CN113755382B - Bacillus aryabhattai NDFY-1 and application thereof - Google Patents
Bacillus aryabhattai NDFY-1 and application thereof Download PDFInfo
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- CN113755382B CN113755382B CN202111126732.1A CN202111126732A CN113755382B CN 113755382 B CN113755382 B CN 113755382B CN 202111126732 A CN202111126732 A CN 202111126732A CN 113755382 B CN113755382 B CN 113755382B
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- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003895 organic fertilizer Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- NESLWCLHZZISNB-UHFFFAOYSA-M sodium phenolate Chemical compound [Na+].[O-]C1=CC=CC=C1 NESLWCLHZZISNB-UHFFFAOYSA-M 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000003516 soil conditioner Substances 0.000 description 1
- 239000006104 solid solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
Classifications
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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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The application discloses bacillus aryabhattai NDFY-1 and application thereof; according to the thallus morphology, colony characteristics, physiological and biochemical characteristics of the NDFY-1 strain and the molecular biology identification result, the strain is identified as bacillus aryabhattai, and is preserved in China general microbiological culture Collection center (CGMCC) No.23131 at 8 and 9 of 2021. The NDFY-1 strain has excellent effects of producing IAA, phosphate and potassium, and producing siderophores; the fermentation performance is good, and the number of viable bacteria in fermentation liquor is high; the strain can achieve the effects of promoting the growth and development of the fertilizer peach and improving the soil fertilizer.
Description
Technical Field
The application relates to the technical field of agricultural microorganisms, in particular to bacillus aryabhattai NDFY-1 and application thereof.
Background
The fertilizer peach is a fruit with high nutritional value and medicinal value, has high demand at home and abroad, has fragrant and sweet taste due to the fragrant smell, contains a plurality of vitamins and is popular with the public. In recent years, under the drive of the market with the first price, peach farmers greatly increase the use of nitrogenous fertilizers, other chemical fertilizers, herbicides, pesticides and other pesticides for increasing the yield of the fertilizer peaches, destroy the ecology of soil, cause the problems of reduction of organic matters in the soil, hardening of the soil, poor air permeability, acidification, lack of trace elements and the like, reduce the quality of the fertilizer peaches and seriously influence the development of the fertilizer peach industry.
Microbial fertilizers are widely used as agricultural materials for increasing yield and improving quality. The microbial fertilizer can improve the nutrients of the rhizosphere soil of the fertilizer peaches and strengthen the enzyme activity, thereby achieving the purpose of promoting the quality improvement of the fertilizer peaches. Therefore, excellent strain materials are provided for the research and development of microbial fertilizers for the fertilizer peaches, and good application potential is displayed.
Bacillus aryabhattai (Bacillus aryabhattai) is a novel species of bacillus found in 2009. In recent years, there are more reports on plant growth promotion of bacillus arvensis, and bacillus arvensis T61 has been proved by Fan Meiyu and the like to be a cadmium-resistant plant growth promoter, and can relieve the stress of cadmium on certain rice varieties (Fan Meiyu and the like, 2021). Paredes-P.liz Karina et al found that Bacillus arvensis RS025 could promote germination of Miscandens seeds in metal contaminated soil with 2.5 fold improvement in germination rate (Paredes-P.liz Karina et al 2016). Bacillus aryabhattai SRB02 isolated from soybean rhizosphere by Park et al was used as a soil conditioner to reduce the toxic effects of nitrite in soil on plants (Park et al, 2017). Muham-mad Shakeel et al found that Bacillus aryabhattai could promote zinc ion uptake by plants (Muhammad et al 2015). Patent CN105985922A discloses a bacillus aryabhattai J5 which has the functions of producing auxin, producing siderophore, producing ammonia, producing protease, resisting salt, antagonizing, dissolving phosphorus and the like, and has wide application prospect in the aspects of improving soil nutrients, eliminating salinization, promoting plant growth, preventing diseases and the like. Patent CN112410244A discloses a bacillus aryabhattai JNJ266 which has the functions of phosphate and potassium dissolving, protease producing, IAA, siderophore and the like, and a potting experiment proves that the strain has good walnut growth promoting and drought resisting effects.
However, it is difficult to expect whether Bacillus aryasis isolated from other environments can stably colonise and act in the rhizosphere soil of nectarine, depending on the rhizosphere soil environment of different plants. At present, no related report of bacillus arvensis for improving the rhizosphere soil nutrient of the fertilizer peaches and promoting the quality improvement of the fertilizer peaches exists.
Disclosure of Invention
Aiming at the prior art, the application aims to provide the bacillus aryabhattai NDFY-1 and the application thereof. The bacillus arvensis NDFY-1 is separated from root soil of a fertilizer urban fertilizer peach healthy plant in Taian city of Shandong province. The strain has excellent effects of producing IAA, dissolving phosphorus and potassium and producing siderophores; the fermentation performance is good, and the number of viable bacteria in fermentation liquor is high; the strain can achieve the effects of promoting the growth and development of the fertilizer peach and improving the soil fertilizer.
Specifically, the application relates to the following technical scheme:
in a first aspect of the present application, there is provided a bacillus aryabhattai (Bacillus aryabhattai) NDFY-1, which has been deposited in the China general microbiological culture collection center (CGMCC, address: institute No. 3, north chen west way No.1, the korean region of beijing) at day 8 and day 9 of 2021, and has a biological deposit number of: CGMCC No.23131.
The colony morphology characteristics of the bacillus aryabhattai NDFY-1 are as follows:
the colony shape on LB solid medium is nearly circular, the colony size is 2-4mm, the surface is flat and moist, the edge is smooth, milky white, opaque, gram-positive.
The physiological and biochemical characteristics of the bacillus aryabhattai NDFY-1 are as follows:
the strain H2S, indole, nitrate reduction, malonate, glucose acidogenesis and sucrose acidogenesis tests are positive; the V-P test and gelatin liquefaction reaction are negative; can be grown in LB liquid medium with NaCl concentration of 7% and 10%.
The molecular biological characteristics of the bacillus aryabhattai NDFY-1 are as follows:
the 16S rRNA sequence of the strain is shown as SEQ ID NO. 1.
In a second aspect of the present application, there is provided a microbial agent comprising the Bacillus aryabhattai NDFY-1.
In the microbial inoculum, the bacillus aryabhattai NDFY-1 can exist in the form of cultured living cells and fermentation liquid of the living cells.
Furthermore, the microbial inoculum can comprise auxiliary materials or carrier matrixes and the like besides the bacillus aryabhattai NDFY-1, such as trehalose, fulvic acid, turf, diatomite, straws of various crops, peanut shells, animal wastes or fertilizers and the like.
The preparation form of the microbial inoculum can be liquid, emulsion, suspending agent, granule, powder, wettable powder or water dispersing agent.
In a third aspect of the application, a preparation method of a bacillus aryabhattai NDFY-1 microbial inoculum is provided, comprising the following steps:
inoculating the seed solution of the bacillus aryabhattai NDFY-1 into a fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: rotating at 180r/min and at 36-38 ℃ for 12-16 h by shake culture to obtain bacillus aryabhattai NDFY-1 fermentation liquor;
each 1L of the fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling for 30min after water boiling, filtering to remove the soybean sprouts, adding 30g of soluble starch, 15g of ammonium chloride and 3g of calcium carbonate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting the pH to 7.0-7.5, and sterilizing.
Preferably, the seed solution of the bacillus aryabhattai NDFY-1 is inoculated in an amount of 8-12% (volume ratio).
Preferably, the viable count in the bacillus aryabhattai NDFY-1 fermentation broth is 8.0X10 9 cfu/ml-9.0×10 9 cfu/ml。
In the preparation method, the prepared bacillus aryabhattai NDFY-1 fermentation liquor can be directly used as a liquid microbial inoculum; furthermore, the bacillus aryabhattai NDFY-1 fermentation liquor can be mixed with auxiliary materials or carrier matrixes to prepare the solid microbial inoculum.
The application method of the prepared bacillus aryabhattai NDFY-1 microbial inoculum comprises the following steps: uniformly mixing the bacillus aryabhattai NDFY-1 liquid microbial inoculum and water according to the volume ratio of 1:29, and irrigating roots; the solid microbial inoculum is diluted by a proper amount of water and then is applied by root irrigation. The two bacterial agents can be used as strains of the biological organic fertilizer and the compound microbial fertilizer.
In a fourth aspect of the present application, there is provided the use of the above Bacillus aryabhattai NDFY-1 or a microbial agent comprising the above Bacillus aryabhattai NDFY-1 in at least one of the following 1) -4):
1) Producing IAA, phosphate and potassium, and producing iron carrier;
2) Promoting the growth and development of crops;
3) Improving the activity of catalase, sucrase and urease in rhizosphere soil of crops;
4) Improving the organic matter, alkaline nitrogen and quick-acting potassium content of the rhizosphere soil of crops.
Preferably, the crop is a fat peach.
In a fifth aspect of the application, a microbial fertilizer for peach is provided, which is prepared from fermentation liquid of bacillus aryabhattai NDFY-1, brevibacterium cold-resistant and Arthrobacter urealyticum in a weight ratio of (2-4): 1:1, compounding; wherein:
the fermentation broth of the bacillus aryabhattai NDFY-1 is prepared by the following method:
inoculating the seed solution of the bacillus aryabhattai NDFY-1 into a first fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: rotating at 180r/min and at 36-38 ℃ for 12-16 h by shake culture to obtain bacillus aryabhattai NDFY-1 fermentation liquor;
each 1L of the first fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling for 30min after water boiling, filtering to remove the soybean sprouts, adding 30g of soluble starch, 15g of ammonium chloride and 3g of calcium carbonate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting the pH to 7.0-7.5, and sterilizing.
The fermentation broth of the cold-resistant Brevibacterium is prepared by the following steps:
inoculating the seed solution of the cold-resistant Brevibacterium into a second fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: rotating at 180r/min, at 30-32 ℃, shake culturing for 12-16 h to obtain cold-resistant Brevibacterium fermentation liquor;
each 1L of the second fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling water, steaming for 30min, filtering to remove the soybean sprouts, adding 30g of glucose, 15g of ammonium sulfate and 3g of magnesium sulfate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting pH to 7.0-7.5, and sterilizing.
The fermentation broth of Arthrobacter urealyticum is prepared by the following method:
inoculating the seed solution of Arthrobacter urealyticum into a second fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: rotating at 180r/min and at 36-38 ℃ for 12-16 h by shake culture to obtain Arthrobacter urealyticum fermentation broth;
each 1L of the second fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling water, steaming for 30min, filtering to remove the soybean sprouts, adding 30g of glucose, 15g of ammonium sulfate and 3g of magnesium sulfate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting pH to 7.0-7.5, and sterilizing.
The application has the beneficial effects that:
(1) The bacillus aryabhattai (Bacillus aryabhattai) NDFY-1 has excellent comprehensive performance and can produce IAA, phosphate and potassium and siderophores.
(2) The bacillus aryasis (Bacillus aryabhattai) NDFY-1 can stably colonize in the rhizosphere soil of the fertilizer peaches, can remarkably improve the activities of catalase, sucrase and urease in the planting soil of the fertilizer peaches, can also improve the contents of organic matters, alkaline nitrogen and quick-acting potassium in the soil, and has the effect of remarkably improving the fertility of the soil.
(3) Under the potting condition, the bacillus aryasis (Bacillus aryabhattai) NDFY-1 fermentation liquid is utilized to irrigate and apply the bacillus aryasis to soil near the root of the fat peach, so that the plant height, the branch number, the leaf number and the chlorophyll content of the fat peach can be remarkably improved, and the growth of the fat peach is promoted.
(4) The bacillus aryabhattai (Bacillus aryabhattai) NDFY-1 has good fermentation property, simple production process, short production period and high viable count in fermentation liquor.
(5) The bacillus aryabhattai (Bacillus aryabhattai) NDFY-1 can survive stably when coexisting with a fertilizer, and can be applied simultaneously with a water-soluble fertilizer or a solid-solution fertilizer.
(6) The bacillus aryabhattai (Bacillus aryabhattai) NDFY-1, the cold-resistant Brevibacterium and the Arthrobacter urealyticum are compounded to prepare the microbial fertilizer for the peach, so that the microbial fertilizer has remarkable synergistic effect and can further improve the growth promoting effect on the peach.
Drawings
Fig. 1: colony morphology of strain NDFY-1.
Fig. 2: bacterial morphology of strain NDFY-1 (magnification 1000X).
Fig. 3: phylogenetic tree constructed based on the 16SrDNA sequence.
Fig. 4: and a potassium-decomposing function diagram of the strain NDFY-1.
Fig. 5: IAA assay standard curve.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments. If experimental details are not specified in the examples, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified. Wherein:
LB liquid medium: tryptone 10.0g, yeast extract 5.0g, nacl 10.0g, deionized water 1000ml, ph=7.0, sterilization conditions: autoclaving at 121℃for 20min.
LB solid medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of NaCl, 15.0-20.0g of agar powder, 1000mL of deionized water, pH=7.0, sterilization conditions: autoclaving at 121℃for 20min.
Bean sprout juice culture medium: 100.0g of soybean sprouts are added into 1000mL of deionized water, after water is boiled for 30min, soybean sprouts are filtered, 20.0g of glucose is added into the liquid, the mixture is complemented to 1000mL by deionized water, and the mixture is stirred uniformly. The pH is adjusted to 7.0-7.5. Sterilization temperature: autoclaving at 121℃for 20min. (added glucose, sterilized at 115℃for 30min, to avoid the occurrence of gelatinization of glucose at too high a temperature.)
Meng Jinna organophosphorus bacteria medium: glucose 10.0g, naCl 0.3g, (NH) 4 ) 2 SO 4 0.5g，KCl 0.3g，MgSO 4 ·7H 2 O 0.3g，MnSO 4 ·4H 2 O 0.03g，FeSO 4 ·7H 2 O 0.03g，CaCO 3 5.0g, lecithin 0.2g, 1000mL of deionized water, and pH 7.0-7.5.
Note that: the solid organic phosphorus culture medium is prepared by adding 1.5% -2% agar powder into liquid culture medium, and packaging uniformly, wherein 1g CaCO is added per 50mL 3 Dissolving lecithin in absolute ethanol, adding deionized water after complete dissolution, heating until ethanol is completely evaporated, and sterilizing: autoclaving at 121℃for 20min.
Silicate bacteria medium: sucrose 5.0g, na 2 HPO 4 2.0g，MgSO 4 ·7H 2 O 0.5g，CaCO 3 0.1g，FeCl 3 0.005g, glass frit 1.0g, deionized water 1000ml, ph=7.0.
Note that: the solid potassium-decomposing culture medium needs to be additionally added with 1.5% -2% of agar in the liquid culture medium, and the glass powder needs to be evenly packaged when the liquid culture medium is prepared, and the sterilization conditions are as follows: autoclaving at 121℃for 20min.
R 2 Medium a: yeast extract 0.5g, glucose 0.5g, tryptone 0.5g, acid casein 0.5g, soluble starch 0.5g, magnesium sulfate heptahydrate 0.05g, L-tryptophan 200mg, K 2 HPO 4 0.3g, sodium pyruvate 0.3g, double distilled water 1000mL, KH was used 2 PO 4 And K 2 HPO 4 Adjusting the pH value to 7.2, and sterilizing: autoclaving at 121℃for 20min.
The preparation method of the Salkowski colorimetric solution comprises the following steps: HClO with volume fraction of 35% 4 50mL of the solution was measured, and 1mL of FeCl with a concentration of 0.5mol/L was added 3 The solution was mixed thoroughly and stored under dark conditions.
SA limited iron culture medium: sucrose 20.0g; 2.0g of L-asparagine; mgSO (MgSO) 4 ·7H 2 O 0.5g；K 2 HPO 4 0.5g; 1000mL of deionized water; autoclaving at 121℃for 20min.
CAS siderophore content detection solution: 6.0mL of the 10mmol/L CTAB solution was added to a 100mL volumetric flask and diluted with double distilled water. 1.5mL of 1mmol/L FeCl 3 After mixing the solution with 7.5mL of 2mmol/L CAS solution, the solution was slowly transferred to the volumetric flask along a glass rod. 4.307g of PIPES (piperazine diethyl sulfonic acid) is weighed and dissolved in 30mL of deionized water, then NaOH solution is slowly added dropwise to adjust the pH, the solution is added into a volumetric flask, and deionized water is used for volume determination.
Example 1: identification of strains
The application screens and obtains a strain NDFY-1 with the effect of promoting growth of the fat peach from root system soil of a healthy plant of the fat peach in Feisha city of Taian, shandong province, and identifies the strain NDFY-1 from aspects of colony morphology, physiological and biochemical indexes, 16S rDNA and the like, and specifically comprises the following steps:
(1) Colony morphology characterization:
the colony morphology was observed by the three-zone streaking method, and single colonies were streaked onto LB solid medium plates, placed in an incubator at 37℃for cultivation for 24 hours, and observed.
Colonies after 24h incubation in a 37℃incubator were stained and pelleted, and the bacterial forms were observed with a microscope, and gram staining was judged to be positive or negative.
The colony morphology graph is shown in FIG. 1; the figure of the bacterial form obtained by microscopic observation after staining is shown in fig. 2. The results show that: bacterial colony morphology of the strain NDFY-1 on LB solid medium is nearly circular, bacterial colony size is 2-4mm, surface is flat and moist, edge is flat, milky white, opaque and gram-positive.
(2) Physiological and biochemical characteristics
The physiological and biochemical index identification uses a bacterial trace physiological and biochemical identification tube provided by Qingdao sea Bo biotechnology Co., ltd, and the physiological and biochemical characteristics of the strain are identified according to the method in the "Berger's bacteria identification handbook", and the results are shown in Table 1.
Table 1: physiological and biochemical characteristics of strain NDFY-1
Note that: + is positive; -negative.
(3) Strain 16S rDNA sequence analysis
The 16S rDNA sequence of the strain NDFY-1 is shown as SEQ ID NO. 1. The phylogenetic tree was constructed using MEGA5.0, as shown in FIG. 3, and was closest to Bacillus aryabhattai in a minimal branch, and the strain NDFY-1 was identified as Bacillus aryabhattai (Bacillus aryabhattai) by combining the colony morphology and physiological and biochemical characteristics thereof.
Based on the morphological and molecular identification results of the strain, the isolated strain NDFY-1 was identified as Bacillus aryabhattai (Bacillus aryabhattai) and designated as Bacillus aryabhattai (Bacillus aryabhattai) NDFY-1. And performing biological preservation on the strain, wherein the preservation information is as follows:
biological material: NDFY-1
Classification naming: bacillus aryabhattai (Bacillus aryabhattai)
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3
Preservation date: 2021, 8 and 9 days
Accession numbers of the preservation center: CGMCC No.23131.
Example 2: bacterial strain NDFY-1 phosphate and potassium dissolving, IAA producing and siderophore producing test
1. Phosphate dissolution test:
the strain NDFY-1 is inoculated into a Meng Jinna organic phosphorus culture medium flat plate by using sterilized toothpicks, placed in a constant temperature electric heating incubator at 28 ℃ for 7d to be cultured, taken out, and whether a phosphate solubilizing ring appears around the strain is observed.
As a result, it was found that a phosphate solubilizing loop appeared around the strain NDFY-1. The ratio of the diameter D of a transparent ring produced by a Meng Jinna organophosphorus medium plate to the diameter D of a bacterial colony is measured to judge the phosphate dissolving function of the strain. The D/D ratio of the strain NDFY-1 was determined to be 2.50.
2. Potassium decomposition test:
the strain NDFY-1 was inoculated with sterilized toothpick points on a plate of silicate detection medium, placed in an incubator at 28℃for 1-2d, taken out, and the plate was observed for the presence or absence of oil drip colonies.
As a result, it was found that oil-drop-like colonies appeared around the strain NDFY-1 (FIG. 4), indicating that the strain NDFY-1 had a good potassium-decomposing function.
3. IAA production test:
inoculating the strain NDFY-1 into a liquid R containing L-tryptophan 2 And (3) placing the culture medium A in a constant temperature shaking box, and culturing for 4d under the condition of 180r/min and 28 ℃. 2ml of the bacterial suspension cultured for 4d was aspirated and centrifuged in a centrifuge tubeCentrifuging at 10000r/min for 10 min, collecting supernatant, adding into test tube, adding equal volume of Salkowski colorimetric solution, standing under light-shielding condition for 30min to wait for color development, and measuring OD 530 Values.
Preparing IAA series concentration standard solution by OD 530 On the abscissa, the IAA working concentration was plotted on the ordinate, and the IAA content per unit volume of fermentation broth was calculated using the standard curve (FIG. 5).
The IAA content of the fermentation broth of the strain NDFY-1 per unit volume was calculated to be 62.5. Mu.g/ml.
4. Iron production carrier test:
a platinum wire inoculating loop is used for inoculating single colony in SA limited iron liquid culture medium, shake culture is carried out for 2d at 180r/min and 37 ℃, bacterial liquid cultured for 2d is transferred to a 10mL centrifuge tube after sterilization, centrifugation is carried out for 15min at 13000r/min, equal volumes of supernatant and quantitative freshly prepared CAS detection liquid are fully and uniformly mixed, the supernatant and quantitative freshly prepared CAS detection liquid are kept stand for 1h, the light absorption value of the wavelength at 630nm is measured and is As, deionized water is used for comparison zeroing, the light absorption value of the SA liquid culture medium which is not inoculated with bacteria at 630nm is read by the same method, and the activity unit of a siderophore is = [ (Ar-As)/Ar ]. Times.100.
The activity of the strain NDFY-1 siderophore reaches 63.2 percent through measurement.
Example 3: fermentation performance test of strain NDFY-1
1. The specific fermentation process comprises the following steps:
(1) Preparing seed liquid:
single colonies were picked from LB solid medium plates using sterile toothpicks, activated, streaked onto another LB solid medium plate, and cultured in an incubator at 37℃for 12h. The cultured strain is inoculated into 50mL of liquid LB culture medium by using an inoculating loop, and shake-cultured for 12h at the temperature of 37 ℃ at 180 r/min.
(2) Fermentation culture:
inoculating the seed solution of the bacillus aryabhattai NDFY-1 into a fermentation culture medium according to the volume ratio of 10% for fermentation culture, wherein the fermentation culture conditions are as follows: the rotation speed is 180r/min, the temperature is 37 ℃, and the bacillus aryabhattai NDFY-1 fermentation liquor is obtained after shake culture for 14 h.
2. Fermentation medium optimization:
the composition of the fermentation medium is a main factor influencing the growth and metabolism of microorganisms, the application takes the bean sprout juice culture medium as a basis, optimizes the carbon source, the nitrogen source and the inorganic salt components in the culture medium, and the optimized fermentation medium is as follows:
each 1L of the fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling for 30min after water boiling, filtering to remove the soybean sprouts, adding 30g of soluble starch, 15g of ammonium chloride and 3g of calcium carbonate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting the pH to 7.0-7.5, and sterilizing.
And (3) taking the bean sprout juice culture medium as a control, carrying out fermentation culture under the same fermentation conditions, and measuring the biomass in the fermentation liquid. The biomass was measured by a dilution-coated plate counting method.
The maximum viable count in the fermentation liquor can reach 8.5 multiplied by 10 after the fermentation culture medium optimized by the application is adopted for fermentation culture 9 cfu/ml; as a control, the maximum viable count of the bean sprout juice culture medium was 1.5X10 7 cfu/ml. The adoption of the optimized fermentation medium can obviously improve the number of viable bacteria in fermentation liquor.
Example 4: survival test of Strain NDFY-1 in fertilizer
The test selects liquid water-soluble fertilizer of Shandong agricultural large fertilizer industry science and technology Co., ltd. To loosen soil No.1 (total humic acid is more than or equal to 90g/L, mineral source fulvic acid is more than or equal to 75g/L, total nutrient is more than or equal to 200g/L, N is more than or equal to 120g/L, NO) 3 - The N is more than or equal to 10g/L, and the H-PSA bioenergy is more than or equal to 10 g/L).
Adding the fermentation liquor of the strain NDFY-1 into the liquid water-soluble fertilizer according to the volume ratio of 10%, uniformly mixing, sealing and storing in a shade place, measuring the number of thalli in the water-soluble fertilizer once every 10 days, and continuously measuring for 60 days. The result shows that the bacterial strain NDFY-1 in the liquid water-soluble fertilizer has no great reduction in bacterial quantity, higher overall level and lower loss rate. After 60 days, the cell viability was 96.7%, and the test results are shown in Table 2.
Table 2: bacterial strain NDFY-1 in liquid water-soluble fertilizer
Example 5: determination of the colonization ability of the Strain NDFY-1 in the rhizosphere of crops
(1) Screening and stability detection of mutant strain of double antibiotics of strain NDFY-1
Scraping 2-loop activated strain NDFY-1 fungus coating in LB liquid medium without rifampicin, culturing at 32 ℃ to logarithmic phase, inoculating seed liquid into LB liquid medium with rifampicin concentration of 0.5 mug/mL at 2% (volume ratio), culturing for 8-24 h, inoculating the culture liquid into LB liquid medium with rifampicin concentration of 1 mug/mL at 2% (volume ratio), culturing for 24h, and transferring sequentially into LB with rifampicin concentration of 5 mug/mL, 10 mug/mL, 20 mug/mL, 50 mug/mL, 100 mug/mL, 150 mug/mL, 200 mug/mL, 250 mug/mL, 300 mug/mL. The obtained anti-rifampicin strain was inoculated into LB liquid medium containing 300. Mu.g/mL of rifampicin and 5. Mu.g/mL of spectinomycin (Spe), and the simultaneous dual-antibacterial strain against both rifampicin and spectinomycin was selected, and during the selection, the concentration of rifampicin in LB liquid medium was 300. Mu.g/mL, and the anti-spectinomycin strain was activated on plates without rifampicin and spectinomycin for 3 passages in the same manner as the increasing and selecting method of the antibiotic concentration of anti-rifampicin strain, and was returned to LB liquid medium containing 300. Mu.g/mL of rifampicin and 300. Mu.g/mL of spectinomycin for cultivation, respectively, to obtain a dual-antibacterial marker strain capable of stably inheriting and simultaneously resisting rifampicin and spectinomycin.
(2) Determination of colonization ability of strain NDFY-1 in root of fat peach
The NDFY-1 double antibiotic mutant strain obtained in the above test was inoculated into LB medium without antibiotic, and shake cultured overnight at 28 ℃. 1mL of the bacterial liquid was transferred to LB medium containing rifampicin (300. Mu.g/mL) and spectinomycin (300. Mu.g/mL), and shake-cultured at 28℃for 12 hours. Transferring 1mL of the grown bacterial liquid into LB liquid culture without antibioticsCulturing for 48h to obtain double-antibody marked strain bacterial liquid, centrifuging the bacterial liquid to remove supernatant to obtain bacterial suspension, and regulating the bacterial concentration to 10 8 CFU/mL。
20 pots were selected for growing primary-fertilized peach seedlings, wherein 10 pots were a treatment group to which double-resistant labeled NDFY-1 strain was applied, and 10 pots were a control group to which inactivated double-resistant labeled NDFY-1 strain was applied. 20mL of double-antibody marked NDFY-1 strain bacterial suspension is filled in each pot of the treatment group, and 15mL of inactivated double-antibody marked NDFY-1 strain bacterial suspension is filled in each pot of the control group.
And (5) 10, 20, 40 and 60 days after root irrigation, recovering the rhizosphere soil of the fat peach, coating and counting on a double-antibiotic flat plate by adopting a gradient dilution coating method, and detecting the colonization condition of the NDFY-1 strain on the rhizosphere of the fat peach. The test results are shown in Table 3. Along with the extension of test time, the number of the double-antibody marker strain of the treated group NDFY-1 in the rhizosphere of the fat peach is reduced to a certain extent, but the number of thalli in each gram of dry soil still reaches 6.0x10 at 60 days 6 CFU/g, whereas the control group had no colony generation, indicating that the NDFY-1 strain was able to stably colonise the root of the Ficus carica.
Table 3: colonization results of NDFY-1 strain on root of fat peach
Example 6: effect of Strain NDFY-1 on fat peach growth
1. Test site:
the test was carried out in the Shandong agricultural industry Co.Ltd greenhouse in Feisha Shandong, taian, 10.1.2020. The area is located in the middle of Shandong province, belongs to a warm-zone semi-moist climate, and has proper precipitation and proper temperature. The plants tested were the fertilizer peach seedlings purchased in the city Liu Taitao city of taan fertilizer. Ph=6.5 of the soil, weakly acidic.
2. Test materials:
the fermentation broth of the strain NDFY-1 is prepared by the following method:
inoculating the seed solution of the bacillus aryabhattai NDFY-1 into a first fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: the rotation speed is 180r/min, the temperature is 37 ℃, and the bacillus aryabhattai NDFY-1 fermentation liquor is obtained after shake culture for 14 h;
each 1L of the first fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling for 30min after water boiling, filtering to remove the soybean sprouts, adding 30g of soluble starch, 15g of ammonium chloride and 3g of calcium carbonate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting the pH to 7.0-7.5, and sterilizing.
The fermentation broth of the cold-resistant Brevibacterium is prepared by the following steps:
inoculating seed solution of Brevibacterium cold-resistant (purchased from China center for type culture collection, accession number ACCC 06432) into a second fermentation medium for fermentation culture, wherein the fermentation culture conditions are as follows: the rotation speed is 180r/min, the temperature is 31 ℃, and shake culture is carried out for 14 hours to obtain cold-resistant Brevibacterium fermentation liquor;
each 1L of the second fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling water, steaming for 30min, filtering to remove the soybean sprouts, adding 30g of glucose, 15g of ammonium sulfate and 3g of magnesium sulfate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting pH to 7.0-7.5, and sterilizing.
The fermentation broth of Arthrobacter urealyticum is prepared by the following method:
inoculating Arthrobacter urealyticus (purchased from China center for type culture collection, accession number ACCC 10474) seed solution into a second fermentation medium for fermentation culture, wherein the fermentation culture conditions are as follows: rotating at 180r/min and at 37 ℃ for shake culture for 14h to obtain Arthrobacter urealyticum fermentation broth;
each 1L of the second fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling water, steaming for 30min, filtering to remove the soybean sprouts, adding 30g of glucose, 15g of ammonium sulfate and 3g of magnesium sulfate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting pH to 7.0-7.5, and sterilizing.
The number of viable bacteria in the fermentation liquid of the strain NDFY-1, the fermentation liquid of the cold-resistant Brevibacterium and the fermentation liquid of the Arthrobacter urealyticum are adjusted to be consistent and are 10 9 cfu/ml。
3. Test treatment:
the test set 5 treatments, respectively:
treatment 1: applying a fermentation broth of the strain NDFY-1; mixing 20ml of fermentation liquor with 580ml of water uniformly for each plant 600ml, and irrigating and applying the mixture into soil near the root of the peach plant;
treatment 2: applying fermentation liquor of cold-resistant Brevibacterium; mixing 20ml of fermentation liquor with 580ml of water uniformly for each plant 600ml, and irrigating and applying the mixture into soil near the root of the peach plant;
treatment 3: applying a fermentation broth of Arthrobacter urealyticum; mixing 20ml of fermentation liquor with 580ml of water uniformly for each plant 600ml, and irrigating and applying the mixture into soil near the root of the peach plant;
treatment 4: applying mixed bacterial liquid, wherein the mixed bacterial liquid is prepared by fermenting the bacillus aryabhattai NDFY-1, the cold-resistant Brevibacterium and the Arthrobacter ureafaciens according to the weight ratio of 3:1:1, compounding to obtain mixed bacterial liquid; each plant is 600ml, 20ml of mixed bacterial liquid is uniformly mixed with 580ml of water, and the mixture is irrigated and applied to soil near the root of the fertilizer peach plant;
treatment 5: CK treatment, watering 600ml of water.
The sizes and growth vigor of the fertilizer peach seedlings selected by each treatment are kept as consistent as possible. Each treatment was set up in 6 groups in parallel. Other culture conditions for each treatment remained consistent.
4. Sample collection:
and collecting the fertilizer peach leaves after 50 days of test, collecting the rhizosphere soil samples treated in each test for 50 days by using a five-point sampling method, and determining the chlorophyll content of the leaves, the enzyme activity and the nutrients of the rhizosphere soil samples.
Leaf chlorophyll content is detected by adopting a SPAD 502Plus chlorophyll meter; the activities of soil catalase, urease and sucrase are respectively measured by adopting a potassium permanganate titration method, a sodium phenolate colorimetric method and a 3, 5-dinitrosalicylic acid colorimetric method. The contents of soil organic matters, quick-acting phosphorus, quick-acting potassium and alkaline hydrolysis nitrogen are respectively measured by adopting a potassium dichromate capacity method, a sodium bicarbonate molybdenum antimony colorimetry, an ammonium acetate flame photometer method and an alkaline hydrolysis diffusion method.
5. Test results:
(1) Plant growth assay results:
the plant height and chlorophyll content of the fat peach plants were measured and the results are shown in table 4.
Table 4: effect of different treatments on plant growth
| Treatment of | Height of plant (cm) | Chlorophyll content (SPAD) |
| Process 1 | 52.4±2.0 | 53.5±1.0 |
| Process 2 | 43.0±1.0 | 50.0±0.5 |
| Process 3 | 41.8±1.5 | 51.3±0.7 |
| Process 4 | 65.3±3.0 | 60.2±1.5 |
| Process 5 | 38.5±1.5 | 48.6±0.8 |
(2) Rhizosphere soil enzyme activity assay results:
the enzyme activity of the rhizosphere soil of the fertilizer peaches is measured, and the result is shown in table 5.
Table 5: influence of different treatments on soil enzyme activity
| Treatment of | Catalase (ml/g) | Urease (mg/g) | Saccharase (mg/g) |
| Process 1 | 3.2±0.04 | 0.82±0.02 | 18.4±0.2 |
| Process 2 | 2.4±0.01 | 0.73±0.02 | 13.5±0.2 |
| Process 3 | 2.6±0.02 | 0.68±0.01 | 12.7±0.1 |
| Process 4 | 4.1±0.05 | 1.20±0.04 | 24.2±0.3 |
| Process 5 | 2.1±0.01 | 0.62±0.01 | 10.8±0.1 |
(3) Rhizosphere soil nutrient measurement results:
the nutrient of the rhizosphere soil of the fertilizer peaches is measured, and the result is shown in table 6.
Table 6: effects of different treatments on soil nutrients
| Treatment of | Organic matter (g/kg) | Alkaline hydrolysis nitrogen (mg/kg) | Quick-acting phosphorus (mg/kg) | Quick-acting potassium (mg/kg) |
| Process 1 | 15.3±0.02 | 42.6±0.6 | 20.4±0.1 | 58.2±1.0 |
| Process 2 | 14.5±0.01 | 38.5±0.2 | 18.5±0.2 | 54.5±0.8 |
| Process 3 | 14.9±0.01 | 40.2±0.3 | 17.9±0.1 | 53.6±0.5 |
| Process 4 | 18.2±0.01 | 55.8±1.0 | 26.2±0.3 | 66.8±1.5 |
| Process 5 | 14.2±0.01 | 34.0±0.5 | 16.8±0.1 | 52.0±0.5 |
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong agricultural fertilizer industry Co.Ltd
<120> A strain of Bacillus aryabhattai NDFY-1 and use thereof
<130> 2021
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1478
<212> DNA
<213> Bacillus aryabhattai (Bacillus aryabhattai) NDFY-1
<400> 1
ggctcaggat gaacgctggc ggcgtgccta atacatgcaa gtcgagcgaa ctgattagaa 60
gcttgcttct atgacgttag cggcggacgg gtgagtaaca cgtgggcaac ctgcctgtaa 120
gactgggata acttcgggaa accgaagcta ataccggata ggatcttctc cttcatggga 180
gatgattgaa agatggtttc ggctatcact tacagatggg cccgcggtgc attagctagt 240
tggtgaggta acggctcacc aaggcaacga tgcatagccg acctgagagg gtgatcggcc 300
acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc 360
aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggctttc gggtcgtaaa 420
actctgttgt tagggaagaa caagtacgag agtaactgct cgtaccttga cggtacctaa 480
ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540
atccggaatt attgggcgta aagcgcgcgc aggcggtttc ttaagtctga tgtgaaagcc 600
cacggctcaa ccgtggaggg tcattggaaa ctggggaact tgagtgcaga agagaaaagc 660
ggaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc 720
ggctttttgg tctgtaactg acgctgaggc gcgaaagcgt ggggagcaaa caggattaga 780
taccctggta gtccacgccg taaacgatga gtgctaagtg ttagagggtt tccgcccttt 840
agtgctgcag ctaacgcatt aagcactccg cctggggagt acggtcgcaa gactgaaact 900
caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960
cgaagaacct taccaggtct tgacatcctc tgacaactct agagatagag cgttcccctt 1020
cgggggacag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080
ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcatttagt tgggcactct 1140
aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1200
ttatgacctg ggctacacac gtgctacaat ggatggtaca aagggctgca agaccgcgag 1260
gtcaagccaa tcccataaaa ccattctcag ttcggattgt aggctgcaac tcgcctacat 1320
gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1380
tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt ggagtaaccg 1440
taaggagcta gccgcctaag tgggacagat gattgggg 1478
Claims (10)
1. Bacillus aryabhattai @ strainBacillus aryabhattai) NDFY-1, with biological accession number: CGMCC No.23131.
2. Comprising the Bacillus alzheimers of claim 1Bacillus aryabhattai) And (3) a microbial inoculum of NDFY-1.
3. The microbial agent according to claim 2, further comprising an auxiliary material and/or a carrier.
4. A bacterial agent according to claim 2 or claim 3 in the form of a liquid, granule, powder, wettable powder or water-dispersible formulation.
5. Bacillus aryabhattai @Bacillus aryabhattai) The preparation method of the NDFY-1 microbial inoculum is characterized by comprising the following steps of:
the bacillus alright of claim 1Bacillus aryabhattai) The seed solution of NDFY-1 is inoculated into a fermentation culture medium for fermentation culture, and the fermentation culture conditions are as follows: rotating at 180r/min and at 36-38 ℃ for 12-16 h by shake culture to obtain bacillus aryabhattai NDFY-1 fermentation liquor;
each 1L of the fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling for 30min after water boiling, filtering to remove the soybean sprouts, adding 30g of soluble starch, 15g of ammonium chloride and 3g of calcium carbonate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting the pH to 7.0-7.5, and sterilizing.
6. The preparation method of claim 5, wherein the bacillus aryabhattai is @ or @ is @Bacillus aryabhattai) The seed liquid of NDFY-1 is inoculated in an amount of 8-12% by volume.
7. The method according to claim 5, wherein the number of viable bacteria in the fermentation broth of Bacillus aryabhattai NDFY-1 is 8.0X10 9 cfu/ml-9.0×10 9 cfu/ml。
8. The bacillus alright of claim 1Bacillus aryabhattai) Use of NDFY-1 or the microbial agent of any one of claims 2-4 in at least one of the following 1) -4):
1) Producing IAA, phosphate and potassium, and producing iron carrier;
2) Promoting the growth and development of crops;
3) Improving the activity of catalase, sucrase and urease in rhizosphere soil of crops;
4) Improving the organic matter, alkaline nitrogen and quick-acting potassium content of the rhizosphere soil of crops;
the crops are fat peaches.
9. A microbial fertilizer for peach is characterized in that the microbial fertilizer for peach is prepared from bacillus aryabhattai according to claim 1Bacillus aryabhattai) The weight ratio of the fermentation liquid of NDFY-1, cold-resistant Brevibacterium and Arthrobacter urealyticum is (2-4): 1:1 are compounded.
10. The microbial fertilizer for peach as claimed in claim 9, wherein the fermentation broth of bacillus aryabhattai NDFY-1 is prepared by the following method:
inoculating the seed solution of the bacillus aryabhattai NDFY-1 into a first fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: rotating at 180r/min and at 36-38 ℃ for 12-16 h by shake culture to obtain bacillus aryabhattai NDFY-1 fermentation liquor;
each 1L of the first fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling water, steaming for 30min, filtering to remove the soybean sprouts, adding 30g of soluble starch, 15g of ammonium chloride and 3g of calcium carbonate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting pH to 7.0-7.5, and sterilizing;
the fermentation broth of the cold-resistant Brevibacterium is prepared by the following steps:
inoculating the seed solution of the cold-resistant Brevibacterium into a second fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: rotating at 180r/min, at 30-32 ℃, shake culturing for 12-16 h to obtain cold-resistant Brevibacterium fermentation liquor;
the fermentation broth of Arthrobacter urealyticum is prepared by the following method:
inoculating the seed solution of Arthrobacter urealyticum into a second fermentation culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: rotating at 180r/min and at 36-38 ℃ for 12-16 h by shake culture to obtain Arthrobacter urealyticum fermentation broth;
each 1L of the second fermentation medium is prepared by the following method:
adding 100g of soybean sprouts into 1000ml of deionized water, boiling water, steaming for 30min, filtering to remove the soybean sprouts, adding 30g of glucose, 15g of ammonium sulfate and 3g of magnesium sulfate into the filtered liquid, supplementing to 1000ml with deionized water, uniformly stirring, adjusting pH to 7.0-7.5, and sterilizing.
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| 赵柏霞 ; 刘浩强 ; 孙丽娜 ; 肖敏 ; 郑玮 ; 赵慧 ; 潘凤荣 ; .甜樱桃根际IAA产生菌的筛选、鉴定及最佳产素条件优化.中国南方果树.2017,(第03期),全文. * |
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