Method for improving the ability of poplar seedlings to resist drought and salt combined stress by using prussian blue nanoparticles

Abstract

The application discloses a method for improving the drought and salt compound stress resistance of 895 poplar seedlings by using Prussian blue nanoparticles, and belongs to the technical field of forest cultivation and improvement of plant stress resistance. The method can significantly alleviate the inhibition of drought and salt compound stress on the growth of the seedlings, effectively improve the net growth of the plant height and ground diameter, improve chlorophyll fluorescence parameters such as initial fluorescence, maximum fluorescence, variable fluorescence, maximum photochemical quantum efficiency and potential activity of photosystem II, protect the structure and function of the photosystem II, and enhance the photosynthetic efficiency and stress resistance stability of the seedlings. The method can significantly improve the tolerance of the 895 poplar seedlings to drought and salt compound stress, and is suitable for large-scale seedling raising and adversity afforestation application.

Description

Technical Field

[0001] This invention belongs to the technical field of forest cultivation and improving plant stress resistance, specifically involving a method for improving the ability of 895 poplar seedlings to cope with combined drought and salt stress using Prussian blue nanoparticles. Background Technology

[0002] Poplar 895 is a fast-growing timber species widely promoted in southern my country. It is characterized by rapid growth and high survival rate, and can adapt to different climates and soil environments, growing normally even in arid, barren, and saline conditions. It is a major species for shelterbelts and timber forests in the plains and sandy areas of northern my country, playing an important role in ecological forest construction. However, with the further expansion of poplar planting areas, abiotic stresses caused by environmental and climatic factors are becoming increasingly frequent, resulting in significant economic losses. Furthermore, drought and soil salinization are major abiotic stress factors in forestry production in northern and coastal areas of my country, often existing as combined stresses. These factors severely negatively impact poplar growth, development, and physiological metabolism, leading to low seedling survival rates and weak growth, thus hindering the promotion and application of Poplar 895 in unsuitable planting areas.

[0003] When plants face abiotic stress, various methods are employed to enhance their resilience, including soil improvement, water and fertilizer management, genetic engineering, and the exogenous addition of plant hormones. Prussian blue nanomaterials possess excellent redox properties, ion adsorption capacity, and biocompatibility. Furthermore, Prussian blue nanomaterials can mitigate the damage caused by salt or drought stress by regulating the redox balance within plants and adsorbing excess salt ions from the soil. In addition, the particle size of nanomaterials is a key factor affecting their bioavailability; smaller particle sizes of Prussian blue nanomaterials are more easily absorbed and transported by plant roots, resulting in more efficient biological activity.

[0004] Abiotic stresses such as drought, salinity, cold, and heat can lead to slowed plant growth and yield loss. Oxidative stress primarily arises from the excessive accumulation of reactive oxygen species (ROS) induced by biotic or abiotic stresses. Excessive ROS accumulation damages cells, mainly through pathways including DNA damage and membrane lipid peroxidation. Prussian blue nanozymes are nanomaterials capable of scavenging ROS, and their unique physicochemical properties make them promising for enhancing crop stress resistance.

[0005] Current research on the stress resistance of Populus 895 seedlings mainly focuses on alleviating single drought or salt stress, while effective methods for alleviating combined drought and salt stress remain unclear. Methods such as soil improvement, water and fertilizer regulation, genetic engineering, and the exogenous addition of plant hormones, when applied to plant stress resistance, may suffer from low root absorption efficiency, weak translocation capacity within the plant, and limited effectiveness in alleviating combined stress. Furthermore, existing agronomic measures, such as soil improvement, are costly and time-consuming, while genetic engineering techniques have drawbacks including operational complexity, controversies regarding biosafety, and poor variety compatibility, making rapid and widespread application in the large-scale cultivation of Populus 895 seedlings difficult. In summary, current technologies have not yet developed an efficient, simple, and low-cost method to significantly improve the tolerance of Populus 895 seedlings to combined drought and salt stress. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a method for improving the ability of 895 poplar seedlings to cope with the combined stress of drought and salt by using Prussian blue nanoparticles. This method significantly promotes the growth and development of 895 poplar seedlings by spraying Prussian blue nanoparticles.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for improving the ability of 895 poplar seedlings to cope with combined drought and salt stress by using Prussian blue nanoparticles is to spray Prussian blue nanoparticles onto the leaves of 895 poplar seedlings.

[0009] Furthermore, the application concentration of the Prussian blue particle nanomaterial is 100-300 μg / mL; the application is carried out by spraying once every 2 days, with 5 mL sprayed per plant each time, for a total of 6 consecutive sprays.

[0010] Furthermore, the Prussian blue particle nanomaterial is a granular Prussian blue nanomaterial, a spherical Prussian blue nanomaterial, or a cubic Prussian blue nanomaterial.

[0011] Furthermore, the preparation method of the granular Prussian blue nanomaterial is as follows: polyvinylpyrrolidone and potassium ferricyanide are dissolved in a 75% ethanol solution containing 0.01M hydrochloric acid, and then heated and stirred in a constant temperature water bath. The reacted solution is placed in a dialysis bag for dialysis, and the ultrapure water is changed every 4 hours. Finally, the ultra-small Prussian blue nanoparticles are obtained by freeze drying.

[0012] The preparation method of the spherical Prussian blue nanomaterial is as follows: Polyvinylpyrrolidone and potassium ferricyanide are placed in a flask, deionized water is added, and the mixture is stirred until completely dissolved and homogeneous. Separately, 4.16 mL of concentrated hydrochloric acid is taken and diluted with deionized water to prepare a dilute hydrochloric acid solution. The dilute hydrochloric acid solution is then added to the flask, and the system is sonicated until homogeneous. Finally, the flask solution is placed in an oil bath in an oil pan, and the resulting solution is washed with a mixture of acetone and water and ethanol. The solution is then centrifuged and collected. The centrifuged precipitate is dried in a vacuum drying oven to obtain spherical Prussian blue.

[0013] The preparation method of the cubic Prussian blue nanomaterial is as follows: Polyvinylpyrrolidone and potassium ferricyanide are placed in a flask, deionized water is added, and the mixture is stirred until completely dissolved and homogeneous. Separately, 84 μL of concentrated hydrochloric acid is taken and diluted with deionized water to prepare a dilute hydrochloric acid solution. The dilute hydrochloric acid solution is then added to the flask, and the system is sonicated until homogeneous. Finally, the flask solution is placed in an oil bath in an oil pan, and the resulting solution is washed with a mixture of acetone and water and ethanol. The solution is then collected by centrifugation, and the centrifuged precipitate is dried in a vacuum drying oven to obtain spherical Prussian blue.

[0014] Application of Prussian blue nanomaterials in improving the biomass of Populus 895 seedlings.

[0015] Furthermore, the Prussian blue nanomaterial is a granular Prussian blue nanomaterial.

[0016] Application of Prussian blue nanomaterials in improving chlorophyll fluorescence parameters of Populus 895 seedlings.

[0017] Furthermore, the Prussian blue nanomaterial is a granular Prussian blue nanomaterial.

[0018] Application of Prussian blue nanomaterials in improving photosynthetic gas exchange parameters of Populus 895 seedlings.

[0019] Furthermore, the Prussian blue nanomaterial is a granular Prussian blue nanomaterial.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) This invention is the first to apply Prussian blue nanoparticles with specific morphology to regulate the stress resistance of 895 poplar seedlings, which significantly improves the stress resistance of seedlings under drought, salt stress and drought + salt combined stress. Compared with the single drought stress group, spraying ultra-small particle Prussian blue nanoparticles (NP200) can increase the net height growth and leaf biomass of poplar seedlings by 41.86%; compared with the single salt stress group, ultra-small particle Prussian blue nanoparticle treatment can increase the leaf biomass of seedlings by 45.41%; compared with the drought + salt combined stress group, NP200 treatment can increase the net height growth by 49.10%, the net diameter growth by 148.00%, and the leaf biomass by 28.93%, which can effectively alleviate the inhibition of seedling growth by single drought and salt stress and combined stress, and provide a reliable guarantee for the normal growth of 895 poplar seedlings in arid and saline environment.

[0022] (2) Under drought, salt stress, and combined stress, the application of Prussian blue nanoparticles to the leaves of Populus tomentosa seedlings significantly optimized the chlorophyll fluorescence parameters and strongly protected the structure and function of photosystem II. Compared with the single drought stress group, the application of ultra-small Prussian blue nanoparticles increased the seedling chlorophyll concentration (Fm) by 16.84% and the Fv / Fm ratio by 3.81%; compared with the single salt stress group, Fm increased by 16.00% and Fv / Fm ratio by 4.07%; compared with the combined drought and salt stress group, Fm increased by 10.74% and Fv / Fm ratio by 3.97%, significantly improving the maximum photochemical efficiency and potential activity of photosystem II, and effectively enhancing the photosynthetic capacity and stress resistance of the seedlings.

[0023] (3) The method of the present invention can effectively alleviate the photoinhibition damage of the photosystem II (PSII) reaction center of the leaves of Populus 895 seedlings under drought and salt stress conditions, and improve the photosynthetic efficiency of seedlings, thereby increasing the photosynthetic yield of seedlings and promoting seedling growth; and enhance the adaptability and stress resistance of Populus 895 seedlings to drought and salt stress. Attached Figure Description

[0024] Figure 1 Transmission electron microscope (TEM) images of three types of ultrasmall Prussian blue nanoparticles with different morphologies prepared in Examples 1-3 of this application;

[0025] Figure 2 The Zeta potential distribution diagrams are for the three morphologies of ultrasmall Prussian blue nanoparticles prepared in Examples 1-3 of this application;

[0026] Figure 3 X-ray diffraction (XRD) patterns of three morphologies of ultrasmall Prussian blue nanoparticles prepared in Examples 1-3 of this application;

[0027] Figure 4This is a diagram showing the effect of different salt stress treatments on the root biomass of Populus 895 seedlings in this application.

[0028] Figure 5 This is a diagram showing the effect of different salt stress treatments on the stem biomass of Populus 895 seedlings in this application.

[0029] Figure 6 Figure showing the effect of different salt stress treatments on leaf biomass of Populus 895 seedlings in this application;

[0030] Figure 7 Figure 1 shows the effect of different morphologies of Prussian blue nanoparticles on the net growth of Populus 895 seedlings under drought and salt stress conditions for screening nanomaterials for this application.

[0031] Figure 8 Figure 1 shows the effect of different morphologies of Prussian blue nanoparticles on the net diameter growth of Populus 895 seedlings under drought and salt stress conditions for screening nanomaterials for this application.

[0032] Figure 9 For the comparative examples and embodiments, the effect of Prussian blue nanoparticle treatment with specific morphology particles on leaf biomass of Populus tomentosa seedlings under drought and salt stress is shown in the figure.

[0033] Figure 10 For the comparative examples and embodiments, the effect of Prussian blue nanoparticle treatment with specific morphology particles on 895 poplar seedlings Fo under drought and salt stress conditions is shown in the figure.

[0034] Figure 11 For the comparative examples and embodiments, the effect of Prussian blue nanoparticle treatment with specific morphology particles on the Fm of 895 poplar seedlings under drought and salt stress is shown in the figure.

[0035] Figure 12 For the comparative examples and embodiments, the effect of Prussian blue nanoparticle treatment with specific morphology particles on Fv / Fm of Populus tomentosa seedlings under drought and salt stress is shown in the figure.

[0036] Figure 13 For the comparative examples and embodiments, the effect of Prussian blue nanoparticle treatment with specific morphology particles on 895 poplar seedlings Pn under drought and salt stress conditions is shown in the figure.

[0037] Figure 14 For the comparative examples and embodiments, the effect of Prussian blue nanoparticle treatment with specific morphology particles on 895 poplar seedlings Gs under drought and salt stress conditions is shown in the figure.

[0038] Figure 15 For the comparative examples and embodiments, the effect of Prussian blue nanoparticle treatment with specific morphology particles on Tr of Populus tomentosa seedlings under drought and salt stress is shown in the diagram.

[0039] Figure 16 The diagram illustrates the effect of Prussian blue nanoparticle treatment with specific morphology particles on Populus 895 seedlings Ci under drought and salt stress conditions in the comparative examples and embodiments. Detailed Implementation

[0040] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0041] The testing method in the following embodiments includes the following steps:

[0042] Seedling morphological index determination: At the beginning of the experiment, the diameter at ground level (mm) of 895 poplar seedlings in each treatment was measured using vernier calipers, and the seedling height (cm) was measured using a ruler. At the end of the experiment, the seedling height and diameter at ground level of each treatment were measured, and the seedling height increment = measured height - initial height, and the diameter at ground level increment = measured diameter at ground level - initial diameter at ground level were calculated.

[0043] Leaf biomass determination: At the end of the experiment, leaves of 895 poplar seedlings in each treatment were sampled and then weighed using an electronic balance (accuracy of 0.001) to obtain leaf biomass.

[0044] Fluorescence parameter determination: The fluorescence parameters of Populus 895 seedlings were measured using a Hansatech FMS-2 portable pulse-modulated fluorometer between 9:30 and 11:00 am. Leaf clips were kept away from leaf veins. The seedlings were allowed to dark adapt for 30 min and the recording time was 2 s. The chlorophyll fluorescence parameters of leaves from 5 Populus 895 seedlings in each treatment were measured, including initial fluorescence (Fo), maximum fluorescence (Fm), and maximum photochemical efficiency (Fv / Fm).

[0045] Photosynthetic gas exchange parameters were measured using a Li-6800 portable photosynthesis meter (Li6800-COR, USA) from 9:00 AM to 11:30 AM, with a light intensity set at 1200 μmol·m⁻¹. -2 ·s -1 CO2 concentration is 400 μmol·mol -1 Net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) of different treatments of poplar seedlings were measured at 25℃ and 60% humidity.

[0046] The polyvinylpyrrolidone (purchased from Sigma-Aldrich Ltd.), potassium ferricyanide (K3[Fe(CN)6]) (purchased from Beijing Innocare Technology Co., Ltd.), hydrochloric acid, acetone (purchased from Sinopharm Chemical Reagent Co., Ltd.) and 75% ethanol used in the following examples were all analytical grade and no additional purification treatment was required.

[0047] Example 1

[0048] Preparation method of granular Prussian blue nanomaterials: 1.5 g of polyvinylpyrrolidone (PVP, MW: 55000) and 0.055 g of potassium ferricyanide (K3[Fe(CN)6]) were dissolved in 20 mL of 75% ethanol solution containing 0.01 M hydrochloric acid. The mixture was then heated and stirred in an 80℃ constant temperature water bath for 3 h. The reacted solution was then dialyzed in a dialysis bag for about 3 days, with the ultrapure water being changed every 4 h. Finally, the solution was vacuum dried at 40℃ for 12 h to obtain ultra-small Prussian blue nanoparticles.

[0049] Example 2

[0050] Preparation method of spherical Prussian blue nanomaterials: 8g of polyvinylpyrrolidone and 0.696g of potassium ferricyanide K3[Fe(CN)6] were placed in a flask, and 50mL of deionized water was added. The mixture was stirred until completely dissolved and homogeneous. Separately, 4.16mL of concentrated hydrochloric acid was diluted to 50mL with deionized water to prepare a dilute hydrochloric acid solution. The 50mL dilute hydrochloric acid solution was added to the flask, and the mixture was sonicated until homogeneous. Finally, the flask solution was placed in an oil bath at 82℃ for 20h. The resulting solution was washed with a 1:1 mixture of acetone and H2O and ethanol, centrifuged, and the collected precipitate was vacuum dried in a vacuum drying oven at 40℃ for 12h to obtain spherical Prussian blue.

[0051] Example 3

[0052] Preparation method of cubic Prussian blue nanomaterials: 8g of polyvinylpyrrolidone and 0.696g of potassium ferricyanide K3[Fe(CN)6] were placed in a flask, and 50mL of deionized water was added. The mixture was stirred until completely dissolved and homogeneous. Separately, 84µL of concentrated hydrochloric acid was diluted to 50mL with deionized water to prepare a dilute hydrochloric acid solution. The 50mL dilute hydrochloric acid solution was added to the flask, and the mixture was sonicated until homogeneous. Finally, the flask solution was placed in an oil bath at 82℃ for 20h. The resulting solution was washed with a 1:1 mixture of acetone and H2O and ethanol, centrifuged, and the collected precipitate was vacuum dried in a vacuum drying oven at 40℃ for 12h to obtain cubic Prussian blue.

[0053] Depend on Figure 1Transmission electron microscopy (TEM) images of the three morphologies of Prussian blue nanoparticles prepared in Examples 1-3 show that the granular Prussian blue nanoparticles are ultra-small nanoparticles with good dispersion, no obvious agglomeration, and extremely small individual particle size, consistent with the scale characteristics of ultra-small nanomaterials, with a size of 3-5 nm. The spherical Prussian blue nanoparticles have a regular spherical structure with clear particle outlines, regular morphology, and uniform individual particle size of about 130 nm. A small number of dispersed spherical particles can be seen in some samples, and the overall morphology conforms to the expected spherical characteristics. The cubic Prussian blue nanoparticles have a regular cubic structure with clear particle outlines, regular morphology, and uniform individual particle size of about 150 nm. A small number of dispersed cubic / near-cubic particles can be seen in some samples, and the overall morphology conforms to the expected cubic morphology characteristics. As can be seen from the figure, Prussian blue nanomaterials with clearly distinguishable morphological features have been successfully prepared. All three materials have successfully formed the expected particle, spherical and cubic structures, indicating that the preparation method can effectively control the morphology and size of Prussian blue nanomaterials.

[0054] Depend on Figure 2 The Zeta potential distribution diagrams of the three Prussian blue (PB) nanomaterials with different morphologies prepared in Examples 1-3 show that all three types of Prussian blue nanomaterials prepared in this application—particulate PB, spherical PB, and cubic PB—exhibit negative potentials in the aqueous dispersion system. Specifically, the Zeta potential of particulate PB nanoparticles is -5.86 mV, that of spherical PB nanoparticles is -6.75 mV, and that of cubic PB nanoparticles is -27.12 mV. All three materials exhibit stable surface negative charge. Zeta potential is a key indicator for evaluating the dispersibility and biocompatibility of Prussian blue nanomaterials in plant-soil systems. Negatively charged Prussian blue nanomaterials can effectively reduce electrostatic repulsion with negatively charged plant roots and soil colloids, reduce non-specific adsorption and aggregation, and more easily migrate with water to the rhizosphere and be absorbed by plants, providing a basis for their role in alleviating drought and salt stress. Compared to granular and spherical Prussian blue (PB), cubic PB exhibits stronger surface negative charge and higher colloidal stability in aqueous dispersion systems. However, its excessive negative charge may also increase electrostatic repulsion with soil colloids, affecting migration efficiency. Granular and spherical PB, on the other hand, show a more moderate negative potential, achieving a better balance between dispersion stability and migration ability. The three Prussian blue nanomaterials prepared in this application all possess suitable surface charge characteristics and demonstrate superior application effects in drought and salt stress treatments of poplar trees. This indicates that the preparation method described in this application can effectively control the surface charge and morphology of Prussian blue nanomaterials, meeting the application requirements for stress-resistant cultivation of forest trees.

[0055] Depend on Figure 3The X-ray diffraction (XRD) patterns of the three Prussian blue nanomaterials with different morphologies prepared in Examples 1-3 show that the cubic PB and spherical PB nanomaterials exhibit obvious sharp diffraction peaks at 2θ positions of 18°, 25°, and 35°, exhibiting typical Prussian blue crystal characteristics. These peaks correspond to the characteristic crystal planes of Prussian blue (PB), indicating that both are well-crystallized crystalline Prussian blue nanomaterials. Furthermore, no obvious impurity peaks were detected, suggesting that the samples are pure-phase cubic and spherical Prussian blue. The XRD pattern of the granular PB, however, shows significantly different characteristics from the previous two. Its diffraction peak intensity is greatly reduced, and the peak shape is broadened and diffused, lacking obvious sharp diffraction peaks, mainly exhibiting broadened diffuse scattering peaks. This phenomenon is consistent with the diffraction characteristics of ultra-small particle-sized nanocrystals. Due to the extremely small particle size and limited crystal domain size, the X-ray diffraction peaks are broadened, and the crystallinity is reduced compared to cubic PB and spherical PB. However, its overall diffraction trend is still consistent with the Prussian blue phase, indicating that it is still an ultra-small nanoparticle with a Prussian blue structure. Comparison of the three samples shows that the preparation method of this application can effectively control the crystal state, size, and morphology of Prussian blue nanomaterials: by controlling the process technology, cubic and spherical Prussian blue with good crystallinity, as well as ultra-small particle-shaped Prussian blue with ultra-small size and broadened diffraction peaks, are achieved. Under the premise that the phases are basically the same, the three materials show significant differences in crystallinity, size, and morphology, demonstrating the controllable controllability of the crystal structure and micromorphology of Prussian blue nanomaterials by this method.

[0056] Example 4

[0057] The granular, spherical, and cubic Prussian blue nanomaterials prepared in Examples 1-3 were formulated to concentrations of 100 μg / mL, 200 μg / mL, and 300 μg / mL for drought and salt stress treatment, including the following steps:

[0058] 1) Select 895 poplar seedlings with basically uniform growth and transplant them into flower pots with a height of 11.5cm and an upper diameter of 8cm. Fill the flower pots with a ratio of humus: loess: vermiculite = 3:1:1. Then place them in a constant temperature climate chamber for 15 days to allow them to recover. The cultivation conditions are: temperature 25℃, photoperiod 16h / 8h. During this period, observe the growth status of the seedlings daily and remove weak and diseased seedlings.

[0059] 2) Salt concentration screening treatment: After the seedlings recovered from transplant shock, the 895 poplar seedlings were randomly divided into four treatment groups with different salt stresses, namely, NaCl concentrations of 0, 50, 100 and 200 mM. When the plants showed yellowing and wilting, the biomass of each organ was measured to screen out the optimal salt concentration (100 mM), laying the foundation for subsequent experiments.

[0060] 3) Nanomaterial concentration screening treatment: After the seedlings of Populus 895 were established, they were randomly divided into different groups: the blank control group CK (soil moisture content maintained at about 80%) and the salt + drought group (salt stress concentration of 100 mmol / L, drought group was natural drought). Prussian blue nanomaterials of granular, spherical and cubic shapes were sprayed on these two groups at concentrations of 0 ug / mL, 100 ug / mL, 200 ug / mL and 300 ug / mL respectively. Finally, granular Prussian blue nanomaterials of 200 ug / mL were selected as the final experimental material and this nanomaterial was designated as NP.

[0061] 4) Drought and salt combined stress treatment: Poplar seedlings after the seedlings recovered were randomly divided into different groups: blank control group CK (soil moisture content maintained at about 80%), CK + granular Prussian blue nanoparticles, drought (natural drought), drought + granular Prussian blue nanoparticles, salt stress (100 mmol / L NaCl solution), salt + granular Prussian blue nanoparticles, drought + salt + granular Prussian blue nanoparticles, drought + salt + salt, and drought + salt + granular Prussian blue nanoparticles.

[0062] 5) Prussian Blue Nano Spraying and Salt Treatment: For each 895 poplar seedling in the Prussian Blue Nano spraying group, apply 5 mL of the solution as a foliar spray, once every two days for a total of 6 sprays. For plants not sprayed with Prussian Blue Nano, apply the same volume of purified water as a foliar spray. Thoroughly irrigate the entire soil with a salt solution, ensuring the solution is fully distributed throughout the soil, using a total of 400 mL of salt solution per pot.

[0063] 6) Once the leaves of Populus 895 show signs of curling, loss of luster, wilting, and yellowing, relevant indicators can be measured. The growth cycle discovered in this invention is about 23 days.

[0064] Comparative Example 1

[0065] Drought management (natural drought)

[0066] In addition to drought treatment, the seedlings treated with drought + granular Prussian blue nanomaterials were foliar sprayed with granular Prussian blue nanomaterials, and the operation was completely consistent with step 4) of Example 4.

[0067] Comparative Example 2

[0068] Salt treatment (100 mmol / L NaCl solution)

[0069] In addition to the salt treatment, the salt + granular Prussian blue nanomaterials were sprayed onto the leaves, and the operation was completely consistent with step 4) of Example 4.

[0070] Comparative Example 3

[0071] Drought + Salt Treatment

[0072] In addition to the drought + salt treatment, the seedlings treated with drought + salt + granular Prussian blue nanomaterials were sprayed with granular Prussian blue nanomaterials on their leaves. The operation was exactly the same as step 4) of Example 4.

[0073] Example 5

[0074] 1. Changes in biomass of various organs in poplar seedlings of variety 895 under different salt stress treatments

[0075] Depend on Figure 4 , Figure 5 , Figure 6 It was found that under treatments of 0–200 mM NaCl, the biomass of roots, stems, and leaves of Populus 895 seedlings showed a continuous decreasing trend with increasing salt concentration. Compared to 0 mM, the decrease in root and stem biomass under 50 mM salt treatment was small (approximately 2% and 1%, respectively), but leaf biomass decreased significantly by about 43%, indicating that leaves were the most sensitive to salt stress. Under 100 mM salt treatment, root biomass decreased by about 42%, stem biomass by about 27%, and leaf biomass by about 69%, all three being significantly inhibited. The largest decreases were observed under 200 mM salt treatment: root biomass decreased by about 69%, stem biomass by about 52%, and leaf biomass by about 81%, indicating that high concentrations of salt stress had a very strong inhibitory effect on the growth of Populus 895 seedlings. Considering both the significant inhibitory effect and the plant's survival ability, a concentration of 100 mM was selected as the concentration for subsequent salt stress experiments.

[0076] 2. Growth of seedling height and diameter at ground level of Populus 895 under Prussian blue nanozyme treatment with different morphologies

[0077] because Figure 7It was found that at 23 days, the height of the control group (CK) 895 poplar seedlings was significantly higher than that of the salt + drought combined stress group and all nanomaterial-treated stress groups, indicating that salt + drought combined stress significantly inhibited the growth of poplar seedlings. The seedling height of the salt + drought combined stress group at 23 days was only 3.87 cm, the lowest among all groups, indicating that salt + drought combined stress had a very strong inhibitory effect on the height growth of poplar seedlings. The introduction of Prussian blue nanomaterials significantly alleviated the growth loss of 895 poplar seedlings under salt + drought combined stress, and the seedling height of all nanomaterial-treated stress groups at 23 days was higher than that of the salt + drought combined stress group. Among the stress treatments, NP nanomaterials showed the best overall mitigation effect. The NP200 (200ug / mL nanoparticles) treatment resulted in a poplar seedling height of 5.77cm, 1.49 times that of the salt + drought combined stress group, the highest among all stress treatments. The NP100 (100ug / mL nanoparticles) treatment followed, with a seedling height of 5.63cm, 1.46 times that of the combined stress group. The NP300 (300ug / mL nanoparticles) treatment also showed a significant mitigation effect. The NS series nanomaterials showed the next best mitigation effect. The NS200 (200ug / mL spherical nanoparticles) treatment resulted in the highest seedling height of 5.36cm, 1.39 times that of the combined stress group. The NS100 (100ug / mL spherical nanoparticles) and NS300 (300ug / mL spherical nanoparticles) treatments also showed significant mitigation effects. The alleviating effect of NC series nanomaterials was relatively weak among the three types of materials. Among them, the seedling height of NC200 (200ug / mL cubic nanomaterials) treatment was the highest, at 5.03cm, which was 1.30 times that of the combined stress group. NC100 (100ug / mL cubic nanomaterials) and NC300 (300ug / mL cubic nanomaterials) treatments also showed certain alleviating effects, and the seedling height was significantly higher than that of the salt and drought combined stress group.

[0078] Depend on Figure 8It was found that the combined stress of salt and drought significantly inhibited the growth of the diameter at breast height (DBH) of poplar seedlings. The introduction of Prussian blue nanomaterials significantly alleviated the DBH loss in Poplar 895 seedlings under combined salt and drought stress. The DBH of all nanomaterial-treated groups (23 days) was significantly higher than that of the combined salt and drought stress group. Among them, NP nanomaterials showed the best overall alleviating effect. The DBH of poplar seedlings treated with NP200 reached 1.22 mm, 2.48 times that of the combined salt and drought stress group, the highest value among all stress treatment groups, and 59.4% of the DBH of the blank control group, demonstrating a very outstanding alleviating effect. NS nanomaterials showed the second best alleviating effect, with NS200 treatment achieving the highest DBH of 1.04 mm, 2.11 times that of the combined stress group. NS300 and NS100 treatments also showed significant alleviating effects, with DBH far exceeding that of the combined salt and drought stress group. The mitigation effect of NC nanomaterials was relatively weak among the three types of materials. Among them, the NC200 treatment had the highest ground diameter, which was 0.88 mm, 1.79 times that of the combined stress group. The NC100 and NC300 treatments also showed some mitigation effect, with ground diameters significantly higher than those of the salt-drought combined stress group.

[0079] In conclusion, Prussian blue nanoparticles with NP200 particles were ultimately selected as the final concentration.

[0080] 3. Leaf biomass

[0081] Depend on Figure 9 It was found that the leaf length of the blank control group (CK) 895 poplar was significantly higher than that of all single or combined stress groups. Drought, salt stress, and the combined stress of the two significantly inhibited poplar growth, with the drought + salt combined stress showing the strongest inhibitory effect, resulting in a leaf biomass of only 1.59g. After introducing ultra-small Prussian blue nanomaterials (NP), the height growth of poplar under all stress conditions was significantly improved: the height growth of the drought, salt, and drought + salt combined stress groups increased to 6.71g, 5.70g, and 2.05g, respectively, representing increases of 41.86%, 45.41%, and 28.93% compared to the stress control group. Meanwhile, the height growth of the CK + NP group (12.89g) under non-stress conditions was also significantly higher than that of the CK group, further demonstrating that the ultra-small Prussian blue nanomaterials prepared in this application can effectively alleviate the inhibitory effects of drought, salt stress, and the combined stress of the two on poplar growth, and have significant application effects in stress-resistant cultivation of forest trees.

[0082] 4. Fo changes

[0083] Depend on Figure 10It was found that the Fo values ​​of poplar seedlings under drought, salt, and combined stress treatments were significantly higher than those of the control group (447.90), indicating that both drought and salt stress damage the structure of the PSII system, leading to a significant increase in initial fluorescence. Among them, the Fo value of the drought + salt combined stress group (698.70) reached the highest, significantly higher than that of the drought-only (602.10) and salt-only (627.80) groups, indicating that the combined stress caused the most severe damage to the PSII system structure, which is consistent with the conclusion that plant height growth was most strongly inhibited. After foliar spraying of poplar seedlings with ultra-small Prussian blue nanomaterials, the Fo values ​​of each stress group decreased significantly: the Fo values ​​of the drought + NP group (555.60), salt + NP group (574.00), and drought + salt + NP group (640.30) were significantly lower than those of the corresponding stress control groups, indicating that the Prussian blue nanomaterials can effectively alleviate the damage of drought, salt, and combined stress to PSII. However, the Fo values ​​of each NP treatment group were still significantly higher than those of the CK group, indicating that the nanomaterial could only alleviate the damage of stress to PSII in poplar seedlings to a certain extent and could not completely reverse the damage. The Fo value was still significantly higher than that of the control group.

[0084] 5. Fm changes

[0085] Depend on Figure 11 It can be seen that, compared with the CK group (2585.8), the maximum fluorescence (Fm) values ​​of Populus 895 seedlings under drought, salt and combined stress treatments were significantly reduced, indicating that stress treatments would damage the electron transfer efficiency of the PSII system and inhibit the function of the photosynthetic system. Among them, the Fm value of the drought + salt combined stress group (1463.4) was the lowest, which was significantly lower than that of the single drought (1771.8) and salt stress (1641.8) groups, indicating that the combined stress had the most severe inhibitory effect on the PSII system. After foliar spraying with ultra-small Prussian blue nanomaterials (NP), the foliar flux (Fm) values ​​of all stress groups were significantly increased: under drought stress, the Fm value increased from 1771.8 to 2070.1, an increase of approximately 16.84%; under salt stress, the Fm value increased from 1641.8 to 1904.4, an increase of approximately 16.00%; and under combined drought and salt stress, the Fm value increased from 1463.4 to 1620.6, an increase of approximately 10.74%. This indicates that the Prussian blue nanomaterials can effectively alleviate the inhibitory effect of different stress conditions on the electron transport efficiency of PSII, improve the maximum photochemical efficiency of the photosynthetic system, and the improvement effect under single stress is better than that under combined stress, showing a significant stress resistance and protective effect.

[0086] 6. Changes in Fv / Fm

[0087] Depend on Figure 12It was found that, compared with the control group, the Fv / Fm values ​​of Populus 895 seedlings significantly decreased and were below normal levels under drought, salt, and combined stress treatments, indicating that the stress had caused irreversible damage to the PSII system and significantly reduced the primary light energy conversion efficiency. Among them, the Fv / Fm value of the drought + salt combined stress group was as low as 0.66, the lowest among all groups, indicating that the inhibitory effect of combined stress on PSII was much stronger than that of single stress, and the photosynthetic system was most severely damaged. After foliar spraying with ultra-small Prussian blue nanomaterials (NP), the Fv / Fm values ​​of all stress groups showed a significant upward trend: under drought stress, Fv / Fm increased from 0.73 to 0.76, an increase of about 3.81%; under salt stress, it increased from 0.71 to 0.74, an increase of about 4.07%; and under drought + salt combined stress, it increased from 0.66 to 0.68, an increase of about 3.97%. Although the Fv / Fm value of the NP treatment group has not fully recovered to the normal range, it has shown a statistically significant improvement compared with the stress control group. This indicates that the Prussian blue nanomaterial prepared in this invention can effectively alleviate the damage of stress to the PSII system, reduce the decline in the original light energy conversion efficiency, and has a significant protective effect on maintaining the basic functional stability of the photosynthetic system. It also shows good stress protection under both single and combined stress conditions.

[0088] 7. Changes in photosynthetic gas parameters

[0089] Depend on Figure 13 , 14 As shown in Figures 15 and 16, compared with the control group, drought, salt, and the combined stress of both significantly reduced the net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) of Populus 895 seedlings, while increasing the intercellular CO2 concentration (Ci). This indicates that stress inhibited photosynthesis through both stomatal and non-stomatal limitation, with the combined drought and salt stress having the most severe inhibitory effect on photosynthetic function, resulting in a Pn of only 1.63 μmol·m⁻¹. -2 ·s -1The value was the lowest among all groups. After foliar spraying with the ultra-small Prussian blue nanomaterial (NP) prepared in this invention, the photosynthetic parameters of each stress group were significantly improved: under drought stress, Pn increased by 52.38%, Gs increased by 34.20%, Tr increased by 37.61%, and Ci decreased from 320.81 to 305.26; under salt stress, Pn increased by 50.24%, Gs increased by 69.80%, Tr increased by 46.57%, and Ci decreased from 331.18 to 315.20; under combined drought and salt stress, Pn increased by 4.99%, Gs increased by 13.84%, Tr increased by 21.46%, and Ci decreased from 343.49 to 320.42. The above results indicate that NP can effectively alleviate stomatal closure and intercellular CO2 accumulation caused by stress, reduce stomatal and non-stomatal limitation damage to the photosynthetic system, significantly improve the photosynthetic gas exchange capacity of poplar under different stress conditions, maintain the stability of photosynthetic system function, and the improvement effect is particularly significant under single stress. It can still play a certain protective role under combined stress, showing excellent stress resistance regulation effect.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for improving the ability of Populus 895 seedlings to cope with combined drought and salt stress using Prussian blue nanoparticles, characterized in that, Prussian blue particle nanomaterials were sprayed onto the leaves of Populus 895 seedlings.

2. The method for improving the ability of Populus tomentosa seedlings 895 to cope with combined drought and salt stress using Prussian blue nanoparticles according to claim 1, characterized in that: The concentration of the Prussian blue granular nanomaterial is 100-300 μg / mL; the application is carried out by spraying once every 2 days, with 5 mL sprayed per plant each time, for a total of 6 sprays.

3. The method for improving the ability of Populus tomentosa seedlings 895 to cope with combined drought and salt stress using Prussian blue nanoparticles according to claim 1, characterized in that: The Prussian blue nanomaterials are granular Prussian blue nanomaterials, spherical Prussian blue nanomaterials, or cubic Prussian blue nanomaterials.

4. The method for improving the ability of Populus tomentosa seedlings 895 to cope with combined drought and salt stress using Prussian blue nanoparticles according to claim 3, characterized in that: The preparation method of the granular Prussian blue nanomaterial is as follows: polyvinylpyrrolidone and potassium ferricyanide are dissolved in a 75% ethanol solution containing 0.01M hydrochloric acid, and then heated and stirred in a constant temperature water bath. The reacted solution is placed in a dialysis bag for dialysis, and the ultrapure water is changed every 4 hours. Finally, the ultra-small Prussian blue nanoparticles are obtained by freeze drying. The preparation method of the spherical Prussian blue nanomaterial is as follows: Polyvinylpyrrolidone and potassium ferricyanide are placed in a flask, deionized water is added, and the mixture is stirred until completely dissolved and homogeneous. Separately, 4.16 mL of concentrated hydrochloric acid is taken and diluted with deionized water to prepare a dilute hydrochloric acid solution. The dilute hydrochloric acid solution is then added to the flask, and the system is sonicated until homogeneous. Finally, the flask solution is placed in an oil bath in an oil pan, and the resulting solution is washed with a mixture of acetone and water and ethanol. The solution is then centrifuged and collected. The centrifuged precipitate is dried in a vacuum drying oven to obtain spherical Prussian blue. The preparation method of the cubic Prussian blue nanomaterial is as follows: Polyvinylpyrrolidone and potassium ferricyanide are placed in a flask, deionized water is added, and the mixture is stirred until completely dissolved and homogeneous. Separately, 84 μL of concentrated hydrochloric acid is taken and diluted with deionized water to prepare a dilute hydrochloric acid solution. The dilute hydrochloric acid solution is then added to the flask, and the system is sonicated until homogeneous. Finally, the flask solution is placed in an oil bath in an oil pan, and the resulting solution is washed with a mixture of acetone and water and ethanol. The solution is then collected by centrifugation, and the centrifuged precipitate is dried in a vacuum drying oven to obtain spherical Prussian blue.

5. Application of Prussian blue nanomaterials in improving the biomass of Populus 895 seedlings.

6. The application according to claim 5, characterized in that: The Prussian blue nanomaterial is a granular Prussian blue nanomaterial.

7. Application of Prussian blue nanomaterials in improving chlorophyll fluorescence parameters of Populus tomentosa seedlings No.

895.

8. The application according to claim 7, characterized in that: The Prussian blue nanomaterial is a granular Prussian blue nanomaterial.

9. Application of Prussian blue nanomaterials in improving photosynthetic gas exchange parameters of Populus tomentosa seedlings No.

895.

10. The application according to claim 9, characterized in that: The Prussian blue nanomaterial is a granular Prussian blue nanomaterial.

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