CN116286437B - Bacillus subtilis 2-20 and application thereof - Google Patents
Bacillus subtilis 2-20 and application thereof Download PDFInfo
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 36
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 36
- 238000004321 preservation Methods 0.000 claims abstract description 15
- 230000000813 microbial effect Effects 0.000 claims abstract description 11
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 4
- 230000001737 promoting effect Effects 0.000 claims abstract description 4
- 238000000855 fermentation Methods 0.000 claims description 19
- 230000004151 fermentation Effects 0.000 claims description 19
- 241000209140 Triticum Species 0.000 claims description 9
- 235000021307 Triticum Nutrition 0.000 claims description 9
- 240000008042 Zea mays Species 0.000 claims description 9
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 9
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 9
- 235000005822 corn Nutrition 0.000 claims description 9
- 239000003337 fertilizer Substances 0.000 claims description 7
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 208000003643 Callosities Diseases 0.000 claims description 3
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 3
- 210000003608 fece Anatomy 0.000 claims description 2
- 239000010871 livestock manure Substances 0.000 claims description 2
- 239000002068 microbial inoculum Substances 0.000 claims description 2
- 240000003768 Solanum lycopersicum Species 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 25
- 230000000694 effects Effects 0.000 abstract description 17
- 239000002689 soil Substances 0.000 abstract description 10
- 238000011161 development Methods 0.000 abstract description 3
- 235000013311 vegetables Nutrition 0.000 abstract description 3
- 244000144972 livestock Species 0.000 abstract description 2
- 238000004382 potting Methods 0.000 abstract description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 15
- 229910052757 nitrogen Inorganic materials 0.000 description 13
- 229930191978 Gibberellin Natural products 0.000 description 11
- 239000004062 cytokinin Substances 0.000 description 11
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 11
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 11
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- HYVABZIGRDEKCD-UHFFFAOYSA-N N(6)-dimethylallyladenine Chemical compound CC(C)=CCNC1=NC=NC2=C1N=CN2 HYVABZIGRDEKCD-UHFFFAOYSA-N 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 description 6
- 229940023877 zeatin Drugs 0.000 description 6
- 241000227653 Lycopersicon Species 0.000 description 5
- 235000010633 broth Nutrition 0.000 description 5
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
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- 239000006228 supernatant Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 3
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 3
- HSFWRNGVRCDJHI-UHFFFAOYSA-N alpha-acetylene Natural products C#C HSFWRNGVRCDJHI-UHFFFAOYSA-N 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 239000002777 nucleoside Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000589151 Azotobacter Species 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
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- 235000011852 gelatine desserts Nutrition 0.000 description 2
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- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 2
- 239000003617 indole-3-acetic acid Substances 0.000 description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 2
- 239000000787 lecithin Substances 0.000 description 2
- 229940067606 lecithin Drugs 0.000 description 2
- 235000010445 lecithin Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- -1 zeatin nucleoside Chemical class 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 241000194103 Bacillus pumilus Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- IXORZMNAPKEEDV-SNTJWBGVSA-N LSM-6641 Chemical compound C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@@]3(C)C(=O)O2 IXORZMNAPKEEDV-SNTJWBGVSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 238000007865 diluting Methods 0.000 description 1
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- 239000000284 extract Substances 0.000 description 1
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- 239000011521 glass Substances 0.000 description 1
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- 239000002054 inoculum Substances 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
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- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 239000000618 nitrogen fertilizer Substances 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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 invention discloses bacillus subtilis 2-20 and application thereof. Relates to the technical field of agricultural microbial agents and application thereof. The preservation number is CGMCC: no.24834. The strain provided by the invention has the effect of promoting the growth of crops and vegetable crops, and both potting experiments and field experiments prove that the strain has the effect of promoting the growth of crops and vegetable crops; moreover, the bacillus subtilis is derived from soil, is safe and environment-friendly, is easy to culture, has no pollution and residue after being used, is harmless to people and livestock, and meets the development requirement of green agriculture.
Description
Technical Field
The invention relates to the technical field of agricultural microbial agents and application thereof, in particular to bacillus subtilis 2-20 and application thereof.
Background
Microbial fertilizers are a specific product containing living microorganisms, and can obtain specific fertilizer effects when applied to agricultural production. It can promote crop growth, raise crop yield, improve farm product quality, improve soil and raise soil fertility. The free-living nitrogen fixing bacteria can convert nitrogen in the air into ammonia which can be absorbed and utilized by plants, thereby achieving the purpose of reducing the use of chemical fertilizers. The bacillus can form spores, has the advantages of strong stress resistance, easy storage and the like, and is commonly used for developing microbial fertilizers. In bacillus with nitrogen fixation, the strain which can produce plant hormone such as auxin (indoleacetic acid), cytokinin, gibberellin and the like and promote growth is screened for agricultural production, and the strain has important significance for reducing nitrogen fertilizer investment and improving plant crop growth.
CN106190887A discloses a bacillus subtilis T400, the activity of the azotase is 600-800 nmol/(mL.h), and the concentration of the indoleacetic acid is 95-103 mg/L.
CN105316268A discloses Bacillus pumilus, in which gibberellin content in fermentation broth is GA 3 51.2mg/L, GA 4 6.9mg/L, GA 7 At 8.7mg/L, there is no report on Bacillus subtilis having a nitrogen fixation enzyme activity and capable of producing cytokinin and gibberellin at high yield.
Therefore, whether a specific bacillus subtilis can overcome the technical obstacle is a problem that needs to be solved by the person skilled in the art.
Disclosure of Invention
In view of the above, the invention provides bacillus subtilis 2-20 and application thereof. Can simultaneously have the activity of the nitrogen fixation enzyme, high yield cytokinin and gibberellin, so that the method can be applied to the preparation of microbial fertilizers and promote the growth and development of plants.
Preservation information: 2-20 of bacillus subtilis (Bacillus subtilis) which is preserved in China general microbiological culture Collection center (China Committee for culture Collection), wherein the preservation address is 1 # 3 of North Chen Xiyu in the Chaoyang area of Beijing city, and the preservation number is CGMCC: no.24834, the date of preservation is 2022, 5, 6.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
2-20 of bacillus subtilis (Bacillus subtilis) with the preservation number of CGMCC: no.24834.
The invention also provides a microbial agent, which comprises bacillus subtilis 2-20 with a preservation number of CGMCC: no.24834.
The invention also provides a microbial fermentation broth, which is fermented by using bacillus subtilis 2-20, and the preservation number is CGMCC: no.24834.
The invention also provides a microbial fertilizer, which comprises bacillus subtilis 2-20 with a preservation number of CGMCC: no.24834.
The invention also provides application of the bacillus subtilis 2-20, or the microbial inoculum, or the fermentation broth, or the bacterial manure in agriculture and industrial production.
Preferably: is applied to tomatoes, wheat and corns.
Compared with the prior art, the invention discloses and provides the bacillus subtilis 2-20 and the application thereof, and the obtained technical effects are that the potting experiment and the field experiment prove that the bacillus subtilis 2-20 has growth promoting effect on crops and vegetable crops; in addition, the bacillus subtilis 2-20 is derived from soil, is safe and environment-friendly, is easy to culture, has no pollution and residue after being used, is harmless to people and livestock, and meets the development requirements of green agriculture.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the determination of gibberellin GA by HPLC method provided by the present invention 3 Standard plot of content versus absorption peak area.
FIG. 2 is a graph showing the standard curve of the zeatin content and the absorption peak area provided by the invention.
FIG. 3 is a graph showing the standard graph of zeatin nucleoside content versus relative peak area provided by the present invention.
FIG. 4 is a graph showing the standard curve of isopentenyl adenine content versus relative peak area provided by the present invention.
FIG. 5 is a diagram showing colonies of the bacillus subtilis 2-20 strain provided by the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses bacillus subtilis 2-20 and application thereof.
In the examples, the experimental materials used were:
LB medium: 10g of peptone, 5g of yeast extract, 10g of sodium chloride and 20g of agar, adding distilled water to a volume of 1L, mixing uniformly, packaging in triangular bottles, and sterilizing at 121 ℃ for 30min.
Azabeth nitrogen-free medium: mannitol 10g, KH 2 PO 4 0.2 g，MgSO 4 ·7H 2 O 0.2g，NaCl 0.2g，CaSO 4 ·2H 2 O 0.1g，CaCO 3 5, g, adding distilled water to a constant volume of 1L, mixing, packaging in triangular flask, and sterilizing at 121deg.C for 30min, wherein 20g of agar is required to be added as solid culture medium.
The bacillus subtilis 2-20 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation address of number 3 of national institute 1, western road, korean, beijing, and a preservation number of CGMCC: no.24834, the date of preservation is 2022, 5, 6; other raw materials and reagents used in the invention are all purchased from the market.
The experimental materials and experimental methods are not mentioned as conventional experimental methods, and are not described herein.
Example 1
Isolation and screening of Strain 2-20
The bacterial strain 2-20 is separated from corn field soil in the pool area of Baoding city in Hebei province, and the specific separation and screening processes are as follows:
weighing 10g of soil, placing into 90mL of sterilized normal saline containing glass beads, sufficiently shaking by using a vortex oscillator, placing into 80 ℃ hot water for 30min, taking out, cooling to room temperature, sucking 1mL of supernatant, and diluting the soil suspension by adopting a gradient dilution method to obtain a suspension 2 、10 3 、10 4 Or 10 5 Doubling, respectively coating 0.1mL of each gradient dilution on Abbe's nitrogen-free culture medium, placing the coated plate in a constant temperature incubator at 30deg.C, and culturing in an inverted mannerCulturing for 5 days, observing colony growth condition on the culture medium every day, obtaining single colony by adopting three dividing lines, and storing the single colony on the inclined plane of the Ababetes nitrogen-free culture medium for later use;
and (3) screening bacteria with the activity of the azotobacter and the high yield of cytokinin and gibberellin by measuring the activity of the azotobacter of each separated strain and the cytokinin content and gibberellin content of each strain after fermentation.
Strain acquisition:
firstly, the activity of the azotase of each strain is measured by adopting an acetylene reduction method to obtain the strain with the activity of the azotase.
The test group activated each strain stored with an Abbe's solid medium, picked the cells with an inoculating loop, inoculated into a 10mL test tube containing 5mL Abbe's solid medium, and sealed with a rubber stopper. The same test tube without the inoculated cells was used as a control group. After the two groups are placed at 30 ℃ for 48 hours for culture, acetylene gas with the volume of 1/10 of that of the test tube is injected into the test tube, the culture is continued for 24 hours, 0.5mL of gas is extracted from the test tube and injected into a gas chromatograph (FL 9790 of Zhejiang Fuli analytical instrument Co., ltd.) for measuring the content of acetylene and ethylene.
The magnitude of the activity of the enzyme is calculated according to the following formula:
C＝(h x ×c×V)/(24.9×h s x t) wherein h x Peak area value of the sample; h is a s Is standard C 2 H 4 Peak area value; c is standard C 2 H 4 Concentration (nmol/m L); v is the culture vessel volume (mL); t is sample incubation time (h); c is C generated 2 H 4 Concentration [ nmol/(m L. H)]。
Secondly, measuring the content of gibberellin and cytokinin in the supernatant of the fermentation broth by adopting a High Performance Liquid Chromatography (HPLC) method.
Inoculating the strain with the activity of the nitrogen fixation enzyme into an LB culture medium, placing the LB culture medium at 30 ℃ and 180r/min for shaking culture for 48 hours to obtain a fermentation liquor, centrifuging the fermentation liquor at 8000r/min for 10 minutes to obtain a fermentation liquor supernatant, passing the supernatant through a microporous filter membrane of 0.45 mu m, and directly measuring gibberellin and cytokinin from the filtrate.
Obtaining gibberellin producing bacteria. GibberellinThe variety is more, and experiments mainly produce GA for strains 3 Capacity was measured. Analytical pure GA was performed on HPLC prior to measurement 3 A standard curve is made as shown in fig. 1.
Obtaining cytokinin-producing bacteria. Cytokinins are more abundant, and screening methods have been used to determine the ability of strains to produce zeatin, zeatin nucleosides and isopentenyl adenine. Standard curves were made using HPLC for analytically pure zeatin, zeatin nucleoside and isopentenyl adenine, respectively, as shown in figures 2-4.
Through the measurement, a strain with the activity of the nitrogen fixation enzyme and the high yield of gibberellin and cytokinin is screened out and named as 2-20.
The activity of the azotase of the strain obtained by the determination method reaches 1210 nmol/(mL.h), gibberellin GA 3 The content reaches 68.45mg/L, and the cytokinin content reaches 42.67mg/L.
Finally, bacteria were identified by determining the bacterial 16S rDNA sequence and the physiological and biochemical properties of the strain.
The strain separated and screened is amplified into 16S rDNA sequence by PCR method, the PCR product is sent to Beijing Liuhua big gene science and technology Co., ltd for sequencing, according to the sequencing result (CGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTT is shown as SEQ ID NO. 1), the on-line inquiry analysis is carried out at http:// www.ncbi.nlm.nih.gov, and the Blast software is utilized to carry out homology comparison with other 16S rDNA sequences in GenBank. According to the sequence comparison result, determining the physiological and biochemical characteristics of the strain (sugar, alcohol fermentation experiment, V-P determination experiment, methyl red experiment, ammonia production experiment, nitrate reduction experiment, starch hydrolysis experiment, indole experiment, lecithin enzyme, contact enzyme, gelatin liquefaction experiment and malonic acid utilization experiment are positive, fluorescent pigment and citric acid utilization is negative, and sodium chloride with the concentration of 7% is tolerated, as shown in table 1), and identifying the strain as bacillus subtilis (B acillus subtilis) through the 16S rDNA sequence and the physiological and biochemical properties, wherein a colony chart of the strain is shown in figure 4; the strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.24834 in 2022, 5 and 6 days.
Table 1 2-20 Strain physiological and Biochemical Properties
| Type of experiment | Experimental results | Type of experiment | Experimental results |
| Anaerobic experiments | Facultative anaerobism | Contact enzyme | + |
| Motility of | + | Fluorescent pigment | - |
| Malonic acid utilization | + | Ammonia production experiment | + |
| Citric acid utilization | - | Indole experiments | + |
| Fermentation of sugar and alcohols | + | Nitrate reduction | + |
| Methyl Red experiment | + | 2% sodium chloride | + |
| V-P assay | + | 5% sodium chloride | + |
| Starch hydrolysis | + | 7% sodium chloride | + |
| Gelatin liquefaction | + | 10% sodium chloride | - |
| Lecithin enzyme | + |
Note that: "+" indicates positive and "-" indicates negative.
Example 2
Application of bacillus subtilis 2-20 fermentation liquor in tomatoes
After the surface of the tomato seeds is disinfected, the seeds are planted in soil. The experiment was set up with 2 treatments, 30 replicates each, each: no inoculation treatment (control group) and inoculation treatment (experimental group), the inoculation amount of inoculation treatment is 5 mL/strain (50 times dilution of fermentation liquor). The emergence rate was measured after 7 days, and the plant height, fresh matter mass and dry matter mass were measured after 21 days as shown in table 2;
TABLE 2 influence of Bacillus subtilis 2-20 on tomato seedling growth
The result shows that compared with a control group without bacteria, the bacillus subtilis can obviously promote the growth of tomato seedlings and increase the biomass of the seedlings.
Example 3
Application of bacillus subtilis 2-20 fermentation liquor in wheat
Selecting full wheat seeds, carrying out surface disinfection by using 70% ethanol, washing for at least 6 times by using sterile water, and coating the sterile water washed for the last time on a beef extract peptone solid culture medium to check whether the surface disinfection of the wheat is thorough. Sterilized wheat seeds were sown in flowerpot soil, 6 per pot, with approximately 1cm of soil covered on the surface. The test set consisted of 2 treatments, each plant of the test group was inoculated with 2-20 inoculum (about 10 8 and/mL) 1mL, and the control group uses an equal amount of clear water, 10 replicates per treatment. The flowerpot is placed in a greenhouse, and the culture conditions are set to be 16h/8h of illumination and 25 ℃/18 ℃. After wheat seedlings emerge, water is poured once every 5 days, and plant height, dry weight and nitrogen content are measured after 60 days. As shown in table 3;
TABLE 3 variation of plant height, dry weight and Nitrogen content before and after wheat inoculation with 2-20 bacterial solutions
The result shows that the bacillus subtilis 2-20 can obviously promote the growth of wheat and increase the nitrogen fixation capacity of plants compared with a control group without bacteria.
Example 4
Application of bacillus subtilis 2-20 fermentation liquor in corn
Selecting full corn seeds and directly planting the seeds in a field. After the corns are planted, the diluted fermentation liquor is used for 1 time and is used once again after 1 month. The experiment adopts a drip irrigation mode, 2 treatments are adopted, each plant of the experiment group averagely uses 5mL of 2-20 bacterial liquid diluted by 50 times, the control group uses equivalent clean water, and each treatment is 1 mu. The effect of corn dry matter accumulation and nitrogen accumulation in the mature period was examined as shown in table 4;
TABLE 4 2-20 Effect of fermentation broths on maturity corn dry matter and Nitrogen accumulation
The results show that the bacillus subtilis 2-20 can obviously promote the growth of corn and the accumulation of nitrogen in plants compared with a control group without bacteria.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. Bacillus subtilis @Bacillus subtilis) 2-20, which is characterized in that the preservation number is CGMCC: no.24834.
2. The microbial agent is characterized by comprising bacillus subtilis 2-20 with a deposit number of CG MCC: no.24834.
3. A microbial fermentation broth is characterized in that bacillus subtilis 2-20 is used for fermentation, and the preservation number is CGMCC: no.24834.
4. The microbial fertilizer is characterized by comprising bacillus subtilis 2-20 with a deposit number of CG MCC: no.24834.
5. Use of bacillus subtilis 2-20 according to claim 1, or a microbial inoculum according to claim 2, or a fermentation broth according to claim 3, or a bacterial manure according to claim 4 for promoting growth of tomatoes, wheat and corns.
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