CN118006518B - Pseudomonas K1 with phosphate and potassium dissolving function and application thereof - Google Patents
Pseudomonas K1 with phosphate and potassium dissolving function and application thereof Download PDFInfo
- Publication number
- CN118006518B CN118006518B CN202410425236.3A CN202410425236A CN118006518B CN 118006518 B CN118006518 B CN 118006518B CN 202410425236 A CN202410425236 A CN 202410425236A CN 118006518 B CN118006518 B CN 118006518B
- Authority
- CN
- China
- Prior art keywords
- pseudomonas
- potassium
- phosphate
- quick
- acting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000589516 Pseudomonas Species 0.000 title claims abstract description 74
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 239000011591 potassium Substances 0.000 title claims abstract description 48
- 229910052700 potassium Inorganic materials 0.000 title claims abstract description 48
- 229910019142 PO4 Inorganic materials 0.000 title claims abstract description 35
- 239000010452 phosphate Substances 0.000 title claims abstract description 35
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 title claims abstract description 35
- 239000002689 soil Substances 0.000 claims abstract description 47
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 37
- 239000011574 phosphorus Substances 0.000 claims abstract description 37
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 37
- 239000003513 alkali Substances 0.000 claims abstract description 28
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 238000009630 liquid culture Methods 0.000 claims abstract description 13
- 238000000855 fermentation Methods 0.000 claims abstract description 12
- 230000004151 fermentation Effects 0.000 claims abstract description 12
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 27
- 150000003839 salts Chemical class 0.000 claims description 22
- 239000000843 powder Substances 0.000 claims description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 239000000725 suspension Substances 0.000 claims description 11
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 10
- 238000004090 dissolution Methods 0.000 claims description 10
- 239000011790 ferrous sulphate Substances 0.000 claims description 8
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 8
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 8
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 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 claims description 7
- 230000003381 solubilizing effect Effects 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 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 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 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 claims description 4
- 239000001506 calcium phosphate Substances 0.000 claims description 4
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 4
- 235000011010 calcium phosphates Nutrition 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000000787 lecithin Substances 0.000 claims description 4
- 229940067606 lecithin Drugs 0.000 claims description 4
- 235000010445 lecithin Nutrition 0.000 claims description 4
- 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 claims description 4
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 16
- 238000004321 preservation Methods 0.000 abstract description 12
- 244000005700 microbiome Species 0.000 abstract description 7
- 239000007787 solid Substances 0.000 description 16
- 108020004465 16S ribosomal RNA Proteins 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 238000012216 screening Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 9
- 230000012010 growth Effects 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 238000012258 culturing Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 229940099596 manganese sulfate Drugs 0.000 description 5
- 239000011702 manganese sulphate Substances 0.000 description 5
- 235000007079 manganese sulphate Nutrition 0.000 description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 5
- 230000035882 stress Effects 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000015784 hyperosmotic salinity response Effects 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000003794 Gram staining Methods 0.000 description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000005304 joining Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 230000008635 plant growth Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 238000013081 phylogenetic analysis Methods 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000586290 Suaeda salsa Species 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000013095 identification testing Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000018343 nutrient deficiency Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 229910001577 potassium mineral Inorganic materials 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to pseudomonas K1 and application thereof, in particular to pseudomonas K1 with a phosphate and potassium dissolving function and application thereof, belonging to the technical field of agricultural microorganisms. The preservation number of the pseudomonas K1 is CGMCC No.29753, and the pseudomonas K1 is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 1 month 24 of 2024. The strain obtained by the invention can obviously increase the contents of quick-acting phosphorus and quick-acting potassium in saline-alkali soil; the invention also provides a liquid culture medium suitable for the fermentation of the pseudomonas K1, and the pseudomonas K1 can be rapidly obtained in large quantity by using the culture medium, so that the strain provides excellent strain resources for the soil restoration of saline-alkali farmland, and the strain and bacterial liquid thereof have higher agricultural application prospect and economic value.
Description
Technical Field
The invention relates to pseudomonas and application thereof, in particular to pseudomonas K1 with a phosphate and potassium dissolving function and application thereof, belonging to the technical field of agricultural microorganisms.
Background
In the actual agricultural production process, the soil is gradually salinized due to excessive use of chemical fertilizers, poor water quality and the like in the traditional planting mode, so that the agricultural cultivation area is reduced year by year. For a long time, how to improve the salt resistance or salt tolerance of crops and the yield of crops in saline-alkali farmlands has been the focus of attention in the agricultural field. In the past studies it has been shown that plant tolerance to abiotic stress is not only related to genes but is also connected to the staggering of various factors in the environment. The interaction of plant root system and soil microbe forms unique micro-domain, and microbe such as bacteria, fungi, actinomycetes and the like forms stable community structure with plant in symbiotic and competitive modes, which is important for plant growth, disease resistance and stress resistance.
Soil salinization causes nutrient deficiency and is one of the main reasons for the decrease of crop yield. The total accumulated amount of nitrogen, phosphorus and potassium of the plant is obviously reduced along with the increase of the salinity of the soil. Research shows that the plant growth promoting bacteria can enhance the absorption and utilization of nutrient substances by plants. In saline-alkali soil, a large amount of phosphorus is fixed, so that the total phosphorus content in the soil is high, but the effective phosphorus content is low, and crops show a 'genetic phosphorus deficiency' symptom. Organic acid and other substances secreted by phosphate solubilizing bacteria can convert the phosphorus which is difficult to be absorbed and utilized by plants into a form which is easy to be absorbed and utilized by the plants through chelating, ion exchange, acidification and other modes, thereby providing more quick-acting phosphorus elements for the plants. The potassium-decomposing bacteria can dissolve potassium minerals in soil by releasing organic acid, so that the minerals are accelerated to be converted into soluble potassium, and the potassium-decomposing bacteria are favorable for absorption and utilization of plants.
Soil microorganisms play an important role in promoting various biological activities of the soil ecosystem and sustainable agricultural development. Plant growth promoting bacteria with salt tolerance property will show great potential in saline-alkali farmland soil improvement and actual production in the future. The salt-tolerant growth-promoting strain is easier to colonize in the saline-alkali soil and plays a growth-promoting function. At present, strain resources applicable to actual production are still relatively poor, and high-salt-tolerance culturable microorganisms with functions of dissolving phosphorus and potassium are not reported, so that the microorganisms can survive in a high-salinity environment, and the utilization rate of the saline-alkali soil can be effectively improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides pseudomonas (Pseudomonas kurunegalensis) K1 with the function of phosphate and potassium dissolution.
The invention also aims to provide application of the pseudomonas K1 with the function of phosphate and potassium dissolution in increasing quick-acting phosphorus and quick-acting potassium in saline-alkali soil.
The technical scheme adopted by the invention for achieving the purpose is as follows:
The invention provides a pseudomonas K1 with a phosphate and potassium dissolving function, wherein the pseudomonas K1 is preserved in China general microbiological culture collection center (CGMCC) No. 29753 at the 24 th month of 2024.
The invention also provides an application of the pseudomonas K1 with the functions of phosphate and potassium dissolution in increasing quick-acting phosphorus and quick-acting potassium in saline-alkali soil under the condition of salt stress.
Further, in the application process, the salt stress refers to saline-alkali soil with the mass ratio of the salt concentration in the soil not lower than 3 per mill.
The invention provides an application process of pseudomonas K1 with a phosphate and potassium dissolving function for increasing quick-acting phosphorus and quick-acting potassium in saline-alkali soil under a salt stress condition, which specifically comprises the following steps:
(1) Inoculating the activated pseudomonas K1 seed solution into a fermentation liquid culture medium for culturing to obtain a pseudomonas suspension;
(2) Diluting the Pseudomonas suspension obtained by fermentation, and applying the diluted Pseudomonas suspension into saline-alkali soil.
In the step (1), the inoculation is carried out by inoculating the activated pseudomonas seed liquid according to the inoculation amount of 1-2% by volume.
Further, in step (1), the OD600 value of the pseudomonas suspension is 0.8-1.0.
Further, in the step (1), the fermentation liquid medium contains 5g g of glucose, 5g g of sucrose, 1g g of yeast powder, 10 g g of sodium chloride, 1g g of potassium feldspar powder, 1g g of calcium phosphate, 1g of lecithin, 0.05 g g of ferrous sulfate, 0.2 g of magnesium sulfate and the pH value is adjusted to 7.5-8.0 in tap water per 1000 mL.
Further, in the step (1), the culture condition is that the culture is carried out for 12-24 hours under the conditions of 28-32 ℃ and 180 rpm.
The Pseudomonas K1 with the function of phosphate and potassium dissolving, which is obtained by screening, has the biological characteristics that: the cell shape is round or oval, the cell size is (0.5 μm to 0.7 μm) × (0.6 μm to 1.0 μm), and no spores are generated. The colony is in a regular round shape, smaller, light yellow, opaque, smooth and moist in surface, easy to pick and neat in edge when in solid culture at 28 ℃; the physiological and biochemical characteristics are as follows: gram staining is negative, aerobic, the optimal growth temperature is 28-32 ℃, the optimal growth pH value is 7.0-8.0, the optimal salt concentration is 1-3%, and the phosphate dissolving and potassium dissolving agent has the characteristics of phosphate dissolving and phosphate dissolving.
The result of measuring the 16S rRNA gene sequence of the pseudomonas K1 with the phosphate and potassium dissolving function shows that the gene length is 1425 bp, and the corresponding nucleotide sequence is shown as SEQ ID NO. 1.
The strain K1 of the present invention was determined to be highly homologous to the model strain Pseudomonas (Pseudomonas kurunegalensis) RW1P2 with sequence similarity as high as 99.44% by using BLASTN program alignment and phylogenetic analysis at the American bioengineering information center (National Center for Biotechnology Information, NCBI). The 16S rDNA sequence of 12 sequences with higher homology is selected as a reference object, and a phylogenetic tree between the strain K1 and a reference strain is constructed by using Mega 7 software by adopting a proximity method (neighbor-Joining). In the evolutionary tree, strain K1 forms a separate intra-cluster evolutionary branch with Pseudomonas (Pseudomonas kurunegalensis) RW1P2 (FIG. 7). Thus, K1 was determined to be a Pseudomonas strain (Pseudomonas kurunegalensis).
The basic method for breeding the pseudomonas K1 strain with the function of phosphate and potassium dissolving comprises the following steps:
Collecting saline-alkali soil at the saline-alkali soil in a natural protection area of yellow river delta, placing 1g of saline-alkali soil in a potassium-dissolving liquid culture medium, carrying out enrichment culture for 48 hours in a 180 rpm constant-temperature shaking incubator at 28-32 ℃, carrying out gradient dilution and coating on a potassium-dissolving solid culture plate, carrying out stationary culture at 28-32 ℃, picking single colony on the same culture plate, purifying twice to obtain a strain, and carrying out glycerol freezing preservation and bacteria preservation.
The invention relates to application of pseudomonas K1 with a function of dissolving phosphorus and potassium in increasing the content of quick-acting phosphorus and quick-acting potassium in soil, wherein the content of quick-acting phosphorus and quick-acting potassium in the soil is respectively increased by 60.7 percent and 43.5 percent, and the phosphorus dissolution index are respectively 2.2 and 1.4.
The beneficial effects of the invention are as follows:
(1) The invention discloses a pseudomonas K1 with a phosphate and potassium dissolving function, which has the characteristics of salt tolerance, phosphate dissolving, potassium dissolving and the like, has a wide salt tolerance range, can maintain normal growth under the condition of 1-3% salt concentration, and has great potential in saline-alkali soil restoration and agricultural production.
(2) The invention obtains a strain with the function of phosphate and potassium dissolution through multiple rounds of screening, and under salt stress, the strain can increase the content of quick-acting phosphorus and quick-acting potassium in soil by 60.7% and 43.5% to the maximum; the invention provides the pseudomonas K1 and a liquid culture medium suitable for the pseudomonas K1 fermentation, and the pseudomonas K1 with the function of phosphate and potassium dissolving can be rapidly obtained in a large amount by using the culture medium.
Preservation information
Preservation time: 2024, 1 month, 24 days;
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);
Preservation number: CGMCC No. 29753;
deposit unit address: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, north Star West way, no. 3, chat.Chao, beijing, city;
Postal code: 100101;
classification naming: pseudomonas kurunegalensis.
Drawings
FIG. 1 shows the cell morphology of Pseudomonas K1 under a microscope;
FIG. 2 shows the colony morphology of Pseudomonas K1;
FIG. 3 shows the growth curves of Pseudomonas K1 under different salt concentrations;
FIG. 4 is a graph showing the effect of Pseudomonas K1 on phosphorus (organophosphorus) dissolution;
FIG. 5 is a graph showing the effect of P.sp.K 1 on phosphorus dissolution (inorganic phosphorus);
FIG. 6 is a graph showing the potassium-decomposing effect of Pseudomonas K1;
FIG. 7 is a phylogenetic tree between Pseudomonas K1 and a reference strain.
Detailed Description
The present invention will be described in detail with reference to the following drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limiting in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
In the following examples, materials, reagents and the like used, unless otherwise specified, were obtained commercially.
EXAMPLE 1 screening of Pseudomonas K1
(1) Collecting root soil sample of Suaeda salsa from saline-alkali soil in natural protection area of yellow river delta, filling into clean sampling bag, marking, placing into ice box, taking back to laboratory, and preserving at-20deg.C for use. 1 g soil samples are weighed in a sterile super clean bench and placed in triangular flasks containing 20 mL potassium-dissolving screening liquid culture medium, and enrichment culture is carried out for 48 hours in a 180 rpm shaking incubator at the temperature of 28 ℃. Under the aseptic condition, respectively sucking 1 mL culture solutions in 9 mL sterile water, fully mixing, then sequentially carrying out gradient dilution to prepare 10 -1、10-3、10-5 sample solutions with different dilutions, respectively sucking 0.2mL sample solutions, coating the sample solutions on a potassium-dissolving screening solid culture medium flat plate, carrying out inversion culture in a constant temperature incubator at 28 ℃ for 1-5 days, and observing whether a degradation transparent ring is generated on the flat plate. And (3) selecting single bacterial colonies on the degrading ring, transferring the single bacterial colonies to the same potassium-degrading screening solid culture medium plate, numbering the single bacterial colonies in sequence, culturing the single bacterial colonies, and transferring and streaking the single bacterial colonies twice to obtain pure culture strains.
(2) Single colonies are picked by an inoculating loop and transferred into a test tube filled with 5 mL salt-tolerant screening liquid culture medium, and the culture is oscillated for 24-36 hours under the conditions of 28 ℃ and 180 rpm, and strain preservation and physiological and biochemical analysis are respectively carried out. The strain preservation method adopts a glycerol tube freezing preservation method, 200 mu L of glycerol (the final concentration of the glycerol is 20%) and 800 mu L of bacterial liquid are respectively added into the freezing preservation tube, and the mixture is placed in an ultralow temperature refrigerator at-80 ℃ for preservation after uniform mixing.
In the screening of the strain, one strain of the strain is circular or elliptical in form, the cell size is (0.5-0.7 μm) x (0.6-1.0 μm), and no spores are generated. The colony is regular round, light yellow, opaque, smooth and moist in surface, easy to pick and neat in edge when in solid culture at 28 ℃; the physiological and biochemical characteristics are as follows: gram staining is negative, aerobic, the optimal growth temperature is 28-32 ℃, the optimal growth pH value is 7.0-8.0, the optimal salt concentration is 1-3%, and the phosphate dissolving and potassium dissolving agent has the characteristics of phosphate dissolving and phosphate dissolving. The strain number was K1, which was initially determined as the selected strain K1 of the present invention.
The potassium-dissolving screening liquid culture medium comprises the following components: every 1000 mL distilled water contains sucrose 5 g, sodium chloride 30 g, ammonium sulfate 0.5 g, yeast powder 0.5 g, magnesium sulfate 0.3 g, disodium hydrogen phosphate 2g, manganese sulfate 0.03 g, ferrous sulfate 0.03 g and potassium feldspar 2g, and the pH is adjusted to 7.5-8.0; the composition of the potassium-dissolving screening solid culture medium is as follows: every 1000 mL distilled water contains 5 g of sucrose, 30 g of sodium chloride, 0.5 g of ammonium sulfate, 0.5 g of yeast powder, 0.3 g of magnesium sulfate, 2g of disodium hydrogen phosphate, 0.03 g of manganese sulfate, 0.03 g of ferrous sulfate, 2g of potassium feldspar and 15 g of agar powder, and the pH is adjusted to 7.5-8.0.
The composition of the salt-resistant screening liquid culture medium is as follows: every 1000 mL distilled water contains 5g of sucrose, 30g of sodium chloride, 0.5 g of ammonium sulfate, 0.5 g of yeast powder, 0.3 g of magnesium sulfate, 2 g of disodium hydrogen phosphate, 0.03 g of manganese sulfate, 0.03 g of ferrous sulfate and 2 g of potassium feldspar, and the pH is adjusted to 7.5-8.0.
Example 2 morphological observations and physiological and Biochemical characterization of Strain K1
The morphology of strain K1 was observed using an oil microscope of Nikon inverted microscope.
The culture temperature of the physiological and biochemical characteristic identification test of the strain K1 is set to 28 ℃. And meanwhile, the optimal temperature, the optimal growth pH value and the optimal salt concentration of the strain K1 are analyzed when the strain K1 grows on an LB solid medium.
The biological characteristics of strain K1 are: the cells were round or oval, and the cell sizes were (0.5 μm to 0.7 μm) x (0.6 μm to 1.0 μm) (FIG. 1). The colony is regular round, protruding, light yellow, opaque, smooth and moist on the surface, easy to pick and neat in edge (figure 2) when solid culture is carried out at 28 ℃.
The physiological and biochemical characteristics of strain K1 are: gram staining is negative, aerobic, the optimal growth temperature is 28-32 ℃, the optimal growth pH value is 7.0-8.0, and the optimal salt concentration is 1-3% (figure 3).
The result of measuring the 16S rRNA gene sequence of the strain K1 obtained by screening shows that the gene length is 1425 bp, and the corresponding nucleotide sequence is shown as SEQ ID NO. 1.
The strain selected was determined to be a Pseudomonas (Pseudomonas kurunegalensis), pseudomonas K1, by using BLASTN program alignment and phylogenetic analysis from the United states Biotechnology information center (National Center for Biotechnology Information, NCBI).
The 16S rDNA sequence of 12 sequences with higher homology is selected as a reference object, and a phylogenetic tree between the strain K1 and a reference strain is constructed by using Mega 7 software by adopting a proximity method (neighbor-Joining). In the evolutionary tree, strain K1 forms a separate intra-cluster clade with the model strain Pseudomonas kurunegalensisstrain RW P2 of Pseudomonas (FIG. 7).
The composition of the liquid culture medium for observing the bacterial forms is as follows: every 1000 mL distilled water contains peptone 10g, yeast powder 5g and sodium chloride 30 g, and the pH is regulated to 7.5-8.0.
The composition of the solid culture medium for observing the bacterial forms is as follows: every 1000 mL distilled water contains peptone 10 g, yeast powder 5g, sodium chloride 30 g and agar powder 15 g, and the pH is regulated to 7.5-8.0.
EXAMPLE 3 analysis of the Protoffee such as phosphate solubilizing and phosphate solubilizing of Strain K1
Transferring the isolated single colony of the strain K1 into a test tube filled with 5 mL liquid culture medium (the same liquid culture medium as in example 2), carrying out shake culture for 12-18 h at 28 ℃ and 180: 180 rpm, taking 10 uL seed liquid spots to connect onto an organophosphorus solid culture medium, culturing in a constant temperature incubator at 28 ℃, and observing whether a degradation transparent ring exists around the colony. The results show that after the strain K1 is cultured in the organophosphorus solid medium for 2 days, obvious degradation transparent rings appear around the colony, the degradation rings are obviously increased after the culture is continued, and the strain K1 is shown to be capable of rapidly dissolving organophosphorus and has strong phosphate dissolving characteristics (table 1 and fig. 4).
The isolated single colony of the strain K1 was transferred to a test tube containing 5 mL liquid medium (the same liquid medium as in example 2), and cultured under shaking at 28℃and 180: 180 rpm for 24 h, 10: 10 uL seed liquid was inoculated onto inorganic phosphorus solid medium, and cultured in a constant temperature incubator at 28 ℃. After 3 days of culture, a clear transparent degradation ring was produced around the colony, indicating that strain K1 was soluble in inorganic phosphorus and had phosphorus-dissolving properties (Table 2 and FIG. 5).
TABLE 1 phosphate solubilizing assay of Strain K1
TABLE 2 phosphorus solubility analysis of Strain K1
The composition of the organic phosphorus solid culture medium is as follows: every 1000 mL distilled water contains glucose 10 g, ammonium sulfate 0.5 g, sodium chloride 30 g, magnesium sulfate 0.3 g, manganese sulfate 0.03 g, potassium chloride 0.3 g, ferrous sulfate 0.03 g, lecithin 2.0 g, agar powder 15 g and pH 7.5-8.0.
The inorganic phosphorus solid culture medium comprises the following components in percentage by weight: every 1000 mL distilled water contains glucose 10 g, ammonium sulfate 0.5 g, sodium chloride 30 g, magnesium sulfate 0.3 g, manganese sulfate 0.03 g, potassium chloride 0.3 g, ferrous sulfate 0.03 g, calcium phosphate 5.0 g, agar powder 15 g and pH 7.5-8.0.
EXAMPLE 4 16S rRNA Gene identification method of Strain K1
The total genomic DNA of isolated and purified strain K1 was extracted according to the instructions of BioTeKe bacterial genome extraction kit. The total genomic DNA extracted was detected by 1% agarose gel electrophoresis at 175V for 20 minutes. The 16S rRNA gene of the isolated strain was amplified using the upstream primer 27F shown in SEQ ID No.2 (5 '-3': AGAGTTTGATCCTGGCTCAG) and the downstream primer 1492R shown in SEQ ID No.3 (5 '-3': GGTTACCTTGTTACGACTT), and the PCR reaction system and conditions are shown in tables 3 and 4.
TABLE 3 16S rRNA Gene PCR reaction System of purified Strain K1
Table 4PCR reaction program settings
The PCR products were detected by 1% agarose gel electrophoresis at 175V for 20 minutes. The PCR of the 16S rRNA gene was observed near the 1.5 kbp Marker band position using a gel imager, and after the desired band was excised, the PCR product was purified and recovered according to the instructions of the agarose gel recovery kit. And (5) sending the purified and recovered PCR product to a sequencing company for sequencing.
The result of measuring the 16S rRNA gene sequence of the strain K1 shows that the gene length is 1425 bp, and the corresponding nucleotide sequence is shown as SEQ ID NO. 1.
The 16S rRNA gene sequence obtained by sequencing is submitted to NCBI database (http:// www.ncbi.nlm.nih.gov) for BLASTN alignment, and the result shows that the similarity of the 16S rRNA of the strain and pseudomonas (Pseudomonas kurunegalensis) RW1P2 is 100%. The 16S rDNA sequence of 12 sequences with higher homology is selected as a reference object, and a phylogenetic tree between the strain K1 and a reference strain is constructed by using Mega 7 software by adopting a proximity method (neighbor-Joining). In the evolutionary tree, strain K1 and Pseudomonas mode strain Pseudomonas kurunegalensisstrain RW P2, therefore, were designated as Pseudomonas K1.
Example 5 application method of Strain K1 in increasing quick-acting phosphorus and quick-acting potassium in saline-alkali soil
(1) And (3) strain selection: pseudomonas strain K1.
(2) Activating strains: inoculating the strain on a strain activated solid culture medium, and standing and culturing for 12-24 hours at the temperature of 28-30 ℃ for later use.
(3) Seed culture: and (3) picking the bacterial colony in the step (2), inoculating the bacterial colony into a liquid seed culture medium containing 5mL, and culturing for 12-18 hours at the temperature of 28-30 ℃ under the condition of 180-rpm.
(4) Fermentation culture: inoculating the seed liquid into a triangular flask (500 mL) containing 100 mL fermentation medium with an inoculum size of 1%, and culturing at 28-30 ℃ under 180 rpm for 24-36 h to obtain a bacterial suspension (OD 600 value is 0.8) containing pseudomonas for later use.
(5) And (3) increasing the detection of quick-acting phosphorus and quick-acting potassium in the saline-alkali soil: filling saline-alkali soil with the salt concentration of 3 per mill and pouring 7 per mill of NaCl solution into a flowerpot (30 cm multiplied by 24 multiplied by cm multiplied by 9 cm), and carrying out different gradient dilution on the Pseudomonas bacteria suspension obtained by fermentation to respectively prepare 10 -1、10-2、10-3 bacteria suspensions; respectively applying two bacterial solutions with different concentrations of 20mL into soil, and simultaneously taking inactivated bacterial solution as a control; and detecting the contents of quick-acting phosphorus and quick-acting potassium in the soil after ten days. Each treatment was repeated three times.
The results show that the contents of the quick-acting phosphorus and the quick-acting potassium in the saline-alkali soil added with the K1 bacterial liquid are obviously increased compared with the control group through the detection of the physical and chemical indexes of the soil (table 5), the contents of the quick-acting phosphorus and the quick-acting potassium are respectively increased by up to 60.7% and 43.5%, and the potassium-decomposing effect is shown in figure 6.
TABLE 5 test results of quick-acting phosphorus in soil
The strain activation solid culture medium comprises the following components: every 1000 mL distilled water contains peptone 10g, yeast powder 5g, sodium chloride 10g and agar powder 15 g, and the pH is regulated to 7.5-8.0.
The liquid seed culture medium comprises the following components: every 1000 mL distilled water contains peptone 10g, yeast powder 5g and sodium chloride 10g, and the pH is regulated to 7.5-8.0.
The composition of the fermentation medium of the pseudomonas K1 is as follows: every 1000 mL tap water contains 5g g g glucose, 5g sucrose g g sucrose, 1g yeast powder g g sodium chloride 10 g g g potassium feldspar powder 1g g g calcium phosphate 1g g g lecithin, 0.05 g ferrous sulfate g g magnesium sulfate 0.2 g and the pH is adjusted to 7.5-8.0.
Claims (8)
1. The pseudomonas K1 with the function of phosphate and potassium dissolving is characterized in that the pseudomonas K1 is preserved in China general microbiological culture collection center (CGMCC) No. 29753 at the 24 th month of 2024.
2. Use of the pseudomonas K1 with a phosphate and potassium solubilizing function according to claim 1 for increasing quick-acting phosphorus and quick-acting potassium in saline-alkali soil under salt stress conditions.
3. The application of pseudomonas K1 with the function of phosphate and potassium dissolving in increasing quick-acting phosphorus and quick-acting potassium in saline-alkali soil under the condition of salt stress, wherein the salt stress refers to saline-alkali soil with the mass ratio of salt concentration in the soil not lower than 3 per mill.
4. Use of pseudomonas K1 with phosphate and potassium solubilizing function according to claim 2 or 3 for increasing the fast-acting phosphorus and fast-acting potassium in saline-alkali soil under salt stress conditions, comprising the steps of:
(1) Inoculating the activated pseudomonas K1 seed solution into a fermentation liquid culture medium for culture to obtain pseudomonas suspension;
(2) Diluting the Pseudomonas suspension obtained by fermentation, and applying the diluted Pseudomonas suspension into saline-alkali soil.
5. The application of pseudomonas K1 with the function of phosphate and potassium dissolution in increasing the quick-acting phosphorus and quick-acting potassium in saline-alkali soil under the condition of salt stress as claimed in claim 4, wherein in the step (1), the inoculation is carried out by using activated pseudomonas seed liquid according to an inoculation amount of 1-2% by volume.
6. The use of pseudomonas K1 with phosphate and potassium solubilizing function according to claim 4 for increasing fast acting phosphorus and fast acting potassium in saline soil under salt stress conditions, wherein in step (1), the pseudomonas suspension has an OD600 value of 0.8-1.0.
7. The application of pseudomonas K1 with the function of phosphate and potassium dissolution according to claim 4, wherein in the step (1), the fermentation liquid culture medium contains glucose 5g, sucrose 5g, yeast powder 1g, sodium chloride 10g, potassium feldspar powder 1g, calcium phosphate 1g, lecithin 1g, ferrous sulfate 0.05 g, magnesium sulfate 0.2 g and pH is adjusted to 7.5-8.0 in tap water per 1000 mL.
8. The application of pseudomonas K1 with the function of phosphate and potassium dissolution in increasing the quick-acting phosphorus and quick-acting potassium in saline-alkali soil under the condition of salt stress as claimed in claim 7, wherein in the step (1), the culture condition is that the culture is carried out for 12-24 hours under the conditions of 28-32 ℃ and 180 rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410425236.3A CN118006518B (en) | 2024-04-10 | 2024-04-10 | Pseudomonas K1 with phosphate and potassium dissolving function and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410425236.3A CN118006518B (en) | 2024-04-10 | 2024-04-10 | Pseudomonas K1 with phosphate and potassium dissolving function and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118006518A CN118006518A (en) | 2024-05-10 |
| CN118006518B true CN118006518B (en) | 2024-06-04 |
Family
ID=90944929
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410425236.3A Active CN118006518B (en) | 2024-04-10 | 2024-04-10 | Pseudomonas K1 with phosphate and potassium dissolving function and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118006518B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022324A (en) * | 2018-08-28 | 2018-12-18 | 西安文理学院 | A kind of Pseudomonas fluorescens and its application |
| CN109182199A (en) * | 2018-09-29 | 2019-01-11 | 中国科学院成都生物研究所 | One plant of rape pseudomonad with Plant growth promotion |
| CN109679858A (en) * | 2018-09-07 | 2019-04-26 | 山东省科学院生态研究所 | The pseudomonas fluorescens strain and its cultural method of one plant of phosphorus decomposing salt tolerant and application |
| CN117025491A (en) * | 2023-10-09 | 2023-11-10 | 山东省农业科学院 | Larens estuary pseudomonas with salt tolerance and growth promoting functions and application thereof |
-
2024
- 2024-04-10 CN CN202410425236.3A patent/CN118006518B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022324A (en) * | 2018-08-28 | 2018-12-18 | 西安文理学院 | A kind of Pseudomonas fluorescens and its application |
| CN109679858A (en) * | 2018-09-07 | 2019-04-26 | 山东省科学院生态研究所 | The pseudomonas fluorescens strain and its cultural method of one plant of phosphorus decomposing salt tolerant and application |
| CN109182199A (en) * | 2018-09-29 | 2019-01-11 | 中国科学院成都生物研究所 | One plant of rape pseudomonad with Plant growth promotion |
| CN117025491A (en) * | 2023-10-09 | 2023-11-10 | 山东省农业科学院 | Larens estuary pseudomonas with salt tolerance and growth promoting functions and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118006518A (en) | 2024-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110129208B (en) | Penicillium oxalicum with broad-spectrum acid production characteristic | |
| CN107236690B (en) | Bacillus pumilus RP01 and application thereof | |
| CN111893074B (en) | Bacillus fusiformis strain and application thereof | |
| CN106167776A (en) | A kind of can bacillus cereus (Bacillus cereus) TH 35 of heavy metal cadmium and application thereof in activating soil | |
| CN107586743B (en) | Bacillus megaterium capable of efficiently dissolving phosphorus at root zone of forest trees and application thereof | |
| CN114875016B (en) | Formulated carrier suitable for pseudomonas fluorescens and microbial inoculum thereof | |
| CN108893421B (en) | Bacillus fusiformis and application thereof in reclamation ecological reconstruction of mining area | |
| CN117025491B (en) | Larens estuary pseudomonas with salt tolerance and growth promoting functions and application thereof | |
| CN108102962B (en) | Bacillus xiamenensis and application thereof | |
| CN116536212B (en) | Serratia AWH-NS6 and application thereof in dissolving phosphorus and promoting plant growth | |
| CN109576171A (en) | Spindle lysine bacillus and its application | |
| CN117106614B (en) | Rhizosphere bacterium pseudomonas solanacearum YIM B08402, microbial agent and application thereof | |
| CN110218677B (en) | Bacillus pumilus S1419 and application thereof in potassium dissolution | |
| CN113897320B (en) | Halophyte with salt-tolerant growth-promoting function and application thereof | |
| CN106591173A (en) | Bacillus flexus HL-37 capable of activating soil heavy metal cadmium, and applications thereof | |
| CN118006518B (en) | Pseudomonas K1 with phosphate and potassium dissolving function and application thereof | |
| CN114164140B (en) | Efficient phosphorus-dissolving bacteria MQR6 and fermentation product and application thereof | |
| CN114045247B (en) | Bacillus seawater and application thereof in salinized farmland production | |
| CN107099486B (en) | Bacillus amyloliquefaciens GN03 and application thereof | |
| CN116064307A (en) | Burkholderia strain capable of efficiently dissolving phosphorus and application thereof | |
| CN102191205A (en) | Bacterial strain B1 for converting insoluble phosphate into soluble phosphate | |
| CN118028177B (en) | Bacillus pricklyash P1 and application thereof | |
| CN117467575B (en) | Brucella lupekinensis K6 with salt-tolerant growth-promoting function and application thereof | |
| CN114231425A (en) | Phosphorus-dissolving potassium-solubilizing bacterium Aspergillus niger Z8 and application thereof | |
| CN116590195B (en) | Pseudomonas, microbial preparation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |