Biostimulant as well as preparation method and application thereof

Abstract

The invention discloses a biostimulant as well as a preparation method and application thereof. The biostimulant disclosed by the invention comprises the following raw materials: PQQ, organic acid, free amino acid and solvent water. The method comprises the following steps: dissolving PQQ, organic acid and free amino acid in solvent water to obtain a mixed solution; and carrying out resonance treatment on the mixed solution to obtain the biostimulant. According to the biostimulant provided by the invention, resonance treatment is utilized, so that the biostimulant has higher stability and better efficiency, is more easily absorbed by plants, has the effects of promoting crop growth, improving the soil state, improving the disease resistance of the plants and the like, and has a wide prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medicine fertilizers, and particularly relates to a biostimulant and its preparation method and application. Background Art

[0002] In the process of agricultural development, the excessive use of chemical wastes and pesticides not only leads to the decline of soil quality and crop quality, but also causes environmental pollution problems and food safety problems. The emergence of plant biostimulants has brought new ideas for solving the problem of "reducing the use of chemical fertilizers and pesticides".

[0003] A biostimulant is a substance containing certain components and / or microorganisms that can improve the nutrition and health status of crops, increase crop yield and improve quality. Due to its origin from natural biological fermentation and having advantages such as little harm to the environment and quick effect, biostimulants are widely used in agricultural production. Currently, the generally recognized types of plant biostimulants mainly include humic acid, seaweed extracts, protein hydrolysates and amino acids, chitin, chitosan and its derivatives, and microbial inoculants. The most commonly used products in the market are compound products containing biostimulants, but the quality of such products is uneven. Many biostimulant products cannot produce the expected effects in actual applications. Summary of the Invention

[0004] The purpose of the present invention is to provide a biostimulant and its preparation method and application. The biostimulant provided by the present invention is prepared by mixing pyrroloquinoline quinone (PQQ for short), organic acids and free amino acids, and then through resonance treatment, and has the effects of promoting seed germination, improving the disease resistance and stress resistance of plants, etc., and has broad prospects.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a biostimulant, the raw materials of which include PQQ, organic acids, free amino acids and solvent water.

[0007] Further, the usage amount of the PQQ is 60 mg / L to 1000 mg / L based on the volume of the solvent water;

[0008] Further, the PQQ is PQQ crystal or a microbial fermentation broth containing PQQ.

[0009] Further, the usage amounts of the organic acids, based on the volume of the solvent water, are: tartaric acid 4.5 - 6.3 mg / ml, malonic acid 1.3 - 1.8 mg / ml, lactic acid 87.4 - 103.8 mg / ml, citric acid 36.7 - 48.2 mg / ml, succinic acid 10.6 - 13.9 mg / ml;

[0010] Further, the usage amounts of the free amino acids, based on the volume of the solvent water, are as follows: 3.7 - 6.1 mg / ml of glutamic acid, 2.1 - 3.2 mg / ml of histidine, 4.2 - 5.7 mg / ml of phenylalanine, 5.9 - 7.8 mg / ml of proline, 2.9 - 4.6 mg / ml of serine, 5.6 - 6.1 mg / ml of alanine, 3.8 - 4.9 mg / ml of valine, 3.6 - 5.9 mg / ml of lysine, and 4.2 - 5.6 mg / ml of arginine.

[0011] Further, the biostimulant is used by means of seed soaking, spraying or root irrigation. When used for seed soaking, it is diluted 50 - 500 times; when used for spraying, it is diluted 200 - 1000 times; when used for root irrigation, it is diluted 100 - 500 times. It is applied 2 - 3 times per season, respectively at the planting stage, the early flowering stage, and the early fruit setting stage.

[0012] The present invention also provides a preparation method of the above biostimulant, which includes the following steps:

[0013] (1) Calculate and weigh PQQ, organic acid and amino acids according to the usage amounts of the biostimulant raw materials, dissolve them in water, and mix well to obtain a mixed liquid;

[0014] (2) Subject the mixed liquid to resonance treatment to obtain the biostimulant.

[0015] Further, the device used for the resonance treatment is an electromagnetic vibration device, whose frequency is 144 - 216 KHz and the treatment time is 10 - 12 min. The maximum power consumption of the electromagnetic vibration device for the resonance treatment ≤ 20 w, the adapter input is AC100 - 24V 1.5A, and the output is DC12V 4A 5A.

[0016] In the present invention, the electromagnetic vibration device is provided by Guangdong Suisen Industry Co., Ltd., and the original supplier is Japan Refine Wave Corp (Japan Refining Wave Company). The photo of the electromagnetic vibration device is shown in Figure 4 as follows.

[0017] When this device is in use, by generating an instantaneous high voltage (up to 12000V) to form an electromagnetic field with a certain frequency, after circulating and treating the liquid product for a certain time, some easily broken covalent bonds or peptide bonds can be broken, or the mixed proteins can be broken into polypeptides, or some covalent bonds of high molecular polysaccharides can be broken into low molecular polysaccharides. At the same time, it promotes the chelation between biological chemical components and promotes the chelation effect. Its working principle is as shown in Figure 5 as follows.

[0018] The mixed liquid will be more stable after resonance treatment, and various components can be better chelated and play their roles more efficiently. At the same time, the increase in the content of hydrogen ions in the solution can also improve the stability and efficacy of the biostimulant.

[0019] The application of the biostimulant in seed germination, soil improvement, crop yield increase, and disease prevention.

[0020] Advantages of the present invention:

[0021] (1) The biostimulant provided by the present invention is treated by resonance, and the biostimulant treated by the electromagnetic vibration device is more conducive to plant absorption.

[0022] (2) After the resonance treatment of the biostimulant provided by the present invention by the electromagnetic vibration device, the content of hydrogen ions is increased, thereby improving the stability and efficacy of the product.

[0023] (3) The biostimulant provided by the present invention has the effects of promoting seed germination, improving soil conditions, increasing crop yields, preventing disease infections, etc., and is green, economical, and pollution-free, solving the problem of reducing the use of chemical fertilizers and pesticides, and has broad prospects. Description of the drawings

[0024] Figure 1 Phylogenetic tree analysis of Acinetobacter calcoaceticus CDWB36;

[0025] Figure 2 Photographs of the morphological characteristics of the cells of Acinetobacter calcoaceticus CDWB36;

[0026] Figure 3 Photographs of the colony morphological characteristics of Acinetobacter calcoaceticus CDWB36;

[0027] Figure 4 Photograph of the electromagnetic vibration device.

[0028] Figure 5 Working principle diagram of the electromagnetic vibration device. Detailed implementation manners

[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1 Preparation of Biostimulant

[0031] Weigh the raw materials according to Table 1 to prepare the biostimulant:

[0032] Table 1 Usage of Each Raw Material Component of the Biostimulant

[0033] Raw material component name Dosage (mg / mL) PQQ 0.06 Tartaric acid 6.1 Malonic acid 1.5 Lactic acid 91.2 Citric acid 41.7 Succinic acid 12.4 Glutamic acid 4.5 Histidine 2.6 Phenylalanine 4.8 Proline 6.7 Serine 3.1 Alanine 5.9 Valine 4.2 Lysine 4.9 Arginine 4.9

[0034] Weigh the above raw material components according to the table and dissolve them in water, mix well, and process the mixed liquid through an electromagnetic vibration device. Among them, the resonance processing frequency is 180 KHz, and it is processed for 10 minutes to obtain the biostimulant.

[0035] Sample and inspect the mixed liquid before and after resonance processing respectively. The test results show that before processing, the pH value of the solution is 7.85, and after processing, the pH value of the solution is 7.51. In addition, the data in Table 2 show that the proportion of small and medium molecular weight substances increases after resonance processing of the mixed liquid.

[0036] Table 2 Molecular Weight Distribution Data of the Mixed Liquid Samples before and after Resonance Processing

[0037]

[0038]

[0039] Comparative Example 1 Preparation of the Mixed Liquid without Resonance Processing

[0040] Compared with Example 1, the difference in this comparative example is only that after mixing the components, resonance processing is not used, and finally the mixed liquid without resonance processing is obtained.

[0041] Comparative Example 2 Preparation of Microbial Fermentation Broth Containing PQQ

[0042] This comparative example involves a strain of Acinetobacter calcoaceticus CDWB36, with the preservation number: CCTCC NO: M2023964, FL04, preserved in the China Center for Type Culture Collection, the preservation date: June 8, 2023, and the preservation address: Wuhan University, China. It can be used for fermenting and producing PQQ.

[0043] Inoculate the Acinetobacter calcoaceticus CDWB36 strain into the LB liquid medium, and under the condition of 30 °C, shake and culture in a shaker at a rotation speed of 180 r / min. Stop culturing when the sampling inspection result OD 600 = 0.8, and obtain the seed liquid. After detection, the viable bacteria concentration in the seed liquid is 2.3×10 4 cfu / mL.

[0044] The obtained seed liquid was then inoculated into the optimized medium at an inoculation amount of 0.5%, the shaking speed of the shaker was set at 180 rpm / min, the pH was adjusted to 6.5 using NaOH, and it was cultured in a shaker at 28°C for 7 days to obtain the Acinetobacter calcoaceticus fermentation broth.

[0045] The formula of the above optimized medium is as follows: yeast powder 10 g / L, mixed nitrogen source (ammonium sulfate:glutamic acid:tyrosine = 2:1:1) 4 g / L, Na2HPO4 2 g / L, KH2PO4 1.4 g / L, MgSO4·7H2O 1 g / L, 0.4258 g / L trace element solution; the trace element solution contains: FeSO4·7H2O 80 mg / L, ZnSO4·7H2O 22.5 mg / L, KI 0.3 mg / L, H3BO3 3 mg / L, CuSO4 5 mg / L, NaCl 15 mg / L, CaCl2·2H2O 400 mg / L.

[0046] The preparation method of LB liquid medium is: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L, dissolve in water, and finally make up the volume to 1000 mL, pH 6.5, autoclave (121°C, 30 min).

[0047] The content of PQQ in the obtained Acinetobacter calcoaceticus fermentation broth was detected to be 60 mg / L.

[0048] Verification experiment on the promotion of tomato seed germination by the biostimulant in Example 2

[0049] Using tomato seeds as experimental materials, the biostimulant obtained in Example 1 was diluted 100 times, soaked for 12 h and then drained for the seed germination experiment. Four liquids, namely water, the fermentation broth obtained in Comparative Example 2, the unresonated mixed liquid obtained in Comparative Example 1 diluted 100 times, and 60 mg / L PQQ solution, were used for soaking the seeds as controls. Seven days after breeding, the seed germination rate and germination potential were counted. The results are shown in Table 3.

[0050] Germination potential (%) = (number of germinated seeds in 4 days / total number of tested seeds) × 100%

[0051] Germination rate (%) = (number of germinated seeds in 7 days / total number of tested seeds) × 100%

[0052] Table 3 Results of the promotion of tomato seed germination by the biostimulant

[0053]

[0054]

[0055] The results showed that, compared with the control group, the germination rate of tomato seeds was improved in each treatment group. Among them, the germination rate of tomato seeds in the group treated with the mixed liquid without resonance treatment (in Comparative Example 1), the fermentation broth containing PQQ (in Comparative Example 2), and the PQQ solution was higher than that of the water control treatment, but lower than that of the biostimulant treatment group. Therefore, compared with other treatments, the biostimulant had the best effect on promoting the germination of tomato seeds.

[0056] Example 3 Verification Experiment on the Improvement of Saline-alkali Soil and Crop Yield Increase by Biostimulant

[0057] The experiment was carried out in a tomato planting base in Dongying City, Shandong Province. Five treatments were set up in the experiment: biostimulant treatment, applying 15 L / ha of the biostimulant obtained in Example 1; water control treatment, applying 15 L / ha of water; treatment with the mixed liquid in Comparative Example 1, applying 15 L / ha of the mixed liquid; treatment with the fermentation broth in Comparative Example 2, applying 15 L / ha of the fermentation broth; treatment with PQQ solution, applying 15 L / ha of 60 mg / L PQQ solution. Each treatment was diluted 200 times with water and then sprayed. It was sprayed on the soil 1 day before planting, and then sprayed once every month after planting tomatoes, and root irrigation and foliar spraying were carried out simultaneously. Samples were taken after 3 months of planting to measure the relevant physical and chemical properties of the soil and crop yield, and the results are shown in Table 4.

[0058] Table 4 Results of the Influence of Biostimulant on Saline-alkali Soil and Tomato Growth

[0059]

[0060] The results showed that the prepared biostimulant had a significant effect on the soil nutrients and the content of water-soluble salts in the saline-alkali soil. The contents of soil nutrients nitrogen, phosphorus and organic matter increased significantly, the content of water-soluble salts and pH value in the soil decreased significantly, and the soil fertility was improved. Applying the biostimulant was beneficial to the improvement of saline-alkali soil and the growth of tomatoes.

[0061] Example 4 Control Effect of Biostimulant on Tomato Root Rot

[0062] Healthy seedlings (tomatoes) with good growth and the same size were selected and transplanted into pots filled with 10 kg of substrate soil, one plant per pot, and conventionally managed in a greenhouse at 26 °C. Five treatments were set up in the experiment:

[0063] Biostimulant treatment: Dilute the biostimulant obtained in Example 1 by 100 times, irrigate 50 ml per plant, and then irrigate the roots of each tomato plant with 10 8 cfu / mL of the root rot pathogen bacterial liquid 20 ml after 15 days;

[0064] Water control treatment: Irrigate 50 ml per plant with water, and then irrigate the roots of each tomato plant with 10 820 ml of the root rot pathogen bacterial solution at cfu / mL;

[0065] Treatment of the mixed liquid in Comparative Example 1: Dilute the mixed liquid obtained in Comparative Example 1 by 100 times, irrigate 50 ml per plant, and then drench the roots of each tomato plant with 10 8 20 ml of the root rot pathogen bacterial solution at cfu / mL;

[0066] Treatment of the fermentation broth in Comparative Example 2: Irrigate 50 ml per plant with the fermentation broth obtained in Comparative Example 2, and then drench the roots of each tomato plant with 10 8 20 ml of the root rot pathogen bacterial solution at cfu / mL;

[0067] Treatment with PQQ solution: Irrigate 50 ml per plant with 60 mg / L PQQ solution, and then drench the roots of each tomato plant with 10 8 20 ml of the root rot pathogen bacterial solution at cfu / mL.

[0068] Each treatment group treated 12 tomato seedlings. Before transplantation, the soil was sprayed and treated once. After the root rot pathogen in the clear water control group colonized and caused tomato disease, no pathogen bacterial solution treatment was carried out for all treatment groups. Other measures were repeated according to the above treatment every month. After 90 days of planting, the incidence rate was counted, and the results are shown in Table 5.

[0069] Table 5 Control effects of different treatments on tomato root rot

[0070] Treatment Number of diseased plants Total number Incidence rate (%) Biostimulant 2 12 16.7 Water control 10 12 83.3 Mixed liquid in Comparative Example 1 6 12 50.0 Fermentation broth in Comparative Example 2 5 12 41.7 PQQ solution 9 12 75.0

[0071] The results showed that the biostimulant had a certain effect on controlling tomato root rot. The incidence rate of tomato root rot in the clear water control group was 83.3%, and after treatment with the biostimulant, the incidence rate decreased to 16.7%. The relative control effect of the biostimulant reached 80.0%, and the biocontrol effect was significant.

Claims

1. A biostimulant, characterized in that, The raw materials of the biostimulant include PQQ, organic acids, free amino acids and solvent water.

2. The biostimulant according to claim 1, characterized in that, The PQQ is PQQ crystal or microbial fermentation broth containing PQQ.

3. The biostimulant according to claim 1, characterized in that, The dosage of the PQQ is 60 mg / L to 1000 mg / L based on the volume of the solvent water.

4. The biostimulant according to claim 1, characterized in that, The dosages of the organic acids are as follows based on the volume of the solvent water: tartaric acid 4.5 - 6.3 mg / ml, malonic acid 1.3 - 1.8 mg / ml, lactic acid 87.4 - 103.8 mg / ml, citric acid 36.7 - 48.2 mg / ml, succinic acid 10.6 - 13.9 mg / ml.

5. The biostimulant according to claim 1, characterized in that, The dosages of the free amino acids are as follows based on the volume of the solvent water: glutamic acid 3.7 - 6.1 mg / ml, histidine 2.1 - 3.2 mg / ml, phenylalanine 4.2 - 5.7 mg / ml, proline 5.9 - 7.8 mg / ml, serine 2.9 - 4.6 mg / ml, alanine 5.6 - 6.1 mg / ml, valine 3.8 - 4.9 mg / ml, lysine 3.6 - 5.9 mg / ml, arginine 4.2 - 5.6 mg / ml.

6. The biostimulant according to claim 1, wherein The biostimulant is used by means of seed soaking, spraying or root irrigation. When soaking seeds, it is diluted 50 - 500 times; when spraying, it is diluted 200 - 1000 times for application; when irrigating roots, it is diluted 100 - 500 times for application, 2 - 3 times per season, and applied respectively at the planting stage, the early flowering stage and the early fruit setting stage.

7. A preparation method of the biostimulant according to any one of claims 1 - 6, comprising the following steps: (1) Calculate and weigh PQQ, organic acids and amino acids according to the usage amounts of the raw materials of the biostimulant, dissolve them in the solvent water, and mix well to obtain a mixed liquid; (2) Subject the mixed liquid to resonance treatment to obtain the biostimulant.

8. The preparation method according to claim 7, characterized in that, In step (2), the resonance treatment uses an electromagnetic vibration device with a frequency of 144 - 216 KHz and a treatment time of 10 - 12 min.

9. An application of the biostimulant according to any one of claims 1 - 6 or the biostimulant prepared by the preparation method according to any one of claims 7 - 8 in seed germination, soil improvement, crop yield increase and disease prevention.

Images