CN109121778B - Extraction method of bradyrhizobium extract and application of bradyrhizobium extract in mango grafting - Google Patents
Extraction method of bradyrhizobium extract and application of bradyrhizobium extract in mango grafting Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G2/00—Vegetative propagation
- A01G2/30—Grafting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G2/38—Holding; Ligating
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses an extraction method of a bradyrhizobium japonicum extract and application of the bradyrhizobium japonicum extract in mango grafting. The extraction method comprises the following steps: (1) selecting and culturing bradyrhizobium: selecting bradyrhizobium lentum from peanut roots, drying and crushing the peanut roots, performing crude extraction by using ethanol to obtain a crude extract, performing supercritical carbon dioxide extraction on the crude extract, and performing supercritical extraction on the crude extract in a supercritical extraction kettle to obtain the bradyrhizobium lentum extract; placing the selected bradyrhizobium in a culture medium for culturing to obtain a culture solution, and filtering and sterilizing the culture solution by using gauze to obtain bradyrhizobium fermentation liquor; and mixing the bradyrhizobium extract with the bradyrhizobium fermentation broth to obtain the bradyrhizobium extract. The healing agent for promoting the grafting wound is prepared by mixing the extract containing the bradyrhizobium and the fermentation broth of the bradyrhizobium, can promote the healing of the grafted mango, and has short healing time of the grafted joint and high grafting survival rate.
Description
Technical Field
The invention relates to the technical field of fruit tree planting, in particular to an extraction method of a bradyrhizobium japonicum extract and application of the bradyrhizobium japonicum extract in mango grafting.
Background
Currently, grafting techniques are widely used on horticultural crops. Factors influencing survival of grafted seedlings in production practice are mainly related to factors such as nutrition conditions, moisture, sterilization and the like of the stock and the scion besides the affinity of the stock and the scion, so that the healing speed of a grafted port is influenced. In order to ensure the survival rate of grafting, people apply various chemical substances to the grafting to promote the healing of the grafting wound as soon as possible, and the effect is obvious. In the existing research, the components of the grafting port healing agent mainly contain plant growth regulators such as naphthylacetic acid, indoleacetic acid, gibberellin and the like, and sterilization components such as carbendazim and the like; nutrient components, water retention components and the like. In the prior art, the research and the utilization of the bradyrhizobium are relatively less. Signal transmission, mutual recognition and sequential expression and regulation of genes between rhizobia and leguminous plants are related to root nodule formation, development and nitrogen fixation, and the research on rhizobia and the promotion of plant cultivation and planting are of great significance.
Disclosure of Invention
The invention overcomes the technical problems of low research utilization rate of bradyrhizobium, unsatisfactory effect of promoting the healing of a grafting opening and low grafting survival rate of the existing plant healing agent in the prior art, and provides an extraction method of bradyrhizobium extract and application thereof in mango grafting.
In order to solve the problems, the invention adopts the following technical scheme:
a method for extracting bradyrhizobium extract comprises the following steps:
(1) selecting and culturing bradyrhizobium: selecting bradyrhizobium rhizobium from the root of peanut, drying and crushing the peanut root, performing crude extraction by using ethanol to obtain a crude extract, performing supercritical carbon dioxide extraction on the crude extract, placing the crude extract in a supercritical extraction kettle to perform supercritical extraction, and extracting for 20min by using a mixed solution of ethanol, ethyl acetate and n-butyl alcohol with equal mass ratio as an entrainer to obtain the bradyrhizobium rhizobium extract; the supercritical carbon dioxide extraction temperature is 35 ℃, the extraction pressure is 35kpa, and the carbon dioxide flow rate is 25L/min;
placing the selected bradyrhizobium in a culture medium, culturing for 5-6 days at 25-28 ℃ to obtain a culture solution, and filtering and sterilizing the culture solution by using gauze to obtain bradyrhizobium fermentation liquor; the formula of the culture medium comprises 2g of peptone, 5g of soybean protein, 6g of agar, 12g of glucose and KH2PO4、MgSO4100ml of water;
and mixing the bradyrhizobium extract with the bradyrhizobium fermentation broth to obtain the bradyrhizobium extract.
Wherein the content of flavone in the bradyrhizobium extract is not less than 84mg/100 g.
The application method of the bradyrhizobium extract on mango grafting comprises the following steps: preparing the bradyrhizobium extract into a grafting wound healing promoting agent, coating the grafting wound healing promoting agent on a mango grafting opening, firmly binding the stock and the scion from bottom to top by using a polyethylene film strip, and then densely binding the scion; the grafting wound healing promoting agent comprises the following components in parts by mass: 80-100 parts of bradyrhizobium rhizogenes extract, 0.1-0.3 part of bactericide, 2-5 parts of trace elements, 1-6 parts of hydrogen peroxide, 2-6 parts of film forming agent, 4-8 parts of humic acid, 0.1-0.3 part of insecticide and 1-3 parts of lauric acid.
Wherein the bactericide is matrine, berberine, rotenone and beauveria bassiana.
Wherein the pesticide is pyrethrin and azadirachtin.
The film forming agent comprises the following components in parts by mass: 1-4 parts of chitosan, 3-6 parts of soybean protein, 1-5 parts of corn starch and 2-4 parts of carrageenan.
Compared with the prior art, the invention has the following beneficial effects:
(1) the slow rhizobium extract and the slow rhizobium fermentation liquor are mixed to prepare the slow rhizobium extract, the slow rhizobium extract contains growth factors for promoting plant growth and absorbing nitrogen, and flavonoid substances contained in the slow rhizobium extract have bacteriostatic and bactericidal effects and can permeate into grafted wounds to induce mangoes to absorb nutrient substances and promote growth and development. The bradyrhizobium fermentation liquor contains a plurality of biological enzymes, and the biological enzymes are matched with growth factors in the bradyrhizobium extract to replace a plant growth regulator commonly used in the prior art, so that the growth of mangoes is effectively promoted, the scab healing of the mangoes at the grafting openings is promoted, and the healing time of the grafting openings is short.
(2) The healing promoting agent for grafting wounds consists of a bradyrhizobium japonicum extract, a bactericide, trace elements, hydrogen peroxide, a film forming agent, humic acid, a pesticide and lauric acid, and the combination of the extracts can effectively promote the healing of mango grafting wounds, and the mango grafting survival rate is high.
Detailed Description
The present invention will be further described with reference to examples and tests.
Example 1
A method for extracting bradyrhizobium extract comprises the following steps:
(1) selecting and culturing bradyrhizobium: selecting bradyrhizobium japonicum from peanut roots, drying and crushing the peanut roots, performing crude extraction by using ethanol to obtain a crude extract, performing supercritical carbon dioxide extraction on the crude extract, placing the crude extract in a supercritical extraction kettle to perform supercritical extraction, and extracting for 20min by using a mixed solution of ethanol, ethyl acetate and n-butanol in equal mass ratio as an entrainer to obtain the bradyrhizobium japonicum extractSaid bradyrhizobium extract; the supercritical carbon dioxide extraction temperature is 35 ℃, the extraction pressure is 35kpa, and the carbon dioxide flow rate is 25L/min. Placing the selected bradyrhizobium in a culture medium, culturing for 5d at 28 ℃ to obtain a culture solution, and filtering and sterilizing the culture solution by using gauze to obtain bradyrhizobium fermentation liquor; the formula of the culture medium comprises 2g of peptone, 5g of soybean protein, 6g of agar, 12g of glucose and KH2PO4、MgSO4100ml of water. And mixing the bradyrhizobium extract with the bradyrhizobium fermentation broth to obtain the bradyrhizobium extract. Wherein the content of flavone in the bradyrhizobium extract is not less than 84mg/100 g.
The application method of the bradyrhizobium extract on mango grafting comprises the following steps: preparing the bradyrhizobium extract into a grafting wound healing promoting agent, coating the grafting wound healing promoting agent on a mango grafting opening, firmly binding the stock and the scion from bottom to top by using a polyethylene film strip, and then densely binding the scion; the grafting wound healing promoting agent comprises the following components in parts by mass: 100 parts of slow rhizobium extract, 0.1 part of bactericide, 5 parts of trace elements, 1 part of hydrogen peroxide, 6 parts of film-forming agent, 4 parts of humic acid, 0.3 part of insecticide and 1 part of lauric acid. The bactericide is matrine, berberine, rotenone and beauveria bassiana, and the insecticide is pyrethrin and azadirachtin. The film forming agent comprises the following components in parts by mass: 4 parts of chitosan, 3 parts of soybean protein, 5 parts of corn starch and 2 parts of carrageenan.
Example 2
A method for extracting bradyrhizobium extract comprises the following steps:
(1) selecting and culturing bradyrhizobium: selecting bradyrhizobium rhizobium from the root of peanut, drying and crushing the peanut root, performing crude extraction by using ethanol to obtain a crude extract, performing supercritical carbon dioxide extraction on the crude extract, placing the crude extract in a supercritical extraction kettle to perform supercritical extraction, and extracting for 20min by using a mixed solution of ethanol, ethyl acetate and n-butyl alcohol with equal mass ratio as an entrainer to obtain the bradyrhizobium rhizobium extract; the supercritical carbon dioxide extraction temperature is 35 ℃, the extraction pressure is 35kpa, and the carbon dioxide flowThe speed was 25L/min. Placing the selected bradyrhizobium in a culture medium, culturing for 6d at 25 ℃ to obtain a culture solution, and filtering and sterilizing the culture solution by using gauze to obtain bradyrhizobium fermentation liquor; the formula of the culture medium comprises 2g of peptone, 5g of soybean protein, 6g of agar, 12g of glucose and KH2PO4、MgSO4100ml of water. And mixing the bradyrhizobium extract with the bradyrhizobium fermentation broth to obtain the bradyrhizobium extract. Wherein the content of flavone in the bradyrhizobium extract is not less than 84mg/100 g.
The application method of the bradyrhizobium extract on mango grafting comprises the following steps: preparing the bradyrhizobium extract into a grafting wound healing promoting agent, coating the grafting wound healing promoting agent on a mango grafting opening, firmly binding the stock and the scion from bottom to top by using a polyethylene film strip, and then densely binding the scion; the grafting wound healing promoting agent comprises the following components in parts by mass: 80 parts of slow rhizobium extract, 0.3 part of bactericide, 2 parts of trace elements, 6 parts of hydrogen peroxide, 2 parts of film-forming agent, 8 parts of humic acid, 0.1 part of insecticide and 3 parts of lauric acid. The bactericide is matrine, berberine, rotenone and beauveria bassiana, and the insecticide is pyrethrin and azadirachtin. The film forming agent comprises the following components in parts by mass: 1 part of chitosan, 6 parts of soybean protein, 1 part of corn starch and 4 parts of carrageenan.
Example 3
A method for extracting bradyrhizobium extract comprises the following steps:
(1) selecting and culturing bradyrhizobium: selecting bradyrhizobium rhizobium from the root of peanut, drying and crushing the peanut root, performing crude extraction by using ethanol to obtain a crude extract, performing supercritical carbon dioxide extraction on the crude extract, placing the crude extract in a supercritical extraction kettle to perform supercritical extraction, and extracting for 20min by using a mixed solution of ethanol, ethyl acetate and n-butyl alcohol with equal mass ratio as an entrainer to obtain the bradyrhizobium rhizobium extract; the supercritical carbon dioxide extraction temperature is 35 ℃, the extraction pressure is 35kpa, and the carbon dioxide flow rate is 25L/min. Placing the selected bradyrhizobium in a culture medium, culturing at 26 deg.C for 5d to obtain a culture solution, and applying gauze to obtain a culture solutionFiltering and sterilizing the culture solution to obtain a bradyrhizobium fermentation broth; the formula of the culture medium comprises 2g of peptone, 5g of soybean protein, 6g of agar, 12g of glucose and KH2PO4、MgSO4100ml of water. And mixing the bradyrhizobium extract with the bradyrhizobium fermentation broth to obtain the bradyrhizobium extract. Wherein the content of flavone in the bradyrhizobium extract is not less than 84mg/100 g.
The application method of the bradyrhizobium extract on mango grafting comprises the following steps: preparing the bradyrhizobium extract into a grafting wound healing promoting agent, coating the grafting wound healing promoting agent on a mango grafting opening, firmly binding the stock and the scion from bottom to top by using a polyethylene film strip, and then densely binding the scion; the grafting wound healing promoting agent comprises the following components in parts by mass: 900 parts of slow rhizobium extract, 0.2 part of bactericide, 3 parts of trace elements, 5 parts of hydrogen peroxide, 5 parts of film-forming agent, 7 parts of humic acid, 0.2 part of insecticide and 2 parts of lauric acid. The bactericide is matrine, berberine, rotenone and beauveria bassiana, and the insecticide is pyrethrin and azadirachtin. The film forming agent comprises the following components in parts by mass: 2 parts of chitosan, 5 parts of soybean protein, 3 parts of corn starch and 3 parts of carrageenan.
Example 4
A method for extracting bradyrhizobium extract comprises the following steps:
(1) selecting and culturing bradyrhizobium: selecting bradyrhizobium rhizobium from the root of peanut, drying and crushing the peanut root, performing crude extraction by using ethanol to obtain a crude extract, performing supercritical carbon dioxide extraction on the crude extract, placing the crude extract in a supercritical extraction kettle to perform supercritical extraction, and extracting for 20min by using a mixed solution of ethanol, ethyl acetate and n-butyl alcohol with equal mass ratio as an entrainer to obtain the bradyrhizobium rhizobium extract; the supercritical carbon dioxide extraction temperature is 35 ℃, the extraction pressure is 35kpa, and the carbon dioxide flow rate is 25L/min. Placing the selected bradyrhizobium in a culture medium, culturing for 5d at 27 ℃ to obtain a culture solution, and filtering and sterilizing the culture solution by using gauze to obtain bradyrhizobium fermentation liquor; the formula of the culture medium comprises 2g of peptone, 5g of soybean protein and agar6g, 12g of glucose, KH2PO4、MgSO4100ml of water. And mixing the bradyrhizobium extract with the bradyrhizobium fermentation broth to obtain the bradyrhizobium extract. Wherein the content of flavone in the bradyrhizobium extract is not less than 84mg/100 g.
The application method of the bradyrhizobium extract on mango grafting comprises the following steps: preparing the bradyrhizobium extract into a grafting wound healing promoting agent, coating the grafting wound healing promoting agent on a mango grafting opening, firmly binding the stock and the scion from bottom to top by using a polyethylene film strip, and then densely binding the scion; the grafting wound healing promoting agent consists of 80 parts by mass of bradyrhizobium japonicum extract, 3 parts by mass of trace elements and 10 parts by mass of film forming agent.
Application example 1
The bradyrhizobium extract extracted in the embodiment 1 is grafted by mango, and the specific application method comprises the following steps: adopting a double-bud cutting and grafting method, selecting a straight smooth part 10-12 cm away from the ground of the stock as a grafting opening, obliquely cutting the back of the grafting opening from bottom to top by using a grafting knife to form a 45-degree angle, longitudinally cutting a cut along a cortex on the inclined plane to reach xylem without damaging the xylem, wherein the length of the cut surface is about 2.8-3.6 cm, and then horizontally cutting the top of the cut surface by a cut to cut off an uneven cut surface of a primary cutter; selecting scion strips with the thickness equivalent to that of the stock, selecting one smooth surface as a cutting surface, downwards beveling the smooth back surface at an angle of 45 degrees by using a knife, then cutting off a cortex layer from top to bottom on the smooth surface by using the knife, slightly bringing xylem, slightly cutting the scion to be longer than the stock, inserting the cut scion into a stock cut, aligning to a cambium, firmly binding the cut scion with a wound grafting healing agent promoting coating mango grafting port from bottom to top after being attached, binding the scion densely, and only binding a single layer at the mango eye position.
Application example 2
The bradyrhizobium extract obtained in the example 2 is grafted by mango, and the specific application method is the same as the application example 1.
Application example 3
The bradyrhizobium extract obtained in the example 4 is grafted by mango, and the specific application method is the same as the application example 1.
Application example 4
The bradyrhizobium extract obtained in the example 4 is grafted by mango, and the specific application method is the same as the application example 1.
In order to illustrate the technical effect of the bradyrhizobium extract obtained by extraction in the application of mango grafting, the following control groups are set.
Control group 1
The application method of the control group 1 was substantially the same as that of the application example 1, except that the grafting wound healing promoting agent of the control group 1 did not contain a bradyrhizobium extract.
Control group 2
The application method of control group 2 was substantially the same as that of application example 1, except that the grafting wound healing promoting agent of control group 2 did not contain bradyrhizobium japonicum extract, but contained indoleacetic acid.
Control group 3
The control group 3 was applied in substantially the same manner as in application example 1, except that the bradyrhizobium extract of the control group 3 contained only the bradyrhizobium extract.
Control group 4
The control group 4 was applied in substantially the same manner as in application example 1, except that the bradyrhizobium extract of the control group 4 contained only bradyrhizobium fermentation broth.
To illustrate the technical effects of the present invention, the following experiments;
according to the application examples 1 to 3 and the comparison groups 1 to 4, the bradyrhizobium extract is applied to mango grafting according to the application method of the bradyrhizobium extract in mango grafting, and the mango grafting survival rate is recorded in table 1.
TABLE 1
As can be seen from table 1, the mango grafting survival rate of application examples 1 to 4 is higher than that of control groups 1 to 4, which indicates that the healing after mango grafting can be promoted by using the grafting wound healing promoting agent containing the bradyrhizobium extract and the bradyrhizobium fermentation broth, the healing time of the grafting opening is short, and the grafting survival rate is high.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (6)
1. The application of the bradyrhizobium extract in mango grafting is characterized in that the bradyrhizobium extract is prepared into a grafting wound healing promoting agent which is coated on a mango grafting opening, wherein the grafting wound healing promoting agent consists of 80-90 parts by mass of the bradyrhizobium extract, 1-5 parts by mass of trace elements and 5-10 parts by mass of a film forming agent;
the extraction method of the bradyrhizobium extract comprises the following steps:
(1) selecting and culturing bradyrhizobium: selecting bradyrhizobium rhizobium from the root of peanut, drying and crushing the peanut root, performing crude extraction by using ethanol to obtain a crude extract, performing supercritical carbon dioxide extraction on the crude extract, placing the crude extract in a supercritical extraction kettle to perform supercritical extraction, and extracting for 20min by using a mixed solution of ethanol, ethyl acetate and n-butyl alcohol with equal mass ratio as an entrainer to obtain the bradyrhizobium rhizobium extract; the supercritical carbon dioxide extraction temperature is 35 ℃, the extraction pressure is 35kpa, and the carbon dioxide flow rate is 25L/min;
placing the selected bradyrhizobium in a culture medium, culturing for 5-6 days at 25-28 ℃ to obtain a culture solution, and filtering and sterilizing the culture solution by using gauze to obtain bradyrhizobium fermentation liquor; the formula of the culture medium comprises 2g of peptone, 5g of soybean protein, 6g of agar, 12g of glucose and KH2PO4、MgSO4100ml of water;
and mixing the bradyrhizobium extract with the bradyrhizobium fermentation broth to obtain the bradyrhizobium extract.
2. The use according to claim 1, wherein the bradyrhizobium extract has a flavone content of not less than 84mg/100 g.
3. The application of the bradyrhizobium extract in mango grafting, which is characterized in that the bradyrhizobium extract is prepared into a grafting wound healing promoting agent to be coated on a mango grafting opening, a stock and a scion are firmly bound by a polyethylene film strip from bottom to top, and then the scion is densely bound; the grafting wound healing promoting agent comprises the following components in parts by mass: 80-100 parts of bradyrhizobium rhizogenes extract, 0.1-0.3 part of bactericide, 2-5 parts of trace elements, 1-6 parts of hydrogen peroxide, 2-6 parts of film forming agent, 4-8 parts of humic acid, 0.1-0.3 part of insecticide and 1-3 parts of lauric acid.
4. The application of the bradyrhizobium rhizogenes extract in mango grafting according to claim 3, wherein the bactericide is matrine, berberine, rotenone, beauveria bassiana.
5. Use of a bradyrhizobium extract according to claim 3 for grafting mangos, wherein the insecticide is pyrethrin and azadirachtin.
6. The application of the bradyrhizobium rhizogenes extract in mango grafting is characterized in that the film forming agent consists of the following components in parts by mass: 1-4 parts of chitosan, 3-6 parts of soybean protein, 1-5 parts of corn starch and 2-4 parts of carrageenan.
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| CN1056959C (en) * | 1994-03-28 | 2000-10-04 | 北京大学 | Efficient peanut yield increasing agent and its preparation |
| CN101743858A (en) * | 2010-01-12 | 2010-06-23 | 王成玉 | Nodule bacteria transferring method by utilizing distant grafting |
| CN102405770B (en) * | 2011-09-19 | 2013-03-13 | 河南农业大学 | Method for promoting growth of pleurotus ostreatus by using bradyrhizobium ddjaponicum |
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| CN106804605B (en) * | 2015-12-02 | 2019-10-25 | 中国科学院大连化学物理研究所 | A kind of wound healing agent for plants and application thereof containing sodium alginate oligosaccharide |
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