CN117965406B - Pond copper bacteria Cupriavidus lacunae and application thereof - Google Patents
Pond copper bacteria Cupriavidus lacunae and application thereof Download PDFInfo
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- CN117965406B CN117965406B CN202410393182.7A CN202410393182A CN117965406B CN 117965406 B CN117965406 B CN 117965406B CN 202410393182 A CN202410393182 A CN 202410393182A CN 117965406 B CN117965406 B CN 117965406B
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- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 241001528480 Cupriavidus Species 0.000 title claims abstract description 29
- 229910052802 copper Inorganic materials 0.000 title claims abstract description 29
- 239000010949 copper Substances 0.000 title claims abstract description 29
- 239000003513 alkali Substances 0.000 claims abstract description 40
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 241000196324 Embryophyta Species 0.000 claims description 29
- 240000006394 Sorghum bicolor Species 0.000 claims description 20
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims description 20
- 241000894006 Bacteria Species 0.000 claims description 17
- 241000209082 Lolium Species 0.000 claims description 17
- 230000008635 plant growth Effects 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 230000001737 promoting effect Effects 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 8
- 230000000813 microbial effect Effects 0.000 claims description 6
- 241000209504 Poaceae Species 0.000 claims 2
- 239000002689 soil Substances 0.000 abstract description 52
- 244000005700 microbiome Species 0.000 abstract description 8
- 241000233866 Fungi Species 0.000 abstract description 7
- 238000009629 microbiological culture Methods 0.000 abstract description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 18
- 230000012010 growth Effects 0.000 description 17
- 239000002609 medium Substances 0.000 description 17
- 239000007788 liquid Substances 0.000 description 14
- 229910052757 nitrogen Inorganic materials 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 12
- 239000011574 phosphorus Substances 0.000 description 12
- 229910052698 phosphorus Inorganic materials 0.000 description 12
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- 235000019319 peptone Nutrition 0.000 description 7
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- 229910019142 PO4 Inorganic materials 0.000 description 6
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- 238000004090 dissolution Methods 0.000 description 6
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- 238000005259 measurement Methods 0.000 description 6
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- 239000010452 phosphate Substances 0.000 description 6
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
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- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
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- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000209072 Sorghum Species 0.000 description 1
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- 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
- RDXARWSSOJYNLI-UHFFFAOYSA-N [P].[K] Chemical compound [P].[K] RDXARWSSOJYNLI-UHFFFAOYSA-N 0.000 description 1
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- 229910000323 aluminium silicate Inorganic materials 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
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- 241000702534 bacterium H9 Species 0.000 description 1
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- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
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- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
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- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
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- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
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- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a copper bacteria Cupriavidus lacunae in a pond and application thereof. The invention aims to provide a new choice for improving the saline-alkali soil. The technical scheme of the invention is that the pond copper fungus Cupriavidus lacunae H is preserved in China general microbiological culture collection center (CGMCC) in the 10 th month 26 of 2023, and the preservation number is CGMCC No.28786.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a copper bacteria Cupriavidus lacunae in a pond and application thereof.
Background
The total area of the salinized lands of different types of the world is up to 1X 10 9 hm2, the salinized lands are widely distributed in Asia, america, europe, australia and the like, the annual rate is increased by 10 percent, the third area of the saline-alkali land area of China is about 9913 ten thousand hectares, the total area is intensively distributed in northwest, north China, northeast and coastal areas, and the total area of 23 provinces and cities is influenced to exceed 5 hundred million mu of cultivated lands, wherein the agricultural utilization value accounts for more than 10 percent of the total area of the Chinese cultivated lands.
Aiming at the defects of high salt content, strong alkalinity, poor physical structure, nutrient deficiency and the like of the saline-alkali soil, a plurality of treatment measures are proposed, and the treatment measures can be specifically classified into physical improvement measures, chemical improvement measures, biological improvement measures and the like, wherein the biological improvement has the effects of lasting and stable desalination, being beneficial to water and soil conservation and ecological balance, and is considered as the measure with the most ecological benefit at present. Biological improvement measures are classified into plant improvement measures and microorganism improvement measures. The biological improvement method has stable effect and strong durability, is increasingly applied to the improvement of saline-alkali soil, and is used for planting halophytes and salt-resistant plants on the saline-alkali soil, so that the saline-alkali soil can be desalted, and microorganisms can directly or indirectly promote the growth of the plants on the saline-alkali soil.
Microorganisms are invisible providers of soil fertility, and soil and root microorganisms interact to regulate the growth and development processes of plants. The rhizosphere growth promoting bacteria can effectively improve the stress resistance of plants by inducing the plants to establish a resistance or tolerance mechanism, wherein nitrogen in the air can be directly used as nitrogen nutrition by the nitrogen fixing bacteria; under the conditions of meta-acid and meta-alkali, the strain can keep stronger growth vigor and higher activity of the azotase, and can adapt to the change of acid and alkali of the environment by regulating self metabolism, so that the soil approaches to neutrality; the potassium decomposing bacteria can decompose inorganic minerals of insoluble aluminosilicate such as potassium feldspar, apatite and the like; the method promotes the conversion of indissolvable nutrient elements such as potassium, phosphorus, magnesium and the like into soluble nutrients, and increases the content of available nutrients in soil; promote the growth and development of crops and increase the yield. The phosphate-solubilizing bacteria can convert the phosphorus which is difficult to be absorbed and utilized by plants into an absorbable and utilized form.
At present, screening and identification of saline-alkali soil improvement microorganisms and studies on soil and restoration mechanisms thereof are still few, saline-alkali resistant and growth promoting bacteria separated from soil are mainly distributed in Bacillus, enterobacter and Pseudomonas, the original microorganisms in inner Mongolia typical salinized soil are not systematically researched, and the bacteria of the greedy copper are concentrated in medicine and research on the influence of heavy metals on soil, but the greedy copper (Cupriavidus) with saline-alkali resistant characteristics and growth promoting functions has not been reported.
Disclosure of Invention
The invention aims to provide a new choice for improving the saline-alkali soil.
The technical scheme of the invention is that the pond copper fungus Cupriavidus lacunaeH is preserved in China general microbiological culture collection center (CGMCC) in the 10 th month 26 of 2023, and the preservation number is CGMCC No.28786. Preservation address: the institute of microbiology, national academy of sciences, north Star west way 1, beijing, chaoyang, post code: 100101.
The invention also provides application of the pond copper fungus Cupriavidus lacunaeH to improvement of saline-alkali resistance of plants.
Furthermore, the invention also provides application of the copper bacteria Cupriavidus lacunaeH in the pond in promoting plant growth.
In particular, the promotion of plant growth is to promote plant height and/or increase yield.
Specifically, the plant is a gramineous plant.
Further, the gramineous plant is sorghum or ryegrass.
The invention also provides a microbial preparation for promoting plant growth, comprising fermentation products or secretions of the copper pond greedy fungus Cupriavidus lacunaeH or the copper pond greedy fungus Cupriavidus lacunaeH.
Further, the concentration of the copper greedy pond Cupriavidus lacunaeH is 10 8 CFU/mL.
In particular, the promotion of plant growth is to promote plant height and/or increase yield.
Specifically, the plant is a gramineous plant.
Further, the gramineous plant is sorghum or ryegrass.
The invention also provides a microbial preparation for improving the saline-alkali tolerance of plants, which comprises fermentation products or secretion of the pond copper bacteria Cupriavidus lacunaeH or the pond copper bacteria Cupriavidus lacunaeH.
Further, the concentration of the copper greedy pond Cupriavidus lacunaeH is 10 8 CFU/mL.
Specifically, the plant is a gramineous plant.
Further, the gramineous plant is sorghum or ryegrass.
The invention also provides application of the pond cupreobacterium Cupriavidus lacunaeH to preparation of a product for regulating the pH of soil.
The invention also provides a microbial preparation for regulating the pH of soil, comprising fermentation products or secretions of the copper greedy pool Cupriavidus lacunaeH or the copper greedy pool Cupriavidus lacunaeH.
Further, the adjusting the pH of the soil is to reduce the pH of the soil.
The invention also provides application of the pond copper bacteria Cupriavidus lacunaeH to preparation of nitrogen fixation, phosphate dissolution or/and potassium dissolution products.
The invention also provides a microbial preparation for fixing nitrogen, dissolving phosphorus or/and removing potassium, which comprises fermentation products or secretions of the copper greedy pool Cupriavidus lacunaeH or the copper greedy pool Cupriavidus lacunaeH.
The invention has the beneficial effects that: the saline-alkali tolerant bacteria in the saline-alkali soil are identified by screening, and the growth promoting function of the saline-alkali tolerant bacteria is explored, so that the saline-alkali tolerant pond copper bacteria is obtained, and the bacteria can survive and propagate in the saline-alkali soil. The strain also has the functions of nitrogen fixation, phosphate and potassium dissolution, and can regulate the pH of soil and improve the nutrients of the soil by regulating the metabolism of the strain; the plant salt and alkali tolerance can be improved, the plant growth can be promoted, and the yield can be improved.
The pond copper fungus Cupriavidus lacunaeH is preserved in China general microbiological culture Collection center (CGMCC) in the 10 th month of 2023 and the preservation number is CGMCC No.28786. Preservation address: the institute of microbiology, national academy of sciences, north Star west way 1, beijing, chaoyang, post code: 100101.
Drawings
FIG. 1 is a view showing colony growth of H9 of the present invention on beef extract peptone medium.
FIG. 2 shows the morphology of the cells after gram staining with H9 according to the invention.
FIG. 3 is a detection profile of the H9 time-of-flight mass spectrometry system of the present invention; in the mass spectrogram, the abscissa represents the mass-to-charge ratio (m/z) value of the ions, the value of the mass-to-charge ratio increases from left to right, and for ions with single charges, the value represented by the abscissa is the mass of the ions; the ordinate indicates the intensity of the ion current, i.e. the strongest ion current intensity is set to 100%, and the intensities of the other ion currents are expressed as percentages thereof.
FIG. 4 is a phylogenetic relationship between the H9 sequence of the invention and a near species.
FIG. 5 is a view showing the growth observation of sorghum seedlings planted in saline-alkali soil after inoculation with H9.
FIG. 6 is a graph showing the results of dry weight and fresh weight measurements of sorghum seedlings and ryegrass seedlings planted in saline-alkali soil after inoculation with H9.
FIG. 7 is a graph showing the measurement results of the plant height of sorghum seedlings and ryegrass seedlings planted in saline-alkali soil after inoculation with H9.
FIG. 8 is a graph showing the results of measurement of SOD activity after inoculation of H9 with sorghum seedlings and ryegrass seedlings planted in saline-alkali soil.
FIG. 9 is a graph showing the results of measuring POD activity after inoculating H9 to sorghum seedlings and ryegrass seedlings planted in saline-alkali soil.
Detailed Description
The media, reagents and equipment sources used in the examples below were as follows:
Gibbons medium: 10.0g of yeast extract, 5.0g of casein, 2.0g of potassium chloride, 3.0g of sodium citrate, 2.0g of magnesium sulfate heptahydrate, 5.0g of tryptone, about 100.0g of sodium chloride, distilled water, 1000mL, 20g of agar powder, and sterilizing by high-pressure steam at 121 ℃ for 20min.
Beef extract peptone medium: beef extract 5g, peptone 10g, naCl5g, agar powder 20g, distilled water 1000mL, pH 7.0-7.2, and steam sterilization at 121 ℃ 20 min.
LB liquid medium: 10g of peptone, 5g of yeast powder, and 10g of NaCl; 1000mL of distilled water, pH 7.0, and autoclaved at 121℃20 min.
LB solid medium: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1000mL of distilled water, 16g of agar powder, and sterilizing by high-pressure steam at 121 ℃ for 20min.
20G of Abbe culture medium :KH2PO40.2g;NaCl 0.2g;Mg/LSO4·7H2O 0.5g;CaSO40.2g;K2SO4·2H2O 0.2g;CaCO35.0g;Glucose 10.0 g/L; agar powder; distilled water, 1000mL, pH 7.0.
Meng Jinna organophosphorus solid medium: glucose 10g;(NH4)2SO40.5g;KCl 0.3g;NaCl 0.3g;FeSO4·7H2O 0.03g;MgSO4·7H2O 0.3g;MnSO4·4H2O 0.03g;CaCO35g; lecithin 0.2g, agar powder 20g, distilled water 1000mL, pH 7.0-7.5, steam sterilization at 115 ℃ for 20min.
Meng Jinna inorganic phosphorus solid medium: glucose 10g,(NH4)2SO40.5g;KCl 0.3g;NaCl 0.3g;FeSO4·7H2O 0.03g;MgSO4·7H2O 0.3g;MnSO4·4H2O 0.03g;Ca3(PO4)210g; agar powder 20g, distilled water 1000mL, pH 7.0-7.5, steam sterilization at 115 ℃ for 20min.
Silicate solid medium: sucrose 5g/L, mgSO 40.5g/L,CaCO30.1g/L,NaHPO42g/L,FeCl3 0.005.005 g/L, glass powder 1g/L, agar powder 20g and pH 7.0; autoclaving at 121℃for 20min.
Reagent instrument and equipment sources: spectrophotometry T6 New century ultraviolet visible spectrophotometer (Beijing Pu analysis general Instrument Co., ltd.), 16S rRNA gene amplification primer is general primer 27F/1492R and bacterial DNA extraction kit (Takara, japan), 2×Taq Plus PCR MASTER Mix (Takara); PCR instrument (berle); ultra clean bench SW-CJ-2F (Soviet A air technologies Co., ltd., centrifuge H2-I6K (Hunan instruments Co., ltd.), electronic balance FA1204B (Shanghai Tianmei balance instruments Co., ltd.).
Example 1 Strain screening and identification
The separation process of the saline-alkali tolerant strain comprises the following steps: collecting saline-alkali soil of Huand Haote City Hubiya village, weighing 5g of saline-alkali soil sample, dissolving in 45mL of sterile water, oscillating for 30min at 30 ℃ and 180r/min, and standing to obtain a soil stock solution. The supernatant was aspirated, diluted 10 -4~10-7 times, and sequentially spread on beef extract peptone medium and Gibbons medium at pH 9, and cultured upside down at 30℃for 5d. After 5d, several single colonies were grown on the solid medium, from which different types of single colonies were picked and repeatedly streaked for purification until purified strains of each single colony were obtained. The purified pure strain was inoculated into 10mL of LB liquid medium and cultured overnight. Mixing LB culture solution with strain and 80% glycerol at a ratio of 2:1, placing into 1.5mL centrifuge tube, and storing in-80deg.C refrigerator. After culturing the remaining strain on the slant medium at 30℃for 2d, the strain was stored in a refrigerator at 4℃for the subsequent test.
Increasing the culture medium salt concentration (NaCl) screen: naCl was added to the LB liquid medium, and the concentration was adjusted to 8 gradients of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8%. The activated strains are inoculated in LB liquid culture media with different concentrations in sequence, and each strain is provided with 3 repetitions. After inoculation, the culture medium is placed on a shaking table for 180r/min, and is cultured overnight at 30 ℃, bacterial liquid is transferred into a 96-well plate, absorbance at 620nm is measured by an enzyme-labeled instrument every 4 hours, and a growth curve is drawn.
Determination of Nitrogen fixation ability of the strain: the separated strain is streaked and inoculated on an Abdominal shell culture medium, and is subjected to inversion culture at 30 ℃. The nitrogen fixation capacity of the strain is denoted by "+". The following day of streaking was started to observe and record the growth, recording of outgrowth colonies for "++++++", one day delay was reduced by one "+", 7d was recorded and the nitrogen fixation capacity of the strain was observed.
Determination of the phosphate-solubilizing ability of the strain: the separated strains are respectively inoculated on Meng Jinna organic phosphorus solid culture medium and Meng Jinna inorganic phosphorus solid culture medium, and after being cultured for 5 days at the temperature of 30 ℃, bacteria which can be used for dissolving organic phosphorus and inorganic phosphorus are obtained through observation and screening. The obtained product is inoculated on Meng Jinna solid culture medium by toothpick, and is cultured upside down at 30deg.C until there is no obvious change in phosphate solubilizing loop. And (3) observing and measuring the size of the phosphate solubilizing ring, recording the diameter (D) of the phosphate solubilizing ring and the diameter (D) of the bacterial colony, calculating the D/D, and judging the strength of the phosphate solubilizing capability. Strains that grew but did not produce transparent circles were noted "+"; strains with transparent circle diameter and colony diameter ratio 1<D/d <1.5 were recorded as "++"; the ratio is greater than or equal to 1.5 Strain memory ++ "; the non-grown strains were excluded.
Determination of the potassium-decomposing ability of the strain: the strain is inoculated on silicate solid culture medium by toothpick, and whether transparent potassium-dissolving ring is generated around colony is observed to judge whether it has potassium-dissolving function.
Strain time-of-flight mass spectrometry system detection: and (3) picking a proper amount of thalli by using a pipetting gun head (10 mu L range, white) or a plastic inoculating loop, uniformly smearing the thalli in a sample hole of a target plate, and naturally airing the thalli at room temperature. And then dripping 1-2 mu L of matrix solution into the sample by using a pipetting gun, naturally airing the sample, and collecting a spectrum. 5-10 mg of fresh thalli are selected by a sterile inoculating loop and put into a 1.5 mL centrifuge tube (without agar); add 300 μl sterile water to suspend well. Adding 900 mu L of absolute ethyl alcohol, and uniformly mixing by vortex oscillation; 12000 Centrifuging at rpm for 2-3 min, discarding supernatant, centrifuging again for 1 min, removing residual supernatant, and drying precipitate 5min (ethanol volatilizing). Adding 80 μl of 70% formic acid aqueous solution (for example, the amount of bacteria is small, formic acid and acetonitrile should be properly reduced), mixing by pipetting, standing at room temperature for 5min (for gram positive bacteria and partial fungi can be properly prolonged); after adding 80. Mu.L of acetonitrile, thoroughly mixing, centrifuging at 12000 rpm for 3: 3 min, transferring the supernatant to a clean centrifuge tube for waiting to sample, or storing at-20deg.C. Sucking 1 mu L of extracted sample point to the target point center, naturally airing, then dripping 1 mu L of matrix solution supernatant to cover the sample, naturally airing or placing the sample in a vacuum drying target box for drying.
And (3) strain PCR identification: the extraction of nucleic acid is completed by using TGuide S-96 magnetic bead method soil genome DNA extraction kit. The detection method comprises the following steps: the concentration of the nucleic acid was measured by using an enzyme-labeled instrument (manufacturer: gene Co. GeneCompang Limited, model syngy HTX), and the integrity was measured by performing detection amplification and electrophoresis of the amplified PCR product using agarose with a concentration of 1.8%. The 16S rRNA full length of the strain was amplified with primers 27F (SEQ ID No.1, AGAGTTTGATCTTGGCTCAG) and 1492R (SEQ ID No.2, GGTTACCTTGTTACGACTT). The amplified sequence (SEQ ID No. 3) was compared with NCBI database to obtain species sequences with higher homology, and the selected sequences were subjected to maximum likelihood analysis Maximum Likelihood (ML) analysis, and phylogenetic tree was constructed using software MEGA 11. The results showed that the gene similarity of strain H9 to Cupriavidus lacunae was up to 98.52%.
SEQ ID No.3 16s rRNA
GGGGGGCTGCCTTACCATGCAAGTCGAACGGCAGCACGGGCTTCGGCCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCTGTAGTGGGGGATAACTAGTCGAAAGATTAGCTAATACCGCATACGACCTGAGGGTGAAAGCGGGGGACCGCAAGGCCTCGCGCTACAGGAGCGGCCGATGTCTGATTAGCTAGTTGGTGGGGTAAAAGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATGGCCTGGGTGAATACCCCGGGTCGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGACAGGCGTGAAATCCCCGAGCTTAACTTGGGAATGGCGCTTGTGACTGCAAGGCTAGAGTATGTCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGACGTCACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCTTCAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAACCTTACCTACCCTTGACATGCCACTAACGAAGCAGAGATGCATCATGTGCCTGAAAGGGAAAGTGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTCTAGTTGCTACGCAAGAGCACTCTAGAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTGCGTACAGAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACGCATCGTAGTCCGGATCGTAGTCTGCAACTCGACTACGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTGCCAGAAGTAGTTAGCCTAACCGCAAGGAGGGCGATACCCAGGGCTTGT.
Of 80 bacteria separated from soil, bacterial strains with salt and alkali resistance and growth promoting capacity are screened by a screening method of increasing the salt concentration (NaCl) of a culture medium, and 30 saline and alkali resistant bacteria are obtained by screening. Further verifying the ability of nitrogen fixation, phosphorus and potassium dissolution to obtain a bacterium H9 (table 1) with the ability of nitrogen fixation, phosphorus dissolution and potassium dissolution, which can grow on a culture medium with the pH value of 9.0 and the salt concentration of 10 percent. Detection by time-of-flight mass spectrometry (FIG. 3) and 16s rRNA phylogenetic tree results (FIG. 4) showed that the bacteria were copper pond (Cupriavidus lacunae).
TABLE 1 bacterial H9 has the ability to fix nitrogen, to dissolve phosphorus and to dissolve potassium
Strain numbering | Nitrogen fixation capacity | Dissolving organic phosphorus | Soluble inorganic phosphorus | Potassium decomposing ability |
H 9 | +++++ | + | + | + |
H9 was cultured on beef extract peptone medium at 30deg.C for 4d, the colony was milky white, the colony surface was smooth, moist, viscous, convex, and the edge was clean (FIG. 1). The morphology of the cells after gram staining is shown in figure 2.
In addition, the ability of H9 strain to adjust soil pH was further examined. The specific operation is as follows:
Treatment group: the ratio of natural saline-alkali soil and flower nutrient soil collected from Huand Haote Tongbiyancun is 2:1, the mixture is put in a flowerpot, the pH value of the planting material (sorghum and ryegrass) and the bacterial liquid is measured before the planting material is treated, the planting material is treated by the bacterial liquid for three times (watering is carried out every 15 days), plants are grown for 45 days, and the pH value of potting soil is measured 90 days after the planting. The method for measuring the pH value of the soil is carried out by referring to Lv Yizhong et al, soil science experiments.
Control group: the ratio of natural saline-alkali soil and flower nutrient soil collected from Huand Haote Tongbiyancun is 2:1, the mixture is put in a flowerpot, the pH value of the mixture is measured before materials (sorghum and ryegrass) are planted, only water is used for watering in the later period, and the pH value of potting soil is measured 90 days after the materials are planted. The results are shown in Table 2.
Table 2 determination of soil pH
Material | PH of soil before planting | 90 Days after planting, the pH value of the soil subjected to watering treatment | 90 Days after planting, the pH value of the soil is treated by bacterial liquid for three times |
Sorghum grain | 8.5 | 8.5 | 7.3 |
Ryegrass (ryegrass) | 8.5 | 8.5 | 7.0 |
Example 2 H9 effect on growth of sorghum and ryegrass
Potting experiment: sorghum seeds were soaked in 0.1% mercuric solution and sterilized for 10min. Washing with sterile water, placing into a culture dish with two layers of filter paper, and culturing at 30deg.C in light incubator under light/dark conditions for 12 hr. And (3) selecting seeds with consistent germination, sowing the seeds in a flowerpot with a proportion of saline-alkali soil to nutrient soil of 2:1, and selecting 40 seedlings with consistent growth vigor for experiments after 10 d.
Preparing bacterial liquid: the strain is inoculated in 5mL of LB liquid medium, cultured for 12h at 30 ℃ under 180r/min in a shaking way, inoculated in 20mL of LB liquid medium according to the inoculum size of 1%, after 12h of culture, centrifuged at 4000rpm for 10min, and the supernatant is poured out and diluted to 20mL with sterile water. Each seedling is inoculated with 2mL of bacterial liquid, no special treatment is carried out on the control, 10 bacterial liquids are arranged in parallel in each treatment, and the bacterial liquid is watered three times at intervals of 15 d.
Determination of morphological indexes: 70 days after sorghum planting, the plant height, stem thickness, fresh/dry weight above ground and fresh/dry weight below ground were measured.
Measurement of plant physiological indexes: plant Peroxidase (POD) activity measurement was carried out on pages 88 to 89 of the "plant physiology experiments guidance (5 th edition)" by Li Xiaofang and Zhang Zhiliang, and superoxide dismutase (SOD) activity measurement was carried out on crops such as Houchen, 2023.
The treatment of ryegrass refers to the treatment of sorghum.
And (3) basic data arrangement and analysis: data statistics and graphing were performed using Excel 2021, and single-factor ANOVA test analysis of variance was performed between different materials in the same treatment using SPSS 27.0.
The results are shown in FIGS. 5 to 9. Under the growth environment of saline-alkali soil, compared with a control, the growth condition of sorghum and ryegrass plants treated by the bacterial liquid of the strain is obviously more advantageous.
The results are shown in FIGS. 5 to 9. And compared with a blank control, the sorghum and ryegrass strain height, the fresh weight and SOD and the POD activity are obviously improved. The strain H9 can obviously improve the tolerance of plant sorghum and ryegrass to saline-alkali stress. Therefore, the strain H9 can promote the growth and development of sorghum in a saline-alkali rib forcing environment, so that the biomass of the sorghum is increased, and the utilization rate of saline-alkali soil is improved.
Claims (8)
1. A strain of pond copper bacteria (Cupriavidus lacunae) H9 with a preservation number of CGMCC No.28786.
2. Use of the copper (Cupriavidus lacunae) H9 of claim 1 in a pond to increase the saline-alkali tolerance of a plant.
3. Use of the copper (Cupriavidus lacunae) H9 in a pond according to claim 1 to promote plant growth in a salt and alkali stress environment.
4. A use according to claim 3, characterized in that: the promotion of plant growth is to promote plant height and/or increase yield.
5. A use according to claim 2 or 3, characterized in that: the plant is Gramineae plant.
6. The use according to claim 5, characterized in that: the Gramineae plant is sorghum or ryegrass.
7. A microbial preparation for promoting plant growth, characterized in that: comprising the copper (Cupriavidus lacunae) th 9 of claim 1.
8. The microbial preparation according to claim 7, wherein: the concentration of the copper bacteria (Cupriavidus lacunae) H9 in the pond is 10 8 CFU/mL.
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