Application of nano-iron atoms in promoting plant growth and nodule nitrogen fixation

Abstract

The application belongs to the field of biotechnology, and particularly relates to application of nano iron atoms in promoting plant growth and nodule fixation of nitrogen, wherein the nano iron atoms significantly improve the plant height, root length, fresh weight of aboveground part, fresh weight of root, dry weight of aboveground part and dry weight of root of alfalfa; the application mode of seed soaking + spraying is more beneficial to the growth of alfalfa; meanwhile, the root activity of alfalfa under the treatment of FeNPs is significantly higher than that of the control; according to the phenotype of root nodule, the total number of root nodules and the number of effective root nodules under the treatment of FeNPs are 2.04 times and 1.42 times higher than those of the control respectively; the data of nitrogen fixation enzyme activity further prove that the nitrogen fixation enzyme activity under the treatment of FeNPs is the highest. The content of urea further shows that FeNPs promote alfalfa to fix nitrogen in the air and produce a large amount of urea in the root nodule, promote the growth of alfalfa, thereby improve the quality and biomass of leguminous forage, and have wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of nano-iron atoms in promoting plant growth and nodulation nitrogen fixation. Background Technology

[0002] Soil infertility is one of the main factors limiting sustainable agricultural development and causing crop yield reduction. To increase crop yields, people use large amounts of chemical fertilizers. However, the widespread use of chemical fertilizers in farmland exacerbates soil pollution, damages the Earth's ecosystem and human living environment, and further affects crop yields. In the past thirty years, many new types of blended fertilizers, such as slow-release and controlled-release fertilizers, have been widely used in agricultural production. New nano-fertilizers, formed by uniformly incorporating nanomaterials into traditional chemical fertilizers, can adsorb nutrients from the environment into plants, providing nutrition for plant growth and increasing crop yields. Compared with traditional chemical fertilizers, new nano-fertilizers have higher nutrient absorption efficiency and lower leaching loss rates. At the same time, nanomaterials can improve plants' resistance and tolerance to various biotic stresses such as pests and diseases and abiotic stresses such as salt, drought, flooding, high temperature, low temperature, and freezing.

[0003] Metal ions are essential trace elements for plant growth. Metals such as nitrogen, phosphorus, potassium, calcium, sodium, iron, and zinc are all essential elements for promoting plant growth. In order to promote plant absorption, those skilled in the art have prepared the above-mentioned trace elements into nanomaterials. For example, invention patent CN110256144B discloses the preparation of biodegradable polymer slow-release organic nano-fertilizer containing multiple nutrients. The organic nano-fertilizer imparts excellent slow-release properties to nitrogen, phosphorus, potassium, or other trace elements, thereby greatly improving fertilizer utilization efficiency. Invention patent CN114478142B discloses a tobacco foliar fertilizer containing nano-rare elements, comprising: 10-40g nano-molybdenum, 2-10g nano-hafnium, 2-10g nano-tantalum, 100-500g soluble nutrients, 2-10g honey, 5-20g humic acid, and 2-10g artemisia annua powder; the soluble nutrients include potassium, phosphorus, calcium, sulfur, magnesium, iron, manganese, zinc, and copper, with a molar ratio of 700-4000:700-4000:90-250:25-80:25-70:0.5-2:1-7:0.5-3:0.2-1.5; the compound nano-foliar fertilizer can increase the potassium concentration of tobacco leaves, reduce nicotine and chlorine content, and improve reducing sugar and total nitrogen, thereby improving the overall quality of flue-cured tobacco; it also enhances the disease resistance of tobacco plants, increases the weight of single leaves, and thus increases the yield of flue-cured tobacco. Regarding iron fertilizers, those skilled in the art typically apply them in the form of nano-iron ions. For example, invention patent CN105110929B discloses the application of nano-iron fertilizer in promoting iron accumulation in crops, wherein the iron fertilizer is FeSO4·7H2O. Invention patent CN115959953A discloses a biochar-nano-iron oxide-humic acid-based fertilizer, wherein the iron ions in the nano-iron oxide are Fe... 3+ However, there is no research in this field on the preparation of nano-iron fertilizer into nano-iron atoms and their application to plants.

[0004] The inventors made an unexpected discovery during their research: preparing iron fertilizer into nano-iron atom fertilizer and applying it to promote plant growth and promote nodule formation and nitrogen fixation had good results. Summary of the Invention

[0005] The primary objective of this invention is to provide the application of nano-iron atoms in promoting plant growth.

[0006] Preferably, the plant is a leguminous forage grass.

[0007] Preferably, the legume forage is one or more of alfalfa, hyacinth bean, arrowleaf pea, birdsfoot root, and red clover.

[0008] A second objective of this invention is to provide the application of nano-iron atoms in promoting plant nodulation and nitrogen fixation.

[0009] Preferably, the plant is a leguminous forage grass.

[0010] Preferably, the legume forage is one or more of alfalfa, hyacinth bean, arrowleaf pea, birdsfoot root, and red clover.

[0011] Preferably, the method of using the nano-iron atoms is: seed soaking and / or foliar spraying.

[0012] Preferably, the nano-iron atoms are used by seed soaking and foliar spraying.

[0013] Preferably, the concentration of the nano-iron atoms applied to the leaves is 10-20 mg / L.

[0014] Preferably, the concentration of the nano-iron atom impregnation is 15 mg / L.

[0015] The beneficial effects of this invention are as follows: This invention provides a novel application of nano-iron atoms in promoting plant growth and nodulation nitrogen fixation. The nano-iron atoms significantly increase the plant height, root length, aboveground fresh weight, root fresh weight, aboveground dry weight, and root dry weight of alfalfa. The application method of seed soaking followed by foliar spraying is more conducive to alfalfa growth. Simultaneously, the root activity of alfalfa treated with FeNPs is significantly higher than that of the control. Phenotypic analysis of the root nodules shows that the total number of root nodules and the number of effective root nodules in the FeNPs-treated area are 2.04 times and 1.42 times higher than those in the control, respectively. Nitrogenase activity data further demonstrates that the nitrogenase activity is highest under FeNPs treatment. The urea content further indicates that FeNPs promote alfalfa's fixation of atmospheric nitrogen and the production of large amounts of urea within the root nodules, thus promoting alfalfa growth and improving the quality and biomass of leguminous forage, demonstrating broad application prospects. Attached Figure Description

[0016] Figure 1 Morphological observation of FeNPs under electron microscopy. Caption: A: Morphological characterization of FeNPs under SEM; B: Morphological characterization of FeNPs under TEM; C: EDS energy dispersive spectroscopy analysis. Figure 2 Effects of different concentrations of FeNPs on alfalfa growth. Note: A: alfalfa potted phenotype; B: alfalfa plant height and root length; C: fresh weight (FW); D: dry weight (DW); values ​​are mean ± standard deviation (n=6), different letters indicate significant differences (P<0.05).

[0017] Figure 3Effects of different FeNPs application methods on alfalfa growth. Note: A: Alfalfa potted phenotype; B: Alfalfa plant height and root length; C: Fresh weight (FW); D: Dry weight (DW); Control; FeNPs seed soaking (Soak); FeNPs spraying (Spray); FeNPs seed soaking + FeNPs spraying (Soak + Spray); Values ​​are mean ± standard deviation (n = 6), different letters indicate significant differences (P < 0.05). Figure 4 Effects of FeNPs on Alfalfa Growth Note: A: Alfalfa potted phenotype; B: Alfalfa plant height and root length; C: Fresh weight (FW); D: Dry weight (DW); Values ​​are mean ± standard deviation (n=6), different letters indicate significant differences (P<0.05).

[0018] Figure 5 Effects of FeNPs on Nitrogen Fixation Capacity in Alfalfa Nodulation Note: A: Control (Control) Alfalfa Rhizobium Morphology; B: Fe 2+ Morphology of alfalfa rhizobia; C: FeNPs; D: Root activity; E: Number of root nodules; F: Nitrogenase activity; G: Urea content. Values ​​are mean ± standard deviation (n = 6). Different letters indicate significant differences (P < 0.05). Detailed Implementation

[0019] To further illustrate the present invention, the scope of protection of the present invention will be described in detail below with reference to the embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0020] In this invention, the nano-iron was purchased from the Institute of Biology, Gansu Academy of Sciences, and the nano-iron is nano-zero-valent iron.

[0021] In the following examples, the concentration of nano-iron was 10 mg / L, and the application methods were seed soaking and spraying.

[0022] Unless otherwise specified, the methods used in the following embodiments are conventional methods in the art, and the instruments and reagents used are commercially available.

[0023] The Hoagland plant nutrient solution formula is as follows: 5 g / L KNO3, 132 g / L CaCl2·2H2O, 120 g / L MgSO4·7H2O, 100 g / L KH2PO4, 150 g / L Na2HPO4·12H2O, 5 g / L Fe-citrate, 2.96 g / L H3BO3, 0.22 g / L ZnSO4·7H2O, 0.08 g / L CuSO4·5H2O, 0.12 g / L Na2MoO4·2H2O, 2.03 g / L MnSO4·4H2O, and 0.09 g / L H2MoO4·H2O.

[0024] Example 1: Different concentrations and application methods of nano-iron promote alfalfa growth

[0025] 1. Characterization of nanomaterials and preparation of their suspensions

[0026] The nanomaterials were iron atom nanoparticles (FeNPs), purchased from the Gansu Academy of Sciences. Approximately 50 mg of FeNPs were placed on a copper grid and observed using a Hitachi S-4800 scanning electron microscope (SEM) and a FEI TALOS F200S transmission electron microscope (TEM) (State Key Laboratory of Functional Organic Chemistry, Lanzhou University). The morphology and elemental composition of the FeNPs were determined by image observation and EDS energy dispersive spectroscopy analysis.

[0027] A high-concentration FeNPs stock solution was prepared for dilution to a lower working concentration. The specific procedure was as follows: Accurately weigh 0.1 g of black FeNPs powder into a 150 mL Erlenmeyer flask, add 100 mL of deionized water, and sonicate for 30 min using an ultrasonic cleaner (BRANSON 5800, Guangdong, China) to obtain a 1 g / L FeNPs stock solution. Dilute the FeNPs stock solution with the corresponding volume of deionized water to obtain the working concentration required for this experiment.

[0028] 2. Cultivation and treatment of plant materials

[0029] The plant material was Medicago sativa cv. Galaxie Max, and the seeds were purchased from the College of Grassland Science, Gansu Agricultural University.

[0030] 3. Potted plant experiment

[0031] The pot experiment was conducted in the plant growth room of the Yifu Biological Building at Lanzhou University.

[0032] After 3 days of cultivation, alfalfa seeds with uniform growth were selected and transferred with tweezers to plastic flowerpots (9cm×10cm) containing sterilized vermiculite, with 5 seeds placed in each flowerpot. These were then placed in the plant growth chamber of the Yifu Biological Building at Lanzhou University for cultivation, initially watered with distilled water. Five days after sowing, the seedlings were thinned and watered with a modified Hoagland plant nutrient solution. Twenty-one days after sowing, different concentrations of FeNPs working solution (0, 10, 20, 50, 100, 200 mg / L) were sprayed on the alfalfa leaves until the water droplets were about to drip off (30 mL per flowerpot). Spraying was repeated every 7 days. Ten days after the third spraying, the alfalfa phenotype was photographed and samples were taken for subsequent determination of relevant indicators.

[0033] After determining the optimal concentration, a pot experiment was conducted to screen the best application method for alfalfa absorption of FeNPs at the optimal concentration. Four treatments were set up: control, FeNPs soaking (soak), FeNPs spraying (spray), and FeNPs soaking + subsequent FeNPs spraying (soak + spray). After seed sterilization, the seeds were evenly divided into four 50mL centrifuge tubes. The soaking treatment involved soaking the seeds in a 15mg / L FeNPs working solution for 8 hours, with sterile water serving as the control. Ten days after the third spraying, alfalfa phenotypic photography was performed, and samples were taken to measure relevant indicators.

[0034] 4. Measurement of relevant indicators

[0035] After phenotypic photography, alfalfa samples were taken from each treatment. The plants were rinsed with running water to remove vermiculite, and phenotypic photographs were taken again before biomass measurement. Subsequent measurements of plant height, root length, and fresh / dry weight were performed using the same methods as in the hydroponic experiment. Six replicates were set up for each treatment.

[0036] 5. Experimental Results

[0037] The morphology of FeNPs was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM images showed that FeNPs possess a typical core-shell structure, namely a nano-iron oxide shell (…). Figure 1 A) Encased in a nano-zero-valent iron core ( Figure 1 A). TEM images revealed that FeNPs exhibited non-uniformity, with many particles dispersed and aggregated together in a chain-like structure. The core-shell structure of single particles was clearly visible in the images. Figure 1 B), meaning a metallic iron core surrounded by a thin outer shell of iron oxide. EDS (Energy Dispersive Spectroscopy) analysis confirmed the chemical purity of the nanoparticles, with iron as their primary chemical component. The figure shows three Fe peaks, with the highest Cu peak originating from the copper mesh used in the EDS measurement. Figure 1 C).

[0038] Compared with the control, low concentrations of FeNPs (10, 20 mg / L) significantly increased alfalfa plant height and root length. Figure 2 A), (P<0.05). Among them, plant height increased significantly by 14.7% and 4.9%, respectively, and root length increased significantly by 31.0% and 12.0%, respectively. However, under high concentrations of FeNPs (50, 100, 200 mg / L), both plant height and root length decreased. Figure 2 B). Compared with the control, the fresh weight of the aboveground parts increased significantly by 45.4% and 3.9% respectively under low concentrations of FeNPs (10, 20 mg / L), and the fresh weight of the roots increased significantly by 99.2% and 2.0% respectively. Figure 2 C), the aboveground dry weight increased significantly by 36.1% and 2.1%, respectively, and the root dry weight increased significantly by 42.3% and 7.6%, respectively. Figure 2 D), (P<0.05); while high concentrations of FeNPs (50, 100, 200 mg / L) reduced alfalfa biomass.

[0039] Different application methods of FeNPs have different effects on alfalfa growth. Figure 3 A). Compared with the control, the plant height of alfalfa in the soaking, spraying, and soaking + spraying treatments increased significantly by 35.4%, 43.8%, and 70.8%, respectively, and the root length increased significantly by 23.9%, 48.8%, and 78.8%, respectively. Figure 3 B), the fresh weight of the aboveground parts increased significantly by 74.4%, 1.3 times, and 1.6 times, respectively, and the fresh weight of the roots increased significantly by 32.7%, 70.9%, and 1.7 times, respectively. Figure 3 C) The aboveground dry weight increased significantly by 84.9%, 1.1 times, and 1.4 times, respectively, and the root dry weight increased significantly by 20.7%, 52.7%, and 1.2 times, respectively (P<0.05). Figure 3 D). It is evident that seed soaking combined with spraying (Soak + Spray) more effectively promotes plant growth. Example 2: Effects of FeNPs on Alfalfa Growth and Nodulation Nitrogen Fixation

[0040] 1. Plant materials and cultivation

[0041] The plant material was Medicago sativa cv. Galaxie Max, and the seeds were purchased from the College of Grassland Science, Gansu Agricultural University.

[0042] The tested bacterial strain was the model strain *Sinorhizobium meliloti*, which our research group had previously frozen at -80℃. The culture media used in the experiment were YMA solid medium and TY liquid medium, with the following formulations:

[0043] YMA solid medium: mannitol 10 g / L, yeast extract 0.4 g / L, K2HPO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.1 g / L, agar powder 15 g / L, Congo red solution 2.5 mL / L.

[0044] TY liquid culture medium: tryptone 5 g / L, yeast extract 3 g / L, anhydrous calcium chloride (CaCl2) 0.665 g / L, pH = 7.4.

[0045] Preparation of bacterial suspension: The strain Sml021, frozen at -80℃, was activated twice consecutively in YMA solid medium. It was then transferred to 20 mL of sterile TY liquid medium and cultured at 28℃ and 180 rpm on a shaker (THZ-300, Shanghai Yiheng Technology) for 48 h. The bacterial concentration was measured using a UV spectrophotometer (V-5800, Shanghai Meipuda Instrument Co., Ltd.). Sterile water was added to adjust the OD600 of the bacterial suspension to 0.1 for later use.

[0046] The pot experiment was conducted in the plant growth room on the 4th floor of the Yifu Biology Building at Lanzhou University. Three treatments were set up: a blank control (Contral), a treatment inoculated with FeCl2 solution, and a treatment inoculated with FeNPs solution. All treatments included the addition of rhizobium Sml021. After sterilization, seeds were aliquoted into five 50mL centrifuge tubes. Seeds for the soaking treatment were soaked in 30mL of their corresponding treatment solution for 8 hours, with sterile water serving as a control. After soaking, the seeds were transferred to plastic pots with vermiculite substrate and watered with distilled water for 3 days. Thinning was then carried out. After thinning, alfalfa seedlings were foliar sprayed with FeNPs, spraying each treatment until the leaves were almost dripping wet (approximately 30mL per pot). Hoagland nutrient solution was used for irrigation during the cultivation period.

[0047] 2. Measurement of relevant indicators

[0048] During the early stage of cultivation, samples were taken at specific time points to measure indicators related to root nodule development. Later, when alfalfa grew to 42 days, phenotypic photographs were taken and samples were collected for the determination of plant growth and physiological indicators.

[0049] (1) Phenotypic index determination

[0050] After phenotypic photography, alfalfa samples were taken from each treatment. The plants were rinsed with running water to remove vermiculite, and phenotypic photographs were taken again before biomass measurement. Subsequent measurements of plant height, root length, and fresh / dry weight were performed using the same methods as in the hydroponic experiment. Six replicates were set up for each treatment.

[0051] (2) Root vitality

[0052] Root activity was determined using the triphenyltetrazolium chloride (TTC) method, with six replicates per treatment. A 1:1 mixture of 0.4% 2,3,5-triphenyltetrazolium chloride solution and 0.15 mol / L PBS (pH 7.0) was used to prepare the reaction solution, which was prepared immediately before use. 0.1 g of alfalfa root tips were weighed, chopped, mixed, and placed into 10 mL centrifuge tubes. One control tube was used for each treatment, with 2 mL of 1M sulfuric acid solution added. The remaining centrifuge tubes were filled with 5 mL of the reaction solution. The tubes were incubated at 37℃ in a benchtop drying oven (202-0, Shanghai Keheng Industrial Development Co., Ltd.) for 3 hours. The reaction was terminated by adding 2 mL of 1M sulfuric acid solution to all centrifuge tubes except the control tube. The root tips were removed from the centrifuge tubes with tweezers, and the surface solution was quickly blotted dry with absorbent paper. All centrifuge tubes were drained of the reaction solution, and the blotted root tips were placed inside. 5 mL of methanol solution was added, and the tubes were incubated in the dark for 24 hours. After the red color faded from the root tips, the absorbance at λ = 485nm was measured using a UV-Vis spectrophotometer (V-5800, Shanghai Meipuda Instrument Co., Ltd.) after zeroing the control tubes for each treatment. The formulas for calculating TTC reduction and root activity are as follows:

[0053] TTC reduction amount = TTC concentration × solution volume

[0054] Root activity = TTC reduction amount / W × incubation time / 1000

[0055] In the formula: W is the fresh weight of the plant root.

[0056] (3) Number of root nodules in plants

[0057] Seven days after treatment, the plants were removed from the pots and rinsed clean, and the number of root nodules was counted. White root nodules were recorded as invalid nodules, and pink root nodules as valid nodules. Each treatment was repeated 10 times.

[0058] (4) Nitrogenase activity

[0059] Nitrogenase activity of each treatment plant was determined using the acetylene reduction method (ARA). Alfalfa roots for each treatment were cut and placed in 50 mL vials, sealed with rubber stoppers, and 20 mL of air was evacuated using a syringe. An equal volume of acetylene gas was then injected, and the injection hole was sealed with a sealing strip and plastic wrap. The incubator was placed at 28°C for 3 hours. The concentration of ethylene obtained from the reduction of acetylene in the vials was determined using a gas chromatograph (GC9720, School of Chemistry and Chemical Engineering, Lanzhou University), and the nitrogenase activity of the strain was calculated accordingly.

[0060] (5) Urea content determination

[0061] The urea content in different tissues of soybean was determined using the plant urea method. The urea assay kit (Sunshine Biotech, Beijing, China) was used according to the supplier's instructions.

[0062] 3. Experimental Results

[0063] like Figure 4 As shown, the growth of alfalfa treated with FeNPs was significantly better than that of the control. The aboveground and underground parts of the plants were 50.99% and 29.58% larger than the control, respectively; the fresh weight of the plants increased by 21.93%, 48.11%, 14.41%, and 40.27% compared to the control; and the dry weight of the plants also increased by 8.17%, 49.63%, 21.17%, and 120.07% compared to the control.

[0064] like Figure 5 As shown, the root activity of alfalfa treated with FeNPs was significantly higher than that of the control. Phenotypic analysis of the root nodules revealed that the total number of nodules and the number of effective nodules in the FeNP-treated group were 2.04 times and 1.42 times higher than those in the control, respectively. Nitrogenase activity data further confirmed that the FeNP-treated group exhibited the highest nitrogenase activity. The urea content further indicates that FeNPs promote the fixation of atmospheric nitrogen by alfalfa and the production of large amounts of urea within the root nodules, thus promoting alfalfa growth.

[0065] In summary, this invention provides a novel application of nano-iron atoms in promoting plant growth and nodulation nitrogen fixation. The nano-iron atoms significantly increased alfalfa plant height, root length, aboveground fresh weight, root fresh weight, aboveground dry weight, and root dry weight. The application method of seed soaking followed by foliar spraying was more beneficial to alfalfa growth. Simultaneously, the root activity of alfalfa treated with FeNPs was significantly higher than the control. Phenotypic analysis of the root nodules showed that the total number of root nodules and the number of effective root nodules in the FeNPs-treated group were 2.04 times and 1.42 times higher than the control, respectively. Nitrogenase activity data further demonstrated that the nitrogenase activity was highest in the FeNPs-treated group. The urea content further indicates that FeNPs promote alfalfa's fixation of atmospheric nitrogen and the production of large amounts of urea within the root nodules, thus promoting alfalfa growth and improving the quality and biomass of leguminous forage, demonstrating broad application prospects.

Claims

1. The application of nano-iron atoms in promoting nitrogen fixation and nodulation in alfalfa, characterized in that, The alfalfa mentioned is Da Yinhe alfalfa ( Medicago sativa (cv. Galaxie Max), the method of using the nano iron atoms is seed soaking and foliar spraying; the concentration of the foliar spraying is 10-20 mg / L, and the concentration of the seed soaking is 10 mg / L.

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