A plant-derived growth regulator and its application in corn planting

Abstract

The present invention discloses a plant-derived growth regulator and its application in corn planting, belonging to the technical field of plant growth regulators. The growth regulator comprises the following raw materials in parts by weight: 30-50 parts of algae plant extract, 5-15 parts of microbial fermentation liquid, 1.5-2.0 parts of adjuvant, 1-3 parts of trace elements, 0.1-0.5 parts of solubilizing synergist, and 50-70 parts of water. The present invention uses spirulina powder and chlorella powder as raw materials to prepare a highly active plant active extract, and simultaneously screens and obtains two functional microorganisms to form a plant growth regulator, which can significantly improve the growth of corn root system, improve corn quality and yield, enhance soil activity, reduce the use of fertilizers and pesticides, reduce environmental pollution, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant growth regulators, and in particular relates to a plant-derived growth regulator and application thereof in corn planting. Background Art

[0002] Corn is one of the main food crops and feed crops, and is also an important chemical, fuel and pharmaceutical raw material. Studying the growth process of corn and increasing the final yield of corn through artificial intervention is one of the effective means to solve resource shortages and the continued growth in demand for corn, and it has far-reaching significance. Crop chemical control cultivation is an important application technology for cost saving, efficiency improvement and yield increase in the planting industry, and is currently widely used in agricultural production. In recent years, the application of plant growth regulators as a means of regulating crop growth and development has received increasing attention. Plant growth regulators are the physical and chemical carriers of crop chemical control, which refer to substances applied to plants from the outside that can cause changes in plant growth and development.

[0003] Common plant growth regulators are mostly chemical substances. The long-term use of such substances will have obvious adverse effects on plants and soil. If chemical plant growth regulators are used excessively or incorrectly, they will cause phytotoxicity to crops. For example, arbitrarily increasing the dosage or using too high a concentration will not only fail to promote plant growth, but will inhibit its growth and even cause leaf deformity, drying and falling, and death of the entire plant. In addition, different crops have different sensitivities to plant growth regulators. Improper use can lead to problems such as reduced production and decreased quality. For example, when applying plant growth regulators such as paclobutrazol to corn, if not handled properly, it may lead to a decrease in grain yield.

[0004] In addition, chemical plant growth regulators also have a negative impact on the environment. These regulators enter water bodies through surface runoff, leakage, etc., which may cause harm to aquatic life. Their persistence and bioaccumulation in the soil make them difficult to degrade, thus having a long-term impact on the soil ecosystem. In addition, the residue problem of plant growth regulators is also worthy of attention. Irregular use can cause harm to human health.

[0005] Therefore, developing a green, efficient and pollution-free plant growth regulator is a technical problem that needs to be solved urgently. Summary of the invention

[0006] The present invention aims at the problems existing in the prior art. The present invention uses spirulina powder and chlorella powder as raw materials to prepare a highly active plant active extract, and simultaneously screens out two functional microorganisms to form a plant growth regulator, which has a significant promoting effect on the growth of corn roots, improves soil activity, and thus greatly improves corn yield and quality.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0008] A plant-derived growth regulator comprises the following raw materials in parts by weight: 30-50 parts of algae plant extract, 5-15 parts of microbial fermentation liquid, 1.5-2.0 parts of auxiliary agent, 1-3 parts of trace elements, 0.1-0.5 parts of solubilizing agent and 50-70 parts of water.

[0009] Furthermore, the microbial fermentation broth is produced by Bacillus subtilis ( Bacillus subtilis ) and Saccharomyces cerevisiae ( Saccharomyces paradoxus ) is prepared by fermentation, and the specific preparation method is as follows:

[0010] (1) Strain activation: Bacillus subtilis and Saccharomyces cerevisiae stored in a refrigerator were transferred into LB medium and placed at 25-28° C. for activation culture for 24 hours to obtain activated strains of Bacillus subtilis and Saccharomyces cerevisiae;

[0011] (2) Seed liquid culture: Take a loop of activated Bacillus subtilis and activated Saccharomyces cerevisiae strains, respectively inoculate into 300 mL conical flasks filled with seed culture medium, fill with 25% liquid, place in a shaking incubator at 25-28°C and 100-180 rpm for 5-10 h to obtain seed liquid of the two bacteria, and mix the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0012] (3) Fermentation culture: The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-28°C and 200-220 r / min for fermentation until the effective viable count was 1-2×10 9 cfu / ml, and the pH value was adjusted to 7.0-7.3 to obtain microbial fermentation broth.

[0013] Furthermore, the Bacillus subtilis has a deposit number of CGMCC No. 12522, a deposit date of January 16, 2023, and is deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration; the strain number of the Saccharomyces cerevisiae is CGMCC No. 2.5691, purchased from the General Microbiology Center of the China National Microbiological Culture Collection Administration, and the original deposit date is December 5, 2016. The Saccharomyces cerevisiae can be purchased through the collection center without repeated deposit.

[0014] Furthermore, the Bacillus subtilis was isolated from the rhizosphere soil of a tomato in an agricultural planting demonstration garden in Linshu County, Linyi City, Shandong Province. The separation and screening method was as follows: 5 g of soil sample was weighed and placed in a 100 mL conical flask containing 45 mL of sterile water, cultured at 25-28°C and 180 r / min for 20 min, and then allowed to stand for 10 min. Then the soil bacterial suspension was heated to 10 −2 −10 −8 After gradient dilution, spread it on LB solid culture medium and culture it in a constant temperature box at 25-28℃ for 24-48h. Finally, pick colonies of different morphologies for strain purification and culture to obtain the corresponding bacterial liquid. The purified strains were stored in a refrigerator at 4℃. The strain separation test was carried out 3 times in total, with 3 repetitions each time. After culturing the purified bacteria in LB liquid culture medium (added with L-tryptophan 100mg / L) for 24h, take 50μL of bacterial liquid and 50μL of Salkowski reagent on a white ceramic plate, keep it away from light for 30min for color reaction, and use sterilized LB liquid culture medium as negative control. The strain with the darkest red is the target strain. After the target strain is cultured on the LB plate at 25-28℃ for 24h, the colony is milky white, opaque, rough and matte, with irregular edges, and there is mucous secretion after picking up the colony. The colony picture is as follows Figure 1 shown.

[0015] Furthermore, the composition of the seed culture medium is: tryptone 11.0 g·L –1 , yeast extract powder 6.0 g•L –1 , NaCl10.5g•L –1 , pH 7.0-7.4, high temperature sterilization at 121℃ for 20min.

[0016] Furthermore, the composition of the fermentation medium is: glucose 25.0 g•L –1 , peptone 12.0 g•L –1 , KH2PO41.0g•L –1 ,MgSO41.0g•L –1 ,MnSO4·H2O 0.2 g·L –1 , sterilize at 115℃ for 20min.

[0017] Furthermore, the preparation method of the algae plant extract is: weigh 1g of Spirulina powder and 1g of Chlorella powder, add 100mL of water to swell, repeatedly freeze and thaw to break the wall, the freeze-thaw method is: freezing at -20°C, thawing at room temperature, repeating 5 times to end the freeze-thaw, and then bathing in 60°C water for 1h, centrifuging at 6000r / min for 10min, collecting the supernatant and vacuum drying to obtain a water extract, the collected water extract is re-dissolved with sterilized water, and passed through a 0.22μm water phase sterilization membrane to obtain an algae plant extract with a water extract concentration of 10mg / mL.

[0018] Furthermore, the auxiliary agent is obtained by mixing ethephon, chlormequat chloride, glucose and citric acid in a mass ratio of 0.1:0.3:10:5.

[0019] Furthermore, the trace element is one or more of CuSO4, ZnSO4, and MgSO4.

[0020] Furthermore, the solubilizing agent is sodium dodecyl sulfate and / or sodium lignin sulfonate.

[0021] A method for preparing a plant-derived growth regulator comprises the following steps:

[0022] (1) preparing microbial fermentation broth;

[0023] (2) Preparation of algae extract:

[0024] (3) adding auxiliary agents and trace elements into water in sequence, mixing and stirring to obtain a premix;

[0025] (4) Finally, the microbial fermentation liquid and the algae plant extract are added to the premix simultaneously, and then the synergist is added, and the mixture is stirred and mixed evenly to obtain the plant-derived growth regulator.

[0026] The invention discloses an application of a plant-derived growth regulator, wherein the plant-derived growth regulator is applied to corn planting to promote the growth of corn root systems and improve the yield and resistance of corn plants.

[0027] The method for using the plant-derived growth regulator of the present invention is as follows: soak corn seeds in the plant-derived growth regulator of the present invention until the solution covers the seeds, take out the corn seeds after standing for 5 minutes, and after sowing, water the roots of the corn seedlings with the plant-derived growth regulator diluted 20 times 7 days after the corn seedlings germinate, with a dosage of 20-30 mL per plant.

[0028] Beneficial effects:

[0029] (1) The present invention screened a strain of Bacillus subtilis from the rhizosphere of tomatoes, which can efficiently secrete plant hormones such as auxin and cytokinin, especially indoleacetic acid compounds, and has growth-promoting functions, including phosphate solubilization, siderophore production, ACC deaminase production, and biofilm production. In particular, it has a significant promoting effect on the growth of corn roots.

[0030] (2) Secondly, the present invention compounded with strange yeast. On the one hand, the active metabolites of strange yeast have multiple functions, including improving soil and promoting growth, resisting stress, and increasing efficiency and yield. These metabolites can act on the entire life cycle of crops from seed germination to maturity and harvest through various pathways, thereby promoting plant growth; in addition, strange yeast may also inhibit the invasion of pathogens by secreting antitoxin proteins and antibacterial substances, thereby indirectly promoting plant growth. For example, it secretes extracellular lytic enzymes and chitinases, which can effectively inhibit the reproduction of pathogens, thereby protecting plants from diseases; more importantly, the combination of the strange yeast of the present invention and the Bacillus subtilis screened by the present invention can significantly improve the growth-promoting properties of Bacillus subtilis, significantly increase the growth rate, root length and fruit number of plants, and improve corn yield and quality;

[0031] (3) The present invention extracts the effective active substances in Spirulina and Chlorella powder by repeated freezing and thawing and water bathing. Spirulina extract contains a variety of plant hormone substances, such as auxin, gibberellin and cytokinin, which can directly affect the primary and secondary metabolism levels of cells, thereby promoting the growth of corn roots; Chlorella extract is rich in polysaccharides, purine substances, alginic acid compounds and other bioactive substances, which can improve the soil, increase the absorption rate of nutrients in the soil, and enhance the stress resistance of crops. Alginic acid can also stimulate microbial activity and enhance the effect of microorganisms. These substances work together and synergistically promote the development and growth of plant roots;

[0032] (4) Finally, the present invention adds a small amount of traditional plant regulators such as ethephon to assist in regulating the plant, adds trace elements to provide supplementary nutrition for the root system, and adds solubilizing agents to improve the overall dissolution and dispersion effect of the system.

[0033] (5) In summary, the plant-derived growth regulator of the present invention can significantly improve the growth of corn roots, improve corn quality and yield, enhance soil activity, reduce the use of chemical fertilizers and pesticides, and reduce environmental pollution, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A colony diagram of the Bacillus subtilis screened for the present invention;

[0035] Figure 2 The effect of algae extract on the growth of Bacillus subtilis;

[0036] Figure 3 The effect of algae extract on the growth of Saccharomyces cerevisiae;

[0037] Figure 4 This is the corn yield of different experimental groups in the field experiment. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0039] Example 1

[0040] A plant-derived growth regulator comprises the following raw materials in parts by weight: 30 parts of algae plant extract, 5 parts of microbial fermentation liquid, 1.5 parts of auxiliary agent, 3 parts of trace elements, 0.1 part of solubility enhancer, and 50 parts of water.

[0041] The microbial fermentation liquid is prepared from Bacillus subtilis ( Bacillus subtilis ) and Saccharomyces cerevisiae ( Saccharomyces paradoxus ) is prepared by fermentation, and the specific preparation method is as follows:

[0042] (1) Strain activation: Bacillus subtilis and Saccharomyces cerevisiae stored in a refrigerator were transferred into LB medium and placed at 25-28° C. for activation culture for 24 hours to obtain activated strains of Bacillus subtilis and Saccharomyces cerevisiae;

[0043] (2) Seed liquid culture: Take a loop of activated Bacillus subtilis and activated Saccharomyces cerevisiae strains, respectively inoculate into 300 mL conical flasks filled with seed culture medium, fill with 25% liquid, place in a shaking incubator at 25-28°C and 100 rpm for 5 h to obtain seed liquid of the two bacteria, and mix the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0044] (3) Fermentation culture: The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-28°C and 200 r / min for fermentation until the effective viable count was 1-2×10 9 cfu / ml, and the pH value was adjusted to 7.0-7.3 to obtain microbial fermentation broth.

[0045] The Bacillus subtilis has a deposit number of CGMCC No. 12522 and a deposit date of January 16, 2023, and is deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration; the strain number of the Saccharomyces cerevisiae is CGMCC No. 2.5691, purchased from the General Microbiology Center of the China National Microbiological Culture Collection Administration, and the original deposit date is December 5, 2016. The Saccharomyces cerevisiae can be purchased through the collection center without repeated deposit.

[0046] The Bacillus subtilis was isolated from the rhizosphere soil of tomatoes in a certain agricultural planting demonstration garden in Linshu County, Linyi City, Shandong Province. The separation and screening method was as follows: 5 g of soil sample was weighed and placed in a 100 mL triangular flask filled with 45 mL of sterile water, cultured at 25-28°C and 180 r / min for 20 min, and then allowed to stand for 10 min. Then the soil bacterial suspension was heated to 10 −2 −10 −8 After gradient dilution, spread it on LB solid medium and culture it in a constant temperature box at 25-28℃ for 24-48h. Finally, pick colonies of different morphologies for strain purification and culture. The purified strains were stored in a refrigerator at 4℃ for use. The strain separation test was carried out 3 times in total, with 3 repetitions each time. After the purified bacteria were cultured in LB liquid medium (added with 100 mg / L of L-tryptophan) for 24 h, 50μL of bacterial solution and 50μL of Salkowski reagent were placed on a white ceramic plate and kept away from light for 30 min for color development. The sterilized LB liquid medium was used as a negative control. The strain with the darkest red color is the target strain. After the target strain was cultured on the LB plate at 25-28℃ for 24h, the colonies were milky white, opaque, rough and matte, with irregular edges, and there was a mucous secretion after picking up the colonies. The colony picture is as follows Figure 1 shown.

[0047] The preparation method of the algae plant extract is as follows: weigh 1g of spirulina powder and 1g of chlorella powder, add 100mL of water to swell, and repeatedly freeze-thaw to break the wall, wherein the freeze-thaw method is: freezing at -20°C, thawing at room temperature, repeating 5 times to end the freeze-thaw, then bathing in 60°C water for 1h, centrifuging at 6000r / min for 10min, collecting the supernatant and vacuum drying to obtain a water extract, re-dissolving the collected water extract with sterilized water, and passing through a 0.22μm water phase sterilization membrane to obtain an algae plant extract with a water extract concentration of 10mg / mL.

[0048] The auxiliary agent is obtained by mixing ethephon, chlormequat chloride, glucose and citric acid in a mass ratio of 0.1:0.3:10:5.

[0049] The trace elements are CuSO4, ZnSO4, and MgSO4 mixed in a mass ratio of 1:1:1.

[0050] The solubility enhancer is sodium lauryl sulfate.

[0051] A method for preparing a plant-derived growth regulator comprises the following steps:

[0052] (1) preparing microbial fermentation broth;

[0053] (2) Preparation of algae extract:

[0054] (3) adding auxiliary agents and trace elements into water in sequence, mixing and stirring to obtain a premix;

[0055] (4) Finally, the microbial fermentation liquid and the algae plant extract are added to the premix simultaneously, and then the synergist is added, and the mixture is stirred and mixed evenly to obtain the plant-derived growth regulator.

[0056] Example 2

[0057] A plant-derived growth regulator comprises the following raw materials in parts by weight: 40 parts of algae plant extract, 7 parts of microbial fermentation liquid, 1.5 parts of auxiliary agent, 2 parts of trace elements, 0.3 parts of solubility enhancer, and 65 parts of water.

[0058] The microbial fermentation liquid is prepared from Bacillus subtilis ( Bacillus subtilis ) and Saccharomyces cerevisiae ( Saccharomyces paradoxus ) is prepared by fermentation, and the specific preparation method is as follows:

[0059] (1) Strain activation: Bacillus subtilis and Saccharomyces cerevisiae stored in a refrigerator were transferred into LB medium and placed at 25-28° C. for activation culture for 24 hours to obtain activated strains of Bacillus subtilis and Saccharomyces cerevisiae;

[0060] (2) Seed liquid culture: Take a loop of activated Bacillus subtilis and activated Saccharomyces cerevisiae strains, respectively, and inoculate them into 300 mL conical flasks filled with seed culture medium, with a liquid volume of 25%, and place them in a shaking incubator at 25-28°C and 180 rpm for 5 h to obtain seed liquid of the two bacteria. The seed liquids of the two bacteria are evenly mixed in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0061] (3) Fermentation culture: The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-28°C and 200 r / min for fermentation until the effective viable count was 1-2×10 9 cfu / ml, and the pH value was adjusted to 7.0-7.3 to obtain microbial fermentation broth.

[0062] The Bacillus subtilis and Saccharomyces cerevisiae are the same as those in Example 1.

[0063] The Bacillus subtilis separation and screening method is the same as that in Example 1.

[0064] The preparation method of the algae plant extract is the same as that of Example 1.

[0065] The auxiliary agent is obtained by mixing ethephon, chlormequat chloride, glucose and citric acid in a mass ratio of 0.1:0.3:10:5.

[0066] The trace element is CuSO4.

[0067] The solubilizing synergist is sodium lignin sulfonate.

[0068] A method for preparing a plant-derived growth regulator comprises the following steps:

[0069] (1) preparing microbial fermentation broth;

[0070] (2) Preparation of algae extract:

[0071] (3) adding auxiliary agents and trace elements into water in sequence, mixing and stirring to obtain a premix;

[0072] (4) Finally, the microbial fermentation liquid and the algae plant extract are added to the premix simultaneously, and then the synergist is added, and the mixture is stirred and mixed evenly to obtain the plant-derived growth regulator.

[0073] Example 3

[0074] A plant-derived growth regulator comprises the following raw materials in parts by weight: 42 parts of algae plant extract, 8 parts of microbial fermentation liquid, 1.5 parts of auxiliary agent, 1 part of trace elements, 0.4 parts of solubilizing agent and 70 parts of water.

[0075] The microbial fermentation liquid is prepared from Bacillus subtilis ( Bacillus subtilis ) and Saccharomyces cerevisiae ( Saccharomyces paradoxus ) is prepared by fermentation, and the specific preparation method is as follows:

[0076] (1) Strain activation: Bacillus subtilis and Saccharomyces cerevisiae stored in a refrigerator were transferred into LB medium and placed at 25-28° C. for activation culture for 24 hours to obtain activated strains of Bacillus subtilis and Saccharomyces cerevisiae;

[0077] (2) Seed liquid culture: Take a loop of activated Bacillus subtilis and activated Saccharomyces cerevisiae strains, respectively inoculate into 300 mL conical flasks filled with seed culture medium, fill with 25% liquid, place in a shaking incubator at 25-28°C and 100 rpm for 10 h to obtain seed liquid of the two bacteria, and mix the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0078] (3) Fermentation culture: The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-28°C and 220 r / min for fermentation until the effective viable bacterial count was 1-2×10 9 cfu / ml, and the pH value was adjusted to 7.0-7.3 to obtain microbial fermentation broth.

[0079] The Bacillus subtilis and Saccharomyces cerevisiae are the same as those in Example 1.

[0080] The Bacillus subtilis separation and screening method is the same as that in Example 1.

[0081] The preparation method of the algae plant extract is the same as that of Example 1.

[0082] The auxiliary agent is obtained by mixing ethephon, chlormequat chloride, glucose and citric acid in a mass ratio of 0.1:0.3:10:5.

[0083] The trace element is ZnSO4.

[0084] The solubility enhancer is sodium lauryl sulfate.

[0085] A method for preparing a plant-derived growth regulator comprises the following steps:

[0086] (1) preparing microbial fermentation broth;

[0087] (2) Preparation of algae extract:

[0088] (3) adding auxiliary agents and trace elements into water in sequence, mixing and stirring to obtain a premix;

[0089] (4) Finally, the microbial fermentation liquid and the algae plant extract are added to the premix simultaneously, and then the synergist is added, and the mixture is stirred and mixed evenly to obtain the plant-derived growth regulator.

[0090] Example 4

[0091] A plant-derived growth regulator comprises the following raw materials in parts by weight: 45 parts of algae plant extract, 10 parts of microbial fermentation liquid, 1 part of auxiliary agent, 2 parts of trace elements, 0.3 parts of solubilizing agent and 60 parts of water.

[0092] The microbial fermentation liquid is prepared from Bacillus subtilis ( Bacillus subtilis ) and Saccharomyces cerevisiae ( Saccharomyces paradoxus ) is prepared by fermentation, and the specific preparation method is as follows:

[0093] (1) Strain activation: Bacillus subtilis and Saccharomyces cerevisiae stored in a refrigerator were transferred into LB medium and placed at 25-28° C. for activation culture for 24 hours to obtain activated strains of Bacillus subtilis and Saccharomyces cerevisiae;

[0094] (2) Seed liquid culture: Take a loop of activated Bacillus subtilis and activated Saccharomyces cerevisiae strains, respectively inoculate into 300 mL conical flasks filled with seed culture medium, fill with 25% liquid, place in a shaking incubator at 25-28°C and 100 rpm for 8 h to obtain seed liquid of the two bacteria, and mix the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0095] (3) Fermentation culture: The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-28°C and 200 r / min for fermentation until the effective viable count was 1-2×10 9 cfu / ml, and the pH value was adjusted to 7.0-7.3 to obtain microbial fermentation broth.

[0096] The Bacillus subtilis and Saccharomyces cerevisiae are the same as those in Example 1.

[0097] The Bacillus subtilis separation and screening method is the same as that in Example 1.

[0098] The preparation method of the algae plant extract is the same as that of Example 1.

[0099] The auxiliary agent is obtained by mixing ethephon, chlormequat chloride, glucose and citric acid in a mass ratio of 0.1:0.3:10:5.

[0100] The trace elements are CuSO4, ZnSO4, and MgSO4 mixed in a mass ratio of 1:2:2.

[0101] The solubility enhancer is sodium lauryl sulfate.

[0102] A method for preparing a plant-derived growth regulator comprises the following steps:

[0103] (1) preparing microbial fermentation broth;

[0104] (2) Preparation of algae extract:

[0105] (3) adding auxiliary agents and trace elements into water in sequence, mixing and stirring to obtain a premix;

[0106] (4) Finally, the microbial fermentation liquid and the algae plant extract are added to the premix simultaneously, and then the synergist is added, and the mixture is stirred and mixed evenly to obtain the plant-derived growth regulator.

[0107] Example 5

[0108] A plant-derived growth regulator comprises the following raw materials in parts by weight: 50 parts of algae plant extract, 15 parts of microbial fermentation liquid, 2 parts of auxiliary agent, 1 part of trace elements, 0.5 parts of solubility enhancer and 60 parts of water.

[0109] The microbial fermentation liquid is prepared from Bacillus subtilis ( Bacillus subtilis ) and Saccharomyces cerevisiae ( Saccharomyces paradoxus ) is prepared by fermentation, and the specific preparation method is as follows:

[0110] (1) Strain activation: Bacillus subtilis and Saccharomyces cerevisiae stored in a refrigerator were transferred into LB medium and placed at 25-28° C. for activation culture for 24 hours to obtain activated strains of Bacillus subtilis and Saccharomyces cerevisiae;

[0111] (2) Seed liquid culture: Take a loop of activated Bacillus subtilis and activated Saccharomyces cerevisiae strains, respectively, and inoculate them into 300 mL conical flasks filled with seed culture medium, with a liquid volume of 25%, and place them in a shaking incubator at 25-28°C and 180 rpm for 10 h to obtain seed liquids of the two bacteria. The seed liquids of the two bacteria are evenly mixed in a volume ratio of 1:1 to obtain a mixed seed liquid;

[0112] (3) Fermentation culture: The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-28°C and 220 r / min for fermentation until the effective viable bacterial count was 1-2×10 9 cfu / ml, and the pH value was adjusted to 7.0-7.3 to obtain microbial fermentation broth.

[0113] The Bacillus subtilis and Saccharomyces cerevisiae are the same as those in Example 1.

[0114] The Bacillus subtilis separation and screening method is the same as that in Example 1.

[0115] The preparation method of the algae plant extract is the same as that of Example 1.

[0116] The auxiliary agent is obtained by mixing ethephon, chlormequat chloride, glucose and citric acid in a mass ratio of 0.1:0.3:10:5.

[0117] The trace elements are CuSO4, ZnSO4, and MgSO4 mixed in a mass ratio of 1:2:1.

[0118] The solubility enhancer is sodium lauryl sulfate.

[0119] A method for preparing a plant-derived growth regulator comprises the following steps:

[0120] (1) preparing microbial fermentation broth;

[0121] (2) Preparation of algae extract:

[0122] (3) adding auxiliary agents and trace elements into water in sequence, mixing and stirring to obtain a premix;

[0123] (4) Finally, the microbial fermentation liquid and the algae plant extract are added to the premix simultaneously, and then the synergist is added, and the mixture is stirred and mixed evenly to obtain the plant-derived growth regulator.

[0124] Comparative Example 1

[0125] A plant-derived growth regulator comprises the following raw materials in parts by weight: 30 parts of algae plant extract, 5 parts of microbial fermentation liquid, 1.5 parts of auxiliary agent, 3 parts of trace elements, 0.1 part of solubility enhancer, and 50 parts of water.

[0126] The microbial fermentation liquid is prepared by fermentation of Bacillus subtilis, and the specific preparation method is as follows:

[0127] (1) Strain activation: The Bacillus subtilis stored in the refrigerator was transferred into LB medium and placed at 25-28° C. for activation culture for 24 hours to obtain an activated strain of Bacillus subtilis;

[0128] (2) Seed liquid culture: Take a loop of activated Bacillus subtilis and inoculate it into a 300 mL conical flask filled with seed culture medium, fill it with 25% liquid, and culture it in a shaking incubator at 25-28°C and 100 rpm for 5 h to obtain seed liquid;

[0129] (3) Fermentation culture: Inoculate the seed liquid at a 1% inoculum into a 1L conical flask containing fermentation medium, fill it with liquid to 50%, and culture it in a shaking incubator at 25-28°C and 200 r / min until the effective viable count is 1-2×10 9 cfu / ml, and the pH value was adjusted to 7.0-7.3 to obtain microbial fermentation broth.

[0130] The Bacillus subtilis is the same as in Example 1.

[0131] The Bacillus subtilis separation and screening method is the same as that in Example 1.

[0132] In this comparative example, except that only Bacillus subtilis was used in the preparation of the microbial fermentation liquid, the other raw materials and preparation method were the same as those in Example 1.

[0133] Comparative Example 2

[0134] A plant-derived growth regulator comprises the following raw materials in parts by weight: 30 parts of algae plant extract, 5 parts of microbial fermentation liquid, 1.5 parts of auxiliary agent, 3 parts of trace elements, 0.1 part of solubility enhancer, and 50 parts of water.

[0135] The microbial fermentation liquid is prepared by fermentation of Saccharomyces cerevisiae, and the specific preparation method is as follows:

[0136] (1) Strain activation: The Saccharomyces cerevisiae stored in a refrigerator was transferred into LB medium and placed at 25-28° C. for activation culture for 24 hours to obtain an activated strain of Saccharomyces cerevisiae;

[0137] (2) Seed liquid culture: Take a loop of activated Saccharomyces cerevisiae and inoculate it into a 300 mL conical flask filled with seed culture medium, fill it with 25% liquid, and culture it in a shaking incubator at 25-28°C and 100 rpm for 5 h to obtain seed liquid;

[0138] (3) Fermentation culture: Inoculate the seed liquid at a 1% inoculum into a 1L conical flask containing fermentation medium, fill it with liquid to 50%, and culture it in a shaking incubator at 25-28°C and 200 r / min until the effective viable count is 1-2×10 9 cfu / ml, and the pH value was adjusted to 7.0-7.3 to obtain microbial fermentation broth.

[0139] The strange yeast is the same as in Example 1.

[0140] In this comparative example, except that only Saccharomyces cerevisiae was used in the preparation of the microbial fermentation liquid, the other raw materials and preparation method were the same as those in Example 1.

[0141] Comparative Example 3

[0142] A plant-derived growth regulator comprises the following raw materials in parts by weight: 30 parts of algae plant extract, 5 parts of microbial fermentation liquid, 1.5 parts of auxiliary agent, 3 parts of trace elements, 0.1 part of solubility enhancer, and 50 parts of water.

[0143] The preparation method of the algae plant extract is as follows: weigh 1g of spirulina powder, add 100mL of water to swell, and repeatedly freeze-thaw to break the wall, wherein the freeze-thaw method is: freezing at -20°C, thawing at room temperature, repeating 5 times to end the freeze-thaw, then bathing in 60°C water for 1h, centrifuging at 6000r / min for 10min, collecting the supernatant and vacuum drying to obtain a water extract, re-dissolving the collected water extract with sterilized water, and passing through a 0.22μm water phase sterilization membrane to obtain an algae plant extract with a water extract concentration of 10mg / mL.

[0144] In this comparative example, except that only spirulina powder was used to prepare the algae plant extract, the other raw materials and preparation process were the same as those in Example 1.

[0145] Comparative Example 4

[0146] A plant-derived growth regulator comprises the following raw materials in parts by weight: 30 parts of algae plant extract, 5 parts of microbial fermentation liquid, 1.5 parts of auxiliary agent, 3 parts of trace elements, 0.1 part of solubility enhancer, and 50 parts of water.

[0147] The preparation method of the algae plant extract is as follows: weigh 1g of chlorella powder, add 100mL of water to swell, and repeatedly freeze and thaw to break the wall, wherein the freeze and thaw method is: freezing at -20°C, thawing at room temperature, repeating 5 times to end the freeze and thaw, then bathing in 60°C water for 1h, centrifuging at 6000r / min for 10min, collecting the supernatant and vacuum drying to obtain a water extract, re-dissolving the collected water extract with sterilized water, and passing through a 0.22μm water phase sterilization membrane to obtain an algae plant extract with a water extract concentration of 10mg / mL.

[0148] In this comparative example, except that only Chlorella powder was used to prepare the algae plant extract, the other raw materials and preparation process were the same as those in Example 1.

[0149] Comparative Example 5

[0150] A plant-derived growth regulator comprises the following raw materials in parts by weight: 30 parts of algae plant extract, 5 parts of microbial fermentation liquid, 1.5 parts of auxiliary agent, 3 parts of trace elements, 0.1 part of solubility enhancer, and 50 parts of water.

[0151] The preparation method of the algae plant extract is as follows: 5g of spirulina powder and 1g of chlorella powder are weighed, 100mL of water is added for swelling, and the algae plant extract is repeatedly frozen and thawed to break the wall, wherein the freezing and thawing method is: freezing at -20°C, thawing at room temperature, repeating the freezing and thawing for 5 times to end the freezing and thawing, and then bathing in a 60°C water bath for 1h, centrifuging at 6000r / min for 10min, collecting the supernatant and vacuum drying to obtain a water extract, and the collected water extract is re-dissolved with sterilized water, and passed through a 0.22μm water phase sterilization membrane to obtain an algae plant extract with a water extract concentration of 10mg / mL.

[0152] In this comparative example, except for increasing the amount of spirulina powder, the other raw materials and preparation process are the same as those in Example 1.

[0153] Comparative Example 6

[0154] A plant-derived growth regulator comprises the following raw materials in parts by weight: 30 parts of algae plant extract, 5 parts of microbial fermentation liquid, 1.5 parts of auxiliary agent, 3 parts of trace elements, 0.1 part of solubility enhancer, and 50 parts of water.

[0155] The preparation method of the algae plant extract is as follows: weighing 1g of spirulina powder and 5g of chlorella powder, adding 100mL of water to swell, repeatedly freezing and thawing to break the wall, the freezing and thawing method is: freezing at -20°C, thawing at room temperature, repeating 5 times to end the freezing and thawing, then bathing in 60°C water for 1h, centrifuging at 6000r / min for 10min, collecting the supernatant and vacuum drying to obtain a water extract, re-dissolving the collected water extract with sterilized water, passing through a 0.22μm water phase sterilization membrane, and obtaining an algae plant extract with a water extract concentration of 10mg / mL.

[0156] In this comparative example, except for the amount of Chlorella powder added, the other raw materials and preparation process are the same as those in Example 1.

[0157] Performance Testing

[0158] Identification of the growth-promoting ability of strains:

[0159] Quantitative test of IAA-producing strains:

[0160] Bacillus subtilis and Saccharomyces cerevisiae strains were inoculated in LB (containing 100 mg·L –1 L-tryptophan) liquid medium, 25-28℃, 180 r·min –1 Culture for 24 h at 10000 r·min –1 The bacterial suspension was centrifuged for 10 min under the condition of , and 2.5 mL of supernatant was taken. At the same time, the concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg mL were prepared. – 1 standard solution, and mixed with Salkowski colorimetric solution at a volume ratio of 1:1, placed at room temperature and away from light for 30 minutes, and measured its OD with distilled water as the control. 530 The corresponding IAA content was calculated according to the standard curve.

[0161] Phosphate determination method:

[0162] Use a pipette to transfer 5 μL of the bacterial solution to the Meng Jinna medium, culture it in a constant temperature incubator at 25-28℃ for 2-3 days, and measure its phosphorus dissolving circle diameter (D) and colony diameter (d). The larger the ratio of the phosphorus dissolving circle diameter (D) of the degrading bacteria to the colony diameter (d), the better the organic phosphorus degradation effect.

[0163] Method for determination of siderophore production capacity:

[0164] 5 μL of the target bacteria solution was applied to the CAS plate and cultured at 25-28°C for 2-3 days to observe whether the colonies produced an orange-yellow halo on the CAS plate. The size of the iron-eating zone was calculated by measuring the colony diameter and the halo diameter. The iron-eating zone size = (halo diameter − colony diameter) / 2. The target strain was inoculated into the MKB medium at a 1% inoculation rate and cultured at 25-28°C and 180 r·min. –1 The culture was carried out for 48 h, and the bacterial solution was taken at 10000 r·min –1 Centrifuge for 10 min, take 3 mL of supernatant in a test tube, add 3 mL of CAS detection solution, mix well and react at room temperature for 1 h, and measure the absorbance As at 630 nm; take another 3 mL of CAS detection solution and mix it with uninoculated MKB medium, and measure the absorbance Ar in the same way. Calculate the iron carrier production according to the formula (As-Ar) / Ar×100%.

[0165] ACC deaminase production capacity determination method:

[0166] The target bacteria were inoculated into ADF medium and DF medium respectively, and the culture medium was kept under the same conditions (25-28℃, 180r·min –1 ) Culture for 24 hours and measure the OD of the bacterial solution with a spectrophotometer 600 The ACC deaminase production capacity of the strain was quantitatively determined according to the method in the literature. The reference is: Penrose Donna M, Glick Bernard R. Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria[J]. Physiologia plantarum, 2003, 118(1): 10−15.

[0167] The ability to produce biofilm was determined using the crystal violet staining method, following the procedure described in the literature: Tang Peijia, Isolation, screening and growth-promoting function of a soybean growth-promoting bacterium Bacillus safensis J2 [D]. Harbin: Northeast Forestry University, 2021.

[0168] Table 1 Growth-promoting properties of strains

[0169]

[0170] From the data in the table, we can see that the Bacillus subtilis and Saccharomyces cerevisiae screened by the present invention both have good growth-promoting properties and secrete various growth-promoting active substances. Among them, biofilm formation is the basis for the successful colonization of PGPR in the rhizosphere of plants, and the stable colonization of PGPR in the rhizosphere of plants is a prerequisite for effectively exerting its growth-promoting effect. The two growth-promoting bacteria of the present invention have good biofilm production capabilities at the same time, and the strains can colonize the root system of plants, thereby continuously and stably exerting a growth-promoting effect.

[0171] Effects of algae extracts on the growth of strains:

[0172] The algae extracts obtained in Example 1 (the preparation method of algae extracts in Examples 2-5 is the same as that in Example 1, so repeated tests are omitted) and Examples 3-6 were tested as follows: the extract to be tested was inoculated into 200 mL of sterilized LB liquid culture medium at a volume ratio of 1:100, and a control without inoculation of the extract was also made. There were 6 treatments in total, and each treatment was repeated three times. The results were averaged. Take a loop of each Bacillus subtilis and Saccharomyces cerevisiae strain on the slope of the test tube, inoculate it into 100 mL of sterilized LB liquid culture medium, activate it at 25-28°C for 24 hours, and then inoculate it into the culture medium at a ratio of 2%. The bacterial solution was cultured at 25-28°C and 150 r / min in a shaking incubator. After 12 hours, a sample was taken and placed in a sterilized test tube, and the absorbance value was measured at 600 nm. From Figures 2 - 3 From the data, we can see that the algae extracts in the examples of the present invention have a synergistic promoting effect on the activity of the strains, while in the comparative examples 3-6 where the extract composition is changed, the synergistic balance is broken, thus resulting in a weakened effect.

[0173] Pot experiment:

[0174] The corn variety used for the test was “Bangyu 519” (Shandong Zhongnong Tiantai Seed Co., Ltd.).

[0175] Experimental design:

[0176] The experiment was conducted in pots with a diameter of 15 cm and a height of 20 cm. Each pot was filled with 300 g of substrate. Before the substrate was filled into the pot, it was disinfected with a 500-fold dilution of Baishiqing bactericide and algaecide (Shanghai Yongtong Ecological Engineering Co., Ltd.), and 5 kg / m2 of vegetable oil-based coated controlled-release urea (nutrient release period 90 days, Shanghai Yongtong Ecological Engineering Co., Ltd.) was used. 3 Mix fertilizer.

[0177] The method of using the plant-derived growth regulator of the embodiment and the comparative example is as follows: soak corn seeds in the plant-derived growth regulator until the solution covers the seeds, take out the corn seeds after standing for 5 minutes, and after sowing, water the roots of the corn seedlings with a 20-fold diluted plant-derived growth regulator 7 days after germination, with a dosage of 20-30 mL per plant.

[0178] Corn was sown on June 20, 2022. The experiment set up 12 treatments, each treatment was repeated 10 times, and the results were averaged. The blank control was an equal amount of sterile water. All results were averaged. The cultivation and management measures were kept consistent. On August 8, the number of leaves, plant height, root volume, number of roots, aboveground dry weight, underground dry weight and other indicators of corn were measured and recorded. Measured using a root scanner (Top Yunnong GXY-A). The test results are shown in Table 2:

[0179] Table 2 Effects of different treatments on the number of leaves, plant height, root volume and number of roots of maize

[0180]

[0181] Table 3 Effects of different treatments on the aboveground and underground dry weights and root-to-shoot ratio of maize

[0182]

[0183] Field experiment:

[0184] Sweet corn Zhetian 19 was used as the test variety. The experiment set up 12 treatments, each treatment was repeated 10 times, and the results were averaged. The treatments were carried out according to the pot experiment, and the blank group was treated with the same amount of sterile water. The treatment groups are as follows:

[0185] T1: every 667m 2 Bottom application of 120 kg organic fertilizer + plant-derived growth regulator of Example 1;

[0186] T2: Every 667m 2 Bottom application of 120 kg organic fertilizer + treatment with plant-derived growth regulator from Example 2;

[0187] T3: every 667m 2 Bottom application of 120 kg organic fertilizer + treatment with plant-derived growth regulator of Example 3;

[0188] T4: every 667m 2 Bottom application of 120 kg organic fertilizer + treatment with plant-derived growth regulator of Example 4;

[0189] T5: every 667m 2 Bottom application of 120 kg organic fertilizer + treatment with plant-derived growth regulator of Example 5;

[0190] T6: every 667m 2 Bottom application of 120kg organic fertilizer + treatment with plant-derived growth regulator of Comparative Example 1;

[0191] T7: every 667m 2 Bottom application of 120 kg organic fertilizer + treatment with plant growth regulator from Comparative Example 2;

[0192] T8: every 667m 2 Bottom application of 120kg organic fertilizer + treatment with plant-derived growth regulator of Comparative Example 3;

[0193] T9: every 667m 2 Bottom application of 120 kg organic fertilizer + treatment with plant growth regulator of Comparative Example 4;

[0194] T10: every 667m 2 Bottom application of 120 kg organic fertilizer + treatment with plant-derived growth regulator of Comparative Example 5;

[0195] T11: every 667m 2 Bottom application of 120 kg organic fertilizer + treatment with plant-derived growth regulator of Comparative Example 6;

[0196] CK: Every 667m 2 Apply 120kg organic fertilizer + sterile water treatment at the bottom.

[0197] When receiving the goods, the yield is counted and the soil properties are tested. The soil property testing method is as follows:

[0198] After corn harvest, the five-point method was used to sample the seed corn plants with the same growth in each experimental plot. The sampling depth was 40 cm, and the samples were packed in envelopes, marked, and brought back to the laboratory and placed in a 4°C cold room to immediately determine the number of microorganisms and enzyme activity. Bacteria were determined using the beef extract peptone medium method, fungi were determined using the PDA medium method, and actinomycetes were determined using the Gao's No. 1 medium method.

[0199] Table 3 Soil microbial activity

[0200]

[0201] from Figure 4 From the soil test data, we can see that the corn yield of the experimental group treated with the conditioner of the present invention was increased and the soil biological activity was effectively improved. However, in the comparative examples 1-6, which changed the treatment method, the synergistic effect of each other was weakened due to the changes in the functional strains and the algae extracts, resulting in a decrease in the effect of the comparative example. This shows that the selection of the microbial strains of the present invention and the selection of the algae extracts are key technical means, and the effect is weak if one of them is missing.

[0202] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A plant-derived growth regulator, characterized in that: The raw materials include the following parts by weight: 30-50 parts of algae plant extract, 5-15 parts of microbial fermentation liquid, 1.5-2.0 parts of auxiliary agent, 1-3 parts of trace elements, 0.1-0.5 parts of solubilizing agent, and 50-70 parts of water; the microbial fermentation liquid is produced by Bacillus subtilis ( Bacillus subtilis ) and Saccharomyces cerevisiae ( Saccharomyces paradoxus ) is prepared by fermentation, and the specific preparation method is as follows: (1) Strain activation: Bacillus subtilis and Saccharomyces cerevisiae stored in a refrigerator were transferred into LB medium respectively, and placed at 25-28° C. for activation culture for 24 hours to obtain activated strains of Bacillus subtilis and Saccharomyces cerevisiae; (2) Seed liquid culture: Take a loop of activated Bacillus subtilis and activated Saccharomyces cerevisiae strains, respectively inoculate into 300 mL conical flasks filled with seed culture medium, fill with 25% liquid, place in a shaking incubator at 25-28°C and 100-180 rpm for 5-10 h to obtain seed liquid of the two bacteria, and mix the seed liquids of the two bacteria in a volume ratio of 1:1 to obtain a mixed seed liquid; (3) Fermentation culture: The mixed seed liquid was inoculated at 1% into a 1L conical flask containing fermentation medium, and the liquid volume was 50%. The flask was placed in a shaking incubator at 25-28°C and 200-220 r / min for fermentation until the effective viable count was 1-2×10 9 cfu / ml, and adjusting the pH value to 7.0-7.3 to obtain microbial fermentation broth; The Bacillus subtilis has a preservation number of CGMCC No. 12522 and a preservation date of January 16, 2023, and is preserved in the General Microbiology Center of the China National Microbiological Culture Collection Administration; the strain number of the Saccharomyces cerevisiae is CGMCC No. 2.5691, which was purchased from the General Microbiology Center of the China National Microbiological Culture Collection Administration, and the original preservation date was December 5, 2016; the preparation method of the algae plant extract is: weigh 1g of Spirulina powder and 1g of Chlorella powder, add 100mL of water to swell, and repeatedly freeze and thaw to break the wall, and the freeze-thaw method is: freezing at -20°C, thawing at room temperature, repeating 5 times to end the freeze-thaw, and then bathing in 60°C water for 1h, centrifuging at 6000r / min for 10min, collecting the supernatant and vacuum drying to obtain a water extract, and the collected water extract is re-dissolved with sterilized water, and passed through a 0.22μm aqueous phase sterilization membrane to obtain an algae plant extract with a water extract concentration of 10mg / mL.

2. The plant-derived growth regulator according to claim 1, characterized in that: The auxiliary agent is obtained by mixing ethephon, chlormequat chloride, glucose and citric acid in a mass ratio of 0.1:0.3:10:

5.

3. The plant-derived growth regulator according to claim 1, characterized in that: The trace elements are one or more of CuSO4, ZnSO4 and MgSO4.

4. The plant-derived growth regulator according to claim 1, characterized in that: The solubilizing synergist is sodium lauryl sulfate and / or sodium lignin sulfonate.

5. A method for preparing the plant-derived growth regulator according to any one of claims 1 to 4, characterized in that: The method comprises the following preparation steps: (1) preparing microbial fermentation broth; (2) Preparation of algae extract: (3) adding auxiliary agents and trace elements into water in sequence, mixing and stirring to obtain a premix; (4) Finally, the microbial fermentation liquid and the algae plant extract are added to the premix simultaneously, and then a solubilizing agent is added, and the mixture is stirred and mixed uniformly to obtain the plant-derived growth regulator.

6. A use of the plant-derived growth regulator according to any one of claims 1 to 4, characterized in that: The plant-derived growth regulator is applied to corn planting to promote the growth of corn root system and improve the yield and resistance of corn plants.

Images