Test method The detection was performed using a malondialdehyde (MDA) detection kit.

[0059] (3) Experimental results Figure 10 The effect of L-gold nanoclusters on malondialdehyde (MDA) content in wheat leaves under stripe rust fungus infection stress. Figure 10 As shown, the average malondialdehyde (MDA) content in wheat leaves treated with L-gold nanoclusters was significantly reduced by 46.51% compared to the control group. This indicates that L-gold nanocluster treatment was significantly more effective than the control group in alleviating cell membrane damage in wheat seedlings under stripe rust infection stress.

[0060] In conclusion, L-gold nanoclusters significantly reduced the MDA content (46.51%) in wheat seedlings under stripe rust infection stress, with a significantly better effect than the control group. This indicates that L-gold nanoclusters enhance wheat resistance to stripe rust by inhibiting cell membrane lipid peroxidation and maintaining membrane structural integrity, thereby preserving selective permeability and signal transduction capabilities.

[0061] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of chiral gold nanoclusters in improving the resistance of wheat seedlings to stripe rust, characterized in that, The chiral gold nanoclusters are L-gold nanoclusters.

2. The application according to claim 1, characterized in that, The L-gold nanoclusters have a diameter of 1.7 nm and a surface charge of -19 mV.

3. The application according to claim 1, characterized in that, The solution of the L-gold nanoclusters was applied to the leaves of wheat seedlings at a concentration of 1 g / L.

4. The application according to claim 3, characterized in that, The solution also contains organosilicon surfactants.

5. The application according to claim 4, characterized in that, The volume concentration of the organosilicon surfactant is 0.05%.

6. The application according to claim 1, characterized in that, The stripe rust is caused by the *Strombus styracifolius* strain.

7. The application according to any one of claims 1-6, characterized in that, The preparation method of the L-gold nanoclusters includes the following steps: L-histidine and chloroauric acid are mixed and reacted at 25°C for 2 h to obtain the L-gold nanoclusters.

8. A method for improving the disease resistance of wheat seedlings under stripe rust infection stress, characterized in that, A solution containing L-gold nanoclusters was applied to the leaves of wheat seedlings. The solution contained 1 g / L of L-gold nanoclusters.

9. Application of L-gold nanoclusters in enhancing the disease resistance of wheat seedlings under stripe rust infection stress.

10. Application of L-gold nanoclusters in improving the antioxidant properties of wheat seedlings under stripe rust infection stress.

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