Microbial humic acid fertilizer for improving relative abundance of Arthrobacter in rhizosphere soil of snap beans

Abstract

The invention relates to a microbial humic acid fertilizer capable of improving relative abundance of Arthrobacter in rhizosphere soil of snap beans, which is prepared by the following steps: pre-decomposing corn steep liquor by using cellulase, alkaline protease and amylase, pre-decomposing by using an auricularia strain, and then pre-decomposing by using a microbial inoculum, so as to obtain the microbial humic acid fertilizer for improving relative abundance of Arthrobacter in rhizosphere soil of snap beans. Diluting the pre-decomposed corn steep liquor containing the microbial fungus residues with water; the diluted corn steep liquor containing the microbial fungus residues is prepared into artificially synthesized microbial source humic acid through three-stage reaction, and the artificially synthesized microbial source humic acid liquid after the reaction is dried to obtain the artificially synthesized microbial source humic acid fertilizer; after being diluted with water, the artificially synthesized microbial humic acid fertilizer is applied to roots of the snap beans in the seedling stage, the relative abundance of arthrobacter in rhizosphere soil of the snap beans reaches 5%-6%, and the length of roots of the snap beans reaches 23-24 cm. The content of arthrobacter rhizosphere (growth-promoting rhizobacteria) of the snap beans can be increased, and the yield and quality of the snap beans are improved.

Description

Technical Field

[0001] The present invention relates to the technology of microbial fertilizer production, and specifically to a synthetic microbial source humic acid that can increase the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans. Background Art

[0002] Beneficial rhizosphere bacteria play an irreplaceable and important role in plant growth, soil improvement, and ecosystem stability. These beneficial bacteria support the healthy growth of plants, optimize the soil environment, and maintain the balance of the ecosystem through various pathways. First of all, beneficial rhizosphere bacteria can significantly promote the nutrient absorption of plants. They can convert atmospheric nitrogen into ammonium nitrogen that plants can utilize through nitrogen fixation, improving the nitrogen nutrition level of plants. At the same time, by secreting organic acids and specific enzymes, beneficial bacteria can convert insoluble phosphates, potassium salts, and certain trace elements (such as iron, zinc, and manganese) in the soil into forms that can be absorbed and utilized by plants, greatly improving the nutrient absorption efficiency of plants. In addition, through the close interaction with plant roots, beneficial bacteria secrete plant hormones such as indole acetic acid and gibberellin, directly stimulating the growth and development of plant roots, thereby enhancing the plant's ability to utilize soil resources.

[0003] Exogenous additives can significantly increase the abundance of beneficial bacteria in plant roots through various mechanisms, thereby optimizing the rhizosphere microbial community and promoting plant growth and health. These additives include organic carbon sources, microorganisms (such as Trichoderma, Arthrobacter, Bacillus), and chemical signaling substances (such as quercetin, flavonoids), etc. They can not only directly provide rich nutrients for beneficial bacteria, improve the rhizosphere microenvironment, but also stimulate the increase of plant root exudates, further attracting and colonizing beneficial bacteria. In addition, soil conditioners such as biochar and humate can also enhance the survival ability of beneficial bacteria by optimizing soil physical and chemical properties and providing a stable microenvironment. Research shows that exogenous additives can increase the relative abundance of beneficial rhizosphere bacteria by 20% - 50%, enhance their disease resistance, nitrogen fixation, and phosphorus solubilization abilities, while improving plant nutrient absorption, inhibiting pathogenic bacteria, and increasing crop yield and quality, showing broad application prospects in green agriculture and soil remediation. Summary of the Invention

[0004] The purpose of the present invention is to provide a microbial source humic acid fertilizer that can increase the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans. This synthetic microbial source humic acid that can increase the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans is used to solve the problem that the relative abundance of Arthrobacter in the rhizosphere soil of traditional kidney bean cultivation is relatively low, affecting the yield and quality of kidney beans.

[0005] To achieve the above purpose, the specific technical solution adopted by the present invention is as follows: This synthetic microbial source humic acid that can increase the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans is prepared by the following method: First, perform pre - decomposition of corn steep liquor. The pre - decomposition is divided into two stages: The first stage: Pre - decompose the corn steep liquor using cellulase, alkaline protease, and amylase; The second stage: Pre - decompose the product of the first stage with Auricularia species, and dilute the corn steep liquor containing microbial fungal residues after pre - decomposition with water; The diluted corn steep liquor containing microbial fungal residues is made into synthetic microbial - derived humic acid through three - stage reactions: The first - stage reaction: The diluted corn steep liquor containing microbial fungal residues is under the condition of pH = 7.0, the temperature is controlled at 140 °C, and the reaction lasts for 2 hours; Add potassium hydroxide and sodium hydroxide, and the mass ratio of potassium hydroxide to sodium hydroxide is 10:1. Control the temperature at 160 °C for the second - stage reaction, and the reaction lasts for 2 hours; Then add sodium dodecyl benzene sulfonate, sodium persulfate, and ferric chloride, and the mass ratio of sodium dodecyl benzene sulfonate, sodium persulfate, and ferric chloride is 3:1:1. Control the temperature at 180 °C for the third - stage reaction, and the reaction lasts for 2 hours. The synthetic microbial - derived humic acid liquid after the reaction is dried to obtain synthetic microbial - derived humic acid fertilizer; After the synthetic microbial - derived humic acid fertilizer is stirred and diluted with water, it is applied to the roots of Phaseolus vulgaris var. hortensis at the seedling stage, once every 7 days for a total of 3 times. After the fourth week, the relative abundance of Arthrobacter in the rhizosphere soil of Phaseolus vulgaris var. hortensis reaches 5% - 6%, and the root length of Phaseolus vulgaris var. hortensis reaches 23 - 24 cm.

[0006] The method for pre - decomposing corn steep liquor in the above - mentioned scheme: In the first stage, pre - decompose the corn steep liquor with cellulase and alkaline protease. The ratio of cellulase to alkaline protease is 3:1, the pre - decomposition temperature is 50 °C, and the pre - decomposition time is 3 hours; Then pre - decompose with amylase, the pre - decomposition temperature is 45 °C, and the pre - decomposition time is 1 hour; In the second stage, pre - decompose the product of the first stage with Auricularia species. The culture temperature for pre - decomposition is 25 °C, and the culture time is 10 days.

[0007] When diluting the corn steep liquor containing microbial fungal residues with water in the above - mentioned scheme, after adding water for dilution, it is further processed by a water processor at a frequency of 20000 Hz for 5 - 20 min.

[0008] In the above - mentioned scheme, the ratio of the total mass of potassium hydroxide and sodium hydroxide to the volume of the diluted corn steep liquor solution containing microbial fungal residues is 0.5% - 1.5%.

[0009] In the above - mentioned scheme, the ratio of the total mass of sodium dodecyl benzene sulfonate, sodium persulfate, and ferric chloride to the volume of the diluted corn steep liquor solution containing microbial fungal residues is 0.2% - 2%.

[0010] When the artificially synthesized microbial source humic acid fertilizer in the above scheme is stirred and diluted with water, the amount of water added is 1000 - 3000 times the amount of the artificially synthesized microbial source humic acid fertilizer. When applying fertilizer, the application amount is 20 - 100 mL / plant. Beneficial effects

[0011] 1. By applying the artificially synthesized microbial source humic acid with corn steep liquor as the raw material to kidney beans, the present invention can increase the content of Arthrobacter in the rhizosphere of kidney beans (plant growth-promoting rhizobacteria), and provides a method for pre-decomposing corn steep liquor and synthesizing artificially microbial source humic acid. As a kind of plant growth-promoting rhizobacteria, Arthrobacter has the function of promoting plant growth and its absorption and utilization of mineral nutrients.

[0012] 2. The artificially synthesized microbial source humic acid prepared by the present invention contains rich active functional groups such as carboxyl, hydroxyl and ketone groups in its molecules, has strong complexing ability and environmental stability, can promote the growth of the roots of kidney beans, make the length of the roots of kidney beans reach 23 - 24 cm, and at the same time make the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans reach 5% - 6%.

[0013] 3. By adding cellulase, alkaline protease and amylase, the present invention decomposes macromolecular substances (such as proteins, polysaccharides, etc.) in corn steep liquor into smaller molecules, such as short peptides, free amino acids and monosaccharides, and then generates a large amount of artificially synthesized microbial source humic acid substances through subsequent reactions. Description of the drawings

[0014] Figure 1 It is a control chart of the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans when the present invention and clear water are applied.

[0015] Figure 2 It is a control chart of the root length of kidney beans when the present invention and clear water are applied. Detailed implementation manners

[0016] The present invention will be further described below with reference to the drawings: Embodiment

[0017] The artificially synthesized microbial source humic acid that can increase the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans is prepared by the following method: The raw material corn steep liquor is subjected to a first-stage pre-decomposition using cellulase, alkaline protease, and amylase. The product of the first stage is subjected to a second-stage pre-decomposition using Auricularia species. The pre-decomposed corn steep liquor containing microbial fungal residues is diluted by treating it with a water processor at a frequency of 20,000 Hz for 10 min. Subsequently, the corn steep liquor containing microbial fungal residues is made into synthetic microbial-derived humic acid through three-stage reactions. The temperature of the first-stage reaction is controlled at 140 °C, and the reaction is carried out for 2 hours under the condition of pH = 7.0. The temperature of the second-stage reaction is controlled at 160 °C, potassium hydroxide and sodium hydroxide are added, the addition ratio of potassium hydroxide to sodium hydroxide is 10:1, and the ratio of the total mass of potassium hydroxide and sodium hydroxide to the volume of the corn steep liquor solution is 0.5%, and the reaction is carried out for 2 hours. The temperature of the third-stage reaction is controlled at 180 °C, and the reaction is carried out for 2 hours. The mass ratio of the catalysts sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride is 3:1:1, and the ratio of the total mass of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride to the volume of the corn steep liquor solution is 0.2%. The reacted liquid is dried to obtain synthetic microbial-derived humic acid fertilizer.

[0018] When the synthetic microbial-derived humic acid fertilizer is applied to the roots of Phaseolus vulgaris var. sesquipedalis, it needs to be diluted 3000 times, and the applied amount after dilution is 80 mL / plant. Example

[0019] This synthetic microbial-derived humic acid that increases the relative abundance of Arthrobacter in the rhizosphere soil of Phaseolus vulgaris var. sesquipedalis is prepared by the following method: The raw material corn steep liquor is subjected to a first-stage pre-decomposition using cellulase, alkaline protease, and amylase. The product of the first stage is subjected to a second-stage pre-decomposition using Auricularia species. The pre-decomposed corn steep liquor containing microbial fungal residues is diluted by treating it with a water processor at a frequency of 20,000 Hz for 5 min. Subsequently, the corn steep liquor containing microbial fungal residues is made into synthetic microbial-derived humic acid through three-stage reactions. The temperature of the first-stage reaction is controlled at 140 °C, and the reaction is carried out for 2 hours under the condition of pH = 7.0. The temperature of the second-stage reaction is controlled at 160 °C, potassium hydroxide and sodium hydroxide are added, the addition ratio of potassium hydroxide to sodium hydroxide is 10:1, and the ratio of the total mass of potassium hydroxide and sodium hydroxide to the volume of the corn steep liquor solution is 1%, and the reaction is carried out for 2 hours. The temperature of the third-stage reaction is controlled at 180 °C, and the reaction is carried out for 2 hours. The mass ratio of the catalysts sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride is 3:1:1, and the ratio of the total mass of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride to the volume of the corn steep liquor solution is 2%. The reacted liquid is dried to obtain synthetic microbial-derived humic acid fertilizer. When the synthetic microbial-derived humic acid fertilizer is applied to the roots of Phaseolus vulgaris var. sesquipedalis, it needs to be diluted 3000 times, and the applied amount after dilution is 100 mL / plant. Example

[0020] The synthetic microbial-derived humic acid that increases the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans is prepared by the following method: The raw material corn steep liquor is pre-decomposed in the first stage using cellulase, alkaline protease, and amylase. The product of the first stage is pre-decomposed in the second stage using the fungus of the genus Auricularia. The pre-decomposed corn steep liquor containing microbial fungal residues is diluted by treatment with a water processor at a frequency of 20000 Hz for 5 minutes. Then, the corn steep liquor containing microbial fungal residues is made into synthetic microbial-derived humic acid through three stages of reaction. The reaction temperature in the first stage is controlled at 140 °C, and the reaction is carried out for 2 hours under the condition of pH = 7.0. The reaction temperature in the second stage is controlled at 160 °C, potassium hydroxide and sodium hydroxide are added, and the addition ratio of potassium hydroxide to sodium hydroxide is 10:1. The ratio of the total mass of potassium hydroxide and sodium hydroxide to the volume of the corn steep liquor solution is 1.5%, and the reaction is carried out for 2 hours. The reaction temperature in the third stage is controlled at 180 °C, and the reaction is carried out for 2 hours. The mass ratio of the catalysts sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride is 3:1:1. The ratio of the total mass of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride to the volume of the corn steep liquor solution is 2%. The liquid after the reaction is dried to obtain the synthetic microbial-derived humic acid fertilizer. When the synthetic microbial-derived humic acid fertilizer is applied to the roots of kidney beans, it needs to be diluted 1000 times, and the applied amount after dilution is 20 mL / plant.

[0021] To verify the effect of the present invention, a confidentiality experiment was carried out, which is specifically as follows:

[0022] This experiment aims to prepare a synthetic microbial-derived humic acid fertilizer according to the method of the invention patent. First, corn steep liquor was selected as the raw material and pretreated. The specific steps are as follows: In the first stage, cellulase and alkaline protease were added to corn steep liquor in a ratio of 3:1 and reacted at 50 °C for 3 hours. Subsequently, amylase was added and secondary pre-decomposition was carried out at 45 °C for 1 hour. In the second stage, the product of the first stage was pre-decomposed with Auricularia species at 25 °C for 10 days. The pre-decomposed corn steep liquor containing microbial fungal residues was passed through a water processor and treated at a frequency of 20000 Hz for 5 - 20 minutes to complete dilution and further activation. Next, the treated corn steep liquor containing microbial fungal residues was made into a synthetic microbial-derived humic acid through three-stage reactions: In the first stage, the temperature was controlled at 140 °C, the reaction was carried out for 2 hours, and the pH value was kept neutral. In the second stage, the temperature was controlled at 160 °C, the reaction was carried out for 2 hours, potassium hydroxide and sodium hydroxide were added, and the mass ratio was 10:1. The ratio of the total mass of potassium hydroxide and sodium hydroxide to the volume of the corn steep liquor solution was 0.5%. In the third stage: the temperature was controlled at 180 °C, the reaction was carried out for 2 hours, sodium dodecylbenzenesulfonate, sodium persulfate and ferric chloride were added, and the mass ratio of the three was 3:1:1. The ratio of the total mass of sodium dodecylbenzenesulfonate, sodium persulfate and ferric chloride to the volume of the corn steep liquor solution was 0.2%. After the reaction, the synthetic microbial-derived humic acid liquid was dried to make a synthetic microbial-derived humic acid fertilizer for subsequent experiments.

[0023] The experiment was carried out in a potting method to study the effect of the synthetic microbial-derived humic acid on the abundance of Arthrobacter in the rhizosphere soil of Phaseolus vulgaris seedlings. An experimental group and a control group were set up. In the experimental group, the synthetic microbial-derived humic acid fertilizer diluted 1000 times was applied, and 50 mL was applied to each plant; the control group was applied with an equal amount of clear water. The experiment was carried out under greenhouse conditions, standard nutrient soil was selected as the culture medium, and the duration was 4 weeks. Fertilization was carried out once every 7 days during the period, and a total of 3 fertilizations were carried out. At the 4th week of the experiment, rhizosphere soil samples of Phaseolus vulgaris were collected and sent to Ovisen Sequencing Company for 16S rRNA high-throughput sequencing, focusing on analyzing the diversity of rhizosphere microbial communities and the relative abundance of Arthrobacter. At the same time, the growth indexes of Phaseolus vulgaris seedlings were statistically analyzed.

[0024] The experimental results show that, referring to Figure 1 , compared with applying clear water, applying the synthetic microbial-derived humic acid provided by the present invention can increase the relative abundance of Arthrobacter in the rhizosphere soil of Phaseolus vulgaris, and the relative abundance of Arthrobacter in the rhizosphere soil of Phaseolus vulgaris reaches 5% - 6%.

[0025] The experimental results show that, compared with applying clear water, applying the synthetic microbial-derived humic acid provided by the present invention can increase the length of the roots of Phaseolus vulgaris, and the length of the roots of Phaseolus vulgaris reaches 23 - 24 cm.

Claims

1. A microbial-source humic acid fertilizer for increasing the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans, characterized in that: The artificially synthesized microbial source humic acid for increasing the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans is prepared by the following method: First, corn steep liquor is pre-decomposed, and the pre-decomposition is divided into two stages: The first stage: The corn steep liquor is pre-decomposed using cellulase, alkaline protease, and amylase; The second stage: The product of the first stage is pre-decomposed using Auricularia species, and the pre-decomposed corn steep liquor containing microbial fungal residues is diluted with water; The diluted corn steep liquor containing microbial fungal residues is made into artificially synthesized microbial source humic acid through three-stage reactions: The first-stage reaction: The diluted corn steep liquor containing microbial fungal residues is reacted at a pH of 7.0, with the temperature controlled at 140 °C for 2 hours; Potassium hydroxide and sodium hydroxide are added, and the mass ratio of potassium hydroxide to sodium hydroxide is 10:

1. The temperature is controlled at 160 °C for the second-stage reaction for 2 hours; Then sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride are added, and the mass ratio of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride is 3:1:

1. The temperature is controlled at 180 °C for the third-stage reaction for 2 hours. The liquid of the artificially synthesized microbial source humic acid after the reaction is dried to obtain the artificially synthesized microbial source humic acid fertilizer; The artificially synthesized microbial source humic acid fertilizer is stirred and diluted with water and applied to the roots of kidney beans at the seedling stage of kidney beans, once every 7 days for a total of 3 times. After the fourth week, the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans reaches 5%-6%, and the length of the roots of kidney beans reaches 23-24 cm.

2. The microbial source humic acid fertilizer for increasing the relative abundance of Arthrobacter in the rhizosphere soil of Phaseolus vulgaris var. hortensis, as claimed in claim 1, is characterized in that: The method for pre-decomposing corn steep liquor: In the first stage, the corn steep liquor is pre-decomposed using cellulase and alkaline protease, and the ratio of cellulase to alkaline protease is 3:

1. The pre-decomposition temperature is 50 °C, and the pre-decomposition time is 3 hours; Then it is pre-decomposed using amylase, the pre-decomposition temperature is 45 °C, and the pre-decomposition time is 1 hour; In the second stage, the product of the first stage is pre-decomposed using Auricularia species, and the pre-decomposition culture temperature is 25 °C, and the culture time is 10 days.

3. The microbial source humic acid fertilizer for increasing the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans according to claim 2, characterized in that: When the corn steep liquor containing microbial fungal residues is diluted with water, it is diluted with water and then processed by a water processor at a frequency of 20000 Hz for 5-20 min.

4. The microbial source humic acid fertilizer for increasing the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans according to claim 3, characterized in that: The ratio of the total mass of potassium hydroxide and sodium hydroxide to the volume of the diluted corn steep liquor solution containing microbial fungal residues is 0.5-1.5%.

5. The microbial-source humic acid fertilizer for increasing the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans according to claim 4, characterized in that: The ratio of the total mass of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride to the volume of the diluted corn steep liquor solution containing microbial fungal residues is 0.2-2%.

6. The microbial source humic acid fertilizer for increasing the relative abundance of Arthrobacter in the rhizosphere soil of kidney beans according to claim 5, characterized in that: When the artificially synthesized microbial source humic acid fertilizer is stirred and diluted with water, the amount of water added is 1000-3000 times the amount of the artificially synthesized microbial source humic acid fertilizer. During fertilization, the application amount is 20-100 mL / plant.

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