Synergistic fertilizer and use and preparation method thereof

Abstract

The invention relates to the technical field of fertilizers, and discloses a synergistic fertilizer and a use and preparation method thereof, and the synergistic fertilizer comprises the following components by mass: 12%-35% of magnesium salt, 10%-60% of exosome, 30%-40% of amino acid, 0.5%-4% of an auxiliary agent, and the balance of water. Compared with a conventional liquid magnesium fertilizer, the synergistic fertilizer has the advantages that magnesium ions can be taken by plant cells more quickly due to the similar compatibility of plant exosomes and the characteristics of the plant exosomes identified by signals, so that the utilization rate of the fertilizer is increased, and compared with a magnesium fertilizer without exosomes, the synergistic fertilizer has obvious yield increase on the premise of the same use amount and use method through tests.

Description

Technical Field

[0001] This invention relates to the field of fertilizer technology, specifically to an enhanced fertilizer, its application, and its preparation method. Background Technology

[0002] Fertilizers, as plant nutrient supplements, are among the most frequently used products in agricultural production to promote healthy crop growth and improve yield and quality. The strength of photosynthesis is a crucial factor in plant growth, and magnesium, as the central atom of chlorophyll molecules involved in photosynthesis, is essential. Furthermore, magnesium is a structural component of ribosomes and an activator of many enzymes, especially those related to carbon dioxide assimilation. Therefore, magnesium deficiency in plants leads to weak photosynthesis and stunted growth.

[0003] In agricultural production, citrus and grapes are important economic crops in my country and globally, and these two crops are typical examples of crops prone to magnesium deficiency. These two crops prefer acidic soils. Furthermore, to promote fruit yield and sugar content, phosphorus and potassium fertilizers are often applied in large quantities. However, this leads to the fixation of magnesium ions in the soil, making them difficult to absorb and utilize. Magnesium deficiency results in interveinal yellowing of leaves, reduced photosynthetic efficiency, and ultimately, a decline in the yield and quality of citrus and grapes. Therefore, developing an efficient magnesium fertilizer is of great significance to agricultural production.

[0004] Magnesium ions are primarily absorbed by plants passively, entering plant cells down their electrochemical potential gradient. However, cell membranes have limited permeability to magnesium ions. Currently available magnesium fertilizers commonly include magnesium sulfate, typically applied to the roots, and are easily fixed by the soil. Chelating agents such as EDTA are also used to chelate magnesium fertilizers, but their utilization rate is similarly low due to the limited permeability of cell membranes to magnesium ions.

[0005] Exosomes are extracellular vesicles, membrane-bound structures with a lipid bilayer actively secreted by cells. They contain various contents derived from primitive cells, such as proteins and RNA, and are the smallest extracellular vesicles, typically between 30-150 nm in diameter. Exosomes possess the ability to precisely transport substances. They are found in various biological fluids and tissues. In 1996, Stoorvogel et al. discovered that exosomes can alter the extracellular microenvironment, participate in immune regulation, and influence overall health, leading to widespread interest in exosomes. American scientists James E. Rothman and Randy W. Schekman, along with German scientist Thomas C. Sudhof, jointly received the 2013 Nobel Prize in Physiology or Medicine for elucidating the regulatory mechanisms of precise substance transport by exosomes and other vesicles.

[0006] Currently, there are published patents that utilize exosomes to prepare pest control compositions, but there is no information available on how to use exosomes to develop an enhanced fertilizer product. Therefore, developing an enhanced magnesium fertilizer is of great significance for reducing excessive fertilizer use, improving fertilizer utilization, promoting agricultural production levels, and reducing negative environmental impacts. Summary of the Invention

[0007] This invention provides an enhanced fertilizer, its application, and a preparation method. It innovatively uses exosomes to prepare the enhanced fertilizer. Tests have shown that the prepared enhanced fertilizer can promote the absorption and utilization of magnesium ions by plants and promote healthy plant growth.

[0008] The present invention provides the following technical solution: an enhanced fertilizer comprising: 12% to 35% magnesium salts, 10% to 60% exosomes, 30% to 40% amino acids, 0.5% to 4% adjuvants, and the remainder being water.

[0009] The present invention also provides a method for using an enhanced fertilizer, which includes: diluting the enhanced fertilizer by 600-1200 times and then spraying it on the leaves or dipping it in the roots, or diluting it by 500-1000 times and mixing it with other non-strong acid or strong alkaline pesticides or fertilizers.

[0010] The present invention also provides a method for preparing an enhanced fertilizer, for preparing any of the enhanced fertilizers described above, comprising:

[0011] Step 1: Obtain exosomes by weight percentage;

[0012] Step 2: Obtain amino acids by mass percentage, and mix the exosomes and amino acids evenly to obtain a medium mixture;

[0013] Step 3: Obtain water and magnesium salt by mass percentage, add them to the intermediate mixture, and stir to dissolve to obtain the intermediate mixture;

[0014] Step 4: Add the additives according to the mass percentage to the intermediate mixture and stir to prepare the enhanced fertilizer;

[0015] This invention also provides a method for preparing exosomes, used to prepare any of the above-described synergistic fertilizers, comprising:

[0016] Step 1: Select new plant tissue, disinfect it, and inoculate it onto induction medium for embryogenic callus induction culture;

[0017] Step 2: Transfer the embryogenic callus tissue to a primary flask containing liquid culture medium for culture to obtain primary seed culture;

[0018] Step 3: Transfer the primary seed culture to a secondary flask containing liquid culture medium to obtain the secondary seed culture;

[0019] Step 4: Transfer the secondary seed culture to a tank containing liquid culture medium for expansion culture. High-density cell culture is carried out by feeding culture medium to obtain cell culture medium.

[0020] Step 5: Add magnesium salt solution to the cell culture medium, and simultaneously increase the light intensity and photoperiod to initially stimulate plant cells to secrete exosomes and exosomes containing magnesium ions;

[0021] Step 6: Increase the light intensity and photoperiod again, while increasing the stirring speed and decreasing the aeration rate to further stimulate the production of exosomes and obtain a cell suspension culture medium;

[0022] Step 7: Extract exosomes from cell suspension culture medium.

[0023] The present invention has the following beneficial effects:

[0024] 1. The enhanced fertilizer prepared in this application, through the combination of exosomes, fertilizer raw materials, amino acids and adjuvants, utilizes the ability of plant-derived exosomes to be similar to and precisely transport substances with plant cells, and the characteristics of changing the cell microenvironment. Magnesium ions in the enhanced fertilizer can be more quickly absorbed by plant cells, improving the utilization rate of fertilizer and thus reducing the amount of fertilizer used.

[0025] 2. Compared with the method of directly extracting exosomes from natural plants, the method of obtaining exosomes through plant suspension cell culture solves the problems of long raw material sources, long culture cycles, and susceptibility to adverse external factors such as diseases, pests, weeds, and climate.

[0026] 3. The method for preparing exosomes in this application involves extracting exosomes from cell suspension culture medium. The remaining suspended plant cell slurry contains a large number of plant cells and can be recycled to produce exosome-rich synergistic fertilizers. Ultimately, it can be used as a raw material for organic fertilizers, with no harmful byproducts.

[0027] 4. The method for preparing exosomes in this application improves the yield of exosomes by combining the addition of magnesium salts, changes in light intensity and period, stirring speed, and aeration rate.

[0028] 5. The method for preparing exosomes in this application utilizes plant cell suspension culture to obtain a high concentration of plant cells, and then adds magnesium salts, increases light intensity and cycle, and treats the process with low air volume and high rotation speed to enhance the uptake of magnesium ions by the suspended plant cells, thereby promoting the production of exosomes containing magnesium ions.

[0029] 6. The plant cells used in the method for preparing exosomes in this application are widely available and easy to obtain, and are not limited by soil, climate, pests and diseases, etc., and are easy to scale up for cultivation. Attached Figure Description

[0030] Figure 1 The total protein concentration is for Example 1 and Comparative Examples 1-5 of this application.

[0031] Figure 2 The particle concentration and particle size of the exosomes purified in Example 1 of this application.

[0032] Figure 3 This is an electron micrograph of the exosomes prepared in Example 1 of this application. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] This application aims to research and develop an enhanced magnesium fertilizer, which is of great significance for reducing excessive fertilizer use, improving fertilizer utilization, promoting agricultural production, and reducing negative environmental impacts. Based on this, an enhanced fertilizer is proposed, comprising, by mass percentage: 12%–35% magnesium salt, 10%–60% exosomes, 30%–40% amino acids, 0.5%–4% adjuvants, and the remainder being water. The water is deionized water. The magnesium salt is selected from one or more of magnesium sulfate, magnesium nitrate, and magnesium sodium ethylenediaminetetraacetate, preferably at a mass percentage of 15%–30%.

[0035] Exosomes include one or a combination of two of the following: exosomes extracted from plant tissues or exosomes obtained from plant cell suspension culture, preferably those obtained from plant cell suspension culture. Compared to directly extracting exosomes from natural plants, this invention obtains exosomes through plant suspension cell culture, solving the problems of long culture cycles and susceptibility to adverse external factors such as pests, diseases, weeds, and climate. Furthermore, by using a composition of exosomes, fertilizer raw materials, amino acids, and adjuvants, the invention leverages the compatibility and precise substance transport capabilities of plant-derived exosomes with plant cells, as well as their ability to alter the cellular microenvironment, thereby improving fertilizer utilization and reducing fertilizer usage.

[0036] Preferably, the plant tissue is selected from the stem tip or bud of a plant.

[0037] Preferably, the plant cells are selected from one or more of the following: citrus, grape, strawberry, codonopsis, tomato, rice, and corn.

[0038] Preferably, the exosomes are in a solution system. The exosome content is not less than 1.0*10 6 particles / mL. The exosome solution mass percentage is more preferably 30%–60%.

[0039] It should be noted that the amino acids are one or more selected from proline, alanine, serine, glycine, isoleucine, cysteine, arginine, threonine, glutamic acid, tryptophan, tyrosine, histidine, valine, glutamine, methionine, aspartic acid, phenylalanine, lysine, and soybean meal hydrolysate. The preferred technical indicators for the soybean meal hydrolysate are a free amino acid content ≥30% and chloride ion content ≤2%. The preferred mass percentage of amino acids is 30%–35%.

[0040] Preferably, the additive is a wetting, spreading, and penetrating functional additive, more preferably a polyether surfactant or alkyl glycoside; the polyether surfactant is a fatty alcohol polyoxyethylene ether, such as AEO-7, preferably Sasol AE07-24S additive, whose main component is polyethoxylated C12-14 fatty alcohol, CAS NO. 68439-50-9, and the main component content (w / w) ≥ 80%; the alkyl glycoside additive is Transfar alkyl glycoside APG0810 or Transfar alkyl glycoside AP0810.

[0041] This invention provides a method for using an enhanced fertilizer, comprising: diluting the enhanced fertilizer by 600-1200 times and then spraying it on the leaves or dipping it in water to the roots, or diluting it by 500-1000 times and mixing it with other non-strong acid or strong alkaline pesticides or fertilizers.

[0042] This invention provides a method for preparing exosomes, comprising:

[0043] Step 1: Select new plant tissues such as tender shoots, stem tips, and fertilized ovules, disinfect them, and inoculate them onto induction medium for embryogenic callus induction culture.

[0044] Step 2: Transfer the embryogenic callus tissue to a primary flask containing liquid culture medium for culture; obtain primary seed culture.

[0045] Step 3: Transfer the primary seed culture to a secondary bottle containing liquid culture medium and culture it to obtain the secondary seed culture.

[0046] Step 4: Transfer the secondary seed culture to a tank containing liquid culture medium for expansion culture; conduct high-density cell culture by feeding culture medium.

[0047] Step 5: Add magnesium salt solution to the cell culture medium from step 4, and simultaneously increase the light intensity and photoperiod to stimulate plant cells to secrete exosomes and exosomes containing magnesium ions.

[0048] Step 6: In the later stages of cell culture in step 5, further increase the light intensity and light cycle, while increasing the stirring speed and reducing the aeration rate to further stimulate the production of exosomes.

[0049] Step 7: Extract a solution containing exosomes from the cell suspension culture medium obtained in Step 6.

[0050] Exosome preparation methods currently employ a combination of techniques, including adding magnesium salts, altering light intensity and duration, stirring speed, and aeration, to increase exosome yield. However, current exosome extraction methods are limited to small-scale production, lacking large-scale manufacturing, and there is no evidence of using plant callus tissue to produce exosomes for enhancing magnesium fertilizer production. This invention utilizes plant callus tissue suspension culture to obtain a high concentration of plant cells. Then, by adding magnesium salts, increasing light intensity and duration, and applying lower aeration and higher rotation speed, the uptake of magnesium ions by the suspended plant cells is enhanced, thereby promoting the production of magnesium-containing exosomes. This provides a method for stimulating exosome loading with fertilizer substances by altering light conditions and adding magnesium salts.

[0051] Preferably, the induction medium in step 1 consists of plant tissue culture medium, sucrose, agar, and plant growth regulators. The plant tissue culture medium is one of B5 medium, MT medium, or MS medium; the sucrose content is 2.5%–3% by mass; the agar content is 0.8% by mass; the plant growth regulators are cytokinins and auxins; cytokinins include, but are not limited to, 6-benzylaminopurine and indolebutyric acid; auxins include, but are not limited to, 1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, and kinetin.

[0052] Furthermore, cytokinin is preferably 6-benzylaminopurine, with a concentration range of 0.5–5 mg / L; auxin is preferably 2,4-dichlorophenoxyacetic acid, with a concentration range of 0.5–5 mg / L.

[0053] The cultivation conditions for both the primary seed culture in step 2 and the secondary seed culture in step 3 are shake culture at a speed of 50–150 rpm, a temperature of 20–28°C, a photoperiod of 10–14 h / 24 h, and a light intensity of 0–400 lx. The pH of the liquid culture medium is 5.0–7.0, preferably 5.5–6.5. The liquid culture medium consists of commonly used plant tissue culture media, sucrose, yeast extract, and plant growth regulators. Commonly used plant tissue culture media are one of B5 medium, MT medium, and MS medium; the sucrose content is 2.5%–3% by mass; the yeast extract is a dry powder of yeast extract. In some specific embodiments of the present invention, the yeast powder is Angel Yeast Extract FM810 or FM802, with a mass content of 0.15%–0.3%; the plant growth regulators are cytokinins and auxins; cytokinins include, but are not limited to, 6-benzylaminopurine and indolebutyric acid; auxins include, but are not limited to, 1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, and kinetin. Furthermore, 6-benzylaminopurine is preferred as the cytokinin, with a concentration range of 0.5–5 mg / L; 2,4-D is preferred as the auxin, with a concentration range of 0.5–5 mg / L.

[0054] In step 4, both the expansion culture and high-density cell culture were conducted under aeration combined with stirring. For expansion culture, the aeration rate was 0.1–1 m³ / h, the stirring speed was 10–50 rpm, the temperature was 20–28°C, the photoperiod was 10–12 h / 24 h, and the light intensity was 0–400 lx. For high-density culture, the aeration rate was 0.5–1.5 m³ / h, the stirring speed was 30–100 rpm, the temperature was 20–28°C, the photoperiod was 10–12 h / 24 h, and the light intensity was 0–800 lx. The nutrient solution was prepared with a pH of 5.0–7.0, preferably 6.0–7.0, and consisted of commonly used plant tissue culture medium, sucrose, yeast extract, and plant growth regulators. Commonly used plant tissue culture media include B5 medium, MT medium, and MS medium; the sucrose content is 9%–12% by mass; the yeast extract is a dry powder of yeast extract, and in some specific embodiments of the present invention, the yeast powder is Angel Yeast Extract FM810 or FM802, with a mass content of 0.15%–0.3%; the plant growth regulators are cytokinins and auxins; cytokinins include, but are not limited to, 6-benzylaminopurine and indolebutyric acid; auxins include, but are not limited to, 1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, and kinetin. Further, 6-benzylaminopurine is preferred as the cytokinin, with a concentration range of 1.5–25 mg / L; 2,4-dichlorophenoxyacetic acid is preferred as the auxin, with a concentration range of 1.5–25 mg / L.

[0055] In step 5, the magnesium salt solution consists of tap water, magnesium sulfate, and sodium ethylenediaminetetraacetate (EDTA), with a mass ratio of magnesium sulfate to EDTA of 3:1, resulting in a total mass content of 0.3%–0.7% for magnesium sulfate and EDTA in the culture medium. The light intensity is 400 lx–1000 lx, and the photoperiod is 12–14 h / 24 h.

[0056] In step 6, the light intensity is 800 lx to 1500 lx. The light cycle is 14 to 24 hours / 24 hours. The stirring speed is 100 to 150 rpm. The aeration rate is 0.05 to 0.5 m³ / h.

[0057] In step 7, the exosome solution is extracted using ceramic membrane filtration with a membrane size of 200 nm and 100 nm. The remaining suspended plant cell slurry after filtration contains a large number of plant cells and can be recycled to produce exosome-rich, enhanced fertilizers, which can ultimately be used as raw materials for organic fertilizers without any harmful byproducts. Furthermore, unlike exosomes extracted from natural plants, the exosome producers of this invention, i.e., plant cells, are widely available and easily obtained, not limited by soil, climate, pests, or other conditions, and are easily cultured on a large scale.

[0058] This invention provides a method for preparing an enhanced fertilizer, comprising: obtaining 12%–35% magnesium salt, 10%–60% amino acids, and 0.5%–4% adjuvants by mass percentage, with the remainder being water. The exosome solution obtained in step 7 above and the amino acids are mixed evenly, then water and magnesium salt are added, stirred to dissolve, and the adjuvants are added and stirred until well mixed to obtain the enhanced fertilizer.

[0059] To better explain the solution of this application, this application provides an enhanced fertilizer and its preparation method, as detailed below:

[0060] Example 1

[0061] A method for preparing exosomes, comprising:

[0062] S1. Select fresh strawberry stem tips as explants.

[0063] S2. After disinfecting the explants, inoculate them onto induction medium for embryogenic callus induction culture.

[0064] S3. Transfer the embryogenic callus tissue to a primary flask containing liquid culture medium for culture; obtain primary seed culture.

[0065] S4. Transfer the primary seed culture to a secondary bottle containing liquid culture medium and culture it to obtain the secondary seed culture.

[0066] S5. Transfer the secondary seed culture to a container containing liquid culture medium for scale-up culture. The aeration rate for scale-up culture is 0.1–0.5 m³ / h, the stirring speed is 10–30 rpm, the culture temperature is 27±1°C, the photoperiod is 10h / 24h, and the light intensity is 0 lx. High-density cell culture is then carried out using a fed-batch culture medium. The aeration rate for high-density culture is 0.5–1 m³ / h, the stirring speed is 30–80 rpm, the culture temperature is 27±1°C, the photoperiod is 10h / 24h, and the light intensity is 400 lx.

[0067] S6. Add sterilized magnesium salt solution to the cell culture medium from S5. The magnesium salt in the solution is magnesium sulfate and sodium ethylenediaminetetraacetate (EDTA), with a mass ratio of 3:1, so that the total mass content of magnesium sulfate and EDTA in the culture medium is 0.5%. Simultaneously, increase the light intensity to 1000 lx and the photoperiod to 14h / 24h to stimulate plant cells to secrete exosomes and exosomes containing magnesium ions.

[0068] S7. In the later stage of S6 cell culture, the light intensity was further increased to 1500 lx and the light cycle was 24h / 24h. At the same time, the stirring speed was increased to 110-150 rpm and the aeration rate was reduced to 0.05-0.2 m³ / h to further stimulate the production of exosomes.

[0069] S8. Filter the cell suspension culture medium from S7 using a ceramic membrane to obtain a solution containing exosomes for later use.

[0070] Comparative Example 1

[0071] A method for preparing exosomes, the difference between this comparative example and Example 1 is that magnesium salt is not added in step S6.

[0072] Comparative Example 2

[0073] Another method for preparing exosomes differs from that in this comparative example and Example 1 in that: after adding magnesium salt in step S6, the light intensity remains unchanged at 400 lx; ​​the light cycle remains unchanged at 10 h / 24 h.

[0074] Comparative Example 3

[0075] A method for preparing exosomes, the difference between this comparative example and Example 1 is that: in step S7, in the later stage of cell culture, the light intensity remains unchanged and is maintained at 1000 lx; ​​the light cycle remains unchanged and is maintained at 14h / 24h.

[0076] Comparative Example 4

[0077] A method for preparing exosomes, the difference between this comparative example and Example 1 is that in step S7, during the later stage of cell culture, the stirring speed remains unchanged and is maintained at 30-80 rpm.

[0078] Comparative Example 5

[0079] A method for preparing exosomes differs from Example 1 in that: in step S7, during the later stage of cell culture, the aeration rate remains unchanged, continuing to be maintained at 0.5–1 m³ / h.

[0080] 160 ml samples were taken from the exosome solutions obtained in Example 1 and Comparative Examples 1-5, and exosomes were extracted and purified by ultracentrifugation. The total protein concentration of Examples 1 and Comparative Examples 1-5 was determined using a BCA protein assay kit (Adamas, catalog number ZJ102). The results are as follows: Figure 1 As shown, Example 1 utilizes a combined process of adding magnesium salt to high-density cell culture medium to simultaneously increase light intensity and light cycle, further increasing light intensity, light cycle, and stirring speed while reducing aeration in the later stages of cell culture. The resulting purified exosomes exhibited the highest total protein concentration, at 7.292 μg / μL, indicating that the combined process of Example 1 can increase exosome yield.

[0081] The particle size of the purified exosomes from Example 1 was further determined using a NanoSight nanoparticle size analyzer (Malvin Instruments Ltd., UK, model NS300). The purified exosome solution was diluted 11.25 times and analyzed using the instrument; the results are as follows. Figure 2 As shown, the measured concentration was 6.75e+08+ / - 6.85e+07 particles / ml, the main particle size was 155.0 nm, and the average particle size was 194.8 nm.

[0082] The appearance of the purified exosomes from Example 1 was further characterized using a transmission electron microscope (JEOL, model JEM1230), such as... Figure 3 As shown, a saucer-like morphology of about 100 nm can be observed, which is consistent with the morphological characteristics of exosomes.

[0083] Example 2

[0084] A method for preparing an enhanced fertilizer, comprising:

[0085] S1. Take 3000g of the exosome solution obtained in Example 1, add 3000g of soybean meal hydrolysate containing 30% free amino acids, and stir evenly.

[0086] S2. Take 2300g of deionized water, add 1500g of magnesium sulfate, stir and mix well, then add it in batches to the mixed solution of exosomes and amino acids, stirring while adding, until the magnesium sulfate is completely dissolved.

[0087] S3. Add 200g of Sasol AE07-24S adjuvant, stir for 30 minutes to mix well, and obtain an enhanced magnesium fertilizer containing exosomes.

[0088] Example 3

[0089] A method for preparing an enhanced fertilizer, comprising:

[0090] S1. Take 4030g of the exosome solution obtained in Example 1, add 3000g of soybean meal hydrolysate containing 30% free amino acids, and stir while adding until well mixed.

[0091] S2. Add 1000g of magnesium sulfate and 1770g of sodium magnesium ethylenediaminetetraacetate in sequence, stirring while adding, until the magnesium salt is completely dissolved.

[0092] S3. Add 200g of Sasol AE07-24S adjuvant, stir for 30 minutes to mix well, and obtain an enhanced magnesium fertilizer containing exosomes.

[0093] Example 4

[0094] A method for preparing an enhanced fertilizer, comprising:

[0095] S1. Take 5100g of the exosome solution obtained in Example 1, and add 2000g of soybean meal hydrolyzed amino acid solution with a free amino acid content of 30%, 500g of glycine with a purity of 99.7%, and 500g of L-alanine with a purity of 99.8% in sequence while stirring until the amino acids are completely mixed and dissolved.

[0096] S2. Add 1000g of magnesium sulfate and 600g of magnesium nitrate in sequence, stirring while adding, until the magnesium salt is completely dissolved.

[0097] S3. Add 400g of Sasol AE07-24S adjuvant, stir for 30 minutes to mix well, and obtain an enhanced magnesium fertilizer containing exosomes.

[0098] Comparative Example 6

[0099] A method for preparing magnesium fertilizer without exosomes, the difference between this comparative example and Example 4 is that: in step S1, the exosome solution obtained in Example 1 is taken and the exosomes are removed by ultracentrifugation to obtain solution A; 5100g of solution A is taken to replace the 5100g of exosome solution in step S1 of Example 4.

[0100] The application effects of the exosome-containing magnesium fertilizer of Example 4 of the present invention and the liquid magnesium fertilizer without exosomes in Comparative Example 6 are compared.

[0101] Applied crop: Citrus fruit of the variety Red Beauty.

[0102] Application area: One connected greenhouse was used as the test plot for each of the two fertilizers. Each greenhouse contained 100 citrus trees, each 6 years old, and the area of ​​each greenhouse was approximately 675 square meters.

[0103] Application period and method:

[0104] First use: In late March, during the new shoot sprouting period, dilute the test fertilizer 600 times and spray it once on both sides of the leaves. That is, dissolve 100g of test fertilizer in 60,000g of water and spray it evenly on both sides of the citrus leaves in the test plot.

[0105] Second application: In late May, during the young fruit development period, dilute the test fertilizer 600 times and spray it on the leaves once. That is, dissolve 100g of test fertilizer in 60,000g of water and spray it evenly on the front and back of the citrus leaves in the test plot.

[0106] Third application: Starting in late July during the fruit enlargement period, spray the leaves three times with the test fertilizer diluted 600 times, with an interval of 14 days between applications. Each time, take 25g of the test fertilizer and dissolve it in 1500g of water. Use 25g of the test fertilizer for 300 square meters.

[0107] During the experiment, the water and fertilizer management was the same in both test plots, except for the application of different test fertilizers. No other magnesium fertilizers were used during the experiment.

[0108] Yield Comparison: In late November, citrus fruits were harvested in the experimental plots, and the weight of citrus fruits in the plots where the two test fertilizers were applied was compared. The total weight of citrus fruits using the synergistic fertilizer containing exosomes (Example 4) was 3307 kg; the total weight of citrus fruits using the liquid magnesium fertilizer without exosomes (Comparative Example 6) was 3024 kg. Compared to the liquid magnesium fertilizer without exosomes used in Comparative Example 6, the synergistic fertilizer containing exosomes (Example 4) increased yield by 9.36%.

[0109] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0110] 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 technical principles 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 synergistic fertilizer, characterized in that, By weight percentage, it comprises: 12%–35% magnesium salts, 10%–60% exosomes, 30%–40% amino acids, 0.5%–4% adjuvants, and the remainder is water.

2. The enhanced fertilizer according to claim 1, characterized in that: Exosomes include at least one of the following: exosomes extracted from plant tissues or exosomes obtained from plant cell suspension culture.

3. The enhanced fertilizer according to claim 2, characterized in that: Plant cells include at least one of citrus, grape, strawberry, codonopsis, tomato, rice, or corn.

4. The enhanced fertilizer according to claim 1 or 2, characterized in that: The exosomes are in a solution system, and the exosome content in the solution is higher than 1.0*10. 6 particles / mL.

5. The enhanced fertilizer according to claim 1 or 2, characterized in that: The amino acids include at least one of the following: proline, alanine, serine, glycine, isoleucine, cysteine, arginine, threonine, glutamic acid, tryptophan, tyrosine, histidine, valine, glutamine, methionine, aspartic acid, phenylalanine, lysine, or hydrolyzed amino acid solution from soybean meal.

6. The enhanced fertilizer according to claim 1 or 2, characterized in that: The additives are wetting, spreading, and penetrating agents.

7. A method of applying a synergistic fertilizer, comprising using the synergistic fertilizer as described in any one of claims 1-6, characterized in that: include: Dilute the synergistic fertilizer 600-1200 times and apply as a foliar spray or root dip, or dilute it 500-1000 times and mix it with other non-strong acid or strong alkaline pesticides or fertilizers.

8. A method for preparing an enhanced fertilizer, used to prepare the enhanced fertilizer as described in any one of claims 1-6, characterized in that: include: Step 1: Obtain exosomes by weight percentage; Step 2: Obtain amino acids by mass percentage, and mix the exosomes and amino acids evenly to obtain a medium mixture; Step 3: Obtain water and magnesium salt by mass percentage, add them to the intermediate mixture, and stir to dissolve to obtain the intermediate mixture; Step 4: Obtain the adjuvants according to the mass percentage, add them to the intermediate mixture, stir and mix well to prepare the enhanced fertilizer.

9. The method for preparing synergistic fertilizer according to claim 8, characterized in that: Methods for preparing exosomes include: Step 1: Select new plant tissue, disinfect it, and inoculate it onto induction medium for embryogenic callus induction culture; Step 2: Transfer the embryogenic callus tissue to a primary flask containing liquid culture medium for culture to obtain primary seed culture; Step 3: Transfer the primary seed culture to a secondary flask containing liquid culture medium to obtain the secondary seed culture; Step 4: Transfer the secondary seed culture to a tank containing liquid culture medium for expansion culture. High-density cell culture is carried out by feeding culture medium to obtain cell culture medium. Step 5: Add magnesium salt solution to the cell culture medium, and simultaneously increase the light intensity and photoperiod to initially stimulate plant cells to secrete exosomes and exosomes containing magnesium ions; Step 6: Increase the light intensity and photoperiod again, while increasing the stirring speed and decreasing the aeration rate to further stimulate the production of exosomes and obtain a cell suspension culture medium; Step 7: Extract exosomes from cell suspension culture medium.

10. The method for preparing synergistic fertilizer according to claim 9, characterized in that: The induction medium in step 1 includes plant tissue culture medium, sucrose, agar, and plant growth regulators; The liquid culture medium for both the primary seed culture in step 2 and the secondary seed culture in step 3 includes plant tissue culture medium, sucrose, yeast extract, and plant growth regulators. The culture conditions are shake culture at 50-150 rpm, temperature 20-28°C, photoperiod 10-14 h / 24 h, light intensity 0-400 lx, and pH of the liquid culture medium 5.0-7.

0. In step 4, both the expansion culture and high-density cell culture were conducted under conditions of aeration combined with stirring. For expansion culture, the aeration rate was 0.1–1 m³ / h, the stirring speed was 10–50 rpm, the temperature was 20–28°C, the photoperiod was 10–12 h / 24 h, and the light intensity was 0–400 lx. For high-density cell culture, the aeration rate was 0.5–1.5 m³ / h, the stirring speed was 30–100 rpm, the temperature was 20–28°C, the photoperiod was 10–12 h / 24 h, and the light intensity was 0–800 lx. During fed-batch culture, the pH of the nutrient solution was 5.0–7.

0. The magnesium salt solution in step 5 includes tap water, magnesium sulfate, and magnesium sodium ethylenediaminetetraacetate. The total mass content of magnesium sulfate and magnesium sodium ethylenediaminetetraacetate is 0.3% to 0.7%, and the light intensity in step 5 is 400 lx to 1000 lx. The light cycle is 12-14 h / 24 h. In step 6, the light intensity is 800 lx to 1500 lx, the light cycle is 14 to 24 h / 24 h, the stirring speed is 100 to 150 rpm, and the aeration rate is 0.05 to 0.5 m³ / h. In step 7, exosomes are extracted from the cell suspension culture medium by using ceramic membrane filtration.

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