CN109576194A - One plant of multifunctional water draws engler bacterium MEM40 and its application - Google Patents
One plant of multifunctional water draws engler bacterium MEM40 and its application Download PDFInfo
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- CN109576194A CN109576194A CN201910038385.3A CN201910038385A CN109576194A CN 109576194 A CN109576194 A CN 109576194A CN 201910038385 A CN201910038385 A CN 201910038385A CN 109576194 A CN109576194 A CN 109576194A
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- 150000001875 compounds Chemical class 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000004900 laundering Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 230000003458 metachromatic effect Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 229960004023 minocycline Drugs 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
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Abstract
The invention discloses one plant of multifunctional waters to draw engler bacterium MEM40 and its application, belongs to microorganisms technical field, and taxology is named as water and draws engler bacterium (Rahnella aquatilis) MEM40, and deposit number is CCTCC NO.M 2018463.The bacterial strain has the function of efficient phosphate-solubilizing, fixed nitrogen, the available phosphorus content and nitrogen element content that plant growth needs in soil can be improved, and the IAA of secretion can promote plant growth, and the bacterium can generate substance and ability metabolism in a variety of soil enzymes quickening soil, and then increase soil fertility, and improve soil conditions, promote plant growth, and Fertilizer application is reduced, especially phosphate fertilizer and nitrogenous fertilizer reduce planting cost.
Description
Technical field
The present invention relates to microorganisms technical field, in particular to one plant of multifunctional water draws engler bacterium MEM40 and its application.
Background technique
Phosphorus is one of nutrient necessary to plant growth.Most P elements are all with slightly solubility in China's soil
Phosphatic form exists, and chemical property is stablized, and is difficult to be absorbed by crops utilization, to meet crop to the need of nutrient
It asks, in China, the vast majority of arable land will impose phosphate fertilizer largely to increase the content of soil available phosphorus, but be applied in soil
Phos can and soil in metal ion combine, formed insoluble phosphate, cause phosphate fertilizer utilization efficiency low；And phosphate fertilizer is indiscriminate
With not only resulting in waste of resources, while serious environmental problem is also brought along, such as soil hardening, acidification, Soil degradation, agricultural production
The reduction of product quality, soil microbe quantity reduction, ecological degeneration, the arable soil ecosystem is unbalance etc..Therefore, it improves
The utilization rate of China farming field Nutrient Elements in Soil is significant.
There are a large amount of microorganisms in soil, plant rhizosphere or plant tissue, plant wherein having diseases prevention, growth-promoting or improving
The bacterial strain of the degeneration-resistant function of object is improving utilization rate of fertilizer, and the use aspect for reducing chemical pesticide has broad prospect of application, gradually
As research and development hot spot, but in terms of the document reported at present, the bacterial strain of development and application at present has a single function mostly, and soil is deposited
Motility rate is not high, therefore, in practical applications, the high efficient strain of a variety of different function is usually mixed and made into composite bacteria agent and goes to give birth to
Generation fertilizer, but the problems such as compound bacteria is there are the antagonism between microorganism, and different strains nutritional need is also not quite similar,
To cause inconvenience to practical application.Therefore, screening obtains, and there are the wild microbial strains of wide spectrum characteristic still to have
It is of great importance.
Summary of the invention
The purpose of the present invention is to provide one plant of multifunctional waters to draw engler bacterium MEM40 and its application, provides plant height effect solution
Phosphorus, fixed nitrogen, the water drawing engler bacterium MEM40 for secreting auxin and a variety of soil enzymes, are reducing fertilizer application amount, are improving soil fertilizer
Effect promotes plant growth etc. to have development prospect well.
Above-mentioned technical purpose of the invention has the technical scheme that
The present invention provides one plant of multifunctional waters to draw engler bacterium MEM40, during which has been preserved on July 11st, 2018
State's Type Tissue Collection (CCTCC), deposit number: CCTCC M2018463, preservation address: wuchang, wuhan Luo Ka
Mountain, taxology are named as water and draw engler bacterium (Rahnella aquatilis) MEM40.
Water draws engler bacterium (Rahnella aquatilis) MEM40, and for 24 hours, bacterium colony is in for 30 DEG C of cultures on LB solid medium
Milky, opaque, surface is smooth, wet, protuberance, and edge is neat, microscopically observation, and cell size is 0.9-1.0 μ m
1.5-2.0 μm, be in subsphaeroidal rod-short, and both ends blunt circle, two sides are parallel, and Gram-negative does not produce gemma, intracellular is no different dye
Grain and lipid particles；MEM40 can be grown within the scope of 1~8% NaCl concentration, contact enzyme positive, oxidase negative, lysine
Decarboxylase and ornithine decarboxylase are negative, and arginine dihydrolase is positive, phenylalanine deaminase weakly positive, using dextrin,
D-Maltose, D- trehalose, D- cellobiose, stachyose, inosine, D-Ser, D-glucitol, PEARLITOL 25C etc., Bu Nengli
With 3- methyl-D-glucose, D- fucose, pectin, there is troleandomycin, rifamycin, minocycline, mould through the ages
Element, aztreonam, lincomycin resistance；The discovery of 16s rDNA sequencing analysis, MEM40 and Rahnella aquatilis CIP
The sequence similarity highest of 78.65 (T), phylogenetic analysis show that MEM40 and Rahnella aquatilis are gathered in same point
Branch, the water draw the 16S rDNA sequence of engler bacterium MEM40 as shown in SEQ ID NO:1；It is raw based on cellular morphology, physiology
Change, the multinomial analysis of 16S rDNA sequence, MEM40 belongs to water and draws engler bacterium (Rahnella aquatilis).
The present invention provides a kind of multifunctional waters to draw application of the engler bacterium MEM40 in Plant growth-promoting effect.
Further, the plant is pakchoi, and the pakchoi fresh weight of addition MEM40 group increases 51.41%；Plant height increases
Add 23.92%；Root long increases 26.02%.
The present invention provides a kind of multifunctional waters to draw application of the engler bacterium MEM40 in insoluble inorganic phosphorus of solving problem.
Further, the slightly solubility Phos is one of calcium phosphate, magnesium phosphate and ground phosphate rock；Dissolving phosphoric acid calcium
When, titanium pigment content is up to 299.545mg/L；When dissolving phosphoric acid magnesium, titanium pigment content is up to 411.56mg/L；Dissolve phosphorus
When miberal powder, titanium pigment content is up to 289.42mg/L.
The present invention provides a kind of multifunctional waters, and engler bacterium MEM40 to be drawn to solve the application in organic phosphorus, has to lecithin
Preferable degradation capability, solvable phosphorus content is up to 28.47mg/L in soybean lecithin culture medium culture 30h, solution.
The present invention provides a kind of multifunctional waters to draw application of the engler bacterium MEM40 in fixed nitrogen, and the bacterial strain is in Nfb culture medium
After cultivating 15d, total nitrogen content in solution is up to 23.478mg/L.
The present invention provides a kind of multifunctional waters, and engler bacterium MEM40 to be drawn to produce the application in heteroauxin, trains in tryptophan
Support base culture for 24 hours, the amount that bacterial strain produces IAA at this time reaches 24.93mg/L.
The beneficial effects of the present invention are:
1. water provided by the invention draws engler bacterium MEM40 to have the function of efficient phosphate-solubilizing, fixed nitrogen, crop in soil can be improved
The available phosphorus content and nitrogen element content needed is grown, and the heteroauxin (IAA) secreted can promote plant growth, and the bacterium
The substance in a variety of soil enzymes quickening soil and ability metabolism can be generated, and then is increase soil fertility, improves soil conditions, promotes
Plant growth, and reduce Fertilizer application, especially phosphate fertilizer and nitrogenous fertilizer reduce planting cost.
2. water provided by the invention draws engler bacterium MEM40 to have while solving Phos and organic phosphorus ability, can be comprehensive
The phosphorus by indissoluble state in soil be converted into the phosphorus that form is directly absorbed and utilized for plant, improve soil available phosphorus and organic matter
Content has good prospects in the conversion aspect for improving soil phosphorus supply performance, phosphorus, can be applied to the agricultures such as biologic phosphorus fertilizer and Plant growth-promoting effect
Industry field.
3. water provided by the invention draws engler bacterium MEM40 to have a variety of growth-promoting abilities, solves multi-cultur es plant growth promoting
The problem inconsistent into antagonism between strain present in agent and nutritional need, growth-promoting effect are more preferable.
4. water provided by the invention draws engler bacterium MEM40 to be isolated from soil, for indigenous bacterium, it is applied in soil, though
It can be competed with soil autochthonous microorganism, but only of short duration, finally by symbiosis in a system, application is harmless to environment
's.
5 water provided by the invention draw engler bacterium MEM40 condition of culture and method is simple, production technology and equipment requirement
It is low.
Detailed description of the invention
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing.
Fig. 1 MEM40 colonial morphology (A) and spore staining (B) figure.
Fig. 2 MEM40 systematic evolution tree, Mycobacterium genavense 2289 are used as outgroup；
The phosphorus decomposing amount of Fig. 3 MEM40 changes with time figure；
Fig. 4 MEM40 degrades lecithin effect picture (A) and degradation lecithin curve (B)；
Upgrowth situation of Fig. 5 MEM40 on Nfb plate；(A) Escherichia coli (negative control), (B) nitrogen-fixing bacteria 20024
(positive control), (C) MEM40, (D) Methylotrophic bacillus (positive control)；
The IAA amount of Fig. 6 MEM40 secretion changes with time figure.
Fig. 7 MEM40 is to pakchoi growth promotion result figure.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with specific embodiment.Obviously, described
Embodiment be only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiment of the present invention, ability
Domain those of ordinary skill every other embodiment obtained without creative efforts, belongs to guarantor of the present invention
The range of shield.
The culture medium prescription and reagent component that embodiment is related to are as follows:
1) indissoluble Phos culture medium (PKO culture medium): glucose 10g, yeast powder 0.5g, (NH4)2SO4 0.5g、NaCl
0.3g、KCl 0.3g、MgSO4·7H2O 0.3g、FeSO4·7H2O 0.03g、MnSO4·H2O 0.03g, Insoluble phosphates
5.0g, deionized water 1L, pH 7.0-7.2 (solid medium adds agar 20g), it is spare after 115 DEG C of sterilizing 20min.
2) soybean lecithin culture medium: glucose 10g, KCl 0.3g, NaCl 0.3g, (NH4)2SO4 0.5g、FeSO4·
7H2O 0.03g、CaCO3 5g、MnSO4·4H2O 0.03g, soybean lecithin 2g, distilled water are settled to 1L, natural pH, and 115 DEG C
Sterilize 30min.
3) nitrogen-free agar (Nfb): malic acid 5g, KOH 4.5g, K2HPO40.5g, MgSO4·7H2O 0.2g,
CaCl2·2H2O 0.02g, NaCl 0.1g, bromthymol blue 2mL, Fe(3)- EDTA (1.64%) 4mL, vitamin solution (VB6
200mg L-1, biotin 100mg L-1) 1mL, microelement (CuSO4 0.4g-1, ZnSO4 0.12g-1, H2BO3 1.4g-1, two
Water sodium molybdate 1g-1, MnSO4 1.5g-1)2mL。
4) A Shi nitrogen-free fluid nutrient medium: sucrose 10g, K is improved2HPO4 0.2g、K2SO4 0.2g、MgSO4·7H2O
0.2g、NaCl 0.2g、CaCO35g, distilled water 1000mL, pH 7.0-7.2,121 DEG C of sterilizing 30min.
5) L-Trp culture medium: tryptone 10g, yeast extract 5g, NaCl 10g, PH 7.4, with thin after sterilizing
L-Trp is added to concentration 100mg/L in bacterium filter.
6) Salkowski developing solution: 50mL 35%HClO4+1mL 0.5mol/L FeCl3。
Embodiment 1
The screening and identification of MEM40
One, isolated from soil using enrichment culture method, it is specific as follows: to acquire 10g from Hubei phosphorizing treatment periphery
Soil sample is added to the PKO culture medium enrichment culture of sterilizing, is then coated on PKO plate, phosphorus decomposing circle is biggish on picking plate
Bacterium colony carries out separating for several times purifying, obtains bacterial strain of the invention, is denoted as MEM40.
Two, strain morphology feature is observed, it is specific as follows: the bacterium of purifying being inoculated in LB culture medium and is activated overnight, is lined
LB solid medium, 30 DEG C of cultures for 24 hours, observe the configuration of surface of bacterium colony, and Gram's staining observes the microscopic morphology of bacterial strain.
Three, identify bacterial strain Physiology and biochemistry property: physiological and biochemical analysis includes utilization of carbon source, produces acid experiment by Beijing
The measurement of Bao Jie los biosynthesis Science and Technology Ltd..
Four, specific as follows: general using the genomic DNA of MEM40 bacterial strain as template to bacterial strain 16S rRNA gene sequencing
Primer 1492R and 27F expand 16S rDNA sequence, are inserted into pMD18-T carrier (Takara) after purification, are transformed into E.coli
DH5 α, obtained positive colony are sent to the sequencing of Wuhan Qing Ke Bioisystech Co., Ltd, by the obtained sequence of sequencing with it is soft online
EzTaxon-e (the http://www.ezbiocloud.net/ of part EzBioCloud's Identify Service module
Identify bacterium database sequence carries out homologous comparison analysis in), using MEGA4.1 software, carries out clustering with N-J method
With the building of systematic evolution tree, sampling 1000 times is repeated, and examines chadogram calculated with (Bootstrap) method of bootstrapping
Topological structure stability, development tree node only show that Bootstrap value is greater than 50% numerical value, upper target " T " intermediate scheme bacterial strain.
Primer sequence is as follows: 1492R:5'-GGTTACCTTGTTACGACTT-3'；
27F:5'-AGAGTTTGATCCTGGCTCAG-3'.
The specific authentication information of bacterial strain MEM40 is as follows:
Morphological feature: for 24 hours, bacterium colony is creamy white for 30 DEG C of cultures on LB solid medium, and surface is smooth, wet, protuberance,
Edge is neat, microscopically observation, cell size be 1.5-2.0 μm of 0.9-1.0 μ m, be in subsphaeroidal rod-short, both ends blunt circle,
Two sides are parallel, and Gram-negative does not produce gemma, no metachromatic granule intracellular and lipid particles, see Fig. 1.
Physiology and biochemistry property: MEM40 can be grown within the scope of 1~8% NaCl concentration, contact enzyme positive, oxidizing ferment yin
Property, lysine decarboxylase and ornithine decarboxylase are negative, and arginine dihydrolase is positive, and phenylalanine deaminase weakly positive can
Utilize dextrin, D-Maltose, D- trehalose, D- cellobiose, stachyose, inosine, D-Ser, D-glucitol, PEARLITOL 25C
Deng be used as carbon source, cannot utilize 3- methyl-D-glucose, D- fucose, pectin, have troleandomycin, rifamycin, diformazan
Amine tetracycline, vancomycin, aztreonam, lincomycin resistance, the test result of specific physiological and biochemical index are as shown in table 1.
The physiological and biochemical property of 1 bacterial strain MEM40 of table
Note :+positive, W weakly positive ,-negative；
16s rDNA sequencing: the 16s rDNA of amplification is made of 1420bp, and homologous comparisons analysis is found, MEM40 and
The sequence similarity of Rahnella aquatilis CIP 78.65 (T) is 99.65%, with Rahnella oryzae J11-6
(T) sequence similarity is 98.52%, and the sequence similarity with Rahnella bruchi FRB 226 (T) is 98.14%.Into
Change tree analysis shows that MEM40 and Rahnella aquatilis are gathered in same branch, sees Fig. 2.
Based on cellular morphology, Physiology and biochemistry, the multinomial analysis of 16S rDNA sequence, MEM40 belongs to water and draws engler bacterium
(Rahnella aquatilis)。
Embodiment 2
The solution Phos effect experiment of water drawing engler bacterium MEM40
The MEM40 bacterium solution being activated overnight is seeded in 200mL PKO culture medium, 30 DEG C, 150rpm constant-temperature shaking culture,
4mL is sampled at regular intervals, and 10000rpm is centrifuged 8min.It is measured according to National Standard Method (GB 11893-89- molybdenum antimony resistance colorimetric method)
Titanium pigment content, not to be inoculated with the culture medium of bacterium solution as control, MEM40 dissolving P capacity result is shown in Fig. 3.
As seen from the figure, it being in the laundering period in 0-8h, MEM40, amount of phosphorus dissolved is lower, and after 12h, bacterial strain is in logarithmic growth phase,
Dissolution slightly solubility Phos ability is stepped up, and in 32h, the amount of MEM40 dissolving phosphoric acid calcium and magnesium phosphate reaches maximum, solution
Middle titanium pigment content respectively reaches 299.5453mg/L and 411.56mg/L；When for 24 hours, the amount of MEM40 dissolution ground phosphate rock reaches
To maximum, titanium pigment content is 289.42mg/L in solution, and later with the reduction of nutriment, thallus vigor declines even
Death, titanium pigment content gradually decreases in solution.This experimental results showed that, MEM40 being capable of slightly solubility in quick-acting dissolution soil
Phosphate such as calcium phosphate, magnesium phosphate and ground phosphate rock are absorbed and utilized with improving available phosphorus content in soil for crop, can be reduced
The usage amount of phosphate fertilizer, and soil fertility can be improved.
Embodiment 3
Water draws engler bacterium MEM40 to solve organic phosphorus effect experiment
By the MEM40 bacterium solution point sample being activated overnight to soybean lecithin plate, 28 DEG C of constant temperature are inverted culture 3d, observe bacterium colony
Whether surrounding there is obvious phosphorus decomposing circle.
The MEM40 being activated overnight is seeded in the conical flask of the fluid nutrient medium of soybean lecithin containing 200mL, 28 DEG C,
150rpm constant-temperature shaking culture, samples at regular intervals, and 8000rpm is centrifuged 5min, is measured using molybdenum antimony resistance colorimetric method solvable
The content of phosphorus, it is each to handle in triplicate, not connect bacterium solution as control.
By Fig. 4 A it is found that occurring obvious hydrolysis circle on lecithin plate around MEM40, illustrate that it has degradation lecithin
Ability, lecithin are hydrolyzed to glycerol, fatty acid, phosphoric acid and choline by the MEM40 phosphatase secreted, and choline is further broken into
Amine, carbon dioxide, organic acid and alcohol so that the thallus cultivated on lecithin plate can be formed around it is certain transparent
Circle, transparent circle diameter and strain enzyme-producing utilize the ability correlation of lecithin, the big and bright explanation of transparent circle
It is stronger that MEM40 solves organic phosphorus ability.According to the degradation lecithin curve of Fig. 4 B it is found that in 0-8h, MEM40 phosphorus decomposing amount drop
It is low；In 12-30h, solvable phosphorus content increases sharply in solution, and solvable phosphorus content is maximum in 30h solution, is
28.47mg/L；Solvable phosphorus content reduces in solution later；Show that MEM40 can use organic phosphorus and is degraded to Leaching Properties of Soluble Phosphorus,
Therefore, the organic phosphorus phosphorus for being converted into utilizing for plant absorption of slightly solubility in soil can be increased available phosphorus in soil by MEM40
Content promotes plant growth.
Embodiment 4
Water draws the nitrogen fixation effect experiment of engler bacterium MEM40
The MEM40 bacterium solution being activated overnight is centrifuged, the PBS of bacterial sediment sterilizing is washed 2-3 times, and it is flat then to line Nfb
Plate, 28 DEG C of constant temperature are inverted culture 3d, can the bacterial strain of normal growth be alternative strains of nitrogen-fixing on Nfb plate, measure it
Nitrogen fixing capacity, using the Escherichia coli of no nitrogen fixing capacity as negative control, in Chinese industrial Microbiological Culture Collection administrative center
(CICC) nitrogen-fixing bacteria bought are positive control.MEM40 is inoculated in improvement A Shi nitrogen-free fluid nutrient medium by 2% inoculation amount
In, 28 DEG C, 180rpm culture 15d take bacterium solution to filter, and not connect bacterium as control, sample is sent to through this biology of Nanjing Cavan
Technology Co., Ltd.'s detection, measuring principle is referring to national standard HJ 636-2012.
By plate result (see Fig. 5) it is found that MEM40 has certain nitrogen fixing capacity in Nfb culture medium well-grown；Training
After supporting 15d, total nitrogen content in solution 23.478mg/L shows that bacterial strain MEM40 has the ability of the nitrogen reduction ammonification of air,
Ammonia, which meets water, becomes ammonium root, and becoming plant can be absorbed the nitrogen utilized, and therefore, MEM40 can be improved the content of nitrogen in soil,
Increase soil fertility, promote plant growth, reduces nitrogenous fertilizer and use, reduce planting cost.
Embodiment 5
The producing IAA effect experiment of water drawing engler bacterium MEM40
The MEM40 bacterium solution being activated overnight is seeded in 200mL L-Trp culture medium, 30 DEG C, 180rpm constant temperature oscillation
Culture, samples at regular intervals, and 8000rpm is centrifuged 3min, and supernatant 3mL is taken to mix with isometric Salkowski developing solution,
Avoid light place 30min at room temperature measures OD530, using the 1:1 mixed liquor of distilled water and Salkowski color solution as blank control,
IAA content in supernatant is calculated according to IAA standard curve；Meanwhile precipitating being taken to be suspended with isometric deionized water, measure OD600,
Characterize the growing state of bacterial strain.
Pinkiness after bacterium solution is mixed with developing solution illustrates that bacterial strain MEM40 has the ability for producing IAA, it will be appreciated from fig. 6 that
0-24h, MEM40 active growth rapidly enter logarithmic phase, and the IAA content generated also extends at any time to be continuously improved, and in
Reach maximum value for 24 hours, the amount that bacterial strain produces IAA at this time reaches 24.93mg/L, and strain growth enters stationary phase after 34h, produces IAA's
Ability is also accordingly declined；It is found that bacterial strain produces the ability of IAA and strain growth is adapted unanimously, and after 24h, strain growth
Amount slowly decline, but the ability for producing IAA but declines rapidly, then is because culture medium nutrition exhausts, the reduction of thallus vigor causes,
This death of rear portion thallus, self-dissolving, release nutriment, breed for newborn thalli growth, and thallus vigor is risen, because
This produces IAA ability and slowly rises.
Embodiment 6
Water draws engler bacterium MEM40 promoting the application in Growth of Cabbage
Mentioning the last week takes several pakchoi seeds to carry out vernalization, is divided into seedlings, is chosen thick when seedling starts " drawing cross "
Strong, the preferable seedling replanting of upgrowth situation enters similar equipped with 3 plants of growing ways, sizes are planted in the soil by grinding, in every basin
Seedling.It is placed in 28 DEG C after transplanted seedling, cultivates in the illumination box of 12h illumination -12h dark, carries out the daily pipe such as water
Reason.It is denoted as 0d with the time for transplanting the same day, applies clear water every 7d control group, experimental group applies MEM40 fermentation liquid.After 35d, survey
Fresh weight, plant height and the root long for measuring plant, so that it is determined that MEM40 growth-promoting effect.
As shown in Figure 7, MEM40 there is preferable growth promoting function to use MEM40 compared to the control group Growth of Cabbage
The pakchoi fresh weight of fermentation liquid increases 51.41%；Plant height increases 23.92%；Root long increases 26.02%, concrete outcome
As shown in table 2.
2 MEM40 of table promotes Growth of Cabbage experimental result
It is basis and the guarantee for improving soil fertility that efficiently solution Phos and organic phosphorus bacterial strain are separated from natural environment,
Conversion and organic phosphorus biomineralization of the Phos of soil slightly solubility to titanium pigment are very heavy to the nutritional need of plant phosphorus
It wants.The application is separated to one plant of water from soil and draws engler bacterium MEM40, which can be with high-efficiency dissolution slightly solubility Phos, such as phosphorus
Sour calcium, magnesium phosphate and ground phosphate rock, while decomposing organic phosphorus ability with good again, thus can comprehensively will be difficult in soil
The phosphorus of solvent is converted into the phosphorus that form is directly absorbed and utilized for plant, improves the content and the content of organic matter of soil available phosphorus,
Promote plant growth；According to we investigate data, about water draw engler bacterium can solve problem simultaneously insoluble inorganic phosphorus with have
The report of machine phosphorus, most microorganism are only worked on one side wherein, and effect is protected from environmental larger, and the application is rich
The rich germ plasm resource of phosphate solubilizing bacteria, the bacterial strain have slightly solubility Phos and organic phosphorus ability simultaneously, are improving soil phosphorus supply
Performance, phosphorus conversion aspect have good prospects, can be applied to the agriculture fields such as biologic phosphorus fertilizer and Plant growth-promoting effect；Moreover, MEM40 has
There is good nitrogen fixing capacity, moreover it is possible to generate natural auxin heteroauxin, plant is promoted to branch out or the top bud-end shape of bud, seedling etc.
At this has also widened phosphate solubilizing bacteria in the application for promoting plant growth field, is applied not only to improve soil available phosphorus content, can also mention
High soil nitrogenous fertilizer content can also secrete heteroauxin and promote crop while promoting plant growth by enhancing soil fertility
Growth preferably improves crop quality and yield.It is raw that MEM40 solves multi-cultur es plant while having a variety of growth-promoting abilities
Antagonism and the inconsistent problem of nutritional need between strain present in long promotor.Importantly, MEM40 is isolated from soil,
For indigenous bacterium, it is applied in soil, though it can be competed with soil autochthonous microorganism, it is only of short duration, finally symbiosis is existed
In one system, application is harmless to environment.
To sum up, the application obtains one plant of efficient phosphate-solubilizing, fixed nitrogen, the water drawing engler bacterium MEM40 for secreting auxin, can be answered
For agriculture fields such as bio-feritlizer and Plant growth-promoting effects, fertilizer application amount, especially phosphate fertilizer and nitrogenous fertilizer are reduced, improves soil fertilizer
Effect promotes plant growth.
Sequence table
<110>Hubei University
<120>one plants of multifunctional waters draw engler bacterium MEM40 and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1420
<212> DNA
<213>water draws engler bacterium (Rahnella aquatilis)
<220>
<221> gene
<222> (1)..(1420)
<400> 1
acacatgcag tcgagcggca gcggaagtag cttgctactt tgccggcgag cggcggacgg 60
gtgagtaatg tctgggaaac tgcctgatgg agggggataa ctactggaaa cggtagctaa 120
taccgcatga cctcgaaaga gcaaagtggg ggatcttcgg acctcacgcc atcggatgtg 180
cccagatggg attagctagt aggtgaggta atggctcacc taggcgacga tccctagctg 240
gtctgagagg atgaccagcc acactggaac tgagacacgg tccagactcc tacgggaggc 300
agcagtgggg aatattgcac aatgggcgca agcctgatgc agccatgccg cgtgtgtgaa 360
gaaggcctta gggttgtaaa gcactttcag cgaggaggaa ggcatcacac ttaatacgtg 420
tggtgattga cgttactcgc agaagaagca ccggctaact ccgtgccagc agccgcggta 480
atacggaggg tgcaagcgtt aatcggaatt actgggcgta aagcgcacgc aggcggtttg 540
ttaagtcaga tgtgaaatcc ccgcgcttaa cgtgggaact gcatttgaaa ctggcaagct 600
agagtcttgt agaggggggt agaattccag gtgtagcggt gaaatgcgta gagatctgga 660
ggaataccgg tggcgaaggc ggccccttgg acaaagactg acgctcaggt gcgaaagcgt 720
ggggagcaaa caggattaga taccctggta gtccacgctg taaaacgatg tcgacttgga 780
ggttgtgccc ttgaggcgtg gcttccggag ctaacgcgtt aagtcgaccg cctggggagt 840
acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg 900
tggtttaatt cgatgcaacg cgaagaacct tacctactct tgacatccac ggaattcgcc 960
agagatggct tagtgccttc gggaaccgtg agacaggtgc tgcatggctg tcgtcagctc 1020
gtgttgtgaa atgttgggtt aagtcccgca acgagcgcaa cccttatcct ttgttgccag 1080
cacgtaatgg tgggaactca aaggagactg ccggtgataa accggaggaa ggtggggatg 1140
acgtcaagtc atcatggccc ttacgagtag ggctacacac gtgctacaat ggcatataca 1200
aagagaagcg aactcgcgag agcaagcgga cctcataaag tatgtcgtag tccggattgg 1260
agtctgcaac tcgactccat gaagtcggaa tcgctagtaa tcgtagatca gaatgctacg 1320
gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt gggttgcaaa 1380
agaagtaggt agcttaacct tcgggagggc gctaccactt 1420
Claims (8)
1. one plant of multifunctional water draws engler bacterium MEM40, which is characterized in that taxology is named as water and draws engler bacterium (Rahnella
Aquatilis) MEM40, deposit number are CCTCC NO.M 2018463.
2. multifunctional water as described in claim 1 draws application of the engler bacterium MEM40 in Plant growth-promoting effect.
3. multifunctional water as claimed in claim 2 draws application of the engler bacterium MEM40 in Plant growth-promoting effect, the plant is small
Chinese cabbage.
4. multifunctional water as described in claim 1 draws application of the engler bacterium MEM40 in insoluble inorganic phosphorus of solving problem.
5. multifunctional water as claimed in claim 4 draws application of the engler bacterium MEM40 in insoluble inorganic phosphorus of solving problem, feature exists
In: the slightly solubility Phos is one of calcium phosphate, magnesium phosphate and ground phosphate rock.
6. multifunctional water as described in claim 1 draws engler bacterium MEM40 solving the application in organic phosphorus.
7. multifunctional water as described in claim 1 draws application of the engler bacterium MEM40 in fixed nitrogen.
8. multifunctional water as described in claim 1 draws engler bacterium MEM40 producing the application in heteroauxin.
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