CN110184220A - One plant height imitates dissolving phosphor and dissolving potassium bacterium and its application - Google Patents
One plant height imitates dissolving phosphor and dissolving potassium bacterium and its application Download PDFInfo
- Publication number
- CN110184220A CN110184220A CN201910475323.9A CN201910475323A CN110184220A CN 110184220 A CN110184220 A CN 110184220A CN 201910475323 A CN201910475323 A CN 201910475323A CN 110184220 A CN110184220 A CN 110184220A
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- CN
- China
- Prior art keywords
- potassium
- dissolving
- culture
- soil
- decomposing
- 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.)
- Granted
Links
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- 229910052700 potassium Inorganic materials 0.000 title claims abstract description 127
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 title claims abstract description 126
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 title claims abstract description 99
- 241000894006 Bacteria Species 0.000 title claims abstract description 66
- 239000002689 soil Substances 0.000 claims abstract description 64
- 239000011574 phosphorus Substances 0.000 claims abstract description 56
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 56
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- 239000008103 glucose Substances 0.000 claims description 9
- 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 9
- 229920001817 Agar Polymers 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
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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 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
The invention discloses a plant heights to imitate dissolving phosphor and dissolving potassium bacterium, its classification naming is Bei Laisi bacillus (Bacillus velezensis), bacterial strain X-P18, it has been preserved in China typical culture collection center, deposit number is CCTCC NO:M 2019317, and the deposit date is on April 29th, 2019.Dissolving phosphor and dissolving potassium bacterium of the invention can convert the Phos of slightly solubility, potassium to high-quality phosphorus, the potassium compound being directly absorbed and utilized for plant, improve insoluble phosphorus, the biological effectiveness of potassium and phosphate fertilizer, potassium utilization rate in soil, with plant growth can be promoted, the advantages that without secondary pollution, applied widely, at low cost, and be of great significance and application value in terms of cultivating and playing soil ecology fertility, keep Agro-ecology balance.
Description
Technical field
The present invention relates to microorganisms technical fields, and in particular to a plant height imitates dissolving phosphor and dissolving potassium bacterium and its application.
Background technique
Phosphorus is one of essential element in plant growth and development process, almost participates in new old generation all in plant
Apologize for having done sth. wrong journey, such as photosynthesis, energy transmission, organic synthesis, signal transduction etc., at the same still cell amplifying nucleic acid, phosphorous enzyme,
The important component of the substances such as ATP, protein.ATP is plant energy i (in vivo) stored substance and referred to as energy currency, albumen
Matter and nucleic acid be form life material base, the substances such as enzyme participate in plant in catalysis reaction guarantee metabolism smoothly into
Row.P elements also have facilitation to the growth and development of the elongation of root system of plant, plant, improve plant to the resistance of environment.
The available P that plant can be absorbed and utilized in soil only accounts for the 2%~3% of full phosphorus amount, and most phosphorus cannot directly be planted
Object absorbs, and needs just be eventually converted into the substance that can be utilized by plant by a series of physics, chemistry and biological respinse.Soil
Phosphorus is non-renewable mineral resource in earth, and there is 1.3 hundred million hectares of farmland in China, and phosphorus content is very high in 74% arable land, but has
The content of phosphorus is imitated less than 5%, and remaining arable land then seriously lacks phosphorus.
The rich content of potassium in the earth's crust, average out to 2.6%, and can reach 3% in mineral soil, the content of potassium is in the earth's crust
The 4th is come in contained all nutrients, and the content of potassium is highest in the nutrient contained by soil.Soil
The form of diverse of potassium in earth, can be divided into mineral K, water-soluble potassium, noncommutative wavelet and exchangeable potassium.Full potassium content in soil
90% or more is to exist in the form of mineral K, and this potassium is only released from mineral and can be just absorbed and used by plants.
In the soil, by that can change in each form with some physics, chemistry or biological activity potassium and there is a kind of dynamic is flat
Weighing apparatus, therefore the existence form of the potassium in soil can be adjusted by way of artificial interference.Under normal circumstances, plant needs potassium amount
It is suitable with nitrogen, or even demand will also be more than nitrogen under special circumstances.However, in some areas, in soil, potassium content deficiency has been
An important factor for as crop yield is restricted.
Phosphate solubilizing microorganism has huge conversion capability to difficultly-soluble phosphates in soil, and can secrete needed for plant growth
Auximone substance promotes plant growth and development.The acidic materials that phosphate solubilizing bacteria generates can accelerate phosphorous insoluble organise
The decomposition of object is closed, phytase, nuclease and the phosphatase etc. of secretion can be chelated with Phos, be made not available inorganic
Phosphorus is converted into available phosphorus, promotes phosphorus element release；It is extracellular that potassium solubilizing bacteria generates a large amount of high-viscosity material one during growth and breeding
Polysaccharide and capsular polysaccharide.These glucides not only have multi-functional physiological activity, but also form soil aggregate structure
Adhesive, generally forms that small crumb structure body is more around root system, is exactly the cementing grogs of polysaccharide of cell secretion as a result, group
The formation of kernel structure make soil become dredging, it is soft, the enhancing of retain water and nutrients performance, water, gas heat are more coordinated.If using dissolving phosphor and dissolving potassium
For bacterium as bio-feritlizer, advantage is obvious: at low cost, effect is good, and sustained release is sustainable, can not only increase after application
Crop yield improves soil quality and structure, moreover it is possible to improve soil organic matter content, improve the effective use of phosphorus, potassium in soil
Rate saves fertilizer volume increase, is of great significance to keeping ecological environment to balance.
Summary of the invention
Technical problem to be solved by the present invention lies in the screenings for passing through dissolving phosphor and dissolving potassium solid medium, and providing one kind has
Degradation soil indissoluble Phos, potassium, a kind of efficient phosphate-solubilizing potassium decomposing of the high-quality phosphorus, potassium that can directly absorb is provided for plant
Bacterium.
The present invention also technical problems to be solved are to provide the application of above-mentioned dissolving phosphor and dissolving potassium bacterial strain.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
One plant height imitates dissolving phosphor and dissolving potassium bacterium, and classification naming is Bei Laisi bacillus (Bacillus velezensis), bacterium
Strain X-P18 has been preserved in China typical culture collection center (abbreviation CCTCC), and address is the Wuhan Wuhan University, China,
Postcode is 430072, and deposit number is CCTCC NO:M 2019317, and the deposit date is on April 29th, 2019.The bacterial strain is hair
Bright people is in the bacterial strain of on the May 15th, 2018 of breeding in the soil sample of farmland area near Ma'an Mountain, Anhui Province potassium mine.
The process of screening, includes the following steps:
A. the preparation of Selective agar medium
B. the primary dcreening operation of dissolving phosphor and dissolving potassium bacterium
C. the secondary screening of dissolving phosphor and dissolving potassium bacterium
D. dissolving phosphor and dissolving potassium bacterial strain ?application in leaf palm-leaf fun Chinese cabbage potting
The step a may further include: preparation phosphorus decomposing solid medium
The step b may further include: 5g soil being taken to be added to enrichment culture in the sterile water of 50ml, condition of culture 20
DEG C, 160r/min cultivate 2h；Take enrichment culture liquid by 10-1、10-2、10-3、10-4、10-5、10-6Gradient dilution is trained in phosphorus decomposing solid
It supports and is coated on base, purifying scribing line culture 5-6 times when growing single colonie is trained by Phos fluid nutrient medium, inorganic potassium liquid
The secondary screening of base is supported, to obtain efficient dissolving phosphor and dissolving potassium bacterial strain.
The step c may further include: by the strain inoculated screened in 100ml Phos fluid nutrient medium and nothing
In machine potassium fluid nutrient medium, 30 DEG C of 160r/min cultivate 4d and 7d respectively, measure the Soluble phosphorus ability of dissolving potassium of bacterial strain.
The step d may further include: the efficient phosphate-solubilizing potassium decomposing bacterial strain that screening obtains being configured to microbial inoculum and is applied to
?in leaf palm-leaf fun Chinese cabbage potting.
Bacterial strain identification method:
16sRNA sequence analysis: PCR amplification uses bacterium 16sRNA universal primer:
27F(5’-AGAGTTTGATCMTGGCTCAG-3’)
1492R(5’-GGTTACCTTGTTACGACTT-3’)
PCR reaction system (2.5 μ L): 5 × amplification buffer (5 μ L), genomic DNA (0.1 μ L),
DNTP (2 μ L), primers F (0.5 μ L), primer R (0.5 μ L), Taq archaeal dna polymerase (0.25 μ L), deionized water
(16.65μL).Reaction condition: 98 DEG C of initial denaturation 10min, 98 DEG C of deformation 10s, 58 DEG C of annealing 10s, 72 DEG C of extension 90s, totally 30
Circulation, 72 DEG C sufficiently extend 10min.
16S rDNA major part sequence is measured, as shown in SEQ ID No:1.
Strain X-P18 has the following properties:
1, colonial morphology feature:
On peptone agar medium 30 DEG C culture 1~2 day after microscope observe vegetative cell be unicellular, bar
Shape has oval gemma.30 DEG C of culture 12h thallus can be with raised growth in above-mentioned culture medium.Bacterium colony is creamy white, round,
Surface is smooth, neat in edge, sticky, intermediate projections.
2, physio-biochemical characteristics:
(1) cultivation temperature: 20-50 DEG C.
(2) it is grown in the range of pH4.5~11；
(3) it is grown in 1%~8% range of salinity
(4) Gram's staining: positive
(5) catalase: positive
(6) V-P reacts: positive
(7) Starch Hydrolysis enzyme reaction: positive
(8) anaerobic condition: positive
(9) citrate utilizes: positive
(10) sugar alcohol ferments: using mannitol, xylose, cellobiose, maltose, lactose
(11) M-R reacts: positive
3,16S rDNA sequence is analyzed
16sRNA sequence analysis: PCR amplification uses bacterium 16sRNA universal primer:
27F(5’-AGAGTTTGATCMTGGCTCAG-3’)
1492R(5’-GGTTACCTTGTTACGACTT-3’)
PCR reaction system (2.5 μ L): 5 × amplification buffer (5 μ L), genomic DNA (0.1 μ L),
DNTP (2 μ L), primers F (0.5 μ L), primer R (0.5 μ L), Taq archaeal dna polymerase (0.25 μ L), deionized water
(16.65μL).Reaction condition: 98 DEG C of initial denaturation 10min, 98 DEG C of deformation 10s, 58 DEG C of annealing 10s, 72 DEG C of extension 90s, totally 30
Circulation, 72 DEG C sufficiently extend 10min.
16S rDNA major part sequence is measured, as shown in SEQ ID No:1.The BLAST of the column website NCBI will be sequenced
The comparison of base sequence is carried out, the phylogenetic tree based on 16S rDNA complete sequence is constructed.The result shows that: bacterial strain and Bei Laisi
It is homologous that bacillus reaches 98%.So assert that the present invention uses Bei Laisi bacillus (Bacillus
Velezensis), specific location Bei Laisi bacillus velezensis X-P18.
Above-mentioned efficient phosphate-solubilizing potassium solubilizing bacteria in soil phosphorus decomposing and/or potassium decomposing application also protection scope of the present invention it
It is interior.
Wherein, efficient phosphate-solubilizing potassium solubilizing bacteria is prepared as follows into the phosphorus decomposing and/or potassium decomposing that microbial inoculum is used for soil, applied
Amount is 1.3-2.0L/m2:
It will be stored in -20 DEG C of dissolving phosphor and dissolving potassium bacterium activation, is coated on phosphorus decomposing solid medium, culture 16~for 24 hours；Picking solution
Strain on phosphorus solid medium is purified on fresh phosphorus decomposing solid medium and is crossed, and culture 16~for 24 hours；Picking phosphorus decomposing is solid
Single colonie on body culture medium, is inoculated in fermentation medium, and 30 DEG C of cultures 16~for 24 hours, expand training in fermentation medium
It supports, is 10 to bacterial concentration8-109When CFU/g, phosphorus decomposing of the fermentation liquid for soil is obtained, alternatively,
It will be stored in -20 DEG C of dissolving phosphor and dissolving potassium bacterium activation, is coated on potassium decomposing solid medium, culture 16~for 24 hours；Picking solution
Strain on potassium solid medium is purified on fresh potassium decomposing solid medium and is crossed, and culture 16~for 24 hours；Picking potassium decomposing is solid
Single colonie on body culture medium, is inoculated in fermentation medium, and 30 DEG C of cultures 16~for 24 hours, expand training in fermentation medium
It supports, is 10 to bacterial concentration8-109When CFU/g, potassium decomposing of the fermentation liquid for soil is obtained.
Wherein, the phosphorus decomposing solid culture based formulas are as follows: glucose 5g, tricalcium phosphate 2g, magnesium sulfate 0.5g, sulfate of ammoniac
0.5g, potassium chloride 1.7g, calcium carbonate 0.1g, ferric sesquichloride 0.005g, agar 20g, water 1000ml.
Fermentation medium are as follows: glucose 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, sulfate of ammoniac 0.5g, calcium carbonate 0.1g,
Ferric sesquichloride 0.005g, water 1000ml.
Potassium decomposing solid culture based formulas are as follows: sucrose 5g, feldspar in powder 3g, magnesium sulfate 0.5g, sulfate of ammoniac 0.5g, biphosphate
Sodium 3.14g, calcium carbonate 0.1g, ferric sesquichloride 0.005g, agar 20g, water 1000ml.
Application of the above-mentioned efficient phosphate-solubilizing potassium solubilizing bacteria in promotion plant growth, reduction fertilizer amount is also in protection of the invention
Within the scope of.
Above-mentioned efficient phosphate-solubilizing potassium solubilizing bacteria can be used in mixed way with charcoal, also can be used alone, and improve rhizosphere microorganism ring
Border promotes plant growth, reduces fertilizer application amount.
Efficient phosphate-solubilizing potassium solubilizing bacteria of the invention is at 30 DEG C, shaken cultivation 4 days under the conditions of 160rpm, can be by 5.0g/L tricresyl phosphate
Calcium phosphorus decomposing, water-soluble phosphorus content are 416.62-534.52mg/L；At 30 DEG C, shaken cultivation 7 days under the conditions of 160rpm, by 3.0g/
L feldspar in powder potassium decomposing, water-soluble potassium content are 2.78-3.48mg/L.
Efficient phosphate-solubilizing potassium decomposing bacterial strain of the invention can survive in the LB culture medium that pH range is 4.5-11, and highest can also
It survives in the LB culture medium of salinity 8wt%, survives under the conditions of 20-50 DEG C of temperature, there is alkali resistance, the salt tolerance of height, energy
It is enough to be applied in salt-soda soil.
The utility model has the advantages that efficient phosphate-solubilizing potassium solubilizing bacteria of the invention and its microbial inoculum being prepared have the advantage that
The present invention filters out the dissolving phosphor and dissolving potassium bacterium Bacillus with degradation slightly solubility Phos, potassium from soil
Velezensis X-P18, and solution is prepared using phosphate solubilizing bacteria Bacillus velezensis X-P18 as strain fermentation culture
Phosphorus potassium decomposing microbial inoculum, the dissolving phosphor and dissolving potassium microbial inoculum can convert slightly solubility Phos, potassium to be directly absorbed and utilized for plant it is excellent
Insoluble phosphorus in soil, the biological effectiveness of potassium and phosphorus, potash fertilizer utilization efficiency can be improved in matter phosphorus, potassium compound, saves fertilizer and increases
It produces, and soil texture can be improved, improve soil organic matter content, to giving full play to soil fertility, keep agroecological environment
Balance etc. all have extremely important meaning and application value.
Dissolving phosphor and dissolving potassium bacterium provided by the invention and its microbial inoculum not only can increase available phosphorus, potassium in soil, but also can promote
Absorption of the plant to other nutrients, dissolving phosphor and dissolving potassium bacterium can adsorb the nutrients such as zinc, copper, calcium around root system of plant,
Improve plant nutrient；But also growth regulatory substance can be secreted, promote root growth；And it after the inoculation of dissolving phosphor and dissolving potassium bacterium, is planting
The breeding of object rhizosphere raised growth, becomes the dominant bacteria of plant rhizosphere, to can inhibit or reduce the micro- life of cause of disease whithin a period of time
The growth machine meeting of object, improves the disease resistance of plant.
Dissolving phosphor and dissolving potassium bacterium provided by the invention and can by slightly solubility Phos in soil, that potassium is degraded to plant is utilizable
Soluble Inorganic Phosphorus, potassium directly discharge high-quality phosphorus, potassic fertilizer into soil, therefore there is no dirty to environmental emission in production process
Object is contaminated, dissolving phosphor and dissolving potassium microorganism is a kind of environment-friendly type fertilizer truly；And dissolving phosphor and dissolving potassium bacterium provided by the invention and
Preparation method, simple production process, high production efficiency are able to achieve industrialized production, have good economic benefits and society
Effect.
Detailed description of the invention
Fig. 1 is X-P18 Gram's staining.
Fig. 2 is X-P18 colonial morphology on LB plate.
Fig. 3 is phosphorus decomposing effect of the X-P18 in phosphorus decomposing fluid nutrient medium.
Fig. 4 is potassium decomposing effect of the X-P18 in potassium decomposing fluid nutrient medium.
Fig. 5 is X-P18 systematic evolution tree
Specific embodiment
The present invention is furture elucidated With reference to embodiment, it should be understood that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the invention, after the present invention has been read, those skilled in the art are to various equivalences of the invention
The modification of form falls within the application range as defined in the appended claims.
The screening of 1: Bei Laisi bacillus X-P18 of embodiment
(1) pedotheque source: nearby farmland is regional for Ma'an Mountain's potassium mine in Anhui city, Jiangsu Province
(2) bacterial strain screening: 5g soil is taken to be added to enrichment culture in the sterile water of 50ml, 20 DEG C of condition of culture, 160r/min
Cultivate 2h；Take enrichment culture liquid by 10-1、10-2、10-3、10-4、10-5、10-6Gradient dilution is coated on phosphorus decomposing solid medium,
Purifying scribing line culture 5-6 times when growing single colonie, by the secondary screening of Phos fluid nutrient medium, inorganic potassium fluid nutrient medium,
To obtain efficient dissolving phosphor and dissolving potassium bacterial strain.
Phos fluid nutrient medium, medium component are glucose 10g, magnesium sulfate 0.5g, sulfate of ammoniac 0.5g, calcium carbonate
0.1g, ferric sesquichloride 0.005g, tricalcium phosphate 5g, water 1000ml.
Inorganic potassium fluid nutrient medium, medium component are sucrose 10g, magnesium sulfate 0.5g, sulfate of ammoniac 0.5g, calcium carbonate
0.1g, sodium dihydrogen phosphate 3.14g, ferric sesquichloride 0.005g, feldspar in powder 3g, water 1000ml.
(3) colony morphological observation of X-P18 bacterial strain
By the X-P18 bacterial strain screened on LB solid medium 30 DEG C culture 1~2 day after carry out Gram's staining, lead to
Cross microscope observe vegetative cell be it is unicellular, it is rod-shaped, have oval gemma, as shown in Figure 1.30 DEG C in above-mentioned culture medium
Cultivating 12h thallus can be with raised growth.Bacterium colony is creamy white, and round, surface is smooth, neat in edge, sticky, intermediate projections, such as
Shown in Fig. 2.
Embodiment 2: the dissolving P capacity measurement of bacterial strain.
100ml Phos fluid nutrient medium is separately added into 250ml triangular flask, medium component is glucose 10g, sulphur
Sour magnesium 0.5g, sulfate of ammoniac 0.5g, calcium carbonate 0.1g, ferric sesquichloride 0.005g, tricalcium phosphate 5g, water 1000ml.Inoculate 2ml use
The bacteria suspension of sterile water preparation, while doing and not being inoculated with control (2ml sterile water is added in culture medium), all processing carry out 3 times
It repeats.At 30 DEG C, 160rpm vibrates 4d respectively, and culture solution is centrifuged 15min at 10000r/min, passes through molybdenum antimony resistance colorimetric method
It measures phosphorus decomposing amount (water-soluble phosphorus content).As shown in figure 3, available phosphorus content was just dramatically increased at second day, incrementss are
Between 420.95-433.94mg/L, third day is little compared with second day increments, incrementss 34.65-111.65mg/L, third
Phosphorus incrementss are basically unchanged in solution after it, and the phosphorus decomposing amount of X-P18 bacterial strain is 416.62-534.52mg/L.Dissolving phosphor and dissolving potassium bacterium X-
P18 dissolving P capacity with higher.
Embodiment 3: the ability of dissolving potassium measurement of bacterial strain.
100ml inorganic potassium fluid nutrient medium is separately added into 250ml triangular flask, medium component is sucrose 10g, sulfuric acid
Magnesium 0.5g, sulfate of ammoniac 0.5g, calcium carbonate 0.1g, sodium dihydrogen phosphate 3.14g, ferric sesquichloride 0.005g, feldspar in powder 3g, water
1000ml.The bacteria suspension that 2ml is prepared with sterile water is inoculated, while doing and not being inoculated with control (2ml sterile water is added in culture medium),
All processing carry out 3 repetitions.At 30 DEG C, 160rpm vibrates 7d respectively, and culture solution is centrifuged 15min at 10000rpm,
Pass through atomic absorption measuring potassium decomposing amount (water-soluble potassium content).As shown in figure 4, a few days ago incrementss are negative value, it may be possible to because
It is greater than the effective potassium total amount for decomposing release for effective potassium total amount that X-P18 bacterium is absorbed and utilized.From third day to the 7th day, potassium increases
Dosage becomes positive value, and curve is in rising trend, increases to 3.48mg/L from 0.12mg/L.Incrementss are 2.78-3.48mg/L.Solution
Phosphorus potassium solubilizing bacteria X-P18 has certain ability of dissolving potassium.
Embodiment 4: strain idenfication.
16sRNA sequence analysis: PCR amplification uses bacterium 16sRNA universal primer:
27F(5’-AGAGTTTGATCMTGGCTCAG-3’)
1492R(5’-GGTTACCTTGTTACGACTT-3’)
PCR reaction system (2.5 μ L): 5 × amplification buffer (5 μ L), genomic DNA (0.1 μ L),
DNTP (2 μ L), primers F (0.5 μ L), primer R (0.5 μ L), Taq archaeal dna polymerase (0.25 μ L), deionized water
(16.65μL).Reaction condition: 98 DEG C of initial denaturation 10min, 98 DEG C of deformation 10s, 58 DEG C of annealing 10s, 72 DEG C of extension 90s, totally 30
Circulation, 72 DEG C sufficiently extend 10min.
16S rDNA major part sequence is measured, as shown in SEQ ID No:1.The BLAST of the column website NCBI will be sequenced
The comparison of base sequence is carried out, constructs the systematic evolution tree based on 16S rDNA complete sequence, as shown in Figure 5.The result shows that: bacterium
It is homologous that strain with Bei Laisi bacillus reaches 98%.So assert that the present invention uses Bei Laisi bacillus (Bacillus
Velezensis), specific location Bei Laisi bacillus velezensis X-P18.
Embodiment 5: the preparation of phosphorus decomposing microbial inoculum
It will be stored in -20 DEG C of phosphate solubilizing bacteria Bacillus velezensis X-P18 activation, is coated on phosphorus decomposing solid medium
On, culture 16~for 24 hours；Strain on picking phosphorus decomposing culture medium purifies scribing line, culture 16 on fresh phosphorus decomposing solid medium
~for 24 hours；Single colonie on picking phosphorus decomposing solid medium, is inoculated in 15ml fermentation medium, and 30 DEG C of cultures 16~for 24 hours, then
Expand culture in fermentation medium, is 10 to bacterial concentration8-109When CFU/g, phosphorus decomposing microbial inoculum, amount of application 1.3-2.0L/ are obtained
m2。
The wherein phosphorus decomposing solid medium, component are as follows: glucose 5g, tricalcium phosphate 2g, magnesium sulfate 0.5g, sulfate of ammoniac
0.5g, calcium carbonate 0.1g, potassium chloride 1.7g, ferric sesquichloride 0.005g, agar 20g, water 1000ml.
Fermentation medium are as follows: glucose 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, sulfate of ammoniac 0.5g, calcium carbonate 0.1g,
Ferric sesquichloride 0.005g, water 1000ml.
Embodiment 6: the preparation of potassium decomposing microbial inoculum
It will be stored in -20 DEG C of potassium solubilizing bacteria Bacillus velezensis X-P18 activation, is coated on potassium decomposing solid medium
On, culture 16~for 24 hours；Strain on picking potassium decomposing solid medium purifies scribing line, training on fresh potassium decomposing solid medium
Feeding 16~for 24 hours；Single colonie on picking potassium decomposing solid medium, is inoculated in 15ml fermentation medium, and 30 DEG C of cultures 16~
For 24 hours, expand culture in fermentation medium, be 10 to bacterial concentration8-109When CFU/g, potassium decomposing microbial inoculum is obtained, amount of application is
1.3-2.0L/m2。
The wherein potassium decomposing solid medium, component are as follows: sucrose 5g, feldspar in powder 3g, magnesium sulfate 0.5g, sulfate of ammoniac
0.5g, sodium dihydrogen phosphate 3.14g, calcium carbonate 0.1g, ferric sesquichloride 0.005g, agar 20g, water 1000ml.
Fermentation medium are as follows: glucose 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, sulfate of ammoniac 0.5g, calcium carbonate 0.1g,
Ferric sesquichloride 0.005g, water 1000ml.
Embodiment 7: the growth characteristics of bacterial strain.
The LB culture medium that salinity mass percent is 2%, 4%, 6%, 8%, 10% is prepared, each gradient is in each test tube
5ml culture medium is dispensed, and is sterilized after doing blank control, X-P18 bacterium solution is then inoculated with, is cultivated in 30 DEG C, 160r/min shaking table
1-3 days, observe whether it can grow.
The NaOH solution for preparing 1mol/L is used to deploy the pH value of LB culture medium, and LB solution and NaOH solution are sterilized, in
The LB solution that pH is 8,9,10,11 is deployed in super-clean bench, each gradient dispenses 5ml culture medium in each sterilizing test tubes, and does sky
White control is cultivated 1-3 days in 30 DEG C, 160r/min shaking table, observes whether it can grow.
100ml LB culture medium is prepared, 5ml culture medium is dispensed in 8 test tubes, in super-clean bench after inoculating strain, respectively
It is put into 20 DEG C, 30 DEG C, 40 DEG C, 160r/min culture 1-3d in 50 DEG C of shaking table with blank control, whether observation bacterial strain can grow.
The growth characteristics of table 1.X-P18
| PH range | Whether can grow | Salinity range | Whether can grow | Temperature (DEG C) | Whether can grow |
| 8 | + | 2% | + | 20 | + |
| 9 | + | 4% | + | 30 | + |
| 10 | + | 6% | + | 40 | + |
| 11 | + | 8% | + | 50 | + |
| 12 | - | 10% | - |
As shown in Table 1, X-P18 bacterial strain can be grown in the case where pH is the range within 8-11, salinity mass percent 8%,
Growth temperature range is larger, can grow at 20-50 DEG C.
Embodiment 8: crop yield can be improved in dissolving phosphor and dissolving potassium microbial inoculum.
Flowerpot bore 140mm, the high 115mm used is tested, every basin fills soil 650g, rate of fertilizer 63.7g/m2Phosphoric acid
Tricalcium, 11.7g/m2Potassium chloride, 13.7g/m2Urea, bacterium solution amount of application are 1.3L/m2, mix fertilizer and soil thoroughly dress basin, then broadcast
Fungus scattering liquid (sterile water wash 3 times) is sowed after standing for 1 night, and every basin pakchoi sows 6 seeds (percentage of seedgermination 98%),
Top earthing 0.5cm, thinning after two weeks to Chinese cabbage growth, adds bacterium solution along plant root again to 3 after germination
(sterile water wash 3 times), bacterium solution amount of application are 1.3L/m2.Using ?leaf palm-leaf fun Chinese cabbage as experimental subjects, wherein
CK group: normal soil+fertilizer
A1 group: normal soil+fertilizer+X-P18 microbial inoculum
Each processing group harvests, plant is cut along root, is eluted with water, biomass is measured for the 30th day in plant growth.
Growth characteristics and yield effect of the 2. dissolving phosphor and dissolving potassium microbial inoculum of table to Chinese cabbage
| Experimental group | Pakchoi plant height (cm) | Pakchoi fresh weight (g/ basin) | Increase production compared with CK |
| CK | 19.63±0.27a | 26.79±0.88a | - |
| A1 | 21.95±0.42b | 32.83±0.57b | 22.55% |
As shown in Table 2, dissolving phosphor and dissolving potassium microbial inoculum can significantly improve pakchoi plant height and fresh weight, increase production with microbial inoculum comparison is not added
22.55%, illustrate that dissolving phosphor and dissolving potassium microbial inoculum can significantly improve Growth of Cabbage characteristic and yield.
Embodiment 9: dissolving phosphor and dissolving potassium microbial inoculum can reduce the application of chemical fertilizer.
A kind of efficient phosphate-solubilizing potassium solubilizing bacteria promotion crop growth, the method for reducing chemical fertilizer application test the flowerpot mouth of use
Diameter 140mm, high 115mm, every basin fill soil 650g, rate of fertilizer 63.7g/m2Tricalcium phosphate, 11.7g/m2Potassium chloride,
13.7g/m2Urea, bacterium solution amount of application are 1.3L/m2, mix fertilizer and soil thoroughly dress basin, then sow bacterium solution (sterile water wash 3
All over), it is sowed after standing for 1 night, every basin pakchoi sows 6 seeds (percentage of seedgermination 98%), top earthing 0.5cm, wait plant
Thinning after two weeks to Chinese cabbage growth, adds bacterium solution (sterile water wash 3 times), bacterium solution along plant root again to 3 after son germination
Amount of application is 1.3L/m2.Using ?leaf palm-leaf fun Chinese cabbage as experimental subjects, wherein
CK group: normal soil+fertilizer
A1 group: normal soil+fertilizer+X-P18 microbial inoculum
A2 group :+70% fertilizer+X-P18 microbial inoculum of normal soil
Each processing group harvests, plant is cut along root, is eluted with water, biomass is measured for the 30th day in plant growth.
3. dissolving phosphor and dissolving potassium microbial inoculum of table and chemical fertilizer are to potting pakchoi characteristic and yield effect
| Experimental group | Pakchoi plant height (cm) | Pakchoi fresh weight (g/ basin) | Increase production compared with CK |
| CK | 19.75±0.44b | 26.34±0.59c | - |
| A1 | 22.66±0.37a | 32.95±0.66a | 25.09% |
| A2 | 20.70±0.6b | 30.70±0.40b | 16.55% |
As shown in Table 3, dissolving phosphor and dissolving potassium microbial inoculum is mixed with fertilizer and is applied, and can promote the root development of pakchoi, to pakchoi strain
High, yield has facilitation, can increase production 25.09%.When subtracting the fertilizer for applying 30% mass, still the yield of Chinese cabbage is mentioned
Height can increase production 16.55%, this illustrates that X-P18 microbial inoculum can effectively reduce the amount of application of chemical fertilizer.
Embodiment 10: dissolving phosphor and dissolving potassium microbial inoculum improves crop yield in salt-soda soil
A kind of efficient phosphate-solubilizing potassium solubilizing bacteria promotes the method for crop growth in salt-soda soil, tests the flowerpot bore of use
140mm, high 115mm, every basin fill soil 650g, rate of fertilizer 63.7g/m2Tricalcium phosphate, 11.7g/m2Potassium chloride, 13.7g/
m2Urea, bacterium solution amount of application are 1.3L/m2, mix fertilizer and soil thoroughly dress basin, then sow bacterium solution (sterile water wash 3 times), stand
It is sowed after 1 night, every basin pakchoi sows 6 seeds (percentage of seedgermination 98%), top earthing 0.5cm, after germination
Thinning after two weeks to Chinese cabbage growth, adds bacterium solution (sterile water wash 3 times) along plant root again, bacterium solution amount of application is to 3
1.3L/m2.Alkaline land soil property is that pH is 8.3, salinity 0.5%.Using ?leaf palm-leaf fun Chinese cabbage as experimental subjects, wherein
CK group: salt affected soil+fertilizer
A1 group: normal soil+fertilizer
A2 group: normal soil+fertilizer+X-P18 microbial inoculum
Each processing group harvests, plant is cut along root, is eluted with water, biomass is measured for the 30th day in plant growth.
4. dissolving phosphor and dissolving potassium microbial inoculum of table and chemical fertilizer are to salt affected soil potting pakchoi characteristic and yield effect
| Experimental group | Pakchoi plant height (cm) | Pakchoi fresh weight (g/ basin) | Increase production compared with CK |
| CK | 17.70±0.15b | 22.68±0.50b | - |
| A1 | 20.00±0.30a | 28.02±0.80b | 23.54% |
| A2 | 20.84±0.48a | 29.94±0.38a | 32.01% |
As shown in Table 4, dissolving phosphor and dissolving potassium microbial inoculum can significantly improve pakchoi plant height and fresh weight in alkaline land soil, and be not added
Microbial inoculum comparison volume increase 32.01%, increases production 6.85% compared with normal soil, illustrates that dissolving phosphor and dissolving potassium microbial inoculum can be in alkaline land soil
Also the growth-promoting functions to pakchoi are played.
Embodiment 11: dissolving phosphor and dissolving potassium microbial inoculum and charcoal combination improve crop yield
A kind of method of efficient phosphate-solubilizing potassium solubilizing bacteria and charcoal combination promotion crop growth, tests the flowerpot bore of use
140mm, high 115mm, every basin fill soil 650g, rate of fertilizer 63.7g/m2Tricalcium phosphate, 11.7g/m2Potassium chloride, 13.7g/
m2Urea, bacterium solution amount of application are 1.3L/m2, mix fertilizer and soil thoroughly dress basin, then sow bacterium solution (sterile water wash 3 times), stand
It is sowed after 1 night, every basin pakchoi sows 6 seeds (percentage of seedgermination 98%), top earthing 0.5cm, after germination
Thinning after two weeks to Chinese cabbage growth, adds bacterium solution (sterile water wash 3 times) along plant root again, bacterium solution amount of application is to 3
1.3L/m2.Charcoal dosage is 1.5kg/m2(charcoal can be with peanut shell, soybean stalk, straw, rice husk, corncob etc.
Raw material are made).Using ?leaf palm-leaf fun Chinese cabbage as experimental subjects, wherein
CK group: normal soil+fertilizer
A1 group: normal soil+charcoal+fertilizer
A2 group: normal soil+fertilizer+X-P18 microbial inoculum
A3 group: normal soil+charcoal+fertilizer+X-P18 microbial inoculum
Each processing group harvests, plant is cut along root, is eluted with water, biomass is measured for the 30th day in plant growth.
5. dissolving phosphor and dissolving potassium microbial inoculum of table and charcoal are combined to pakchoi characteristic and yield effect
| Experimental group | Pakchoi plant height (cm) | Pakchoi fresh weight (g/ basin) | Increase production compared with CK |
| CK | 19.90±0.30b | 27.34±1.00c | - |
| A1 | 20.21±0.31b | 29.37±0.75b | 7.43% |
| A2 | 22.45±0.35a | 33.70±0.30a | 23.26% |
| A3 | 22.78±0.45a | 36.99±1.24a | 35.30% |
As shown in Table 5, charcoal can increase the yield of pakchoi to a certain extent, and dissolving phosphor and dissolving potassium microbial inoculum and charcoal
It is mixed to apply, it can more promote the root development of pakchoi, have facilitation to yield of pakchoi, 35.30% can be increased production, than individually applying
It is more significant with dissolving phosphor and dissolving potassium bacterial manure effect.
Sequence table
<110>Nanjing University of Technology
<120>one plant heights imitate dissolving phosphor and dissolving potassium bacterium and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 988
<212> DNA
<213>Bei Laisi bacillus (Bacillus velezensis)
<400> 1
ttgatgttat ctgcaagtcg agcggacaga tgggagcttg ctccctgatg ttagcggcgg 60
acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccggg 120
gctaataccg gatggttgtt tgaaccgcat ggttcaaaca taaaaggtgg cttcggctac 180
cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggca 240
acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 360
gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagta 420
ccgttcgaat agggcggtac cttgacggta cctaaccaga aagccacggc taactacgtg 480
ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg 540
ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 600
ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 660
gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 720
gaggagcgaa agcgtggggg agcgaacagg attagatacc ctggtagtcc acgccgtaaa 780
cgatgagtgc taagtgttag ggggtttccg ccccttagtg ctgcagctaa cgcattaagc 840
actccgccct ggggagtacg gtcgcaagac tggaaactca aggaattgac ggggggcccg 900
cacaagcgtg gagcatgtgg tttaattcga accacgcgaa gaaccttacc agtcttgaca 960
tcctctgaca atccctagag atagacgc 988
Claims (7)
1. a plant height imitates dissolving phosphor and dissolving potassium bacterium, classification naming is Bei Laisi bacillus (Bacillus velezensis), bacterial strain
Number X-P18, has been preserved in China typical culture collection center, and deposit number is CCTCC NO:M 2019317, preservation date
For on April 29th, 2019.
2. application of the efficient phosphate-solubilizing potassium solubilizing bacteria described in claim 1 in soil phosphorus decomposing and/or potassium decomposing.
3. application according to claim 2, which is characterized in that efficient phosphate-solubilizing potassium solubilizing bacteria is prepared as follows into microbial inoculum
For the phosphorus decomposing and/or potassium decomposing of soil, amount of application 1.3-2.0L/m2:
It will be stored in -20 DEG C of dissolving phosphor and dissolving potassium bacterium activation, is coated on phosphorus decomposing solid medium, culture 16~for 24 hours；Picking phosphorus decomposing is solid
Strain on body culture medium is purified on fresh phosphorus decomposing solid medium and is crossed, and culture 16~for 24 hours；The training of picking phosphorus decomposing solid
The single colonie on base is supported, is inoculated in fermentation medium, culture 16~for 24 hours, expand culture in fermentation medium, to bacterium solution
Concentration is 108-109When CFU/g, phosphorus decomposing of the fermentation liquid for soil is obtained, alternatively,
It will be stored in -20 DEG C of dissolving phosphor and dissolving potassium bacterium activation, is coated on potassium decomposing solid medium, culture 16~for 24 hours；Picking potassium decomposing is solid
Strain on body culture medium is purified on fresh potassium decomposing solid medium and is crossed, and culture 16~for 24 hours；The training of picking potassium decomposing solid
The single colonie on base is supported, is inoculated in fermentation medium, culture 16~for 24 hours, expand culture in fermentation medium, to bacterium solution
Concentration is 108-109When CFU/g, potassium decomposing of the fermentation liquid for soil is obtained.
4. application according to claim 3, which is characterized in that the phosphorus decomposing solid culture based formulas are as follows: glucose 5g,
Tricalcium phosphate 2g, magnesium sulfate 0.5g, sulfate of ammoniac 0.5g, potassium chloride 1.7g, calcium carbonate 0.1g, ferric sesquichloride 0.005g, agar
20g, water 1000ml.
Fermentation medium are as follows: glucose 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, sulfate of ammoniac 0.5g, calcium carbonate 0.1g, chlorination
High-speed rail 0.005g, water 1000ml.
Potassium decomposing solid culture based formulas are as follows: sucrose 5g, feldspar in powder 3g, magnesium sulfate 0.5g, sulfate of ammoniac 0.5g, sodium dihydrogen phosphate
3.14g, calcium carbonate 0.1g, ferric sesquichloride 0.005g, agar 20g, water 1000ml.
5. efficient phosphate-solubilizing potassium solubilizing bacteria described in claim 1 is promoting plant growth, is reducing the application in fertilizer amount.
6. application according to claim 5, which is characterized in that after being uniformly sprinkled into vegetable seeds into soil, top earthing
0.5cm。
7. application according to claim 5 or 6, which is characterized in that into soil, sowing fertilizers are 63.7g/m2Tricresyl phosphate
Calcium, 11.7g/m2Potassium chloride, 13.7g/m2Urea.
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