Application of mofs2@fe3o4 nanoparticles as an additive for promoting soybean nodule nitrogen fixation and preparation method thereof

Abstract

The application provides application of MoS2@Fe3O4 nanoparticles in promotion of soybean nodule fixation nitrogen additives and a preparation method thereof, and relates to the field of special nanofertilizer for soybean production. The MoS2@Fe3O4 nanoparticles can improve the activity oxygen buffer capacity of soybean root nodules, promote the growth and reproduction of the bacteria in the root nodules, significantly improve the nitrogen fixation enzyme activity of the soybean, promote the accumulation of nitrogen in the root nodules and the leaves, optimize the transport efficiency of nitrogen from the roots to the leaves, and increase the dry matter accumulation of the aboveground part of the soybean. Meanwhile, the nanoparticles can also improve the development state of the soybean root nodules, increase the number and fresh weight of the root nodules. The MoS2@Fe3O4 nanoparticles are synthesized by a self-assembly hydrothermal method, the preparation method is simple and low in cost, the nanoparticles can be applied in the preparation of special nanofertilizer for soybean production, and a new, efficient and stable method is provided for solving the problem of low nitrogen fixation efficiency in soybean production.

Description

Technical Field

[0001] This invention relates to the field of nano-fertilizers for soybean production, specifically to the application of MoS2@Fe3O4 nanoparticles as an additive to promote soybean nodulation and nitrogen fixation, and its preparation method. Background Technology

[0002] Improving soybean yield and quality has always been a crucial goal in agricultural production. As an important food and oilseed crop, soybeans' nitrogen fixation has a decisive impact on both yield and quality. However, the nitrogen fixation efficiency of soybeans is limited by various factors, such as soil conditions, fertilization methods, and the genetic characteristics of soybeans themselves, leading to low nitrogen fixation efficiency and becoming a key factor restricting soybean yield.

[0003] In recent years, the application of nanomaterials in agriculture has provided new ideas for the efficient delivery of trace elements. Nanomaterials have great potential in enhancing biological nitrogen fixation. By improving the biological nitrogen fixation capacity of legumes such as soybeans, nanomaterials can help reduce dependence on synthetic nitrogen fertilizers, thereby reducing the carbon and nitrogen footprint of agricultural production, while increasing crop yields and food security. Molybdenum, as a key trace element in soybean nitrogen fixation, has a crucial impact on the activity of nitrogenase, a molybdenum-containing enzyme that catalyzes the conversion of atmospheric nitrogen into ammonia nitrogen available to plants. It is the core enzyme in soybean symbiotic nitrogen fixation. In some molybdenum-deficient areas, the nitrogen fixation efficiency of soybeans is severely limited; therefore, molybdenum plays an indispensable role. The colonization of soybean rhizobia and the activity of nitrogenase not only depend on a sufficient supply of molybdenum but are also closely related to the dynamic balance of reactive oxygen species, carbohydrate distribution, and the activity of nitrogen metabolism enzymes in the rhizosphere microenvironment.

[0004] CN113924935A discloses a novel soybean-specific nano-molybdenum fertilizer that promotes nodulation, nitrogen fixation, and growth, proposing that this nano-molybdenum fertilizer has a significant promoting effect on the growth of soybean root nodules and nitrogen fixation efficiency. The study shows that MoS2 nanoparticles can significantly increase root activity by 143.7%, increase the number of effective nodules by 204.4%, increase the total number of nodules by 144.8%, and increase nitrogenase activity by 90.6%. Furthermore, the presence of Fe3O4 nanoparticles in root nodules can enhance the symbiotic relationship between rhizobia and legumes, promoting nodulation and nitrogen fixation. CN115885800A discloses a method for promoting enhanced nodulation and increased yield in soybeans through foliar application of iron-based nanomaterials. This invention involves foliar spraying of a suspension of iron-based nanomaterials at a certain concentration (5-20 mg / L), achieving a 35.4%-50.0% increase in soybean nodulation and a yield increase of approximately 40-90%. Moreover, the yield-increasing effect of applying NMs is more significant compared to applying non-nanomaterial (NMs) iron-containing materials (containing the same amount of iron). These studies collectively reveal the important role and application potential of nanoparticles in regulating the symbiotic relationship between plants and rhizobia and promoting nitrogen fixation. Summary of the Invention

[0005] Based on the above technical background, this invention uses molybdenum (Mo) as the core element and transition metal element Fe as the key element to enhance the electron transport efficiency of nanomaterials, constructing a MoS2@Fe3O4 nanoparticle, aiming to enhance its interaction with soybean roots, thereby more effectively promoting nitrogen fixation in soybeans.

[0006] The application of MoS2@Fe3O4 nanoparticles provided by this invention as an additive to promote nodulation and nitrogen fixation in soybeans is specifically as follows: the prepared MoS2@Fe3O4 nanoparticles are dispersed in water and uniformly applied to soybean leaves by spraying, and multiple spraying treatments are carried out during the key growth stages of soybeans.

[0007] Furthermore, the spraying treatment is performed once when the first cotyledon of the soybean is fully expanded, once when nodulation begins, and once when nodulation is completed.

[0008] Furthermore, the concentration of the MoS2@Fe3O4 nanoparticles in the water is 30-80 mg / L.

[0009] The MoS2@Fe3O4 nanoparticles provided by the present invention have a particle size of 1-100 nm. The MoS2@Fe3O4 nanoparticles include Fe3O4 nanoparticles and the flower-like structure MoS2 coated on their surface.

[0010] The present invention also provides a method for preparing the MoS2@Fe3O4 nanoparticles, comprising the following steps:

[0011] S1: Raw material processing: Molybdenum source and sulfur source are accurately weighed in a specific molar ratio, dissolved in deionized water, and a homogeneous solution is prepared by ultrasonic treatment technology; then, Fe3O4 nanoparticles are added to the solution and dispersed again by ultrasonic treatment technology.

[0012] S2: Hydrothermal synthesis: The above uniformly dispersed mixed solution is transferred to a liner-lined autoclave and subjected to a hydrothermal reaction at a certain temperature for 10 hours.

[0013] S3: Washing and Drying: After the reaction is complete, allow the autoclave to cool naturally to room temperature, remove the reaction product, and wash to remove impurities; then, dry the product in a vacuum environment until it is completely dry.

[0014] The hydrothermal temperature is 180°C; the drying temperature is 60°C.

[0015] Furthermore, the molybdenum source is either sodium molybdate or ammonium molybdate.

[0016] Furthermore, the sulfur source is one of thiourea, thioacetamide, or L-cysteine.

[0017] Furthermore, the MoS2@Fe3O4 nanoparticles can significantly improve the nitrogenase activity of soybeans, promote nitrogen accumulation in root nodules and leaves, optimize nitrogen transport efficiency from roots to leaves, and increase the fresh weight, dry weight, and plant height of soybean aboveground parts.

[0018] Furthermore, the MoS2@Fe3O4 nanoparticles can improve the development of soybean root nodules, increase the number and fresh weight of root nodules, enhance the reactive oxygen species (ROS) buffering capacity of soybean root nodules, and promote the growth and reproduction of bacteria within the root nodules.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) Promote growth: Compared with the control group, soybeans treated with MoS2@Fe3O4 nanoparticles showed a significant increase in aboveground fresh weight, dry weight and plant height, and increased dry matter accumulation, which provided a guarantee for increased yield.

[0021] (2) Significantly promotes soybean nodulation: It can significantly increase the number and fresh weight of soybean root nodules, improve the nodulation status, and create favorable conditions for nitrogen fixation.

[0022] (3) Enhance nitrogen fixation: It can significantly increase the activity of soybean nitrogenase, promote nitrogen accumulation in root nodules and leaves, and optimize the efficiency of nitrogen transport from roots to leaves.

[0023] (4) Optimize nitrogen metabolism: Increase the activity of various key enzymes in nitrogen metabolism in soybean leaves, making soybean fixation, absorption and utilization of nitrogen more efficient.

[0024] (5) Stabilize the root nodule environment: After treatment, the ROS buffering capacity of soybean root nodules becomes stronger, which helps to maintain a stable microenvironment inside the root nodules.

[0025] (6) It is conducive to the growth of rhizobia: it makes the bacteria in the root nodules grow well, and makes the absorption and utilization of carbohydrates more efficient, thus enhancing the growth and reproduction capacity of rhizobia in the root nodules. Attached Figure Description

[0026] Figure 1 In the image, a represents Fe3O4 nanoparticles, b represents MoS2 nanoparticles, and C1~C3 are transmission electron microscope (TEM) images of the prepared MoS2@Fe3O4 nanoparticles.

[0027] Figure 2 X-ray photoelectron spectroscopy (XPS) of MoS2@Fe3O4 nanoparticles;

[0028] Figure 3 Raman spectrum and X-ray diffraction (XRD) pattern of MoS2@Fe3O4 nanoparticles.

[0029] Figure 4 The results showed the fresh weight and dry weight of soybean aboveground parts and roots, as well as the soybean plant height, after spraying with different nanoparticles.

[0030] Figure 5 The number of soybean root nodules and fresh weight of root nodules after spraying with different nanoparticles;

[0031] Figure 6 Photographs of soybean root nodules at harvest;

[0032] Figure 7 The study investigated the nitrogenase activity in soybeans and the cumulative amount of nitrogen in soybean leaves, roots, and root nodules after spraying with different nanoparticles.

[0033] Figure 8 To measure the activity of soybean nitrogen metabolism-related enzymes after spraying with different nanoparticles;

[0034] Figure 9 The study investigated the ROS fluorescence intensity, cell morphology, and glycogen accumulation in soybean root nodules after spraying with different nanoparticles. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0036] The reagents used in the examples are as follows: Fe3O4 nanoparticles (purity >99.5%, Ф=50 nm) were purchased from Xidian Laboratory, and ammonium molybdate and thiourea were purchased from China National Pharmaceutical Group.

[0037] Example 1: Preparation of MoS2@Fe3O4 nanoparticles by self-assembly hydrothermal synthesis includes the following steps:

[0038] S1: Raw material processing: Accurately weigh ammonium molybdate (NH4)6Mo7O at a molar ratio of 1:15. 24 H₂O and thiourea SC(NH₂)₂ were dissolved in 80 mL of deionized water and a homogeneous solution was prepared using ultrasonic treatment. Subsequently, 128 mg of Fe₃O₄ nanoparticles were added to the solution and dispersed again using ultrasonic treatment for 10 minutes.

[0039] S2: Hydrothermal synthesis: The above uniformly dispersed mixed solution was transferred to a 100 mL stainless steel autoclave with a liner and subjected to a hydrothermal reaction at a constant temperature of 180°C for 10 hours.

[0040] S3: Washing and Drying: After the reaction is complete, allow the autoclave to cool naturally to room temperature, remove the reaction product, and wash it thoroughly with water to remove impurities. Then, dry the product in a vacuum environment at 60°C until it is completely dry.

[0041] Example 2: Characterization of MoS2@Fe3O4 nanoparticles

[0042] XRD is used to characterize the surface crystal composition of MoS2@Fe3O4 nanoparticles; XPS is used to analyze the elemental properties, occurrence state and structure of MoS2@Fe3O4 nanoparticles; TEM is used to observe the microstructure of the material and test the lattice fringes; Raman spectroscopy is extremely sensitive to the vibration of chemical bonds in the material, and the crystal properties of the material are determined by the changes in peak intensity, peak width and intensity.

[0043] TEM characterization results show that Figure 1 C1 and Figure 1 The MoS2 flower-like structure shown in C2 grows anisotropically on the Fe3O4 substrate surface. The two are well bonded together, forming an effective heterostructure. The particle size of MoS2@Fe3O4 nanoparticles is about 50 nm.

[0044] Figure 2XPS characterization results showed that a characteristic Fe-S bond peak was detected at a binding energy of 707.88 eV in MoS2@Fe3O4 nanoparticles. This spectroscopic evidence can serve as a criterion for the formation of chemical bonds at the composite interface, thus confirming the successful construction of the Fe3O4 / MoS2 heterostructure. High-resolution spectral analysis of the Mo 3d orbitals revealed the presence of a mixed 1T and 2H crystalline phase on the surface of the MoS2@Fe3O4 nanoparticles: the characteristic peak at a binding energy of 229.00 eV belongs to the 3d5 / 2 orbital of Mo 3d in the 1T phase MoS2, while the doublets at 230.12 eV and 232.30 eV correspond to the 3d5 / 2 and 3d3 / 2 orbitals of Mo 3d in the 2H phase MoS2, respectively. Furthermore, the S 2p orbital splitting peak (doublet spacing ~1.18 eV) and the Fe 2p orbital spin orbital splitting peak (Fe 3+ with Fe 2+ Quantitative peak fitting of characteristic peaks confirmed that Fe3O4 and MoS2 achieved interfacial coupling at the atomic scale.

[0045] Figure 3 B represents the XRD characterization results of MoS2@Fe3O4 nanoparticles. The characteristic diffraction peaks of Fe3O4 and MoS2 are clearly shown in the diffraction pattern, and no characteristic peaks of heterocrystalline phases are detected, indicating that the composite system has high phase purity and chemical compatibility of interfacial bonding. Figure 3 Figure A shows the Raman spectral characterization results of MoS2@Fe3O4 nanoparticles. It can be seen from the figure that in the low-frequency region (100-450 cm⁻¹),... -1 Three characteristic vibrational modes, J1, J2, and J3, were observed, and they are respectively related to the structure-activity relationship of the lattice distortion of the two-dimensional basal plane of MoS2. Specifically, the J1 vibrational mode can be attributed to the coupling effect of the out-of-plane vibrational modes of Mo atoms within the two-dimensional basal plane and the interlayer shear vibration; the J2 vibrational mode originates from the interlayer breathing vibration of the sulfur atom layer relative to the Mo atom basal plane; and the J3 vibrational mode corresponds to the quantized characteristics of the out-of-plane bending vibration in the Z-shaped chain configuration. Furthermore, located at 283 cm⁻¹... -1 The characteristic peaks (identified as Eg vibrational modes) correspond to the octahedral coordination configuration of the 1T metallic phase MoS2, while the typical Raman fingerprint peaks of the 2H semiconductor phase are located at 376 cm⁻¹. -1 (belonging to E) 12 (in-plane vibration mode) and 400 cm -1 (Attributed to the A1g out-of-plane vibrational mode). The above multidimensional spectroscopic evidence jointly indicates that the prepared material possesses the cross-scale heterojunction structure characteristics of 1T-2H mixed-phase MoS2 and Fe3O4, which suggests that the lattice matching degree and interface coupling effect of the composite material meet the design requirements.

[0046] Example 3: Effect of MoS2@Fe3O4 nanoparticles on nitrogen fixation in soybean symbiosis

[0047] The soybean variety tested was Zhonghuang 13. To eliminate the influence of other factors, vermiculite was used to cultivate the soybeans.

[0048] Transplant uniformly sized soybean (Zhonghuang 13) seedlings (5 days old) into pots, using vermiculite as the culture medium, with one seedling per pot. On the day of transplanting, inoculate each pot with 5 mL (OD200). 600 The soybean seedlings were treated with a low-nitrogen, low-molybdenum nutrient solution (Bradyrhizobium japonicum USDA110) containing 0.2 g of rhizobium. During the soybean growth process, 100 mL of the solution was applied weekly. The nutrient solution formula is shown in Table 1. Subsequently, the seedlings were placed in a greenhouse at Huazhong Agricultural University, with a daytime and nighttime temperature of 25℃ and a humidity of 70%. At 7 days (first cotyledon unfolds), 17 days (beginning of nodulation), and 25 days (nodulation complete), the soybeans were sprayed with a 50 mg / L MoS2@Fe3O4 nanoparticle aqueous solution. During spraying, the solution was evenly applied to the soybean leaves until completely wet. To prevent the solution from dripping onto the roots, the rim of the pot was completely covered with aluminum foil during spraying.

[0049] Table 1. Nutrient solution formula for low-nitrogen and low-molybdenum nutrient solution

[0050]

[0051] Comparative Example 1: The MoS2@Fe3O4 nanoparticles sprayed in Example 1 were replaced with CK solution, while other culture conditions remained unchanged.

[0052] Comparative Example 2: The MoS2@Fe3O4 nanoparticles sprayed in Example 1 were replaced with a 40.30 mg / L Na2MoO4 (denoted as IonMo) solution, while other culture conditions remained unchanged.

[0053] Comparative Example 3: The MoS2@Fe3O4 nanoparticles sprayed in Example 1 were replaced with a 12.82 mg / L ferric citrate solution (denoted as IonFe), while other culture conditions remained unchanged.

[0054] Comparative Example 4: The MoS2@Fe3O4 nanoparticles sprayed in Example 1 were replaced with an aqueous solution of 26.65 mg / L MoS2 nanoparticles, while other culture conditions remained unchanged.

[0055] Comparative Example 5: The MoS2@Fe3O4 nanoparticles sprayed in Example 1 were replaced with an aqueous solution of 4.20 mg / L Fe3O4 nanoparticles, while other culture conditions remained unchanged.

[0056] Comparative Example 6: The MoS2@Fe3O4 nanoparticles sprayed in Example 1 were replaced with a mixed aqueous solution of 26.65 mg / L MoS2 nanoparticles + 4.20 mg / L Fe3O4 nanoparticles (denoted as MixFeMo), while other culture conditions remained unchanged.

[0057] The substance content in the other spray treatment solutions in the comparative example was calculated based on the elemental content of MoS2@Fe3O4 nanoparticles, and was expressed as an equal amount of Mo or Fe.

[0058] Example 4: Determination of soybean and root nodule phenotypic indices cultured in Examples 3 and Comparative Examples 2-6:

[0059] Data statistics and one-way ANOVA were performed using Excel 2019 and SPSS 18. The significance test between treatments was performed using Duncan (t<0.05). Plotting was performed using OriginPro 2021.

[0060] (1) Plant height: The height of the main stem of soybeans was measured with a measuring tape at harvest;

[0061] (2) Fresh weight: Weigh the aboveground parts, roots and root nodules of the harvested soybean samples immediately;

[0062] (3) Dry weight: The aboveground and root samples of soybeans were blanched at 105℃ for 30 min and then dried at 60℃ to constant weight. The dry weight was recorded.

[0063] Data statistics and one-way ANOVA were performed using Excel 2019 and SPSS 18. The significance test between treatments was performed using Duncan (t<0.05). Plotting was performed using OriginPro 2021.

[0064] The results of soybean aboveground and root fresh weight, dry weight, and soybean plant height are as follows: Figure 4As shown, based on phenotypic observation data, there was no statistically significant difference in overall morphological characteristics between the CK and other experimental groups. By the harvest date of day 30, although there were no significant changes in root dry weight and fresh weight among the treatment groups (P>0.05), the aboveground agronomic traits of plants treated with IonMo, IonFe, MoS2 nanoparticles, Fe3O4 nanoparticles, MixFeMo, and MoS2@Fe3O4 nanoparticles were significantly improved. Specifically, compared with the control group, the aboveground fresh weight of the above-ground treatment groups increased by 35.71%, 12.71%, 42.90%, 41.94%, 44.03%, and 51.25%, respectively; the aboveground dry weight increased by 5.15%, 10.69%, 23.96%, 21.22%, 25.99%, and 36.13%, respectively; and the plant height increased by 1.94%, 7.89%, 18.29%, 15.12%, 24.63%, and 12.09%, respectively. Among them, the treatment with MoS2@Fe3O4 nanoparticles showed the best performance in terms of the increase in fresh weight (51.25%) and dry weight (36.13%) of the aboveground parts of the plant, which comprehensively indicates that the MoS2@Fe3O4 nanoparticles have a significant enhancement effect on the accumulation of dry matter in soybean plants.

[0065] Soybean root nodule number and root nodule results as follows Figure 5 As shown in the figure, MoS2@Fe3O4 nanoparticle treatment significantly enhances the formation of soybean root nodules. Specifically, MoS2@Fe3O4 nanoparticles exhibit the best biological response in terms of both nodule number and fresh weight. Compared with the control group (CK), IonMo, IonFe, MoS2 nanoparticles, Fe3O4 nanoparticles, and MixFeMo, the fresh weight of soybean root nodules treated with MoS2@Fe3O4 nanoparticles increased by 34.59%, 19.13%, 25.13%, 6.86%, 14.75%, and 5.50%, respectively. This data indicates that MoS2@Fe3O4 nanoparticles demonstrate synergistic effects in promoting symbiotic nitrogen fixation in legumes, and their technical effect is significantly better than that of single ion or nanomaterial treatment groups and the physical mixture control group.

[0066] Figure 6 The image shows a phenotypic image of soybean root nodules. It is evident that the number of soybean nodules and the nodules are more plump under the treatment of MoS2@Fe3O4 nanoparticles, confirming that this nanocomposite material can effectively promote the development of nitrogen-fixing organs in the legume-rhizobium symbiotic system.

[0067] Example 5: Determination of nitrogenase activity and metabolism-related enzyme activity in soybeans cultured in Examples 3 and Comparative Examples 2-6:

[0068] (1) Nitrate reductase, nitrite reductase, glutamine synthase, glutamate synthase, and glutamate dehydrogenase were measured using a kit (Beijing Solarbio Science & Technology Co., Ltd.);

[0069] (2) Xanthine dehydrogenase was measured using a kit (ZCIBIO Technology Co., Ltd.);

[0070] (3) Nitrogenase activity was determined using the acetylene reduction method (ARA).

[0071] Data statistics and one-way ANOVA were performed using Excel 2019 and SPSS 18. The significance test between treatments was performed using Duncan (t<0.05). Plotting was performed using OriginPro 2021.

[0072] Results of soybean nitrogenase activity and cumulative nitrogen levels in soybean leaves, roots, and root nodules are as follows: Figure 7 As shown, where, Figure 7 A represents nitrogenase activity. Figure 7 B represents nitrogen accumulation. The figure shows that MoS2@Fe3O4 nanoparticles significantly enhance the nitrogen fixation capacity of soybeans. Nitrogenase activity treated with MoS2@Fe3O4 nanoparticles increased by 75.98%, 43.49%, 72.51%, 40.93%, 52.71%, and 19.81% compared to those treated with CK, IonMo, IonFe, MoS2 nanoparticles, Fe3O4 nanoparticles, and MixFeMo, respectively. Furthermore, MoS2@Fe3O4 nanoparticle treatment significantly promoted nitrogen accumulation in root nodules and leaves. Compared to the CK group, nitrogen accumulation in root nodules and leaves increased by 89.32% and 75.32%, respectively. Notably, no significant difference was observed in nitrogen accumulation in roots among the different treatments, indicating that MoS2@Fe3O4 nanoparticle treatment effectively improved the efficiency of nitrogen translocation from roots to leaves.

[0073] Figure 8 This study demonstrates the further transformation and utilization of nitrogen-containing compounds in the underground parts of the plant, with key enzymes including nitrate reductase (NR), nitrite reductase (NiR), glutamine synthase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH). As shown in the figure, in soybean leaves treated with MoS2@Fe3O4 nanoparticles, the enzyme activities of NR and NiR were the most prominent, increasing by 125.25% and 5.98% respectively compared to the control (CK). Inside the leaf, NR and NiR work together to convert acylurea compounds into ammonium nitrogen (NH4+).+ - Therefore, under the treatment of MoS2@Fe3O4 nanoparticles, NH4 + - The cumulative amount of N reached its peak, increasing by 146.96% compared to the CK group. Subsequently, GS, GOGAT, and GDH further increased NH4. + - Nitrogen is converted into nitrogen-containing compounds such as proteins. Notably, the activities of GS, GOGAT, and GDH enzymes treated with MoS2@Fe3O4 nanoparticles were significantly higher than those in the control group, increasing by 56.33%, 42.85%, and 26.13%, respectively. These results clearly demonstrate that MoS2@Fe3O4 nanoparticles significantly promote the fixation, absorption, and utilization efficiency of nitrogen in soybeans.

[0074] Example 6: Observation of ROS fluorescence intensity, cell morphology and glycogen accumulation in soybean root nodules

[0075] (1) ROS in root nodules were stained with ethidium dihydrogen fluorescein (DHE) staining solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining solution. The fluorescence intensity of ROS in root nodules was detected by laser confocal microscopy (Zeiss LSM800). Fluorescence images were acquired at an excitation wavelength of 488 nm and an emission wavelength of 610 nm.

[0076] (2) Toluidine blue staining was used to observe the state of root nodule cells and mycoplasma, while PAS staining was used to observe the accumulation of glycogen or starch-based carbon sources. Finally, paraffin sections were observed under an optical microscope (Leica 2500), and relevant phenotypes were recorded by photographing.

[0077] Figure 9The results of specific staining of reactive oxygen species (ROS) in soybean root nodules treated with DHE and DCFDA on day 25 after the third application are presented, and their fluorescence intensity was detected by laser confocal microscopy. The results showed that, compared with the control (CK), treatments with IonMo, IonFe, MoS2 nanoparticles, Fe3O4 nanoparticles, MixFeMo, and MoS2@Fe3O4 nanoparticles significantly enhanced the fluorescence intensity of ROS in the root nodules, suggesting that these treatments may induce some kind of adaptive response in soybean root nodules, leading to changes in ROS levels. Furthermore, the fluorescence intensity of ROS in the root nodules was measured 48 hours after treatment, revealing a decrease in fluorescence intensity for all treatments, with the most significant decrease observed in the MoS2@Fe3O4 nanoparticle treatment. This indicates that soybean root nodules treated with MoS2@Fe3O4 nanoparticles possess the strongest ROS buffering capacity.

[0078] The growth of bacteria within rhizobia has a crucial impact on nitrogen fixation. To visually demonstrate the growth of bacteria under different treatments, this study used toluidine blue to stain the bacteria within the rhizobia. The staining results showed that the bacteria treated with MoS2@Fe3O4 nanoparticles had a higher density and more robust morphology within the rhizobia. Notably, the energy within the rhizobia primarily comes from carbohydrates provided by the host plant, which is one of the key factors influencing the growth of rhizobia.

[0079] To investigate the absorption and utilization of carbohydrates by rhizobium under different treatments, this study used PAS staining to stain glycogen within the rhizobia. The staining results showed that the accumulation of starch granules in the rhizobia and cortical cells treated with MoS2@Fe3O4 nanoparticles was significantly lower, indicating that the rhizobium under this treatment could more effectively absorb and utilize carbohydrates, thereby promoting its growth and reproduction. In contrast, other treatments showed greater starch granule accumulation, further highlighting the superiority of the MoS2@Fe3O4 nanoparticle treatment.

[0080] In summary, MoS2@Fe3O4 nanoparticles effectively promote the symbiotic nitrogen fixation process in soybeans through the following mechanisms: First, the nanoparticles can optimize the microaerobic environment within the root nodules, creating a favorable microenvironment for nitrogen fixation by rhizobia, thereby enhancing the activity of nitrogenase; Second, under suitable microaerobic conditions, MoS2@Fe3O4 nanoparticles promote the uptake and utilization of glycogen by bacteria within the root nodules, accelerating bacterial growth and reproduction.

Claims

1. The application of MoS2@Fe3O4 nanoparticles as an additive to promote nitrogen fixation and nodulation in soybeans, characterized in that, This includes dispersing the MoS2@Fe3O4 nanoparticles in water and applying them evenly to soybean leaves by spraying. The preparation method of the MoS2@Fe3O4 nanoparticles includes the following steps: S1: Raw material processing: Molybdenum source and sulfur source are accurately weighed in a specific molar ratio, dissolved in deionized water, and a homogeneous solution is prepared by ultrasonic treatment technology; then, Fe3O4 nanoparticles are added to the solution and dispersed again by ultrasonic treatment technology. S2: Hydrothermal synthesis: The above uniformly dispersed mixed solution is transferred to a liner-lined autoclave and subjected to a hydrothermal reaction at a certain temperature for 10 hours. S3: Washing and Drying: After the reaction is complete, allow the autoclave to cool naturally to room temperature, remove the reaction product, and wash to remove impurities; then, dry the product in a vacuum environment until it is completely dry. The hydrothermal temperature is 180℃.

2. The application according to claim 1, characterized in that, The spraying treatment is performed once when the first cotyledon of the soybean is fully expanded, once when nodulation begins, and once when nodulation is completed.

3. The application according to claim 1, characterized in that, The concentration of the MoS2@Fe3O4 nanoparticles in water is 30-80 mg / L.

4. The application according to claim 3, characterized in that, The MoS2@Fe3O4 nanoparticles have a particle size of 1-100 nm and include Fe3O4 nanoparticles and the flower-like structure MoS2 coated on their surface.

5. The application according to claim 4, characterized in that, The drying process is performed at a temperature of 60°C.

6. The application according to claim 5, characterized in that, The molybdenum source is either sodium molybdate or ammonium molybdate.

7. The application according to claim 5, characterized in that, The sulfur source is one of thiourea, thioacetamide, or L-cysteine.

Images