CN116814479B - Bacillus GM-12-PT and method for promoting germination of Pterocarpus marsupium seeds by using same - Google Patents
Bacillus GM-12-PT and method for promoting germination of Pterocarpus marsupium seeds by using same Download PDFInfo
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- 240000008976 Pterocarpus marsupium Species 0.000 title claims abstract description 61
- 235000010453 Pterocarpus marsupium Nutrition 0.000 title claims abstract description 61
- 230000035784 germination Effects 0.000 title claims abstract description 47
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 31
- 230000001737 promoting effect Effects 0.000 title claims abstract description 18
- 238000002791 soaking Methods 0.000 claims abstract description 50
- 230000001580 bacterial effect Effects 0.000 claims abstract description 31
- 239000000725 suspension Substances 0.000 claims abstract description 29
- 230000001954 sterilising effect Effects 0.000 claims abstract description 17
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 33
- 238000005286 illumination Methods 0.000 claims description 20
- 238000004659 sterilization and disinfection Methods 0.000 claims description 18
- 238000012258 culturing Methods 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 12
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 12
- 238000007865 diluting Methods 0.000 claims description 9
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 claims description 7
- 238000009629 microbiological culture Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 4
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- 244000005700 microbiome Species 0.000 claims description 3
- 230000000977 initiatory effect Effects 0.000 abstract description 32
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 abstract description 16
- 239000004382 Amylase Substances 0.000 abstract description 16
- 108010065511 Amylases Proteins 0.000 abstract description 16
- 102000013142 Amylases Human genes 0.000 abstract description 16
- 235000019418 amylase Nutrition 0.000 abstract description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 12
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- 238000004519 manufacturing process Methods 0.000 abstract description 11
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 abstract description 6
- 229940106157 cellulase Drugs 0.000 abstract description 6
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 6
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 6
- 229910052698 phosphorus Inorganic materials 0.000 abstract description 6
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- 241000196324 Embryophyta Species 0.000 abstract description 3
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- 239000001888 Peptone Substances 0.000 description 22
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- 235000015278 beef Nutrition 0.000 description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 19
- 238000009630 liquid culture Methods 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- 241000894006 Bacteria Species 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229930191978 Gibberellin Natural products 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 235000009984 Pterocarpus indicus Nutrition 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 108010019077 beta-Amylase Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 description 3
- 229910052564 epsomite Inorganic materials 0.000 description 3
- 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 3
- 239000003448 gibberellin Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 2
- 229910052603 melanterite Inorganic materials 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
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- 238000012163 sequencing technique Methods 0.000 description 2
- 241000894007 species Species 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
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 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
- 108020004414 DNA Proteins 0.000 description 1
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- 102000005548 Hexokinase Human genes 0.000 description 1
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- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000522652 Ormosia <angiosperm> Species 0.000 description 1
- 241001423217 Ormosia henryi Species 0.000 description 1
- 244000086363 Pterocarpus indicus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- DLHONNLASJQAHX-UHFFFAOYSA-N aluminum;potassium;oxygen(2-);silicon(4+) Chemical compound [O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[Al+3].[Si+4].[Si+4].[Si+4].[K+] DLHONNLASJQAHX-UHFFFAOYSA-N 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
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- 238000000576 coating method Methods 0.000 description 1
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- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 229910052593 corundum Inorganic materials 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
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- 239000011521 glass Substances 0.000 description 1
- 239000011121 hardwood Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
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- 239000005720 sucrose Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229910000391 tricalcium phosphate Inorganic materials 0.000 description 1
- 229910000404 tripotassium phosphate Inorganic materials 0.000 description 1
- 229910001845 yogo sapphire Inorganic materials 0.000 description 1
Classifications
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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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/60—Flowers; Ornamental plants
- A01G22/67—Dwarf trees, e.g. bonsai
-
- A—HUMAN NECESSITIES
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Wood Science & Technology (AREA)
- Chemical & Material Sciences (AREA)
- Environmental Sciences (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Plant Pathology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Botany (AREA)
- Organic Chemistry (AREA)
- Pest Control & Pesticides (AREA)
- Biochemistry (AREA)
- Agronomy & Crop Science (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Dentistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Soil Sciences (AREA)
- Pretreatment Of Seeds And Plants (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of plant seed germination. The invention provides bacillus GM-12-PT and a method for promoting germination of Pterocarpus marsupium seeds, which comprises the following steps: (1) Cleaning and sterilizing Pterocarpus marsupium seeds, and soaking the seeds in bacillus GM-12-PT bacterial suspension; (2) The soaked Pterocarpus marsupium seeds are alternately cultivated for 15-25d in a dark state. The bacillus GM-12-PT has better amylase production, cellulase production, organic phosphorus dissolution and nitrogen fixation capabilities, and the strain is used for biologically triggering the Pterocarpus marsupium seeds, so that the germination rate of the seeds can be remarkably improved; the activity of the storage substances and amylase after initiation is obviously improved, the content of the seed abscisic acid and cellulose is obviously reduced, a material foundation is laid for the growth of seedlings, the seedlings are more robust in the growth process, and the stress resistance, the seeding emergence rate and the seedling quality of the seedlings are improved.
Description
Technical Field
The invention relates to the technical field of plant seed germination, in particular to bacillus GM-12-PT and a method for promoting germination of Pterocarpus marsupium seeds.
Background
Pterocarpus marsupium (Ormosia HENRYI PRAIN, identified by Yang Chengzi at 6.26.2009) belongs to the genus Pterocarpus, papilionaceae, ormosia, also called rosewood, a secondary protective plant in China, and a special species in China. The tree is excellent in material, the tree is straight and attractive, the tree is common rare hardwood, and is also an excellent landscaping tree species, and meanwhile, roots, barks, stems and leaves of the tree can be used as medicines, so that the tree has higher market economic value, ornamental value and ecological value.
The seeds of the Pterocarpus marsupium are typical hard seeds, the germination rate of the seeds of the Pterocarpus marsupium sowed without any treatment is extremely low, the hard and compact seeds can be broken to germinate by soaking the seeds in hot water at the temperature of more than 90 ℃ or carrying out acid etching treatment by 95% concentrated sulfuric acid in sowing and raising seedlings, but the physical and mechanical damages are extremely easy to be caused by adopting the methods, a large amount of nutrient substances are lost, the problems of low vigor of the seeds and seedlings, poor growth vigor, poor stress resistance and the like are caused, and the forestation effect and quality are influenced. Therefore, it is necessary to explore a method which can effectively promote seed germination and improve seed vigor without affecting the Pterocarpus marsupium seeds themselves.
Disclosure of Invention
The invention aims to provide bacillus GM-12-PT and a method for promoting germination of Pterocarpus marsupium seeds, wherein the bacillus GM-12-PT has better amylase production, cellulase production, organic phosphorus dissolution and nitrogen fixation capabilities, and the strain is used for biologically triggering Pterocarpus marsupium seeds, so that the germination rate of the seeds can be remarkably improved; the activity of the storage substances and amylase after initiation is obviously improved, the content of the seed abscisic acid and cellulose is obviously reduced, a material foundation is laid for the growth of seedlings, the seedlings are more robust in the growth process, and the stress resistance, the seeding emergence rate and the seedling quality of the seedlings are improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides Bacillus sp.GM-12-PT which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 4 th month and 28 th month of 2023, wherein the preservation address is CGMCC NO:27240, and the preservation number is that of China general microbiological culture Collection center, which is also the national institute of sciences No. 3, of West-1, north Star, towards the YangKorea of Beijing.
The invention provides application of bacillus GM-12-PT serving as growth promoting bacteria in promoting plant growth.
The invention provides application of bacillus GM-12-PT in promoting germination of Pterocarpus marsupium seeds.
The invention also provides a method for promoting germination of Pterocarpus marsupium seeds, which comprises the following steps:
(1) Cleaning and sterilizing Pterocarpus marsupium seeds, and soaking the seeds in bacillus GM-12-PT bacterial suspension;
(2) And (5) placing the soaked Pterocarpus marsupium seeds into an illumination incubator for light-dark alternate culture for 15-25d.
Preferably, the preparation process of the bacillus GM-12-PT bacterial suspension in the step (1) comprises the following steps: inoculating the activated bacillus GM-12-PT strain into a culture medium, shake culturing at 25-32 ℃ for 12-18h, collecting thalli, and diluting to obtain (0.5-1.5) multiplied by 10 8 CFU/mL bacterial suspension.
Preferably, the shaking speed is 120-180rpm, the bacterial cells are collected by adopting a centrifugal mode, and the centrifugal speed is 3500-4500rpm.
Preferably, the sterilization in the step (1) comprises the steps of sequentially performing sodium hypochlorite solution sterilization and alcohol sterilization; the mass concentration of the sodium hypochlorite solution is 2-6%, and the sterilizing time of the sodium hypochlorite solution is 8-12min; the volume concentration of the alcohol is 75%, and the disinfection time of the alcohol is 20-40s.
Preferably, the seed soaking frequency in the step (1) is 1-3 times, the seed soaking frequency is 8-12 d/time, and the seed soaking time is 2-6 h/time.
Preferably, the conditions for the alternate light and dark culture in the step (2) include: the daytime temperature is 23-28 ℃, the illumination is 10-13h, and the illumination intensity is 1300-1800Lux; culturing for 10-13h at night without illumination.
By adopting the technical scheme, the invention has the following beneficial effects:
1. The bacillus GM-12-PT screened by the invention is a functional beneficial strain and has the capabilities of producing amylase, cellulose, dissolving organic phosphorus and fixing nitrogen.
2. The invention utilizes the strain suspension to biologically trigger the Pterocarpus marsupium seeds, can rapidly improve germination rate and seed activity of Pterocarpus marsupium seeds, obviously improves the activity of storage substances and amylase after initiation, obviously reduces the content of seed abscisic acid and cellulose, lays a material foundation for the growth of seedlings, makes the seedlings more robust in the growth process, and improves stress resistance, seeding emergence rate and seedling quality of the seedlings.
3. Compared with the initiation of non-initiation and sterile beef extract peptone liquid culture medium, the strain suspension is used for biologically initiating the Pterocarpus marsupium seeds, and has the obvious improvement in the aspects of germination rate, germination vigor, gibberellin content, abscisic acid content, GA/ABA ratio, beta amylase content, total amylase content, BCA protein content, glucose content and the like.
Drawings
FIG. 1 is a phylogenetic tree of Bacillus GM-12-PT.
Description of biological preservation
The bacillus (bacillus sp.) GM-12-PT provided by the invention is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 28 of 4 of 2023, and the preservation address is CGMCCNO:27240 of China academy of sciences of China, no. 3 of Xila 1, north Star, the Korean area of Beijing.
Detailed Description
The invention provides a bacillus (bacillus sp.) GM-12-PT which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 4 months and 28 days in 2023, wherein the preservation address is CGMCCNO:27240 of China academy of sciences of China, no. 3, north Star West Lu 1 in Chao Yangjia of Beijing.
In the invention, the bacillus GM-12-PT is derived from the interplanting soil of the Pterocarpus marsupium and belongs to soil-borne functional bacteria (not belonging to foreign strains), so that the problem of sample pollution and soil environment is avoided when the Pterocarpus marsupium seeds are inoculated by the bacteria.
The invention also provides application of the bacillus GM-12-PT serving as a growth promoting bacterium in promoting plant growth.
The invention also provides application of the bacillus GM-12-PT in promoting germination of Pterocarpus marsupium seeds.
The invention also provides a method for promoting germination of Pterocarpus marsupium seeds, which comprises the following steps:
(1) Cleaning and sterilizing Pterocarpus marsupium seeds, and soaking the seeds in bacillus GM-12-PT bacterial suspension;
(2) The soaked Pterocarpus marsupium seeds are alternately cultivated for 15-25d in a dark state.
The sources of the Pterocarpus marsupium seeds are not particularly limited in the invention, and any Pterocarpus marsupium seeds can be used, and in the implementation process of the invention, the Pterocarpus marsupium seeds are preferably obtained from the same Pterocarpus marsupium tree of Qinglong county (105 degrees 13 '23.07' W,26 degrees 0 '53.97' N) at the end of 2021, and the thousand seed weight is preferably 362.35 +/-8.38 g. The invention selects full, healthy and uniform Pterocarpus marsupium seeds, and cleans the surface dirt with clean water and then carries out disinfection treatment. The disinfection treatment comprises the steps of sequentially carrying out sodium hypochlorite solution disinfection and alcohol disinfection, wherein the mass concentration of the sodium hypochlorite solution is preferably 2-6%, more preferably 3-5%, still more preferably 4%; the time for sterilizing the sodium hypochlorite solution is preferably 8-12min, more preferably 9-11min, still more preferably 10min; the volume concentration of alcohol used was 75%; the alcohol sterilization time is preferably 20 to 40 seconds, more preferably 25 to 35 seconds, still more preferably 30 seconds. In the present invention, it is preferable to perform a corresponding sterilization treatment for the germination apparatus, equipment and appliances involved in the germination process.
In the invention, disinfected Pterocarpus marsupium seeds are washed and then placed in bacillus GM-12-PT bacterial suspension for seed soaking. The washing is preferably carried out for 2-3 times by adopting distilled water. The preparation process of the GM-12-PT bacterial suspension comprises the following steps: inoculating the activated bacillus GM-12-PT strain into a culture medium, shake culturing at 25-32 ℃ for 12-18h, collecting thalli, and diluting to obtain (0.5-1.5) multiplied by 10 8 CFU/mL strain suspension. The inoculation culture medium used in the invention is preferably beef extract peptone liquid culture medium, and the culture medium comprises the components of 3.0g of beef extract, 10g of peptone, 5g of sodium chloride and 1000mL of distilled water; the pH of the medium is preferably 7.0 to 7.2. After the preparation of the beef extract peptone liquid culture medium is finished, sterilization treatment is needed, wherein the sterilization temperature is preferably 118-125 ℃, more preferably 120-123 ℃, still more preferably 121 ℃; the sterilization time is preferably 25 to 35min, more preferably 28 to 32min, still more preferably 30min. Preferably, shake cultivation is performed by a shaking table, wherein the shaking speed is preferably 120-180rpm, more preferably 140-160rpm, and still more preferably 150rpm; the temperature of the shake culture is preferably 25-32 ℃, more preferably 28-31 ℃, still more preferably 30 ℃; the time of the shake culture is preferably 12 to 18 hours, more preferably 14 to 17 hours, still more preferably 16 hours. In the present invention, after completion of shake culture, the bacterial liquid is collected and centrifuged, and the speed of centrifugation is preferably 3500 to 4500rpm, more preferably 3800 to 4200rpm, still more preferably 4000rpm, and the time of centrifugation is preferably 5 to 15min, more preferably 8 to 12min, still more preferably 10min. The bacillus GM-12-PT bacterial suspension is obtained by collecting bottom bacterial cells after centrifugal treatment and fully diluting the bacterial cells with PBS buffer solution, wherein the concentration of the bacterial suspension is preferably (0.5-1.5) multiplied by 10 8 CFU/mL, more preferably (0.8-1.2) multiplied by 10 8 CFU/mL, and even more preferably 1.0 multiplied by 10 8 CFU/mL. In the present invention, the number of times of seed soaking is preferably 1 to 3 times, and more preferably 2 times; the seed soaking frequency is preferably 8-12 d/time, more preferably 9-11 d/time, still more preferably 10 d/time; the seed soaking time is preferably 2 to 6 hours/time, more preferably 3 to 5 hours/time, still more preferably 4 hours/time.
In the invention, the soaked Pterocarpus marsupium seeds are preferably placed in a germination box on sterile absorbent cotton, the absorbent cotton is soaked in sterile water to be saturated in advance, and then the seeds are placed in the germination box for alternate cultivation under light and dark conditions. The alternate light and dark culture is preferably carried out in an illumination incubator, and the temperature of the alternate light and dark culture in the daytime is preferably 23-28 ℃, more preferably 24-26 ℃, still more preferably 25 ℃; the illumination time of the daytime culture is preferably 10-13h, more preferably 11-12.5h, still more preferably 12h; the illumination intensity of the daytime culture is preferably 1300-1800Lux, more preferably 1400-1600Lux, and even more preferably 1500Lux. The temperature of the night culture in the present invention is preferably 18 to 23 ℃, more preferably 19 to 21 ℃, still more preferably 20 ℃; the night time is 10-13 hours, more preferably 11-12.5 hours, still more preferably 12 hours. In the present invention, the time for the alternate light and dark cultivation is preferably 15 to 25d, more preferably 18 to 22d, still more preferably 21d. In the present invention, the water loss is periodically and quantitatively replenished every day.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The screening process of bacillus GM-12-PT bacteria comprises the following steps:
1. Preparing a suspension of Pterocarpus marsupium seed soil. 10g of soil in a soil area of 1-10mm around germinated seeds of Pterocarpus marsupium is taken and placed in a 250mL conical flask, 90mL of sterile water and 6 glass beads are added, and the mixture is placed in a shaking table at 180rpm for shaking for 30min, so that a Pterocarpus marsupium seed soil suspension is obtained.
2. Screening the purified strain. Diluting the Pterocarpus marsupium interplanting soil suspension into soil solutions with different dilutions of 10 3、104、105、106、107 by adopting a gradient dilution method, respectively sucking 0.1mL of the soil solutions with different dilutions by using a pipettor, adding the soil solutions into a beef extract peptone culture medium plate, uniformly coating, culturing for 4 days at 28 ℃, respectively picking single colonies with different forms, inoculating the single colonies onto a new culture medium plate, carrying out four-region streaking, and continuously repeating the operation process until single colonies appear on the surface of the culture medium and the phenotype characteristics are stable, thus obtaining the purified strain;
3. Screening for functional beneficial strains. The purified strains are respectively inoculated into a cellulase-producing detection medium, an amylase-producing medium, an inorganic phosphorus-dissolving medium, an organic phosphorus-dissolving solid medium and an Ashby nitrogen-free solid medium, and are cultured for 5 days at 30 ℃, each treatment is repeated for 3 times, and then functional beneficial strains are screened according to amylase-producing, cellulase-producing, phosphorus-dissolving capacity and nitrogen-fixing capacity. The cellulose production capacity, amylase production capacity, phosphorus dissolution capacity, nitrogen fixation capacity and potassium dissolution capacity were primarily and qualitatively judged according to the ratio of cellulose production ring to colony diameter, amylase production ring diameter to colony diameter, phosphorus dissolution ring diameter to colony diameter, nitrogen fixation ring diameter to colony diameter, and potassium dissolution ring diameter to colony diameter (screening results are shown in table 1).
The components of the cellulase production detection medium comprise yeast powder 0.1g, peptone 0.5g, congo red dye 30mL of 0.4g/L, sodium carboxymethylcellulose 5.0g, agar 20g and distilled water 1000mL;
The components of the amylase-producing culture medium comprise 20g of starch, 0.5g of KCl, 0.5g of NaNO 32.0g、K3PO41.0g、MgSO4·7H2 O, 5.0g of NaCl, 20.0g of agar and 1000mL of deionized water;
the inorganic phosphorus decomposing culture medium comprises 20g of glucose 10g、(NH4)2SO40.5g、KCl 0.3g、MgSO4·7H2O 0.3g、FeSO4·7H2O 0.03g、MnSO4·4H2O 0.03g、Ca3(PO4)210g、NaCl 0.3g、 agar and 1000mL of distilled water, wherein the pH value is 7.0, and the culture medium is sterilized for 20min at 121 ℃;
the components of the organic phosphorus-dissolved solid culture medium comprise glucose 10g、(NH4)2SO40.5g、KCl0.3g、MgSO4·7H2O 0.3g、FeSO4·7H2O 0.03g、MnSO4·4H2O 0.03g、KCl0.3g、NaCl 0.3g、CaCO35g、 g lecithin 0.2g, agar 20g and distilled water 1000mL, wherein the pH value is 7.0, and the culture medium is sterilized for 20min at 121 ℃;
The components of the Ashby nitrogen-free solid culture medium comprise KH 2PO40.2g、MgSO4·7H2O0.2g、NaCl 0.2g、CaCO3 g, mannitol 10g, caSO 4 0.1.1 g, agar 20g and distilled water 1000mL, wherein the pH is 7.0, and the sterilization is carried out for 20min at 121 ℃.
The solid culture medium for decomposing potassium comprises 1.0g of sucrose 10.0g、MgSO4·7H2O 1.0g、NaHPQ42.0g、(NH4)2SO42.0g、CaCO31.0g、FeCl30.005g、 potassium feldspar (K 2O·Al2O3·6SiO2) powder, 20g of agar and 1L of distilled water, wherein the pH value is 7.0.
TABLE 1 functional assay of functional beneficial strains
Note that: "+" represents positive; "-" represents negative.
4. Screening the strain with the best germination rate. Inoculating the screened functional beneficial bacteria into beef extract peptone liquid culture medium for culture, collecting bacterial liquid, diluting with PBS buffer solution to obtain bacterial suspension of 1.0X10 8 CFU/mL, soaking seeds for 4h each time, soaking seeds for 2 times each time for 10d, and then culturing for 20d (day temperature 25 ℃/12h, illumination intensity 1500Lux; night non-illumination culture 20 ℃/12 h) alternately under light and dark, and screening out the bacterial strain with the best effect according to the germination rate.
5. And (5) carrying out molecular identification on the strain with the best germination rate effect. Extracting DNA sequence (NCBI, accession Number: OQ 859968) with bacterial genome DNA extraction kit (purchased from biological engineering Co., ltd., shanghai), amplifying by PCR with ：GAGCATGGCGCAGCTATAATGCAGTCGAGCGAATGGATTGAGAGCTTGCTCTCAAGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGAGCCAGCCGCTAAGGTACCTAACCCG. shown in SEQ ID NO:1, purifying the PCR amplified product with 2× Bench TopTMTaq Master Mix (purchased from BIOMIGA Chinese official), sequencing the purified target fragment with sequencing company, comparing the obtained sequences in Genbank database, constructing phylogenetic tree (as shown in FIG. 1) of the strain by using MEGA7.0 and other software, and identifying classification status of the strain.
The primer for PCR amplification comprises a 27F sequence and a 492R sequence, wherein the 27F sequence is shown as SEQ ID NO.2 and 5'-AGAGTTTGATCCTGGCTCAG-3'; the 492R sequence is shown in SEQ ID NO. 3, 5'-GGTTACCTTGTTACGACTT-3'. The PCR amplification system comprises: 2. Mu.L of DNA template, 2. Mu.L of SEQ ID NO:2, 2. Mu.L of SEQ ID NO:3, 25. Mu.L of 2 XMaster Mix, and finally, the volume was fixed to 50. Mu.L with ddH 2 O. The PCR amplification program conditions of the invention are as follows: pre-denaturation (1 min30s at 94 ℃); denaturation (30 s at 94 ℃); annealing (30 s at 57 ℃); extension (60 s at 72 ℃); final extension (5 min at 72 ℃); preserving at 4 ℃. Wherein the denaturation, annealing and extension are continuously cycled 30 times. In the present invention, the strain was identified as Bacillus (Bacillus sp), and the inventors named the strain as Bacillus GM-12-PT.
Example 2
A method of promoting germination of Pterocarpus marsupium seeds, comprising the steps of:
(1) Washing full, healthy and uniform-size Pterocarpus marsupium seeds, soaking for 10min by using 4% sodium hypochlorite solution, and soaking for 30s by using 75% alcohol to obtain sterilized Pterocarpus marsupium seeds;
(2) Inoculating the activated bacillus GM-12-PT strain to a beef extract peptone liquid culture medium, shake culturing for 16h at 30 ℃ and 150rpm, collecting bacterial liquid, centrifuging for 10min at 4000rpm, collecting bottom bacterial liquid, and fully diluting with PBS buffer to obtain bacillus GM-12-PT bacterial suspension with the concentration of 1.0X10 8 CFU/mL;
(3) Washing sterilized Pterocarpus marsupium seeds with distilled water for 2 times, soaking seeds in bacillus GM-12-PT bacteria suspension with concentration of 1.0X10 8 CFU/mL for 4h each time, soaking seeds once every 10d, and soaking seeds for 2 times;
(4) Placing the soaked Pterocarpus marsupium seeds into a germination box on sterile absorbent cotton, soaking the absorbent cotton into sterile water in advance, placing the seeds into the germination box of a bed, and transferring the seeds into an illumination incubator for alternate light and dark culture for 21d: the daytime temperature is 25 ℃/12h, and the illumination intensity is 1500Lux; culturing at 20 ℃/12h without illumination at night.
Example 3
A method of promoting germination of Pterocarpus marsupium seeds, comprising the steps of:
(1) Washing full, healthy and uniform-size Pterocarpus marsupium seeds, soaking for 12min by using a 2% sodium hypochlorite solution, and soaking for 40s by using 75% alcohol to obtain sterilized Pterocarpus marsupium seeds;
(2) Inoculating the activated bacillus GM-12-PT strain to a beef extract peptone liquid culture medium, shake culturing for 18 hours at 25 ℃ and 120rpm, collecting bacterial liquid, centrifuging for 10min at 3500rpm, collecting bottom bacterial liquid, and fully diluting with PBS buffer to obtain bacillus GM-12-PT bacterial suspension with the concentration of 0.5 multiplied by 10 8 CFU/mL;
(3) Washing sterilized Pterocarpus marsupium seeds with distilled water for 2 times, soaking seeds in bacillus GM-12-PT bacteria suspension with concentration of 0.5X10 8 CFU/mL for 2h each time, soaking seeds once every 12d, and soaking seeds for 1 time;
(4) Placing the soaked Pterocarpus marsupium seeds into a germination box on sterile absorbent cotton, soaking the absorbent cotton into sterile water in advance, placing the seeds into the germination box of a bed, and transferring the seeds into an illumination incubator for alternate culturing for 15d under light and dark conditions: the daytime temperature is 28 ℃/11h, and the illumination intensity is 1800Lux; the culture was carried out at 23℃for 13h without light at night.
Example 4
A method of promoting germination of Pterocarpus marsupium seeds, comprising the steps of:
(1) Washing full, healthy and uniform-size Pterocarpus marsupium seeds, soaking for 8min by using a 6% sodium hypochlorite solution, and soaking for 20s by using 75% alcohol to obtain sterilized Pterocarpus marsupium seeds;
(2) Inoculating the activated bacillus GM-12-PT strain to a beef extract peptone liquid culture medium, shake culturing for 12 hours at 32 ℃ under 180rpm, collecting bacterial liquid, centrifuging for 10min at 4500rpm, collecting bottom bacterial body, and fully diluting with PBS buffer to obtain bacillus GM-12-PT bacterial suspension with the concentration of 1.5X10 8 CFU/mL;
(3) Washing sterilized Pterocarpus marsupium seeds with distilled water for 2 times, soaking seeds in bacillus GM-12-PT bacteria suspension with concentration of 1.5X10 8 CFU/mL for 6h each time, soaking seeds once every 8d, and soaking seeds for 1 time;
(4) Placing the soaked Pterocarpus marsupium seeds into a germination box on sterile absorbent cotton, soaking the absorbent cotton into sterile water in advance, placing the seeds into the germination box of a bed, and transferring the seeds into an illumination incubator for alternate culturing for 25d under light and dark conditions: the daytime temperature is 23 ℃/13h, and the illumination intensity is 1300Lux; culturing at 18 ℃/11h without illumination at night.
Test example 1
Based on the sterilized Pterocarpus marsupium seeds obtained in example 2 and the Bacillus GM-12-PT bacteria suspension. Three groups were set, each group set with 3 replicates: non-priming group (CK 1), sterile beef extract peptone liquid medium priming group (CK 2), bacillus GM-12-PT bacteria suspension priming group. After soaking sterilized Pterocarpus marsupium seeds in the three sets of solutions, and culturing for 20d according to the soaking, light-dark alternate culturing procedure of example 1. The germination rate and physiological index (GA content, ABA content, GA/ABA ratio, beta amylase, total amylase content, BCA protein content, cellulose content, and glucose content) of each group of Pterocarpus marsupium seeds were examined, and the average was calculated, and the results were shown in table 2.
Seed stock material was determined by reference to the method in the guidelines for seed chemistry experiments (Liu Zifan et al, 2010): the index measurement of gibberellin and abscisic acid adopts an enzyme-linked immunosorbent assay; the glucose content is measured by adopting a hexokinase method; the BCA protein content is measured by adopting a Coomassie brilliant blue method; the determination of the cellulose content adopts an anthrone colorimetric method; the amylase activity was measured using a 3, 5-dinitrosalicylic acid chromogenic method.
Germination percentage (GR;%) = (number of germinated seeds/number of seeds tested) ×100%
Germination Index (GI) = Σgt/dt=number of germination per day/number of germination per day+number of germination per day+ … …, gt: number of sprouting on day t; dt: number of germination days; sigma: sum up.
Germination vigor (GP;%) = (number of germinated seeds/number of seeds tested in a predetermined time) ×100
That is, when the number of germinated seeds reaches the highest peak during germination, the percentage of germinated seeds to the number of samples to be tested is generally based on the percentage of the number of germinated seeds to be tested in the first 1/3 period (7 days) of the specified period of the Pterocarpus test.
TABLE 2 Effect of different groups of treatments on Pterocarpus marsupium seed germination Rate and physiological index
Note that: one-way anova test has significant differences at P <0.05 level.
As can be seen from Table 2, the seed vitality effect of the seed soaking initiation treatment by using Bacillus sp bacterial suspension is best, the gibberellin content can reach 55.62pg/mL, and the seed soaking initiation treatment is improved by 9.15% compared with the non-initiation treatment and is improved by 20.28% compared with the aseptic beef extract peptone liquid culture medium seed soaking initiation treatment; the abscisic acid content is 26.98 mug/L, which is 9.77% lower than that of the non-initiation treatment and 11.34% lower than that of the initiation treatment of the aseptic beef extract peptone liquid culture medium seed soaking; GA/ABA can reach 2.06, which is improved by 20.72 percent compared with the non-initiation treatment and 35.57 percent compared with the seed soaking initiation treatment of the aseptic beef extract peptone liquid culture medium; the beta amylase can reach 7913.72 mu g/min/g, 73.57% is improved compared with the non-initiation treatment, and 51.87% is improved compared with the seed soaking initiation treatment of the aseptic beef extract peptone liquid culture medium; the total amylase can reach 8717.63 mu g/min/g, which is 59.25 percent higher than that of the non-initiation treatment and 48.47 percent higher than that of the aseptic beef extract peptone liquid culture medium seed soaking initiation treatment; the BCA protein content can reach 26.23mg/g, which is 53.56% higher than that of the non-initiation treatment and 26.65% higher than that of the initiation treatment of the aseptic beef extract peptone liquid culture medium seed soaking; the cellulose content is 58.18mg/g, which is reduced by 22.98 percent compared with the non-initiation treatment and 12.18 percent compared with the seed soaking initiation treatment of the aseptic beef extract peptone liquid culture medium; the glucose content can reach 0.44mg/g, which is improved by 22.71% compared with the non-initiation treatment and 19.01% compared with the initiation treatment of the aseptic beef extract peptone liquid culture medium seed soaking.
The germination rate after seed soaking initiation treatment by using bacillus sp bacterial suspension has the best effect, the germination rate can reach 76.19 percent, and is 14.76 percent higher than that of no initiation treatment and 16.19 percent higher than that of the initiation treatment by using a sterile beef extract peptone liquid culture medium; the germination potential can reach 50.95%, which is 14.29% higher than that of the non-initiation treatment, and is 11.90% higher than that of the initiation treatment of the aseptic beef extract peptone liquid culture medium seed soaking; the germination index can reach 103.02, 59.56% is improved compared with the non-initiation treatment, and 43.94% is improved compared with the initiation treatment of the aseptic beef extract peptone liquid culture medium seed soaking.
As can be seen from the above examples and test examples, the bacillus GM-12-PT obtained by screening of the invention has better amylase production, cellulase production, organophosphorus dissolution and nitrogen fixation capabilities. The strain is utilized to biologically trigger the Pterocarpus marsupium seeds, so that the germination rate and the seed activity of the seeds can be remarkably improved; the synthesis and accumulation of the substances after initiation also lay a material foundation for the growth of the seedlings, so that the seedlings are more robust in the growth process, and the stress resistance, the seeding emergence rate and the seedling quality of the seedlings are improved.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. A bacillus (bacillus sp.) strain GM-12-PT is characterized in that the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 4 months and 28 days in 2023, and the preservation address is CGMCCNO:27240 at the institute of microorganisms, national academy of sciences No. 3, north Star West Lu 1 in Chao Yangjia of Beijing.
2. Use of bacillus GM-12-PT according to claim 1 for promoting germination of Pterocarpus marsupium seeds.
3. A method of promoting germination of Pterocarpus marsupium seeds, comprising the steps of:
(1) Cleaning and sterilizing Pterocarpus marsupium seeds, and soaking the seeds in the bacillus GM-12-PT bacterial suspension according to claim 1;
(2) The soaked Pterocarpus marsupium seeds are alternately cultivated for 15-25d in a dark state.
4. A method according to claim 3, wherein the preparation of the bacillus GM-12-PT suspension according to step (1) comprises the steps of: inoculating the activated bacillus GM-12-PT strain into a culture medium, shake culturing at 25-32 ℃ for 12-18h, collecting thalli, and diluting to obtain (0.5-1.5) multiplied by 10 8 CFU/mL bacterial suspension.
5. The method according to claim 4, wherein the shaking speed is 120-180rpm, and the collecting of the bacterial cells is performed by centrifugation at 3500-4500rpm.
6. A method according to claim 3, wherein the disinfection of step (1) comprises sequential sodium hypochlorite solution disinfection and alcohol disinfection; the mass concentration of the sodium hypochlorite solution is 2-6%, and the sterilizing time of the sodium hypochlorite solution is 8-12min; the volume concentration of the alcohol is 70-85%; the alcohol sterilization time is 20-40s.
7. A method according to claim 3, wherein the number of seed soaking in step (1) is 1 to 3, the frequency of seed soaking is 8 to 12 d/time, and the time of seed soaking is 2 to 6 h/time.
8. A method according to claim 3, wherein the conditions of the alternate light and dark culture of step (2) comprise: the daytime temperature is 23-28 ℃, the illumination is 10-13h, and the illumination intensity is 1300-1800Lux; culturing for 10-13h at night without illumination.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006124280A (en) * | 2004-10-26 | 2006-05-18 | Kumiai Chem Ind Co Ltd | Agent for improving germination rate of plant seed |
| WO2009031874A1 (en) * | 2007-09-04 | 2009-03-12 | Jorge Olmos Soto | Strain of bacillus subtilis for agricultural use |
| CN105165627A (en) * | 2015-10-22 | 2015-12-23 | 贵州大学 | Tissue culture disinfection and sterilization formula of ormosia henryi prain and tissue culture method of ormosia henryi prain |
| CN106942283A (en) * | 2017-03-20 | 2017-07-14 | 广西中烟工业有限责任公司 | A kind of tobacco seed sprouts strengthening agent and its preparation, application method |
| CN111484951A (en) * | 2019-01-28 | 2020-08-04 | 福建省农业科学院农业生物资源研究所 | Bacillus for dissolving phosphorus and fixing nitrogen and application thereof in growth promotion |
| CN111484953A (en) * | 2019-01-28 | 2020-08-04 | 福建省农业科学院农业生物资源研究所 | Bacillus capable of promoting growth and dissolving phosphorus and application thereof |
| CN117356221A (en) * | 2023-11-23 | 2024-01-09 | 西藏自治区农牧科学院草业科学研究所 | Method for improving germination of Phaffia scirpa seeds by utilizing soil-promoting bacteria |
-
2023
- 2023-06-08 CN CN202310678021.8A patent/CN116814479B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006124280A (en) * | 2004-10-26 | 2006-05-18 | Kumiai Chem Ind Co Ltd | Agent for improving germination rate of plant seed |
| WO2009031874A1 (en) * | 2007-09-04 | 2009-03-12 | Jorge Olmos Soto | Strain of bacillus subtilis for agricultural use |
| CN105165627A (en) * | 2015-10-22 | 2015-12-23 | 贵州大学 | Tissue culture disinfection and sterilization formula of ormosia henryi prain and tissue culture method of ormosia henryi prain |
| CN106942283A (en) * | 2017-03-20 | 2017-07-14 | 广西中烟工业有限责任公司 | A kind of tobacco seed sprouts strengthening agent and its preparation, application method |
| CN111484951A (en) * | 2019-01-28 | 2020-08-04 | 福建省农业科学院农业生物资源研究所 | Bacillus for dissolving phosphorus and fixing nitrogen and application thereof in growth promotion |
| CN111484953A (en) * | 2019-01-28 | 2020-08-04 | 福建省农业科学院农业生物资源研究所 | Bacillus capable of promoting growth and dissolving phosphorus and application thereof |
| CN117356221A (en) * | 2023-11-23 | 2024-01-09 | 西藏自治区农牧科学院草业科学研究所 | Method for improving germination of Phaffia scirpa seeds by utilizing soil-promoting bacteria |
Non-Patent Citations (2)
| Title |
|---|
| 接种不同根瘤菌对花榈木幼苗光合生理的影响及促生效应;段如雁;韦小丽;安常蓉;;浙江农林大学学报;20181130(第06期);115-123 * |
| 花榈木的组织培养和快速繁殖;姚军;李洪林;杨波;;植物生理学通讯;20070220(第01期);123-124 * |
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