CN114703081A - Brevundimonas ST3CS3 and application thereof - Google Patents
Brevundimonas ST3CS3 and application thereof Download PDFInfo
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- CN114703081A CN114703081A CN202210000216.2A CN202210000216A CN114703081A CN 114703081 A CN114703081 A CN 114703081A CN 202210000216 A CN202210000216 A CN 202210000216A CN 114703081 A CN114703081 A CN 114703081A
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- 241000131407 Brevundimonas Species 0.000 title claims abstract description 58
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 42
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000011574 phosphorus Substances 0.000 claims abstract description 35
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 35
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000003337 fertilizer Substances 0.000 claims abstract description 28
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 21
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000011591 potassium Substances 0.000 claims abstract description 16
- 229910052700 potassium Inorganic materials 0.000 claims abstract description 16
- 239000003617 indole-3-acetic acid Substances 0.000 claims abstract description 15
- 241000244558 Brevundimonas olei Species 0.000 claims abstract description 5
- 241000196324 Embryophyta Species 0.000 claims description 50
- 230000008635 plant growth Effects 0.000 claims description 11
- 230000001737 promoting effect Effects 0.000 claims description 9
- 241001263604 Stellera chamaejasme Species 0.000 claims description 6
- 241001576300 endophytic bacterium Species 0.000 claims description 3
- 239000003124 biologic agent Substances 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 27
- 230000012010 growth Effects 0.000 abstract description 17
- 239000000126 substance Substances 0.000 abstract description 15
- 229930002875 chlorophyll Natural products 0.000 abstract description 5
- 235000019804 chlorophyll Nutrition 0.000 abstract description 5
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 abstract description 5
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 3
- 239000012880 LB liquid culture medium Substances 0.000 description 3
- WYWFMUBFNXLFJK-UHFFFAOYSA-N [Mo].[Sb] Chemical compound [Mo].[Sb] WYWFMUBFNXLFJK-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
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- 239000012895 dilution Substances 0.000 description 3
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000012353 t test Methods 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- JCRIDWXIBSEOEG-UHFFFAOYSA-N 2,6-dinitrophenol Chemical compound OC1=C([N+]([O-])=O)C=CC=C1[N+]([O-])=O JCRIDWXIBSEOEG-UHFFFAOYSA-N 0.000 description 2
- 229930192334 Auxin Natural products 0.000 description 2
- 241000186146 Brevibacterium Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 2
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 241001310698 Shangrila Species 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002363 auxin Substances 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 239000010451 perlite Substances 0.000 description 2
- 235000019362 perlite Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 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 2
- 229960004799 tryptophan Drugs 0.000 description 2
- 244000018217 Artocarpus elasticus Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 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 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 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
- WZLMXYBCAZZIRQ-UHFFFAOYSA-N [N].[P].[K] Chemical compound [N].[P].[K] WZLMXYBCAZZIRQ-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002411 adverse Effects 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
- 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
- 230000003851 biochemical process Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 210000003495 flagella Anatomy 0.000 description 1
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- 239000008103 glucose Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229930195732 phytohormone Natural products 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 150000003112 potassium compounds Chemical class 0.000 description 1
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- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
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
-
- 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
Abstract
The invention discloses brevundimonas (Brevundimonas)Brevundimonasolei) ST3CS3, wherein the preservation number of the general microbiological center of China Committee for culture Collection of microorganisms is CGMCC No. 24140; the strain has the capability of fixing nitrogen and dissolving potassium, and higher capability of dissolving phosphorus and producing indoleacetic acid; the bacterial strain is colonized in a plant body, can obviously promote the growth of the plant, increases the chlorophyll and nitrogen content of the plant, further reduces the use of chemical fertilizers, improves the crop yield, and has important significance in agricultural production.
Description
Technical Field
The invention belongs to the field of growth-promoting bacteria biotechnology, and particularly relates to brevundimonas and application thereof in promoting plant growth.
Background
With the development of industrialization and modernization in China, the cultivated land area is sharply reduced year by year. Since the 60 s of the 20 th century, the use of the fertilizer guarantees the national food safety and plays an important role in increasing the yield and income of agriculture in China. However, excessive use of chemical fertilizers not only affects the quality of agricultural products, but also causes great pressure on the rural ecological environment. In recent years, the traditional conventional chemical fertilizers (such as nitrogen, phosphorus and potassium compound fertilizers) have the problems of low utilization rate and abuse, because the chemical fertilizers generally contain heavy metals, the unreasonable application of the chemical fertilizers in successive years can cause the accumulation of the heavy metals in soil and harm the soil structure and the sustainable development of human society, the blind application of the conventional chemical fertilizers can cause a series of negative effects such as yield reduction, and the like, and the reasonable application and even the reduced application of the conventional chemical fertilizers are urgent.
Plant growth-promoting bacteria (PGPR) refer to an autogenous bacterium that is beneficial to crops. The action of the growth-promoting bacteria for directly promoting the growth of crops means that certain plant growth-promoting bacteria can synthesize certain substances (such as auxin and the like) which have direct effects on the growth and development of the crops and (or) change the forms of certain ineffective elements in soil, so that the effective substances are further beneficial to the absorption of the crops (such as nitrogen fixation, phosphorus dissolution and the like). The growth-promoting bacteria can also indirectly act on certain plants to inhibit or reduce the adverse effect of certain plant diseases on the growth, development and yield of crops. This has prompted the scientists around the world to actively explore and develop microbial fertilizers centered on microorganisms to replace (or replace part of) the use of fertilizers and pesticides.
The microbial fertilizer takes plant growth-promoting bacteria as a core, is a novel green and environment-friendly fertilizer, has the characteristics of long acting, no toxicity, no pollution, energy conservation, low cost and the like, and has the effects of promoting the growth of crops, improving the yield and quality of the crops, reducing the content of nitrate in crop products, improving the disease resistance of the crops, enhancing the soil fertility, reducing the using amount of chemical fertilizers, purifying the environment, maintaining ecological balance and the like. The microbial fertilizer mainly depends on the life activities of microorganisms to produce enzymes and beneficial substances to play a role, nitrogen, phosphorus and potassium are necessary elements for plant growth, nitrogen in the air, inorganic phosphorus in soil and potassium in the form of silicate minerals are difficult to be directly absorbed by plants, and the microorganisms can produce the enzymes and the beneficial substances to convert the unavailable substances into elements which can be directly utilized; indoleacetic acid (IAA) is a plant endogenous auxin and is involved in regulation and control of various physiological and biochemical processes such as growth of plant cells, and the IAA produced by microorganisms plays an important role in the plant micro-ecological environment. Under the current situation that China faces energy crisis, resource shortage and environmental pollution, the importance of microbial fertilizers to the modern agricultural development of China is self-evident, the development of the microbial fertilizers can be regarded as an important base stone for green safety of grains and guaranteeing a few cultivated land environments of China, the microbial fertilizers are truly novel pollution-free fertilizers, the sustainable development concept of China is met, the microbial fertilizers have wide application scenes with the increasingly high awareness and demand of people on green pollution-free agricultural products, and the microbial fertilizers play an important role in the sustainable agricultural development of China.
Disclosure of Invention
The invention provides a plant endophytic bacterium brevundimonas ST3CS3, which is named by classificationBrevundimonas oleiThe strain is preserved in China general microbiological culture Collection center (CGMCC) at 20 months 12 and 2021, the preservation number is CGMCC No.24140, and the preservation address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing. The invention also aims to provide a new application of the plant endophytic bacterium brevundimonas ST3CS3, namely the brevundimonas ST3CS3 can be used for preparing a biological microbial agent or a microbial fertilizer for promoting plant growth when being applied to promoting plant growth, and the brevundimonas ST3CS3 has nitrogen fixation and potassium dissolution capabilities and higher phosphorus dissolution and IAA production capabilities; the strain is colonized in a plant body, can obviously promote the growth of the plant, increases the chlorophyll and nitrogen contents of the plant, and further reduces the use of chemical fertilizers.
In order to achieve the above purpose, the invention adopts the following technical measures:
1. collecting radix Euphorbiae Fischerianae (radix Euphorbiae Fischerianae) as invasive plant in Shangrila pasture of Yunnan provinceStellera chamaejasme L.) washing the plant sample clean under tap water;
2. respectively carrying out surface disinfection on the plant sample divided into an overground part and an underground part, firstly soaking for 3-5 min by using an ethanol solution with the volume concentration of 75%, washing for 3-5 times by using sterile water, then soaking for 2-3 min by using a sodium hypochlorite solution with the effective chlorine concentration of 5%, washing for 3-5 times by using the sterile water, and placing the plant sample on sterile filter paper to absorb water after washing; performing bacterial separation by adopting a mixed dilution plate method, grinding 1g of plant sample with a sterilized surface into powder in sterile mortar, fully mixing the powder with 9mL of sterile water, transferring the mixture after gradient dilution to an LB culture medium for culture, culturing for 10 days at 37 ℃, observing every other day, picking when bacterial colonies grow out, obtaining a plurality of endophytic bacterial strains after separation and purification, and preparing the endophytic bacterial strains into bacterial suspension;
3. inoculating the separated endophytic strain to an inorganic phosphorus culture medium, and judging the phosphorus dissolving capacity of the strain by a hydrolysis ring;
4. selecting a bacterial strain with stronger phosphorus dissolving capacity, storing the bacterial strain on an LB inclined plane for later use, and naming the bacterial strain as ST3CS 3;
5. identification of Strain ST3CS3
combining morphological characteristics and molecular identification results, and finally, obtaining the strainIdentified as Brevundimonas (Brevundimonas olei) (ii) a The culture medium used for the preservation and activation of the strain is an LB culture medium.
The invention carries out pot experiment on brevundimonas ST3CS3 separated from stellera chamaejasme, discusses the influence of the brevundimonas ST3CS3 on the growth of tobacco seedlings, namely, researches the influence of brevundimonas ST3CS3 inoculation on the growth of potted tobacco, and provides bacterial strains and theoretical research basis for microbial fertilizer.
Compared with the prior art, the invention has the following beneficial effects:
the brevundimonas ST3CS3 provided by the invention is derived from stellera chamaejasme, has the capabilities of fixing nitrogen, dissolving phosphorus, dissolving potassium and producing IAA, can be effectively colonized in plants, effectively dissolves insoluble potassium in plant rhizosphere soil, improves the content of nitrogen in plants, further promotes the growth of plants, reduces the use of chemical fertilizers, is simple in culture operation, low in cost, safe to environment, and suitable for industrial production and market popularization and application.
Drawings
FIG. 1 is the colony morphology of Brevundimonas ST3CS3 on LB medium;
FIG. 2 shows the growth of Brevundimonas ST3CS3 on inorganic phosphorus solid medium;
FIG. 3 is a standard working curve of a phosphorus standard solution;
FIG. 4 shows the determination results of the phosphate-solubilizing ability of Brevundimonas ST3CS 3;
FIG. 5 shows the results of measurement of nitrogen-fixing ability of Brevundimonas ST3CS 3;
FIG. 6 shows the results of measurement of potassium-releasing ability of Brevundimonas ST3CS 3;
FIG. 7 is a standard working curve of an IAA standard solution;
FIG. 8 shows the results of measurement of IAA secretion ability of Brevundimonas ST3CS 3;
FIG. 9 is the effect of Brevundimonas ST3CS3 on tobacco seedling growth;
FIG. 10 is a graph showing the effect of Brevundimonas ST3CS3 on chlorophyll content of tobacco seedling leaves.
Detailed Description
The following detailed description will be made with reference to specific embodiments and accompanying drawings, but the present invention is not limited to the following embodiments. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Those skilled in the art can implement the invention by referring to various conventional tool books, technical and scientific documents or relevant specifications, manuals, etc. before the filing date of the present application.
Example 1: separation, screening and identification of brevundimonas ST3CS3
(1) Collecting a plant sample of a dominant plant stellera chamaejasme in Shangrila pasture of Yunnan province, and washing the plant sample clean under tap water;
(2) respectively disinfecting the surfaces of the overground part and the underground part of a plant sample, firstly soaking the plant sample in 75% ethanol solution by volume concentration for 5min, washing the plant sample with sterile water for 5 times, then soaking the plant sample in 5% sodium hypochlorite solution by effective chlorine concentration for 2min, washing the plant sample with sterile water for 4 times, and placing the plant sample on sterile filter paper to absorb water after washing; performing bacteria separation by mixing dilution plate method, grinding 1g plant sample with surface sterilized in sterile mortar into powder, mixing with 9mL sterile water, and diluting the mixture to 10-3、10-4、10-5Transferring the diluted solution to LB culture medium (5 g yeast extract, 10g tryptone, 10g sodium chloride, 14g agar, 1L distilled water, pH 7.0-7.2), inversely placing in a biochemical incubator at 37 ℃ for 10 days, observing every other day, picking out single colony when bacterial colony grows out, separating and purifying to obtain multiple endophytic bacterial strains, and preparing the endophytic bacterial strains into bacterial suspensions;
(3) inoculating the separated endophytic strain to an inorganic phosphorus culture medium (10 g of glucose, 0.5g of ammonium sulfate, 0.3g of sodium chloride, 0.3g of potassium chloride, 0.3g of magnesium sulfate heptahydrate, 0.03g of ferrous sulfate heptahydrate, 1g of manganese sulfate tetrahydrate, 5g of tricalcium phosphate, 14g of agar, 1L of distilled water and pH of 7.0-7.5), and judging the phosphorus dissolving capacity of the strain by a hydrolysis loop;
(4) selecting the bacterial strain with the strongest phosphorus dissolving capacity, storing the bacterial strain on an LB inclined plane for later use, and naming the bacterial strain as ST3CS 3;
(5) identification of the Strain ST3CS3
Example 2: determination of phosphate-solubilizing ability of Brevundimonas ST3CS3
Dissolving a phosphorus ring: inoculating the shortwave monad ST3CS3 obtained by separation to an inorganic phosphorus solid culture medium, repeating the inoculation for three times at 4 inoculation points per dish, culturing at constant temperature of 30 ℃ for 5 days, observing and recording the growth condition of the strain, a phosphorus dissolving ring (D) and the colony diameter (D), wherein the larger the D/D is, the stronger the phosphorus dissolving capacity is, the result is shown in figure 2, the shortwave monad ST3CS forms a more obvious phosphorus dissolving ring on the inorganic phosphorus culture medium, the diameter of the phosphorus dissolving ring is 0.75cm, and the diameter of the colony is 0.3 cm.
Phosphorus dissolving capacity: carrying out shake culture on brevundimonas ST3CS in an LB liquid culture medium at 30 ℃ and 180rpm for 15h, then inoculating the brevundimonas ST3CS into an inorganic phosphorus liquid culture medium by 4% of inoculum size, taking the LB liquid culture medium with the same amount as the inoculated strain as a blank control, setting three times of repetition for each treatment, and carrying out shake culture on the shaker at 30 ℃ and 180rpm for 6 d; the content of soluble phosphorus (the wavelength of a spectrophotometer is 700 nm) is measured by adopting a molybdenum-antimony colorimetric method, and the method comprises the following specific steps:
firstly, centrifuging the fermentation liquor at 10000rpm for 10min, putting 2.5mL of supernatant into a 50mL volumetric flask, adding 2 drops of 2, 6-dinitrophenol indicator, adjusting the pH value of the solution to be light yellow by using 10% sodium hydroxide or 5% dilute sulfuric acid, adding 5mL of molybdenum-antimony anti-color-developing agent, and adding deionized water to the volume of 50 mL;
② after standing for 30min, carrying out color comparison at 700nm of a spectrophotometer, and simultaneously measuring a blank control group;
thirdly, simultaneously drawing a phosphorus standard curve in an experiment, respectively sucking 0, 2, 4, 6, 8 and 10mL of 5 mug/mL phosphorus standard solution into a 50mL volumetric flask, adding 2 drops of 2, 6-dinitrophenol indicator, adjusting the pH value with 10% sodium hydroxide or 5% dilute sulfuric acid until the solution is just yellowish, adding 5mL of molybdenum-antimony color-developing resisting agent, adding deionized water to the solution to fix the volume to obtain 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mug/mL phosphorus standard series solutions, carrying out colorimetry in a spectrophotometer at 700nm after standing for 30min, drawing a standard working curve (figure 3), then substituting the absorbance value measured in the step II into the standard working curve to obtain 51mg/L of phosphorus-dissolved amount of brevundimonas ST3CS in inorganic phosphorus culture solution (figure 4), which shows that brevundimonas ST3CS has the capability of converting inorganic phosphorus into organic phosphorus, so that plants can absorb phosphorus more easily, thereby promoting the growth of plants.
Example 3: determination of nitrogen fixation capability of brevundimonas ST3CS3
Inoculating brevundimonas ST3CS3 to an arbuscular mycorrhizal solid culture medium (0.2 g of disodium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 0.2g of sodium chloride, 5g of calcium carbonate, 10g of mannitol, 0.1g of calcium sulfate, 14g of agar, 1L of distilled water and pH 7.0), streaking in three zones, repeating for three times, and culturing at constant temperature of 30 ℃ for 5 days; if the strain can grow on the culture medium, the strain is proved to have the nitrogen fixing capacity, and otherwise, the strain has no nitrogen fixing capacity.
The results are shown in FIG. 5, from which it can be seen that Brevundimonas ST3CS3 can grow on the culture medium of Artocarpus, demonstrating that Brevundimonas ST3CS3 can fix N in the air2The nitrogen is converted into effective nitrogen, and the nitrogen is absorbed by the plants, so that the growth of the plants is promoted.
Example 4: determination of potassium-dissolving capacity of Brevundimonas ST3CS3
Shortwave monospora ST3CS3 is inoculated on a silicate solid culture medium (2 g of disodium hydrogen phosphate, 5g of cane sugar, 0.5g of magnesium sulfate heptahydrate, 0.005g of ferric chloride, 0.1g of calcium carbonate, 1g of potassium feldspar powder, 14g of agar and 1L of distilled water, the pH value is 7.0-7.5), 4 inoculation points are arranged in each dish, the three times are repeated, and the constant temperature culture is carried out for 5 days at the temperature of 30 ℃; if the strain forms a potassium-decomposing ring on the strain, the strain is proved to have the potassium-decomposing capability, otherwise, the strain does not have the potassium-decomposing capability.
As a result, as shown in FIG. 6, Brevundimonas ST3CS forms a potassium-releasing ring on a silicate solid culture medium, the diameter of the potassium-releasing ring is 0.45cm, and the diameter of a bacterial colony is 0.35cm, which indicates that Brevundimonas ST3CS3 can convert insoluble potassium into effective potassium, help plants to absorb the potassium and further promote plant growth.
Example 4: determination of IAA secretion Capacity of Brevundimonas ST3CS3
Culturing Brevundimonas ST3CS3 in LB liquid culture medium (containing L-tryptophan 0.1 mg/mL) in the dark on a shaking table at 30 ℃ and 120rpm for 24h, taking 1.5mL of bacterial liquid and centrifuging at 12000rpm for 10min, removing precipitates, taking 0.5mL of supernatant, adding an equivalent amount of Salkowski's reaction solution (1 mL of 0.5mol/L ferric chloride and 49mL of 35% perchloric acid), reacting in the dark for 30min, measuring the light absorption value at 530nm, repeating each group for three times, and adjusting zero by taking the treatment of the non-inoculated culture medium and the upper part as a control; the IAA standard solution with the concentration of 0, 5.5, 11, 22, 44 and 88 mug/mL is used for measuring the absorbance value and drawing a standard curve (figure 7) by adopting the method, then the absorbance value of an experimental group inoculated with brevundimonas is substituted into the standard curve to obtain the IAA secreted by the brevundimonas ST3CS3 (figure 8), the IAA secreted by the brevundimonas ST3CS3 synthesizes the plant hormone IAA by taking L-tryptophan as a precursor, the growth and the proliferation of plant cells are stimulated, water and nutrients are effectively absorbed, and the life activity of the plant body is adjusted.
Example 5: growth promotion experiment of brevundimonas ST3CS3 on tobacco seedlings
This example aims to demonstrate the growth-promoting effect of brevundimonas ST3CS3 on plants; with tobacco (Nicotiana tabacumL.) test plants, the experimental procedure was as follows:
A. preparing a tobacco sterile seedling: randomly selecting tobacco seeds (MSK 326), firstly soaking in 75% ethanol solution for 2min, and washing with sterile water for 4 times; then soaking in NaClO solution with effective chlorine concentration of 5% for 1min, washing with sterile water for 5 times, and placing on sterile filter paper to absorb water for use; then autoclaving (121 deg.C, 15psi, 15 min; 3 times) the tray containing Canadian peat and perlite (volume ratio 7: 3), placing tobacco seeds in the tray for germination at 25 deg.C and relative humidity of 60%, and watering the plants with distilled water once every 3 days to maintain water content;
B. preparation of an endophytic bacteria inoculant and an inactivator: inoculating brevundimonas ST3CS3 into a 250mL conical flask, and culturing for 15h in a constant temperature shaking table at 28 ℃ and 180 rpm; the bacterial liquid is divided into A, B parts, A part is used as an endophytic bacteria inoculant,
sterilizing part B at 121 deg.C under 15psi for 15min to kill endophyte as inactivator;
C. pot experiment: after 30 days of tobacco seed planting, 20 seedlings of consistent growth were selected and transplanted into flowerpots (10.5X 8.5 cm; 1 seedling/pot) containing 600g of field soil, perlite (7: 3, v/v) and 3g of calcium phosphate per pot. The pots were randomly divided into two groups (group I and group II), each of 10 pots, and then the endophytic bacteria inoculant and inactivator of step B were sprayed onto the leaf surface and roots of seedlings of group I and group II, respectively, each 3mL of inoculum, until wet (group I: E +, ST3CS3 inoculum; group II: E-, ST3CS3 no inoculum). Inoculating bacterial liquid on the 1 st day and the 9 th day after transplantation, inoculating for 2 times, placing the seedlings at room temperature (18-25 ℃) for natural illumination culture, pouring sterile water once every 3 days during culture, pouring 100mL of sterile water in each pot (preferably, the water is poured through soil without overflowing the bottom of the pot), and closely observing the growth condition of each group of tobacco seedlings in the experimental process. To determine the colonization of the bacteria in the inoculated plants, 3 plants were randomly collected in groups E + and E-5 days after the last inoculation, respectively, washed under tap water and surface sterilized; then, the seedlings were homogenized by grinding, and the diluted suspension was cultured in LB medium to detect the inoculated strain. This strain was re-isolated from E + plants, but not from E-plants, confirming the colonization by Brevundimonas. After 25 days of culture, plants were harvested and the height, dry weight, root length, leaf width, leaf length and chlorophyll content of the plants, nitrogen phosphorus potassium content in the plants and soil were determined.
The results are shown in table 1, table 2, fig. 9 and 10, and show that brevundimonas ST3CS3 has obvious promotion effect on tobacco seedlings, and significant difference is generated in the experimental group and the control group (p is less than 0.01, t test). At 25 days, compared with a control, the plant height, the leaf length, the leaf width, the above-ground dry weight, the underground dry weight, the plant nitrogen content and the chlorophyll content are respectively increased by 43.36 percent, 27.63 percent, 25.17 percent, 56.90 percent, 74.84 percent, 20.20 percent and 15.06 percent;
TABLE 1 Effect of Brevundimonas ST3CS3 on tobacco Biomass
Note: taking a as a standard, b represents that a is significantly different from a (p <0.01, t test);
TABLE 2 influence of Brevundimonas ST3CS3 on the content of nitrogen, phosphorus and potassium in tobacco and its soil
Note: taking a as a standard, b represents that a is significantly different from a (p <0.01, t test);
the results of the above examples show that brevundimonas ST3CS3 obtained by separation in the present invention has the ability of fixing nitrogen, dissolving phosphorus and dissolving potassium, thereby promoting the absorption of nitrogen, phosphorus and potassium by plants, and also has the ability of producing IAA, providing phytohormones to promote the growth of plants. Pot experiments prove that the growth of host plants can be improved after the brevundimonas sp ST3CS3 is inoculated, and the method can be widely applied to the preparation of microbial fertilizers and reduce the use and pollution of the fertilizers.
Sequence listing
<110> university of Kunming science
<120> brevundimonas ST3CS3 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1020
<212> DNA
<213> Stellera chamaejasme (Stellera chamaejasme L.)
<400> 1
gggggaaggg ctagaatgca gtcgaacgaa ctcttcggag ttagtggcgg acgggtgagt 60
aacacgtggg aacgtgcctt ttggttcgga ataactcagg gaaacttgtg ctaataccga 120
atgtgccctt cgggggaaag atttatcgcc attagagcgg cccgcgtctg attagctagt 180
tggtgaggta acggctcacc aaggcgacga tcagtagctg gtctgagagg atgatcagcc 240
acactgggac tgagacacgg cccggactcc tacgggaggc agcagtgggg aatcttgcgc 300
aatgggcgaa agcctgacgc agccatgccg cgtgaatgat gaaggtctta ggattgtaaa 360
attctttcac cggggacgat aatgacggta cccggagaag aagccccggc taacttcgtg 420
ccagcagccg cggtaatacg aagggggcta gcgttgctcg gaattactgg gcgtaaaggg 480
cgcgtaggcg gatcgttaag tcaggggtga aatcccgggg ctcaacctcg gaattgccct 540
tgatactggc gatcttgagt atgagagagg tatgtggaac tccgagtgta gaggtgaaat 600
tcgtagatat tcggaagaac accagtggcg aaggcgacat actggctcat tactgacgct 660
gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac 720
gatgattgct agttgtcggg ctgcatgcag ttcggtgacg cagctaacgc attaagcaat 780
ccgcctgggg agtacggtcg caagattaaa actcaaagga attgacgggg gcccgcacaa 840
gcggtggagc atgtggttta attcgaagca acgcgcagaa ccttaccacc ttttgacatg 900
cctggaccgc cacggagacg tggctttccc ttcggggact aggacacagg tgctgcatgg 960
ctgtcgccag ctcgggcctg ggaatgttgg gttaagtccc gaacgagcgc accctcgcct 1020
Claims (3)
1. Brevundimonas (Brevundimonas) ((Brevundimonasolei) ST3CS3, a plant endophytic bacterium separated from stellera chamaejasme, is preserved and managed by China Committee for culture Collection of microorganismsCGMCC No.24140 of common microorganism center.
2. The use of brevundimonas ST3CS3 in promoting plant growth according to claim 1, wherein: brevundimonas ST3CS3 has the functions of dissolving phosphorus, potassium and nitrogen, and producing indoleacetic acid.
3. Use according to claim 2, characterized in that: brevundimonas ST3CS3 is used for preparing biological agents or fertilizers for promoting plant growth.
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