CN114525217B - Potassium-dissolving growth-promoting pyrrolburkholderia as well as microbial inoculum and application thereof - Google Patents
Potassium-dissolving growth-promoting pyrrolburkholderia as well as microbial inoculum and application thereof Download PDFInfo
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- 239000002068 microbial inoculum Substances 0.000 title claims description 14
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 78
- 241000208125 Nicotiana Species 0.000 claims abstract description 63
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims abstract description 52
- 239000011591 potassium Substances 0.000 claims abstract description 52
- 229910052700 potassium Inorganic materials 0.000 claims abstract description 52
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 241001453380 Burkholderia Species 0.000 claims abstract description 33
- 230000001580 bacterial effect Effects 0.000 claims abstract description 29
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 230000000813 microbial effect Effects 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims description 20
- 241000866604 Burkholderia pyrrocinia Species 0.000 claims description 18
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 claims description 14
- 230000012010 growth Effects 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- 230000001737 promoting effect Effects 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 5
- 239000008223 sterile water Substances 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000008107 starch Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 230000003698 anagen phase Effects 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims 2
- 244000061176 Nicotiana tabacum Species 0.000 abstract description 15
- 239000002028 Biomass Substances 0.000 abstract description 9
- 238000000354 decomposition reaction Methods 0.000 abstract description 6
- 230000008635 plant growth Effects 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 6
- 239000003337 fertilizer Substances 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
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- 238000009331 sowing Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
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- 241000238631 Hexapoda Species 0.000 description 2
- 238000010876 biochemical test Methods 0.000 description 2
- 238000002485 combustion reaction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000705 flame atomic absorption spectrometry Methods 0.000 description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 2
- 230000002015 leaf growth Effects 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- SXQXMCWCWVCFPC-UHFFFAOYSA-N aluminum;potassium;dioxido(oxo)silane Chemical compound [Al+3].[K+].[O-][Si]([O-])=O.[O-][Si]([O-])=O SXQXMCWCWVCFPC-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 238000013095 identification testing Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 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 1
- 239000010445 mica Substances 0.000 description 1
- 229910052618 mica group Inorganic materials 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 229940072033 potash Drugs 0.000 description 1
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 1
- 235000015320 potassium carbonate Nutrition 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000019086 sulfide ion homeostasis Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- C12N1/02—Separating microorganisms from their culture media
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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
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses potassium-dissolving and growth-promoting Burkholderia pyrroctis as well as a microbial agent and application thereof, and belongs to the technical field of microorganisms. Wherein, the potassium-decomposing and growth-promoting pyrrolidovorax DY211 strain is preserved in China center for type culture Collection, with the preservation registration number: cctccc M20211436. The microorganism bacterial agent of the Burkholderia pyrrocina and the preparation method thereof are also provided, and can be applied to potassium decomposition and plant growth promotion. The bacterial liquid prepared by DY211 can effectively improve the biomass of tobacco plants, the stem height of the tobacco plants in the mature period is increased by 12.0%, the leaf number is increased by 12.9%, the stem diameter is increased by 16.9%, the middle leaf length is increased by 28.8%, and the middle leaf width is increased by 18.5%. The bacterial liquid prepared by DY211 can effectively improve the potassium content of the top and middle leaves of the tobacco, is beneficial to improving the quality of the tobacco and reducing the use of chemical fertilizers, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to potassium-dissolving and growth-promoting Burkholderia pyrrocinia and a microbial inoculum and application thereof.
Background
Tobacco (Nicotiana tabacum) belongs to annual herbaceous plants of the Solanaceae family and is one of important commercial crops. The tobacco yield in China accounts for 1/3 of the total world yield, and the tobacco has a wide planting area in many areas. The potassium is required in the tobacco growing process. Potassium is used as one of the quality elements of flue-cured tobacco, is closely related to the combustion completeness, combustion uniformity, taste, aroma, chemical components and tobacco safety of tobacco, and has remarkable effects of improving the combustibility and smoldering fire holding property of tobacco. In nature, potassium elements for tobacco growth absorption and utilization mainly come from soil. The total potassium content of the soil in China is 0.5% -2%, the insoluble potassium mainly comprising potassium aluminosilicate such as potassium feldspar and mica accounts for 95% of the total amount, and potassium resources which can be directly absorbed and utilized by crops are extremely limited. In order to improve the quality of tobacco leaves and the potassium content of tobacco, a large amount of quantitative potassium fertilizer is often used in tobacco planting, and the applied potassium fertilizer is very easy to fix in soil to become invalid or slow-acting potassium, so that the method has little effect on the growth of annual tobacco. The application of a large amount of potash fertilizer not only increases the tobacco cultivation cost, but also seriously affects the ecological environment of a planting area, such as the increase of the drug resistance of plant diseases and insect pests, soil hardening, the exceeding of heavy metal content, water pollution and the like, so that the environmental pollution and the resource waste are caused. Therefore, finding new methods to increase the absorption of potassium elements in the soil by crops has become an urgent need to solve the problem. The potassium decomposing bacteria are also called silicate bacteria, and are microorganisms capable of decomposing minerals such as silicate in soil, converting elements such as insoluble potassium, phosphorus and the like into a soluble state for plant absorption and utilization. The potassium decomposing bacteria can slowly convert ineffective potassium in soil into effective potassium through acidolysis and other ways, so that the potassium is slowly absorbed by crops and the potassium content in the crops is improved. Thereby improving the lodging resistance, drought resistance, disease and insect resistance and the like of crops, increasing the yield of crops and improving the economic benefit. The research obtains the pyrrol burkholderia DY211 with excellent potassium-decomposing ability from tobacco rhizosphere soil in a tobacco planting area of Luzhou Sichuan, the potassium-decomposing ability of the strain in different periods is detected through an indoor experiment, the influence of the strain on the tobacco yield and quality is further examined through a field experiment, and the pyrrol burkholderia DY211 has a good growth promoting effect and can improve the potassium content of top and middle tobacco leaves.
Disclosure of Invention
1. Problems to be solved
The first aim of the invention is to provide a potassium-decomposing bacterium-pyrrol burkholderia (Burkholderia pyrrocinia) DY211 separated from tobacco rhizosphere soil in Sichuan Luzhou, which enriches the strain resources of potassium-promoting bacteria and lays a foundation for research and development of the growth-promoting bacteria agent.
The second object of the invention is to provide the application of the Burkholderia pyrrocinia DY211 in potassium decomposition and plant growth promotion.
The third object of the present invention is to provide a microbial agent produced by using the strain DY 211.
The fourth object of the present invention is to provide a method for producing the microbial agent.
The fifth object of the invention is to provide the application of the microbial agent for potassium decomposition and plant growth promotion.
The sixth object of the present invention is to provide a method for screening and identifying the strain DY 211.
2. Technical proposal
In order to solve the problems, the invention adopts the following technical scheme.
The Latin chemical name of the potassium-decomposing and growth-promoting Burkholderia is Burkholderiapyrrocinia, the Latin chemical name is DY211, and the Latin chemical name is preserved in China Center for Type Culture Collection (CCTCC) M20211436 in the year 11 and 17.
A microbial agent comprising burkholderia pyrrocinia as described above.
In the microbial inoculum, the active ingredients of the microbial inoculum are bacterial liquid of Burkholderia pyrrocinia and metabolic supernatant fluid thereof.
The preparation method of the microbial inoculum comprises the following steps:
(1) Preparing a solid potassium-decomposing culture medium;
(2) Preparing a liquid potassium-decomposing culture medium;
(3) Preparation of activated strains: picking single bacterial colony of pyrrol burkholderia in a solid potassium-decomposing culture medium plate, preparing seed liquid, inoculating the seed liquid into a liquid potassium-decomposing culture medium, culturing the seed liquid in a shake flask with the temperature of 28 ℃ and the rotating speed of 160r/min, and diluting the bacterial liquid by using sterile water when the bacterial strain grows to a logarithmic growth phase to obtain the bacterial agent.
In the preparation method of the microbial inoculum,
The solid potassium-decomposing culture medium in the step (1) comprises the following components in parts by weight: 10g/L of glucose, 0.4g/L of yeast extract, 0.2g/L of magnesium sulfate, 0.5g/L of dipotassium hydrogen phosphate, 0.2g/L of sodium chloride, 1g/L of calcium carbonate and 20g/L of agar.
In the preparation method of the microbial inoculum,
The liquid potassium-decomposing culture medium in the step (1) comprises the following components in parts by weight: starch 5g/L, yeast extract 1g/L, magnesium sulfate 0.5g/L, disodium hydrogen phosphate 2g/L, calcium carbonate 1g/L and ferric trichloride 5mg/L.
The application of potassium-dissolving growth-promoting Burkholderia pyrrocinia in promoting tobacco growth.
The potassium-dissolving and growth-promoting Burkholderia pyrroctis is applied to the promotion of tobacco growth,
The application of promoting tobacco growth comprises the steps of increasing biomass of tobacco plants in the maturity stage and increasing potassium content of tobacco leaves.
The microbial inoculum is applied to the promotion of tobacco growth,
The preparation form of the Burkholderia pyrrocina is a pharmaceutically acceptable preparation form.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
The strain is obtained from tobacco rhizosphere soil in a tobacco planting area of Luzhou Sichuan with the number DY211 through screening, and can dissolve insoluble potassium, promote plant growth and improve leaf potassium content. The morphological characteristics are as follows: gram-negative, circular colony, transparent white, moist and glossy surface, regular edge and water drop shape. And combining various physiological and biochemical test results and molecular biological analysis, and identifying the strain as Burkholderia pyrrocinia. The application of the Burkholderia pyrroctis to potassium decomposition and plant growth promotion is not disclosed in the prior art, and the Burkholderia pyrroctis DY211 is different from the disclosed Burkholderia pyrroctis. As can be seen from the functional identification test, burkholderia DY211 can dissolve insoluble potassium (potassium feldspar powder); meanwhile, in a potassium-decomposing test, the strain liquid and the strain body of the Burkholderia pyrroctis DY211 have higher potassium-decomposing capacity. The application of the DY211 bacterial agent of Burkholderia pyrroctis can improve the biomass of tobacco plants in the maturity stage and the potassium content of tobacco leaves. The stem height of the tobacco plant is increased by 12.0%, the leaf number is increased by 12.9%, the stem diameter is increased by 16.9%, the middle leaf length is increased by 28.8%, the middle leaf width is increased by 18.5%, and the potassium content in the top and middle tobacco leaves can be obviously improved.
Drawings
FIG. 1 is a macroscopic morphology of Burkholderia DY211 provided by the invention;
FIG. 2 is a macroscopic morphology of a known Burkholderia A12;
FIG. 3 is a graph showing the potassium-decomposing performance of Burkholderia DY211 according to the present invention in solid culture;
FIG. 4 is a graph showing the potassium-decomposing performance of the known Burkholderia A12 in solid culture;
FIG. 5 is a graph showing the dynamic change of soluble potassium in liquid culture of Burkholderia DY211 and Burkholderia A12 according to the present invention;
FIG. 6 is a schematic diagram of the amplification of the 16SrDNA gene sequence of Burkholderia DY211 according to the present invention;
Fig. 7 is a phylogenetic tree constructed based on a 16SrDNA gene sequence of Burkholderia DY211 provided by the invention.
Detailed Description
The invention is further described below in connection with specific embodiments.
The following description of the embodiments of the present invention will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, 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.
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 invention belongs.
In the present invention, burkholderia pyrroctis (Burkholderia pyrrocinia) strain DY211 or simply referred to as strain DY211. The Burkholderia pyrrocina provided by the invention can be used as a liquid microbial inoculum.
In the invention, the pyrrolburkholderia A12 is selected from Shandong university plant protection college, and the relativity between the strain A12 and Burkholderia pyrrocinia strain DQ360828 existing in GenBank is nearest, and the similarity of the 16S rDNA sequence reaches 99.85%.
Example 1 isolation and screening of Strain DY211
Collecting soil samples from tobacco rhizosphere soil in a Luzhou tobacco planting area of Sichuan, separating different microorganisms from the soil samples, treating the soil samples by adopting a gradient dilution method, absorbing 200 mu L of dilution liquid with proper gradient onto a flat plate for coating, and inversely culturing at 28 ℃. The morphological characteristics of bacterial colonies are observed day by day, strains with transparent potassium-decomposing rings or bacterial plaques are selected, purification is carried out through a plate streaking method, and the purified strains are stored on a solid potassium-decomposing culture medium for later use.
And (3) carrying out shake flask culture, activating and propagating on the strain to be used, preparing a potassium-decomposing solid culture medium, preparing a sterilized flat plate, pouring the plate after sterilizing the culture medium, sucking 1 mu L of bacterial liquid onto the flat plate after solidification, placing the sealed plate at 28 ℃ for culture, and observing an experimental result when a transparent potassium-decomposing ring or bacterial plaque exists. Referring to FIGS. 1 and 2, the strain DY211 with the strongest potassium-decomposing ability is selected for further research, and compared with the known potassium-decomposing ability of the potassium-decomposing bacteria A12, the experimental operation is consistent with that of the strain DY211, the potassium-decomposing ability of the strain DY211 and the strain A12 are shown in Table 1, and the diameter ratio of the strain DY211 reaches 4.37 and is stronger than that of the strain A12. As shown in FIGS. 3 and 4, the strain DY211 of the present example is capable of dissolving poorly soluble potassium, and has a large diameter of the potassium-dissolving ring and a large diameter of the bacterial plaque. The silicate solid medium comprises the following components:
2.0g of high calcium phosphate, 0.005g of magnesium sulfate, 0.1g of ferric trichloride, 2.0g of calcium carbonate, 5.0g of sucrose, 0.5g of yeast extract, 2.0g of potassium feldspar powder, 15.0g of agar powder, 1.0L of deionized water and pH 7.2-7.5. Potassium solution evaluation criteria: diameter ratio = potassium-solution circle diameter/plaque diameter 100%;
TABLE 1 evaluation of Potassium-decomposing ability of DY211 and known Potassium-decomposing bacteria A12
EXAMPLE 2 determination of Potassium-decomposing Capacity of Strain DY211
Preparing culture medium according to the formula of seed liquid culture medium, subpackaging in 250mL triangular bottles, each bottle having 100mL, wrapping the bottle mouth with sealing film with filter pore diameter of 0.22 μm, sterilizing at 121deg.C for 20min, and cooling. Under the aseptic condition, inoculating the activated strain into a seed liquid culture medium by using an inoculating loop, inoculating one loop for each bottle, repeating three times, wrapping a sterile sealing film on a bottle opening, performing shake culture at 28 ℃, sampling every 24 hours of aseptic operation, counting the number of bacteria in fermentation liquor, and taking the strain as seed liquid when the number of viable bacteria is increased to about 10 8 cfu/mL. Sterilizing one bottle of seed liquid at 121deg.C for 20min to obtain inactivated seed liquid.
Preparing culture medium according to the formula of fermentation culture medium, subpackaging in 250mL triangular bottles, each bottle having 100mL, wrapping the bottle mouth with sealing film with filter pore diameter of 0.22 μm, sterilizing at 121deg.C for 20min, and cooling. Under aseptic conditions, 1mL of seed solution was added to each flask for inoculation of the fermentation medium. Adding normal seed liquid into experimental groups (DY 211 group and A12 group respectively), adding inactivated seed liquid into control group (CK group), repeating the experimental groups and CK group for 6 times, wrapping the bottle mouth with a sterile sealing film, shake culturing at 28 ℃, sampling 10mL and preserving in a sterile centrifuge tube respectively at 3, 7, 14, 21 and 28d, preliminarily filtering impurities by adopting filter paper, centrifuging the filtrate at 8500rpm/min for 10min, taking the supernatant, and determining the effective potassium content according to GB 5009.91-2017.
2.0G of disodium hydrogen phosphate, 0.5g of magnesium sulfate, 0.1g of calcium carbonate, 5.0mg of ferric trichloride, 0.5g of yeast extract, 5.0g of soluble starch, 1.0L of deionized water and pH of 7.0-7.2.
The fermentation medium comprises 0.2g of ammonium sulfate, 0.5g of magnesium sulfate, 0.1g of calcium carbonate, 0.1g of sodium chloride, 5.0mg of ferric trichloride, 10.0g of sucrose, 5.0g of potassium feldspar powder, 1.0L of deionized water and pH of 7.0-7.2.
As shown in fig. 5, the DY211 group had significantly higher potassium-decomposing ability than the CK group and the a12 group.
Example 3 identification of Strain DY211
1. Morphological identification
As shown in FIG. 1, the result of the gram staining shows that the strain DY211 is gram-negative and spherical. The colony is round, transparent and white, the surface is moist and glossy, the edge is regular, and the water drops are raised.
2. Physiological and biochemical identification
Referring to the handbook of common bacterial System identification, the potassium-decomposing and growth-promoting bacterial strain is primarily identified through a physiological and biochemical test, and the identification result shows that: gram negative, indole reaction negative, V-P experiment reaction negative, methyl red reaction positive, hydrogen sulfide production negative, oxidase experiment negative, indole experiment negative, and rhamnose, glucose and sucrose utilization experiment positive.
According to the identification result, the potassium-decomposing and growth-promoting bacterial strain DY211 is initially identified as Burkholderia.
3. Molecular biological identification
As shown in FIG. 4, the strain DY211 was used as a template for DNA extraction, and the 16S rDNA was amplified using the upstream primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3' downstream primer 1499R: 5'-TACGG TTACCTTGTTACGACTT-3', and the amplified fragment was directly sequenced. The reaction conditions of the 16SrDNAPCR system are shown in tables 2 and 3.
Table 216S rDNA PCR reaction system
| Component (A) | Reaction system (mu L) |
| 2×TaqMasterMix | 26 |
| 27F primer | 1 |
| 1492R primer | 1 |
| Genomic DNA | 1 |
| ddH2O | 21 |
| Total volume of | 50 |
Table 316S rDNA PCR reaction procedure
The sequencing result is input into BLAST program on NCBI website for comparison, and the result shows that the homology of the 16SrDNA nucleotide sequence of the strain with the 16srDN A sequence of Burkholderia pyrrocinia in GenBank gene library is highest, and the homology rate reaches 99.58%, as shown in figures 6 and 7; the result of genetic evolution analysis of the 16SrDNA sequence of the existing Burkholderia in Genbank by DNAMAN6.0 shows that the homology of the potassium-decomposing and growth-promoting strain DY21116S rDNA and Burkholderia pyrrocinia is highest, so that the strain DY211 can be primarily judged to be the Burkholderia pyrrole.
The strain is pyrrol burkholderia and named pyrrol burkholderia DY211, and is preserved in China center for type culture collection (China) on 11-month 17 of 2021, and the preservation registration number is shown by morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis: cctccc M20211436.
Example 4 Effect of Strain DY211 on tobacco seed emergence
Use of the strain DY211 to promote tobacco plant growth, comprising the steps of:
(1) Seed treatment, namely placing an appropriate amount of untreated tobacco seeds into a culture dish for disinfection and sterilization, placing the selected seeds into a fume hood for chlorine disinfection, placing 50mL of sodium hypochlorite and 10mL of hydrogen chloride into a beaker, closing a shop window, disinfecting for 2 hours, placing into an ultra-clean workbench after disinfection, and ventilating for later use
(2) Sterilizing nutritional soil (potassium source is mainly insoluble potassium) at 121deg.C for 20min, cooling, placing into trays, placing into sterilized water, and placing two sterilized tobacco seeds into each tray after sufficiently absorbing sterilized water
(3) Preparing a growth promoting bacterial liquid: fermenting the activated strain, culturing at 28deg.C, periodically sampling to detect strain concentration, diluting the strain with sterile water, continuously fermenting the strain with concentration not reaching standard, ensuring effective concentration of applied bacterial liquid is 10 8 cfu/mL during seedling culture,
(4) To explore the effect of different dosages and different periods of application of DY211 bacterial liquid on tobacco seed emergence, the following experimental treatments were designed: a1, 1mL of bacterial liquid/hole is applied during sowing, A2, 2 mL/hole is applied during sowing, A3: 5 mL/hole is applied during sowing, 1 mL/hole is applied during sowing and seedling emergence period, 2 mL/hole is applied during sowing and seedling emergence period, 5 mL/hole is applied during seedling emergence period, 1 mL/hole is applied during seedling emergence period, 8 mL/hole is applied during seedling emergence period, 5 mL/hole is applied during seedling emergence period, corresponding amount of sterile water is applied during seedling emergence period, 4 columns are treated each, 8 repeated regular intervals of each column are counted, three times are counted, the first time is 25d of treatment, the second time is 50d of treatment, and the third time is 80d,
(5) In order to compare the effect of the strain DY211 with the effect of the existing strain A12 on tobacco emergence, the existing strain A12 and the strain DY211 are treated in the same way, the experimental treatment numbers are sequentially B1, B2, B3, B4, B5, B6, B7, B8 and B9, the emergence situation is counted periodically, three times of statistics are counted, the first time is 25d, the second time is 50d and the third time is 80d.
TABLE 4 Effect of the DY211 strain on plug experiment on tobacco seed emergence
TABLE 5 Effect of Strain A12 on plug tray experiments on tobacco seed emergence
As shown in tables 4 and 5, the strain DY211 has obvious advantages compared with the existing strain A12 in the emergence experiments, and particularly, the emergence rates of the two groups A7 and A9 are highest. According to the influence condition of the strain DY211 on the emergence of tobacco seeds, the treatment groups A1, A4, A7 and A9 are selected for carrying out subsequent field experiments, and in order to maintain the same conditions, the existing strain is also selected for carrying out the subsequent field experiments by the same treatments B1, B4, B7 and B9.
Example 5 Effect of Strain DY211 on tobacco plant biomass
Transplanting tobacco seedlings into a field in a transplanting period, and respectively measuring the stem height increase, the leaf number, the stem diameter, the middle leaf length and the middle leaf width of tobacco plants after the tobacco grows to a mature period. Ten replicates per plant, and by OriginLa b OriginPro software analysis, it was found that the bacterial liquid prepared by using DY211 can increase the content of soluble potassium in soil and increase the biomass of tobacco, and as shown in table 6, treatment group A1 increased the stem height of the mature tobacco plants by 8.4%, the leaf number by 12.5%, the stem diameter by 15.3%, the middle leaf length by 23.3%, and the middle leaf width by 12.1%. And other treatment groups are also generally significantly improved compared to CK; as shown in tables 6 and 7, the strain DY211 has obvious advantages over the existing strain A12 in terms of stem height, stem diameter, middle leaf length and middle leaf width, and the promotion of tobacco seedlings by the strain A12 is mainly reflected in tobacco stem diameter. Meanwhile, it is obvious that the two groups A7 and A9 are relatively high in various indexes of tobacco biomass (stem height, leaf number, stem diameter, middle leaf length and middle leaf width), for example, the treatment group A9 increases the stem height of the tobacco plants in the maturity stage by 12.0%, the leaf number by 12.9%, the stem diameter by 16.9%, the middle leaf length by 28.8% and the middle leaf width by 18.5%.
TABLE 6 Effect of the DY211 strain on tobacco biomass
Note that: * Representing significant/representing very significant
TABLE 7 Effect of Strain A12 on tobacco biomass
Note that: * Representing significant/representing very significant
Example 6 Effect of Strain DY211 on tobacco Potassium content
Taking middle and top leaves of mature tobacco plants, repeating each leaf ten times, and measuring potassium content in the leaves by flame atomic absorption spectrometry (GB 5009.91-2017) after drying. Analysis of the data by OriginLab Origi nPro 8.5.8 software found a significant increase in potassium content in both the top and middle leaves compared to the control. As shown in Table 8, the potassium content of the middle leaf and the top leaf of the treatment group A7 is improved most obviously, the potassium content increase rate reaches 22.22% and 72.48% respectively, and the effect on the emergence of tobacco seeds is small, as shown in Table 8 and Table 9, the strain DY211 has a remarkable effect on the tobacco content relative to the strain A12, and the strain A12 has a certain promotion effect on the tobacco leaves at the top of tobacco plants, but the increase rate is not large. Specifically, the indices of group A9 account for significant differences, such as a central leaf growth rate of 20.98%, and a top leaf growth rate of 59.63%.
TABLE 8 Effect of the DY211 strain on tobacco middle and top lamina Potassium content
Note that: * Representing significant/representing very significant
TABLE 9 Effect of Strain A12 on tobacco middle and top lamina Potassium content
Note that: * Representing significant/representing very significant
The invention screens potassium-decomposing bacteria strain DY211 from tobacco rhizosphere soil in a tobacco planting area of Luzhou Sichuan, and determines morphological characteristics, physiological and biochemical characteristics and 16s rDNA sequences of the strain to determine that the strain is Burkholderia pyrrocina DY211 (Burkholderia pyrrocinia). The content of soluble potassium in the metabolic supernatant of the strain DY211 is measured by adopting a flame atomic absorption spectrometry (GB 5009.91-2017), and then the growth promotion effect of tobacco is tested in field experiments, and the result shows that the strain can utilize indissolvable potassium under the conditions of solid and liquid culture, the content of quick-acting potassium in the metabolic supernatant is 5.48mg/L in the liquid culture for 14d, and the potassium decomposition rate reaches 292.86%, so that the strain has excellent potassium decomposition capability. Meanwhile, the bacterial liquid prepared by utilizing the Burkholderia pyrrocinia DY211 can be used for biomass of tobacco plants and potassium content of tobacco leaves. The stem height of the tobacco plant is increased by 12.0%, the leaf number is increased by 12.9%, the stem diameter is increased by 16.9%, the middle leaf length is increased by 28.8%, the middle leaf width is increased by 18.5%, and the potassium content in the top and middle tobacco leaves can be obviously improved. Thereby promoting the growth of tobacco, improving the quality of tobacco leaves and reducing the use of chemical fertilizers, and the potassium-dissolving growth-promoting strain and bacterial liquid thereof have wide application prospects.
The foregoing is a further elaboration of the present invention in connection with the detailed description, and it is not intended that the invention be limited to the specific embodiments shown, but rather that a number of simple deductions or substitutions be made by one of ordinary skill in the art without departing from the spirit of the invention, should be considered as falling within the scope of the invention as defined in the appended claims.
Claims (6)
1. A potassium-decomposing growth-promoting pyrrolizhou-de bacterium, which is characterized in that:
The Latin school name of the Burkholderia pyrroctis is Burkholderia pyrrocinia, the Latin school name is DY211, and the Latin school name is preserved in China Center for Type Culture Collection (CCTCC) No. M20211436 in 2021, 11 months and 17 days.
2. A microbial inoculum, characterized in that:
comprising the Burkholderia pyrrocinia of claim 1.
3. The microbial agent of claim 2, wherein:
The active ingredient of the microbial inoculum is bacterial liquid of Burkholderia pyrrocinia.
4. The microbial agent of claim 2, wherein:
the preparation method of the microbial inoculum comprises the following steps:
(1) Preparing a solid potassium-decomposing culture medium; the solid potassium-decomposing culture medium comprises the following components: 10g/L of glucose, 0.4g/L of yeast extract, 0.2g/L of magnesium sulfate, 0.5g/L of dipotassium hydrogen phosphate, 0.2g/L of sodium chloride, 1g/L of calcium carbonate and 20g/L of agar;
(2) Preparing a liquid potassium-decomposing culture medium; the liquid potassium-decomposing culture medium comprises the following components: 5g/L of starch, 1g/L of yeast extract, 0.5g/L of magnesium sulfate, 2g/L of disodium hydrogen phosphate, 1g/L of calcium carbonate and 5mg/L of ferric trichloride;
(3) Preparation of activated strains: picking single bacterial colony of pyrrol burkholderia in a solid potassium-decomposing culture medium plate, preparing seed liquid, inoculating the seed liquid into a liquid potassium-decomposing culture medium, culturing the seed liquid in a shake flask with the temperature of 28 ℃ and the rotating speed of 160r/min, and diluting the bacterial liquid by using sterile water when the bacterial strain grows to a logarithmic growth phase to obtain the bacterial agent.
5. Use of potassium-releasing growth-promoting burkholderia pyrrocinia according to claim 1 for promoting tobacco growth.
6. Use of a microbial agent according to claim 2 or 3 for promoting tobacco growth, wherein: the dosage form of the microbial inoculum is a pharmaceutically acceptable dosage form.
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| CN104911122A (en) * | 2015-04-13 | 2015-09-16 | 华南农业大学 | Burkholderia kururiensis strain and application thereof |
| CN111387209A (en) * | 2020-05-21 | 2020-07-10 | 湖北大学 | Composite antagonistic bacterium agent for preventing and controlling tobacco diseases and application thereof |
| KR102231012B1 (en) * | 2019-11-04 | 2021-03-23 | 동아대학교산학협력단 | Purple acid phosphatase 9 gene from Burkholderia pyrrocinia CH-67 improving phosphate uptake efficiency in plant and uses thereof |
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| CN104911122A (en) * | 2015-04-13 | 2015-09-16 | 华南农业大学 | Burkholderia kururiensis strain and application thereof |
| KR102231012B1 (en) * | 2019-11-04 | 2021-03-23 | 동아대학교산학협력단 | Purple acid phosphatase 9 gene from Burkholderia pyrrocinia CH-67 improving phosphate uptake efficiency in plant and uses thereof |
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