CN110938581A - Culture medium for cultivating high-activity plant growth-promoting bacteria and preparation method thereof - Google Patents
Culture medium for cultivating high-activity plant growth-promoting bacteria and preparation method thereof Download PDFInfo
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Abstract
The invention provides a culture medium for culturing high-activity plant growth-promoting bacteria, which contains beef extract, peptone, sodium chloride, uranyl acetate dihydrate, glycerol and 1-aminocyclopropane-1-carboxylic acid, wherein the plant growth-promoting bacteria with higher saline-alkali resistance can be cultured by adding the uranyl acetate dihydrate, the plant growth-promoting bacteria cultured by adding the 1-aminocyclopropane-1-carboxylic acid have higher activity, can promote plants to produce more PGPB capable of secreting more ACC deaminase (ACCdeamidase, ACCD) to reduce the ethylene level by metabolizing the precursor ACC for producing ethylene, and can influence the endogenous steady state of the plant ethylene and promote root elongation by changing genes for coding the ethylene synthetase, the ACC synthase and the ACC oxidase.
Description
Technical Field
The invention belongs to the field of culture medium preparation, and particularly relates to a culture medium for culturing high-activity plant growth-promoting bacteria and a preparation method thereof.
Background
Plant growth-promoting bacteria (PGPR) are a class of microorganisms that promote plant growth, control diseases, and increase crop yield. PGPR has certain biological control effect on harmful pathogenic microorganisms in soil and non-parasitic rhizosphere harmful microorganisms (DRMO for short), and also has promotion effect on the absorption and utilization of mineral nutrients by plants, and can produce metabolites beneficial to the growth of plants, thereby promoting the growth and development of the plants. In recent years, studies of a plurality of scholars show that the plant growth promoting bacteria can indirectly inhibit or reduce the adverse effects of plant diseases on plants by producing antibiotics and secreting siderophores, and can directly stimulate and regulate the growth of plants by dissolving phosphorus, fixing nitrogen, producing phytohormones, reducing the ethylene level by enzymolysis and the like. Many plant growth-promoting rhizobacteria can produce indoleacetic acid (IAA), vitamins (antibiotic) and the like, and utilize and degrade 1-aminocyclopropane-1-carboxylate (ACC), which is an ethylene synthesis precursor produced by plants, to decompose ACC into A-tetronic acid and ammonia, so that a large amount of ethylene produced in plants under adverse conditions is reduced, and the growth and development of the plants are promoted. Plant growth-promoting bacteria, particularly those containing ACC deaminase, have received much attention, and are considered to be effective in promoting plant growth under stress conditions.
Plant growth promoting bacteria (PGPR) refer to beneficial bacteria that live in the range of the plant root circle, and that promote plant growth or antagonize pathogenic bacteria. The microbial fertilizer can be used independently or in combination with microbial fertilizers, so that the growth promoting effect, the biocontrol effect and the fertilizer efficiency are better combined to improve and enhance the application effect, and the microbial fertilizer can be widely applied to biological fertilizer composite preparations, green food production, organic food production and development of ecological agriculture. However, the existing culture medium for culturing plant growth-promoting bacteria (PGPR) has the defects of poor pertinence and low number of cultured viable plant growth-promoting bacteria.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium for culturing high-activity plant growth-promoting bacteria and a preparation method thereof, wherein the culture medium contains plant growth-promoting bacteria obtained by culturing uranyl acetate dihydrate and 1-aminocyclopropane-1-carboxylic acid, and the plant growth-promoting bacteria can improve the saline-alkali resistance of plants and the ACC secretion of plant seeds when being used for the plants, and the technical scheme of the invention is as follows:
the culture medium for cultivating the high-activity plant growth-promoting bacteria contains 8-12 g of beef extract, 10-20 g of peptone, 4-5 g of sodium chloride, 4-6 g of uranyl acetate dihydrate, 8-12 g of glycerol and 70-90 mg of 1-aminocyclopropane-1-carboxylic acid in every 1000mL of the culture medium for cultivating the high-activity plant growth-promoting bacteria.
In some embodiments of the invention, the weight of the beef extract is 100wt%, and the solid content of the beef extract is 75-85 wt%.
In some embodiments of the present invention, the 1-aminocyclopropane-1-carboxylic acid is prepared by: the method comprises the steps of taking nitroacetate and 1, 2-dihalogenated ethane as raw materials, carrying out alkylation cyclization, nitro reduction and carboxyl hydrolysis reaction to obtain crude 1-aminocyclopropane-1-carboxylic acid, and then carrying out separation, purification and crystallization on the crude 1-aminocyclopropane-1-carboxylic acid to obtain high-content and high-purity 1-aminocyclopropane-1-carboxylic acid.
In a particular embodiment of the invention, the peptone comprises, per 100g of said peptone, 20g of tryptone, 5g of lactose, 2.75g of dipotassium hydrogenphosphate chloride, 2.75g of potassium dihydrogenphosphate, 5.0g of sodium chloride and 0.1g of sodium lauryl sulfate.
In some embodiments of the invention, the pH of the culture medium for culturing the high-activity plant growth-promoting bacteria is 7.0-7.2, and the culture medium is sterilized at 120 ℃ for 15min after being uniformly mixed.
Another object of the present invention is to provide a method for preparing a culture medium for cultivating high-activity plant growth-promoting bacteria, comprising the steps of:
step 1: sequentially adding beef extract and peptone into water, stirring, performing microwave treatment, adding glycerol and sodium chloride, and stirring;
step 2: mixing uranyl acetate dihydrate, 1-aminocyclopropane-1-carboxylic acid and water uniformly, and sterilizing by high-pressure steam to obtain the culture medium for culturing the high-activity plant growth-promoting bacteria.
In some embodiments of the invention, the stirring time in the step 1 is 10-16 min, and the stirring temperature is 70-80 ℃.
In some embodiments of the invention, the high-pressure steam sterilization time in the step 2 is 20-30 min, and the sterilization temperature is 110-120 ℃.
Compared with the prior art, the invention has the beneficial effects that:
1. the culture medium formula system for cultivating the high-activity plant growth-promoting bacteria contains beef extract, peptone, sodium chloride, uranyl acetate dihydrate, glycerol and 1-aminocyclopropane-1-carboxylic acid, wherein the plant growth-promoting bacteria with higher salt and alkali resistance can be cultivated by adding the uranyl acetate dihydrate, and the plant growth-promoting bacteria cultivated by using the culture medium have better pertinence.
2. The formula system of the culture medium for culturing the high-activity plant growth-promoting bacteria contains 1-aminocyclopropane-1-carboxylic acid, so that the plant growth-promoting bacteria obtained by culturing the culture medium have higher activity, the plant growth-promoting bacteria can promote the plant to secrete more ACC deaminase (ACCdeamidase, ACCD), and the plant growth-promoting bacteria can reduce the ethylene level by metabolizing to produce the precursor 1-aminocyclopropane-1-carboxylic acid of ethylene, thereby influencing the endogenous steady state of the plant ethylene and promoting the root elongation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below so that those skilled in the art can better understand the advantages and features of the present invention, and thus the scope of the present invention will be more clearly defined. The embodiments described herein are only a few embodiments of the present invention, rather than all embodiments, and all other embodiments that can be derived by one of ordinary skill in the art without inventive faculty based on the embodiments described herein are intended to fall within the scope of the present invention.
Example 1
A culture medium for culturing high-activity plant growth-promoting bacteria, wherein each 1000mL of the culture medium for culturing high-activity plant growth-promoting bacteria contains 10g of beef extract, 15g of peptone, 4.5g of sodium chloride, 5g of uranyl acetate dihydrate, 10g of glycerol and 80mg of 1-aminocyclopropane-1-carboxylic acid.
Wherein the weight of the beef extract is 100wt%, and the solid content of the beef extract is 80 wt%.
The preparation method of the 1-aminocyclopropane-1-carboxylic acid comprises the following steps: the method comprises the steps of taking nitroacetate and 1, 2-dihalogenated ethane as raw materials, carrying out alkylation cyclization, nitro reduction and carboxyl hydrolysis reaction to obtain crude 1-aminocyclopropane-1-carboxylic acid, and then carrying out separation, purification and crystallization on the crude 1-aminocyclopropane-1-carboxylic acid to obtain high-content and high-purity 1-aminocyclopropane-1-carboxylic acid.
Wherein, each 100g of the peptone contains 20g of tryptone, 5g of lactose, 2.75g of dipotassium chlorophosphate, 2.75g of potassium dihydrogen phosphate, 5.0g of sodium chloride and 0.1g of sodium dodecyl sulfate.
Wherein the pH value of the culture medium for culturing the high-activity plant growth-promoting bacteria is 7.1, and the culture medium is sterilized for 15min at 120 ℃ after being uniformly mixed.
A preparation method of a culture medium for culturing high-activity plant growth-promoting bacteria comprises the following steps:
step 1: sequentially adding beef extract and peptone into water, stirring, performing microwave treatment, adding glycerol and sodium chloride, and stirring;
step 2: mixing uranyl acetate dihydrate, 1-aminocyclopropane-1-carboxylic acid and water uniformly, and sterilizing by high-pressure steam to obtain the culture medium for culturing the high-activity plant growth-promoting bacteria.
Wherein the stirring time in the step 1 is 13min, and the stirring temperature is 75 ℃.
Wherein the high-pressure steam sterilization time in the step 2 is 25min, and the sterilization temperature is 115 ℃.
Example 2
A culture medium for culturing high-activity plant growth-promoting bacteria, wherein each 1000mL of the culture medium for culturing high-activity plant growth-promoting bacteria contains 8g of beef extract, 20g of peptone, 4g of sodium chloride, 4g of uranyl acetate dihydrate, 8g of glycerol and 70mg of 1-aminocyclopropane-1-carboxylic acid.
Wherein the weight of the beef extract is 100wt%, and the solid content of the beef extract is 80 wt%.
The preparation method of the 1-aminocyclopropane-1-carboxylic acid comprises the following steps: the method comprises the steps of taking nitroacetate and 1, 2-dihalogenated ethane as raw materials, carrying out alkylation cyclization, nitro reduction and carboxyl hydrolysis reaction to obtain crude 1-aminocyclopropane-1-carboxylic acid, and then carrying out separation, purification and crystallization on the crude 1-aminocyclopropane-1-carboxylic acid to obtain high-content and high-purity 1-aminocyclopropane-1-carboxylic acid.
Wherein, each 100g of the peptone contains 20g of tryptone, 5g of lactose, 2.75g of dipotassium chlorophosphate, 2.75g of potassium dihydrogen phosphate, 5.0g of sodium chloride and 0.1g of sodium dodecyl sulfate.
Wherein the pH value of the culture medium for culturing the high-activity plant growth-promoting bacteria is 7.1, and the culture medium is sterilized for 15min at 120 ℃ after being uniformly mixed.
A preparation method of a culture medium for culturing high-activity plant growth-promoting bacteria comprises the following steps:
step 1: sequentially adding beef extract and peptone into water, stirring, performing microwave treatment, adding glycerol and sodium chloride, and stirring;
step 2: mixing uranyl acetate dihydrate, 1-aminocyclopropane-1-carboxylic acid and water uniformly, and sterilizing by high-pressure steam to obtain the culture medium for culturing the high-activity plant growth-promoting bacteria.
Wherein the stirring time in the step 1 is 13min, and the stirring temperature is 75 ℃.
Wherein the high-pressure steam sterilization time in the step 2 is 25min, and the sterilization temperature is 115 ℃.
Example 3
A culture medium for culturing high-activity plant growth-promoting bacteria, wherein each 1000mL of the culture medium for culturing high-activity plant growth-promoting bacteria contains 12g of beef extract, 10g of peptone, 5g of sodium chloride, 6g of uranyl acetate dihydrate, 12g of glycerol and 90mg of 1-aminocyclopropane-1-carboxylic acid.
Wherein the weight of the beef extract is 100wt%, and the solid content of the beef extract is 80 wt%.
The preparation method of the 1-aminocyclopropane-1-carboxylic acid comprises the following steps: the method comprises the steps of taking nitroacetate and 1, 2-dihalogenated ethane as raw materials, carrying out alkylation cyclization, nitro reduction and carboxyl hydrolysis reaction to obtain crude 1-aminocyclopropane-1-carboxylic acid, and then carrying out separation, purification and crystallization on the crude 1-aminocyclopropane-1-carboxylic acid to obtain high-content and high-purity 1-aminocyclopropane-1-carboxylic acid.
Wherein, each 100g of the peptone contains 20g of tryptone, 5g of lactose, 2.75g of dipotassium chlorophosphate, 2.75g of potassium dihydrogen phosphate, 5.0g of sodium chloride and 0.1g of sodium dodecyl sulfate.
Wherein the pH value of the culture medium for culturing the high-activity plant growth-promoting bacteria is 7.1, and the culture medium is sterilized for 15min at 120 ℃ after being uniformly mixed.
A preparation method of a culture medium for culturing high-activity plant growth-promoting bacteria comprises the following steps:
step 1: sequentially adding beef extract and peptone into water, stirring, performing microwave treatment, adding glycerol and sodium chloride, and stirring;
step 2: mixing uranyl acetate dihydrate, 1-aminocyclopropane-1-carboxylic acid and water uniformly, and sterilizing by high-pressure steam to obtain the culture medium for culturing the high-activity plant growth-promoting bacteria.
Wherein the stirring time in the step 1 is 13min, and the stirring temperature is 75 ℃.
Wherein the high-pressure steam sterilization time in the step 2 is 25min, and the sterilization temperature is 115 ℃.
Example 4
A culture medium for culturing high-activity plant growth-promoting bacteria, wherein each 1000mL of the culture medium for culturing high-activity plant growth-promoting bacteria contains 10g of beef extract, 15g of peptone, 4.5g of sodium chloride, 5g of uranyl acetate dihydrate, 10g of glycerol and 80mg of 1-aminocyclopropane-1-carboxylic acid.
Wherein the weight of the beef extract is 100wt%, and the solid content of the beef extract is 75 wt%.
The preparation method of the 1-aminocyclopropane-1-carboxylic acid comprises the following steps: the method comprises the steps of taking nitroacetate and 1, 2-dihalogenated ethane as raw materials, carrying out alkylation cyclization, nitro reduction and carboxyl hydrolysis reaction to obtain crude 1-aminocyclopropane-1-carboxylic acid, and then carrying out separation, purification and crystallization on the crude 1-aminocyclopropane-1-carboxylic acid to obtain high-content and high-purity 1-aminocyclopropane-1-carboxylic acid.
Wherein, each 100g of the peptone contains 20g of tryptone, 5g of lactose, 2.75g of dipotassium chlorophosphate, 2.75g of potassium dihydrogen phosphate, 5.0g of sodium chloride and 0.1g of sodium dodecyl sulfate.
Wherein the pH of the culture medium for culturing the high-activity plant growth-promoting bacteria is 7.0, and the culture medium is sterilized for 15min at 120 ℃ after being uniformly mixed.
A preparation method of a culture medium for culturing high-activity plant growth-promoting bacteria comprises the following steps:
step 1: sequentially adding beef extract and peptone into water, stirring, performing microwave treatment, adding glycerol and sodium chloride, and stirring;
step 2: mixing uranyl acetate dihydrate, 1-aminocyclopropane-1-carboxylic acid and water uniformly, and sterilizing by high-pressure steam to obtain the culture medium for culturing the high-activity plant growth-promoting bacteria.
Wherein, the stirring time in the step 1 is 10min, and the stirring temperature is 70 ℃.
Wherein the high-pressure steam sterilization time in the step 2 is 20min, and the sterilization temperature is 110 ℃.
Example 5
A culture medium for culturing high-activity plant growth-promoting bacteria, wherein each 1000mL of the culture medium for culturing high-activity plant growth-promoting bacteria contains 10g of beef extract, 15g of peptone, 4.5g of sodium chloride, 5g of uranyl acetate dihydrate, 10g of glycerol and 80mg of 1-aminocyclopropane-1-carboxylic acid.
Wherein the weight of the beef extract is 100wt%, and the solid content of the beef extract is 85 wt%.
The preparation method of the 1-aminocyclopropane-1-carboxylic acid comprises the following steps: the method comprises the steps of taking nitroacetate and 1, 2-dihalogenated ethane as raw materials, carrying out alkylation cyclization, nitro reduction and carboxyl hydrolysis reaction to obtain crude 1-aminocyclopropane-1-carboxylic acid, and then carrying out separation, purification and crystallization on the crude 1-aminocyclopropane-1-carboxylic acid to obtain high-content and high-purity 1-aminocyclopropane-1-carboxylic acid.
Wherein, each 100g of the peptone contains 20g of tryptone, 5g of lactose, 2.75g of dipotassium chlorophosphate, 2.75g of potassium dihydrogen phosphate, 5.0g of sodium chloride and 0.1g of sodium dodecyl sulfate.
Wherein the pH value of the culture medium for culturing the high-activity plant growth-promoting bacteria is 7.2, and the culture medium is sterilized for 15min at 120 ℃ after being uniformly mixed.
A preparation method of a culture medium for culturing high-activity plant growth-promoting bacteria comprises the following steps:
step 1: sequentially adding beef extract and peptone into water, stirring, performing microwave treatment, adding glycerol and sodium chloride, and stirring;
step 2: mixing uranyl acetate dihydrate, 1-aminocyclopropane-1-carboxylic acid and water uniformly, and sterilizing by high-pressure steam to obtain the culture medium for culturing the high-activity plant growth-promoting bacteria.
Wherein, the stirring time in the step 1 is 16min, and the stirring temperature is 80 ℃.
Wherein the high-pressure steam sterilization time in the step 2 is 30min, and the sterilization temperature is 120 ℃.
The formula system of the invention contains beef extract, peptone, sodium chloride, uranyl acetate dihydrate, glycerol and 1-aminocyclopropane-1-carboxylic acid, wherein the addition of the uranyl acetate dihydrate can culture plant growth-promoting bacteria with higher saline-alkali tolerance, the pertinence of the plant growth-promoting bacteria cultured by using the culture medium is better, the plant growth-promoting bacteria cultured by using the culture medium has higher activity due to the addition of the 1-aminocyclopropane-1-carboxylic acid, the cultured plant growth-promoting bacteria can promote plants to secrete more ACC deaminase, and the plant growth-promoting bacteria reduce the ethylene level by metabolizing to produce the precursor 1-aminocyclopropane-1-carboxylic acid of ethylene to influence the endogenous homeostasis of plant ethylene and promote root elongation.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A culture medium for culturing high-activity plant growth-promoting bacteria is characterized in that: each 1000mL of the culture medium for culturing the high-activity plant growth-promoting bacteria contains 8-12 g of beef extract, 10-20 g of peptone, 4-5 g of sodium chloride, 4-6 g of uranyl acetate dihydrate, 8-12 g of glycerol and 70-90 mg of 1-aminocyclopropane-1-carboxylic acid.
2. The culture medium for cultivating high-activity plant growth-promoting bacteria according to claim 1, wherein: the weight of the beef extract is 100wt%, and the solid content of the beef extract is 75-85 wt%.
3. The culture medium for cultivating high-activity plant growth-promoting bacteria according to claim 1, wherein: the preparation method of the 1-aminocyclopropane-1-carboxylic acid comprises the following steps: the method comprises the steps of taking nitroacetate and 1, 2-dihalogenated ethane as raw materials, carrying out alkylation cyclization, nitro reduction and carboxyl hydrolysis reaction to obtain crude 1-aminocyclopropane-1-carboxylic acid, and then carrying out separation, purification and crystallization on the crude 1-aminocyclopropane-1-carboxylic acid to obtain high-content and high-purity 1-aminocyclopropane-1-carboxylic acid.
4. The culture medium for cultivating high-activity plant growth-promoting bacteria according to claim 1, wherein: each 100g of the peptone contained 20g of tryptone, 5g of lactose, 2.75g of dipotassium hydrogenphosphate chloride, 2.75g of potassium dihydrogenphosphate, 5.0g of sodium chloride and 0.1g of sodium laurylsulfate.
5. The culture medium for cultivating high-activity plant growth-promoting bacteria according to claim 1, wherein: the pH value of the culture medium for culturing the high-activity plant growth-promoting bacteria is 7.0-7.2, and the culture medium is sterilized for 15min at 120 ℃ after being uniformly mixed.
6. A preparation method of a culture medium for culturing high-activity plant growth-promoting bacteria is characterized by comprising the following steps: the preparation method of the culture medium for culturing the high-activity plant growth-promoting bacteria comprises the following steps:
step 1: sequentially adding beef extract and peptone into water, stirring, performing microwave treatment, adding glycerol and sodium chloride, and stirring;
step 2: mixing uranyl acetate dihydrate, 1-aminocyclopropane-1-carboxylic acid and water uniformly, and sterilizing by high-pressure steam to obtain the culture medium for culturing the high-activity plant growth-promoting bacteria.
7. The method for preparing a culture medium for cultivating high-activity plant growth-promoting bacteria according to claim 6, wherein: in the step 1, the stirring time is 10-16 min, and the stirring temperature is 70-80 ℃.
8. The method for preparing a culture medium for cultivating high-activity plant growth-promoting bacteria according to claim 6, wherein: and 2, sterilizing by high-pressure steam for 20-30 min at 110-120 ℃.
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