CN116711741A - Application of microbial preparation in astragalus membranaceus planting - Google Patents

Application of microbial preparation in astragalus membranaceus planting Download PDF

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CN116711741A
CN116711741A CN202310926798.1A CN202310926798A CN116711741A CN 116711741 A CN116711741 A CN 116711741A CN 202310926798 A CN202310926798 A CN 202310926798A CN 116711741 A CN116711741 A CN 116711741A
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bacillus
microbial preparation
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astragaloside
culture solution
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CN116711741B (en
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靳颖华
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Inner Mongolia Polypeptide Technology Co ltd
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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    • A01N63/23—B. thuringiensis
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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    • A01N63/22—Bacillus
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J53/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by condensation with a carbocyclic rings or by formation of an additional ring by means of a direct link between two ring carbon atoms, including carboxyclic rings fused to the cyclopenta(a)hydrophenanthrene skeleton are included in this class
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Abstract

The invention provides an application of a microbial preparation in astragalus planting, in particular to an application in improving the content of active ingredients in astragalus planting. The microorganism is selected from the group consisting of Bacillus subtilis, bacillus thuringiensis, bacillus licheniformis, and Bacillus megaterium. The content of the astragaloside planted by adopting the microbial preparation is improved by more than 100%, and the content of the calycosin, the astragaloside and the astragaloside I is improved by more than 50%.

Description

Application of microbial preparation in astragalus membranaceus planting
Technical Field
The invention relates to the field of microorganisms, in particular to application of a microbial preparation in astragalus planting.
Background
Astragalus root is dried root of Astragalus mongholicus Astragalus membranaceus (Fisch.) bge. Var. Mongholicus (bge.) Hsiao or Astragalus membranaceus Astragalus membranaceus (Fisch.) bge. In Leguminosae. In the large-scale planting process of astragalus, a large amount of chemical fertilizers and pesticides are applied, so that the number of a plurality of biological populations is drastically reduced or even is extinct, the ecological balance is seriously destroyed, the soil acidification and hardening are caused, the air permeability is reduced, the physical and chemical properties of the soil are deteriorated, and the quality of the astragalus is reduced.
2020. The Chinese pharmacopoeia of the edition provides that the astragalus medicinal material takes astragaloside IV and calycosin as index components, the content of the astragaloside IV is not less than 0.04 percent (0.4 mg/g), and the content of the calycosin is not less than 0.02 percent (0.2 mg/g). The radix astragali contains, in addition to calycosin and astragaloside IV, formononetin, and astragaloside, wherein the formononetin and astragaloside have indispensable effects in the therapeutic effect of radix astragali.
The formononetin can obviously inhibit proliferation of human breast cancer cells, lead the cell cycle to be blocked in the G0/G1 phase, inhibit DNA replication activity, and improve cognitive dysfunction caused by mouse sepsis, the mechanism of the formononetin is possibly related to inhibiting inflammatory reaction and oxidative stress reaction of hippocampal tissues, and the formononetin also has obvious improvement effect on type 2 diabetic nephropathy rat kidney injury. Zhang Shujuan and the like screen 3 components as potential quality markers of astragalus medicinal materials by adopting a network pharmacology and fingerprint spectrum method, and sequentially comprise astragaloside IV, calycosin and formononetin (astragalus quality marker prediction based on network pharmacology and fingerprint spectrum, chinese journal of Chinese traditional medicine, 2691-2698 pages, volume 46, 11 th and 2021). Therefore, not only the content of the calycosin and the astragaloside in the astragalus root needs to be improved, but also the content of the formononetin needs to be improved.
Xin Zhongyao and the like show that the bacillus subtilis can produce growth hormone substances, so that the quality of the astragalus membranaceus is improved (the bacillus subtilis B1B2 strain has disease prevention and growth promotion effects on angelica sinensis and astragalus membranaceus, plant protection, pages 142-144, volume 34, 6 th period and 2008). However, it is not explicitly stated what quality, or what quality index, of astragalus root is improved.
CN112438171a discloses that bacillus subtilis can effectively increase the content of effective components including calycosin glucoside and astragaloside in astragalus, can increase the content of calycosin glucoside by 35.14% -63.51%, and can increase the content of astragaloside by 26.39% -45.83%. However, it is not mentioned whether the contents of formononetin and astragaloside can be increased.
Disclosure of Invention
The invention provides an application of a microbial preparation in astragalus planting, in particular to an application in improving the content of active ingredients in astragalus planting.
The microorganism is selected from the group consisting of Bacillus subtilis, bacillus thuringiensis, bacillus licheniformis, and Bacillus megaterium.
The bacillus subtilis strain is from ACCC No.10242 of China center for agricultural microorganism culture collection, the bacillus thuringiensis is from ACCC No.01599 of China center for agricultural microorganism culture collection, the bacillus licheniformis is from ACCC No.02975 of China center for agricultural microorganism culture collection, and the bacillus megaterium is from ACCC No.04314 of China center for agricultural microorganism culture collection.
Inoculating bacillus subtilis, bacillus thuringiensis, bacillus licheniformis and bacillus megaterium into an LB culture medium respectively, and culturing at 30-32 ℃ for 24-48 hours to obtain bacillus subtilis, bacillus thuringiensis, bacillus licheniformis and bacillus megaterium culture solutions respectively. The culture solution is used according to a certain proportion, and the weight ratio of the culture solution of bacillus subtilis, bacillus thuringiensis, bacillus licheniformis and bacillus megaterium is (0.5-5): (0.5-5): (0.5-5): (0.5-5), preferably 1:1:1:1.
The microbial preparation also comprises shrimp and crab shells, collagen, sodium chloride, compound amino acid, carbohydrate and compound fertilizer, wherein the weight ratio of the strain culture solution to the shrimp and crab shells, the collagen, the sodium chloride, the compound amino acid, the carbohydrate and the compound fertilizer is (0.1-1): (0.01-1): (0.01 to 9): (1-50): (1-20): (1-10).
Shrimp and crab shells are derived from any shell of aquatic animals rich in chitin in rivers, lakes and seas, and the dried shell is crushed and then passes through fine particle powder of 20-100 meshes. Preferably shrimp shell meal, crab shell meal or shrimp crab meal.
The collagen is selected from one or more of pigskin collagen, fish skin collagen and cow hide collagen.
The compound amino acid consists of kelp fermentation liquor, zinc sulfate monohydrate and ammonium heptamolybdate tetrahydrate, wherein 20-40 g of zinc sulfate monohydrate and 1-3 g of ammonium heptamolybdate tetrahydrate are added into each 80-120 ml of kelp fermentation liquor.
The carbohydrate is selected from one or more of glucose, fructose and sucrose.
The weight ratio of nitrogen, phosphorus and potassium in the compound fertilizer is (10-20): (10-20): (10-20), preferably 15:15 (15-20).
The preparation method of the microbial preparation comprises the following steps:
(1) Adding the strain culture solution, shrimp and crab shells, collagen and sodium chloride into water, mixing, fermenting at 25-35 ℃ for 3-5 days, bubbling and aerating to obtain a fermentation solution;
(2) Adding amino acid, carbohydrate and compound fertilizer into the fermentation liquid in the step (1), wherein the fermentation temperature is 25-35 ℃, the fermentation time is 3-5 days, and bubbling and ventilation are carried out to obtain the microbial preparation.
The number of viable bacteria in the microbial preparation obtained in the step (2) is not less than 1 multiplied by 10 9 ~2×10 10 The number of viable bacteria per ml is preferably not less than 1X 10 10 And each ml.
The application method of the microbial preparation comprises the following steps: diluting the microbial preparation with water by 1-10 times, adding soapberry extract with the volume of 0.5-1.5% (v/v) of the microbial preparation, and root irrigation of astragalus mongholicus. The dosage is 100L-1000L/mu.
The preparation method of the soapberry extract comprises the following steps: drying and pulverizing seed coat of fructus Sapindi Mukouossi, extracting with water at 60deg.C for 2 hr for 3 times, filtering, and concentrating the filtrate to a weight ratio of fructus Sapindi Mukouossi to 4:1.
The content of the astragaloside planted by adopting the microbial preparation is improved by more than 100%, and the content of the calycosin, the astragaloside IV and the astragaloside I is improved by more than 50%.
Drawings
FIG. 1 is an HPLC diagram of a control solution of calycosin and formononetin (Peak 1 calycosin, peak 2 formononetin) prepared by the method of the control solution under test example 4 (1).
FIG. 2 is an HPLC chart of a test solution (Peak 1 Calycosin, peak 2 Mannheim glycoside) prepared by the test solution preparation method of test example 4 (1).
FIG. 3 is an HPLC chart of a control solution of astragaloside IV and astragaloside I (peak 3 astragaloside IV, peak 4 astragaloside I) prepared by the control solution preparation method under test example 4 (2).
FIG. 4 is an HPLC chart of a sample solution (peak 3 astragaloside IV, peak 4 astragaloside I) prepared by the sample solution preparation method of test example 4 (2).
Description of the embodiments
Example 1
0.5Kg of bacillus subtilis culture solution, 0.5Kg of crab meal, 1Kg of pigskin collagen, 0.1Kg of sodium chloride, 10Kg of amino acid, 5Kg of fructose and 5Kg of compound fertilizer (nitrogen-phosphorus-potassium ratio 15:15:15).
The amino acid is that 40g of zinc sulfate monohydrate and 3g of ammonium heptamolybdate tetrahydrate are added into each 120ml of kelp fermentation broth.
The preparation method of the microbial preparation comprises the following steps:
(1) 250L of water, adding a bacillus subtilis culture solution, crab meal, collagen, sodium chloride and bubbling ventilation for culture, wherein the culture temperature is controlled at 28 ℃, and the culture time is 4 days to obtain a fermentation broth;
(2) Adding amino acid, fructose and compound fertilizer into the fermentation broth in the step (1), continuously bubbling and ventilating for culturing at the temperature of 32 ℃ for 3 days to obtain a microbial preparation for later use.
Example 2
0.5Kg of bacillus subtilis culture solution, 0.5Kg of bacillus thuringiensis culture solution, 0.5Kg of bacillus licheniformis culture solution and 0.5Kg of bacillus megaterium culture solution, 0.5Kg of crab meal, 1Kg of pigskin collagen, 0.1Kg of sodium chloride, 10Kg of amino acid, 5Kg of fructose and 5Kg of compound fertilizer (nitrogen-phosphorus-potassium ratio 15:15:15).
The amino acid is that 40g of zinc sulfate monohydrate and 3g of ammonium heptamolybdate tetrahydrate are added into each 120ml of kelp fermentation broth.
The preparation method of the microbial preparation comprises the following steps:
(1) 1000L of water, adding a bacillus subtilis culture solution, a bacillus thuringiensis culture solution, a bacillus licheniformis culture solution and a bacillus megaterium culture solution, crab meal, collagen, sodium chloride and bubbling ventilation for culture, wherein the culture temperature is controlled at 28 ℃ and the culture time is 4 days, so as to obtain a fermentation broth;
(2) Adding amino acid, fructose and compound fertilizer into the fermentation broth in the step (1), continuously bubbling and ventilating for culturing at the temperature of 32 ℃ for 3 days to obtain a microbial preparation for later use.
Example 3
0.3Kg of bacillus subtilis culture solution, 0.2Kg of bacillus thuringiensis culture solution, 0.7Kg of bacillus licheniformis culture solution and 0.3Kg of bacillus megaterium culture solution, 1Kg of crab meal, 0.05Kg of pigskin collagen, 0.05Kg of sodium chloride, 7Kg of amino acid, 10Kg of glucose and 5Kg of compound fertilizer (nitrogen-phosphorus-potassium ratio 15:15:20).
The amino acid is 20g of zinc sulfate monohydrate and 1g of ammonium heptamolybdate tetrahydrate are added into each 100ml of kelp fermentation broth.
The preparation method of the microbial preparation comprises the following steps:
(1) 1000L of water, adding a bacillus subtilis culture solution, a bacillus thuringiensis culture solution, a bacillus licheniformis culture solution, a bacillus megaterium culture solution, crab meal, collagen, sodium chloride and bubbling ventilation for culture, wherein the culture temperature is controlled at 32+/-2 ℃ and the culture time is 4 days, so as to obtain a fermentation broth;
(2) Adding amino acid, fructose and compound fertilizer into the fermentation liquid, continuously bubbling and aerating for culturing at 28+/-2 ℃ for 3 days to obtain the microbial preparation for later use.
Example 4
0.25Kg of bacillus subtilis culture solution, 0.5Kg of bacillus thuringiensis culture solution, 0.25Kg of bacillus licheniformis culture solution, 0.5Kg of bacillus megaterium culture solution, 0.5Kg of crab meal, 0.5Kg of pigskin collagen, 0.5Kg of sodium chloride, 1Kg of amino acid, 5Kg of sucrose and 10Kg of compound fertilizer (nitrogen-phosphorus-potassium ratio 15:15:20).
The amino acid is 20g of zinc sulfate monohydrate and 1g of ammonium heptamolybdate tetrahydrate are added into each 100ml of kelp fermentation broth.
The preparation method of the microbial preparation comprises the following steps:
(1) 1000L of water, adding a bacillus subtilis culture solution, a bacillus thuringiensis culture solution, a bacillus licheniformis culture solution, a bacillus megaterium culture solution, crab meal, collagen, sodium chloride and bubbling ventilation for culture, wherein the culture temperature is controlled at 32+/-2 ℃ and the culture time is 4 days, so as to obtain a fermentation broth;
(2) Adding amino acid, fructose and compound fertilizer into the fermentation liquid, continuously bubbling and aerating for culturing at 28+/-2 ℃ for 3 days to obtain the microbial preparation for later use.
Test example 1 seed Density detection
The microbial preparations obtained in examples 1 to 4 were subjected to strain density measurement, and the results are shown in Table 1:
TABLE 1 seed Density for each example
Strain Density (cfu/ml)
Example 1 2.1×10 11
Example 2 1.9×10 11
Example 3 2.6×10 11
Example 4 8.7×10 10
As is clear from Table 1, the microbial preparations obtained in examples 1 to 4 have a very high cell density, which is higher than 1X 10 10 cfu/ml。
Test example 2 microbial preparation dilution
Diluting the obtained microbial preparation with water, adding the soapberry extract, and mixing to obtain the microbial preparation diluted solution. The lean ratios are shown in table 2:
table 2 microbial preparation liquid-diluted proportion example
Group of Microbial preparation Water and its preparation method Soapberry extract
Example 1 1L 9L 10ml
Example 2 1L 9L 10ml
Example 3 1L 9L 10ml
Example 4 1L 9L 10ml
The preparation method of the soapberry extract comprises the following steps: drying and pulverizing seed coat of fructus Sapindi Mukouossi, extracting with water at 60deg.C for 2 hr for 3 times, filtering, and concentrating the filtrate to a weight ratio of fructus Sapindi Mukouossi to 4:1.
Test example 3 microbial preparation diluted formulation application
Selecting astragalus seedlings with similar sizes, randomly selecting 9m of sample formulas of the same continuous cropping land block, conventionally ploughing, applying base fertilizer, irrigating different microbial preparation diluted solutions to roots, placing the seedlings according to a row spacing of 40cm, applying the microbial preparation diluted solutions obtained in the embodiments 1-4 every 7-30 days according to the required water supplementing condition, and about 50ml each plant. An additional equivalent amount of water treatment was set as a reference group and observed for 2 years.
Test example 4 determination of the content of active ingredient in Astragalus membranaceus
The content of calycosin, formononetin, astragaloside I and astragaloside IV in the radix astragali obtained in test example 3 was measured.
(1) HPLC-UV method for determining content of calycosin and formononetin
C18 column (250 mm x 4.6mm,5 μm), mobile phase a: methanol, mobile phase B:0.1% phosphoric acid aqueous solution, flow rate of 1.0ml/min, column temperature of 30 ℃, detection wavelength: 254nm. The mobile phase elution ratios are shown in table 3:
TABLE 3 elution ratio of mobile phases
Time Mobile phase A ratio
0~35min 25%~60%
35~45min 60%
Preparing a reference substance solution:
weighing appropriate amounts of calycosin reference substance and formononetin reference substance, precisely weighing, and diluting with methanol to obtain solution with certain concentration.
Sample solution preparation:
4.0g of astragalus powder is precisely weighed, placed in a round bottom flask, 50ml of methanol is added, and precisely weighed. Reflux for 3 hours under heating, standing at room temperature to compensate for the lost liquid mass, filtering, concentrating the solution to dryness, transferring to a 5ml measuring flask with methanol, diluting to scale with methanol, and filtering with 0.45 μm microporous membrane.
(2) HPLC-ELSD method for determining content of astragaloside IV and astragaloside I
C18 column (150 mm x 4.6mm,5 μm), mobile phase a: acetonitrile, mobile phase B: the flow rate of water was 1.0ml/min and the column temperature was 30 ℃. ELSD detection conditions: the temperature of the drift tube is 90 ℃ and the air flow rate is 2.8L/min. The mobile phase elution ratios are shown in table 4:
TABLE 4 elution ratio of mobile phases
Time Mobile phase A ratio
0~8min 30%~40%
8~12min 40%~45%
12~14min 45%
14~16min 45%~65%
16~18min 65%
18~20min 65%~30%
Preparing a reference substance solution:
weighing appropriate amounts of astragaloside IV reference substance and astragaloside I reference substance, precisely weighing, and diluting with methanol to obtain solution with certain concentration.
Sample solution preparation:
adding 3g of radix astragali powder into a Soxhlet extractor, adding appropriate amount of methanol, cold soaking for 12h, adding methanol to 150ml, heating and refluxing in water bath at 80deg.C for 6h, filtering, concentrating the filtrate under reduced pressure to dryness, adding 70% methanol solution into a 10ml measuring flask, and diluting to scale.
(3) Content measurement results
The results of the content measurements of the samples obtained in the different examples are shown in Table 5:
TABLE 5 determination of the content of Calycosin, formononetin, astragaloside I and Astragaloside IV (mg/g)
Group of Calycosin glycoside Ardisia glabra glycoside Astragalus saponin I Astragaloside IV
Clean water group 0.31 0.19 0.76 0.78
Example 1 group 0.49 0.29 1.04 1.11
Example 2 group 0.48 0.42 1.19 1.25
Example 3 group 0.49 0.39 1.21 1.18
Example 4 group 0.47 0.38 1.15 1.21
(4) Data analysis
The percentage of active ingredient increase compared to the fresh water group is shown in table 6:
TABLE 6 percentage of active ingredient increase compared to the fresh water group
Group of Calycosin glycoside Ardisia glabra glycoside Astragalus saponin I Astragaloside IV
Example 1 group 58.1% 52.6% 36.8% 42.3%
Example 2 group 54.8% 121.1% 56.5% 60.3%
Example 3 group 58.1% 105.3% 59.2% 51.2%
Example 4 group 51.6% 100.0% 51.3% 55.1%
As can be seen from Table 6, the contents of effective components including acteoside, formononetin, astragaloside I and astragaloside IV in Astragalus membranaceus can be obviously improved in each of the groups 1 to 4 relative to the clear water group.
In the prior art, it is mentioned that the bacillus subtilis can increase the content of the acteoside and the astragaloside in the astragalus, and compared with the group of the example 1 (bacillus subtilis alone), the group of the examples 2-4 (bacillus subtilis, bacillus thuringiensis, bacillus licheniformis and bacillus megaterium are combined) has the same lifting amplitude of the content of the acteoside as the group of the example 1, and the lifting amplitude of the content of the astragaloside is obviously higher than that of the group of the example 1.
The unexpected finding of the present invention is that the improvement of formononetin and astragaloside I content of the groups of examples 2-4 is significantly higher than that of the group of example 1. In particular, the increasing range of the content of the formononetin is 1.90-2.30 times of that of the embodiment 1, and the increasing range of the content of the astragaloside I is 1.39-1.54 times of that of the embodiment 1.
The foregoing describes in detail preferred embodiments of the present invention. However, the present invention is not limited to the specific details of the above-described embodiments, and various simple modifications may be made to the technical solution of the present invention within the scope of the technical concept of the present invention. These simple variants are all within the scope of protection of the present invention.
In addition, the respective specific features described in the above embodiments may be combined in any manner without contradiction. The various possible combinations of the invention are not described in detail in order to avoid unnecessary repetition.
Moreover, any combination of the various embodiments of the invention may be made without departing from the spirit of the invention, which should also be considered as being disclosed herein.

Claims (10)

1. The application of the microbial preparation in astragalus root planting is characterized in that the strain in the microbial preparation consists of bacillus subtilis, bacillus thuringiensis, bacillus licheniformis and bacillus megaterium.
2. The use according to claim 1, wherein the microbial preparation is used for increasing the content of active ingredients in astragalus membranaceus cultivation.
3. The use according to claim 2, wherein the active ingredients are calycosin, formononetin, astragaloside and astragaloside I.
4. The use according to any one of claims 1-3, characterized in that bacillus subtilis, bacillus thuringiensis, bacillus licheniformis and bacillus megaterium are inoculated into an LB medium respectively and cultured for 24-48 hours at 30-32 ℃ to obtain bacillus subtilis, bacillus thuringiensis, bacillus licheniformis and bacillus megaterium culture solutions; the culture solution is used according to a certain proportion, and the weight ratio of the culture solution of bacillus subtilis, bacillus thuringiensis, bacillus licheniformis and bacillus megaterium is (0.5-5): (0.5-5): (0.5-5): (0.5-5).
5. The use according to claim 4, wherein the bacillus subtilis, bacillus thuringiensis, bacillus licheniformis and bacillus megaterium are in a weight ratio of 1:1:1:1.
6. The use according to claim 4, wherein the microbial preparation further comprises shrimp and crab shells, collagen, sodium chloride, compound amino acids, carbohydrates and compound fertilizers.
7. The application of the compound fertilizer according to claim 6, wherein the weight ratio of the culture solution to shrimp and crab shells, collagen, sodium chloride, compound amino acid, carbohydrate and compound fertilizer is (0.1-1): (0.01-1): (0.01 to 9): (1-50): (1-20): (1-10).
8. The use according to claim 6, wherein the microbial preparation is prepared by the steps of:
(1) Adding the strain culture solution, shrimp and crab shells, collagen and sodium chloride into water, mixing, fermenting at 25-35 ℃ for 3-5 days, bubbling and aerating to obtain a fermentation solution;
(2) Adding amino acid, carbohydrate and compound fertilizer into the fermentation liquid in the step (1), wherein the fermentation temperature is 25-35 ℃, the fermentation time is 3-5 days, and bubbling and ventilation are carried out to obtain the compound fertilizer.
9. The use according to claim 8, wherein the microbial preparation is applied by the following method: diluting the microbial preparation with water by 1-10 times, adding soapberry extract with the volume of 0.5-1.5% (v/v) of the microbial preparation, and root irrigation of astragalus mongholicus.
10. The use according to claim 9, wherein the soapberry extract is prepared by the following method: drying and pulverizing seed coat of fructus Sapindi Mukouossi, extracting with water at 60deg.C for 2 hr for 3 times, filtering, and concentrating the filtrate to a weight ratio of fructus Sapindi Mukouossi to 4:1.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118077719A (en) * 2024-04-25 2024-05-28 赤峰市乾铭农业科技发展有限公司 Application of microbial preparation in platycodon grandiflorum planting

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106316544A (en) * 2015-06-15 2017-01-11 陈兴春 Special biological composite fertilizer for Radix Astragali, and preparation method thereof
CN107287139A (en) * 2017-08-03 2017-10-24 北京泰克美高新技术有限公司 A kind of microorganism formulation and preparation method thereof and the application in continuous cropping obstacle is improved
CN111226793A (en) * 2020-03-02 2020-06-05 内蒙古自治区生物技术研究院 Method for producing astragalus membranaceus hairy roots in large scale
CN112438171A (en) * 2019-09-02 2021-03-05 北京中科金株生物科技有限公司 Application of bacillus subtilis HF1 in astragalus planting
CN116410902A (en) * 2023-04-22 2023-07-11 上药(宁夏)中药资源有限公司 Biological microbial agent for preventing and treating root rot of astragalus and preparation method thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106316544A (en) * 2015-06-15 2017-01-11 陈兴春 Special biological composite fertilizer for Radix Astragali, and preparation method thereof
CN107287139A (en) * 2017-08-03 2017-10-24 北京泰克美高新技术有限公司 A kind of microorganism formulation and preparation method thereof and the application in continuous cropping obstacle is improved
CN112438171A (en) * 2019-09-02 2021-03-05 北京中科金株生物科技有限公司 Application of bacillus subtilis HF1 in astragalus planting
CN111226793A (en) * 2020-03-02 2020-06-05 内蒙古自治区生物技术研究院 Method for producing astragalus membranaceus hairy roots in large scale
CN116410902A (en) * 2023-04-22 2023-07-11 上药(宁夏)中药资源有限公司 Biological microbial agent for preventing and treating root rot of astragalus and preparation method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
宋庆燕等: ""氮磷钾元素对黄芪生长和干物质积累的影响"", 《中国现代中药》, pages 1157 - 1158 *
祖未希等: ""芽孢杆菌R57 对黄芪的防病提质作用及其鉴定"", 《中国农学通报》, pages 129 - 135 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118077719A (en) * 2024-04-25 2024-05-28 赤峰市乾铭农业科技发展有限公司 Application of microbial preparation in platycodon grandiflorum planting
CN118077719B (en) * 2024-04-25 2024-07-09 赤峰市乾铭农业科技发展有限公司 Application of microbial preparation in platycodon grandiflorum planting

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