CN112680376A

CN112680376A - Rice rhizosphere Klebsiella and application thereof

Info

Publication number: CN112680376A
Application number: CN202011588422.7A
Authority: CN
Inventors: 白洋; 张婧赢; 白波; 刘永鑫; 曲宝原
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-04-20
Anticipated expiration: 2040-12-29
Also published as: CN112680376B

Abstract

The invention discloses a Klebsiella and application thereof. The Klebsiella sp strain is Klebsiella sp (Klebsiella sp.), the strain number of the Klebsiella sp strain is R1452, and the registration number of the Klebsiella sp strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 21199. The strain can hydrolyze inorganic insoluble phosphorus and organic phosphorus, promote plant to absorb phosphorus, and can be used for soil improvement. The strain is safe to human and livestock, has no environmental pollution problem, simple culture condition, easy preservation and is suitable for development and application.

Description

Rice rhizosphere Klebsiella and application thereof

Technical Field

The invention relates to a rice rhizosphere Klebsiella and phosphorus dissolving characteristics thereof in the technical field of biology.

Background

Phosphorus is one of the essential major elements for plant growth and development, is an important element for forming nucleic acid, cytoplasmic membranes and the like, and is seriously inhibited from plant growth due to phosphorus deficiency, so that crop yield is seriously influenced due to phosphorus deficiency in agriculture. In recent years, in order to increase the grain yield, a large amount of phosphate fertilizers are applied, and due to the fact that the utilization efficiency is too low, a large amount of phosphate fertilizers which cannot be absorbed and utilized by crops are fixed in an unavailable form or enter the environment, so that environmental pollution and ecological imbalance are caused, systemic risks exist, the using amounts of chemical fertilizers and pesticides need to be reduced, residues of the chemical fertilizers and the pesticides are reduced, the environmental pollution is reduced, the food safety is guaranteed, and the ecological environment is protected.

The inorganic nutrient elements required by the plant are absorbed by the root system growing in the soil, the soil is a carrier with the most abundant microorganism content, the plant root system and part of the microorganisms form a reciprocal and beneficial symbiotic relationship in the long-term evolution process, 10-30% of the plant photosynthesis products are secreted into the soil through the root system, part of the plant photosynthesis products are used as a carbon source of the soil microorganisms, and conversely, the microorganisms play an important role in plant growth, disease resistance, stress resistance and nutrient absorption. Recent researches show that the fungus symbiotic with root systems of arabidopsis thaliana can promote the growth of arabidopsis thaliana in a culture medium only containing insoluble phosphorus, arabidopsis thaliana PHR1 coordinates phosphorus deficiency response and immune response, and mutation of some phosphorus deficiency response genes can obviously change root system microorganism groups, so that the important role of the symbiotic microorganisms with root systems in phosphorus absorption of plants is implied, and the manipulation of the symbiotic microorganisms with root systems is a potential application technology capable of improving the phosphorus absorption efficiency of the plants. In addition to fungi, bacteria also play an important role in the uptake of phosphorus by plants. A large number of bacteria exist in the soil rhizosphere, and a plurality of bacteria can play a role of growth-promoting bacteria (PGPR), and promote the growth of plants by synthesizing IAA, fixing nitrogen, dissolving phosphorus and the like. Pseudomonas (Pseudomonas), Bacillus (Bacillus), Burkholderia (Burkholderia), Rhizobium (Rhizobium), Achromobacter (Achromobacter), Agrobacterium (Agrobacterium), Aerobacter (Aerobacter), Micrococcus (Micrococcus), Flavobacterium (Flavobacterium) and Erwinia (Erwinia) are all reported to haveThe function of dissolving phosphorus. These bacteria lower the pH of the soil by releasing organic acids, via H⁺Ions, apatite or metal ions (Al)³⁺,Fe³⁺,Ca²⁺Etc.) the chelated phosphate radical is replaced for the absorption and utilization of plants. PGPR also secretes phosphatase to hydrolyze the organic form of phosphorus, which is converted to a form available to plants, thereby promoting phosphorus uptake by plants.

Due to the limitations of microorganism isolation culture and artificial recombination technology of microorganism populations, a large number of plant root system symbiotic microorganisms cannot be purified and cultured, and thus the functions of target microorganisms cannot be researched. Bai and colleagues combine a metagenome sequencing technology and systematically culture bacterial populations with 65% of root systems and 54% of leaves of arabidopsis thaliana by using a high-throughput microorganism culture system; and a microbial flora recombination system is established, and the composition and the formation process of the plant root system and leaf microorganisms under natural conditions are successfully simulated in a laboratory. The achievement breaks through the research bottleneck, makes the research on the functions of root system microorganisms possible from the level of microbial population, promotes the transformation of plant microbiome from descriptive research to functional research, and provides a basis and unprecedented opportunity for the application of microorganisms in agriculture. On the basis, more growth-promoting bacteria resources, such as phosphate solubilizing bacteria resources, can be developed.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the efficiency of phosphorus absorption of plants and/or how to improve soil.

In order to solve the technical problem, the invention firstly provides a Klebsiella sp R1452 strain.

The registration number of the Klebsiella (Klebsiellas) R1452 provided by the invention in the China general microbiological culture Collection center is CGMCC No. 21199. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 18 days in 2020, and the preservation address is No. 3 Xilu No. 1 Beijing, Chaoyang, and is named as Beijing. Hereinafter abbreviated to Klebsiella R1452.

The shape of the R1452 thallus of Klebsiella sp is short rod, the average size of the thallus is 1-2 μm × 0.5-0.8 μm, the gram stain is negative, no spore, no flagellum and no fluorescence characteristic. The bacterium is cultured on a TSB solid culture medium for 24 hours, the bacterial colony is wet, the surface is smooth and opaque, the edge is irregular, the bacterial colony is milky white, and the center is slightly yellowish. The growth temperature range of the strain is 15-40 ℃, the optimum growth temperature is 25-35 ℃, and the growth pH value is 7.0-7.6. Klebsiella (Klebsiellas) R1452 has the 16S rDNA shown in sequence 1 of the sequence listing.

Any of the following uses of the metabolites of Klebsiella R1452 or/and Klebsiella R1452 also fall within the scope of the present invention:

use of U1, klebsiella R1452 or/and a metabolite of klebsiella R1452 for hydrolysing inorganic insoluble phosphorus and organophosphorus;

the application of U2, Klebsiella R1452 or/and Klebsiella R1452 metabolites in promoting plant phosphorus absorption;

use of U3, klebsiella R1452 or/and a metabolite of klebsiella R1452 for improving soil.

In order to solve the technical problems, the invention also provides a product which contains the Klebsiella R1452 or/and the metabolites of the Klebsiella R1452.

The product can be a microbial inoculum, a microbial ecological agent containing the microbial inoculum, or a biological fertilizer containing the microbial inoculum or the microbial ecological agent.

The product can be any one of the following products:

v1, products of hydrolysing inorganic insoluble phosphorus and organic phosphorus;

v2, product for promoting phosphorus absorption of plants;

v3, product for improving soil.

The active ingredient of the product can be Klebsiella R1452 or/and metabolites of Klebsiella R1452, and the active ingredient of the product can also contain other biological or non-biological components, and the other active ingredients of the product can be determined by those skilled in the art according to the effect of the product.

The product may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material may be at least one of grass peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material is at least one of straws, pine shells, rice straws, peanut shells, corn flour, bean flour, starch, grass peat and animal manure of various crops; the liquid carrier can be water; in the product, the Klebsiella R1452 or/and the metabolite of Klebsiella R1452 may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and filtrate. The preparation formulation of the product can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the product can also be added with surfactant (such as Tween 20, Tween 80, etc.), binder, stabilizer (such as antioxidant), pH regulator, etc.

Any of the following applications of the above products also fall within the scope of the present invention:

x1, the use of said products for hydrolyzing inorganic insoluble phosphorus and organic phosphorus;

x2, the use of said product for promoting phosphorus uptake in plants;

x3, and the application of the product in soil improvement.

The metabolite of the Klebsiella R1452 may be a fermentation broth of Klebsiella R1452, as described above. The fermentation broth of Klebsiella R1452 can be prepared as follows: the Klebsiella R1452 is cultured in a liquid fermentation medium, and a fermentation broth (containing the Klebsiella R1452 and substances secreted into the liquid medium) which is a metabolite of the Klebsiella R1452 is collected.

A culture of Klebsiella R1452 is also within the scope of the present invention. The culture of Klebsiella R1452 is a substance obtained by culturing Klebsiella R1452 in a microorganism culture medium (e.g., a fermentation broth containing Klebsiella R1452 and a substance secreted into a liquid medium, or a solid medium containing Klebsiella R1452 and a substance secreted into a solid medium).

The culture of the Klebsiella R1452 has at least one of the following functions W1-W3:

w1, hydrolyzed inorganic insoluble phosphorus and organic phosphorus;

w2, promoting plant phosphorus absorption;

w3, improving soil.

The method for culturing the Klebsiella R1452 also belongs to the protection scope of the invention.

The method for culturing the Klebsiella R1452 comprises the step of culturing the Klebsiella R1452 in a culture medium.

The method for preparing the product also belongs to the protection scope of the invention.

The method for preparing the product comprises the step of taking the Klebsiella R1452 and/or the metabolites of the Klebsiella R1452 as the components of the product to obtain the product.

In the method, the product can be a liquid microbial inoculum. In the method, the Klebsiella R1452 can be cultured in a fermentation medium to obtain a fermentation liquid, and the fermentation liquid is mixed with a carrier to obtain the liquid microbial inoculum. The fermentation medium may consist of: tryptone 17.0g/L, soybean papain digest 3.0g/L, sodium chloride 5.0g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, pH 7.0.

Experiments prove that the Klebsiella R1452 can hydrolyze both inorganic insoluble phosphorus and organic phosphorus, can promote plants to absorb phosphorus, and can be used for soil improvement. The Klebsiella R1452 is safe to human and livestock, has no environmental pollution problem, simple culture condition, easy preservation, and is suitable for development and application.

Biological material preservation instructions

Taxonomic nomenclature of biological materials: klebsiella sp

Latin literature name of biomaterial: klebsiella sp.

Strain number of biological material: r1452

The preservation unit is called as follows: china general microbiological culture Collection center

The preservation unit is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: year 2020, 11 and 18

The preservation number is: CGMCC No.21199

Drawings

FIG. 1 shows the phosphorus dissolution circle and the phosphorus dissolution curve of Klebsiella R1452 for dissolving calcium phosphate.

FIG. 2 shows the phosphorus-solubilizing circle and phosphorus-solubilizing curve of Klebsiella R1452 for solubilizing phytin.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.

In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Example 1 isolation and characterization of Klebsiella sp R1452 CGMCC No.21199

First, the separation of Klebsiella sp R1452 CGMCC No.21199

Klebsiella sp R1452 CGMCC No.21199 is isolated from the rhizosphere of rice. The rice rhizosphere sample is collected from 2016 and 10 months in China Beijing Chang plain area.

1. Isolation culture of rice root system microorganism

Digging a rice plant which grows to about 8 weeks from the field, cutting root system tissues which are about 15cm away from a root-stem joint, washing the root system with deionized water, sucking water on the surface of the root system with filter paper, shearing the root system into small sections of 2mm, uniformly mixing, putting 0.02g of the root system small sections into a 1.5mL centrifugal tube, adding 200uL of sterile water, grinding the root system tissues into homogenate with a grinding rod, performing gradient dilution on the homogenate by 10 times, 100 times, 1000 times, 10000 times and 100000 times, transferring 160uL of diluent into a bacterial culture plate with a pipettor, diluting 45 culture plates with each gradient, covering a cover for sealing, and culturing at room temperature.

The bacterial culture plate is placed for about 3 weeks, and the gradient of turbidity of 30-40% of the wells of each plate is reserved for identification. A part of the cultured bacteria in each well was taken out for identification of the bacteria, and 80% (v/v) glycerol was added to the remaining cultured bacteria at a volume ratio of 1:1, and the mixture was stored at-80 ℃ for further use.

2. Identification and strain comparison of isolated bacteria

6uL of cultured bacteria is taken out and put into a 96-hole PCR plate, 10uL of buffer solution I (the solutes are 25mM NaOH and 0.2mM EDTA; the solvent is water; pH is 12) is added, after uniform mixing, DNA is extracted in a PCR instrument at the temperature of 95 ℃ for 30min, and 10uL of buffer solution II (the solutes are 40mM Tris-HCl; the solvent is water; pH is 7.5) is added, so that template DNA is obtained by extraction.

Two-step PCR was performed on template DNA, the first step using 799F and 1193R as primers, the second step using 799F containing one of 96 well barcodes and 1193R containing a plate barcode as primers (ligation Zhang, Yong-Xin Liu, NaZhang, Bin Hu, Tao Jin, HaoranXu, Yuan Qin, Pengxu Yan, XialongZhang, Xiaoxuan Guo, Jinghui, Shouyun Cao, Xin Wang, Hui Wang, Baoyuuan Qu, Guangyi Fan, Lixing Yuan, Bengan Garrido-Oter, Chengcai Chu, Yang Bai, NRT 1.1is tissue and transforming gene 7), and the bacterial library was identified by amplification of Nature S-12 rRNA.

Sequencing on Hiseq 2500 platform. Sequencing the 16S rRNA gene sequence of the bacteria in each hole, and performing Blast comparison at NCBI to obtain the comparison strain of each bacteria. The sequence of the 16S rRNA gene of one of the bacteria with the strain number R1452 (hereinafter referred to as the strain R1452) is shown as sequence 1 in the sequence table.

Identification of Klebsiella sp R1452 CGMCC No.21199

The strain R1452 separated above is identified by biological characteristics and morphological observation, and the method is as follows:

the specific method for observing the morphology, size, roughness and edges of bacterial colonies, gram staining and testing physiological and biochemical characteristics refers to the handbook for identifying common bacterial systems. The results showed that the strain R1452 had a short rod-like shape, an average size of 1-2. mu. m.times.0.5-0.8. mu.m, a gram-negative stain, no spores, no flagella, and no fluorescence. The bacterium is cultured on a TSB solid culture medium for 24 hours, the bacterial colony is wet, the surface is smooth and opaque, the edge is irregular, the bacterial colony is milky white, and the center is slightly yellowish. The growth temperature range of the strain is 15-40 ℃, the optimum growth temperature is 25-35 ℃, and the growth pH value is 7.0-7.6. Klebsiella sp R1452 has 16S rDNA shown in sequence 1 in the sequence table, and the similarity of the R145216S rDNA sequence of the strain and the strain of Klebsiella reaches 99%. Strain R1452 uses mainly glucose as carbon source.

Blast alignment identified strain R1452 as klebsiella (Klebsiellasp.) by the above morphological characteristics, culture characteristics, physiobiochemical characteristics, 16S rDNA sequence. Klebsiella sp (Klebsiella sp.) R1452 has been deposited at CGMCC (CGMCC) at 11/18/2020, with the accession number of CGMCC No. 3, which is CGMCC No.21199 at the CGMCC. Hereinafter abbreviated to Klebsiella R1452.

Example 2 Klebsiella R1452 hydrolysis of inorganic insoluble phosphorus

The Klebsiella R1452 is streaked and activated on a 1/2TSB solid culture medium plate, sealed and placed in an incubator at 28 ℃ for about 3 days, then a single colony is picked up and placed in a 1/2TSB liquid culture medium, and the single colony is placed in a shaker at 28 ℃ and cultured at 180rpm for about 3 days, so as to obtain fermentation liquor. Subjecting the fermentation broth to fermentation at a temperature higher thanCentrifuging at 2900g speed for 10min, discarding supernatant in a super clean bench, adding 25ml sterile water, and mixing by vortex. Repeating the above steps for 2 times, re-suspending with 15ml sterile water for the last time, vortex, taking out 1ml, and measuring bacterial OD with spectrophotometer_600nmValue, recorded and diluted to OD_600nmA bacterial solution of klebsiella R1452 was obtained at 0.5, and the content of klebsiella R1452 in the bacterial solution of klebsiella R1452 was 5 × 10⁸cfu/mL。

Wherein, the 1/2TSB solid culture medium is prepared according to the following method: 7.5g of tryptone, 2.5g of soybean peptone, 2.5g of sodium chloride and 20g of agar, wherein the volume is determined to be 1L by using distilled water, the pH value is adjusted to be 7.2 +/-0.2, and the mixture is used after being sterilized by pressure steam at the temperature of 121 ℃.

Wherein, the 1/2TSB liquid culture medium is prepared according to the following method: 7.5g of tryptone, 2.5g of soybean peptone and 2.5g of sodium chloride, wherein the volume is determined to be 1L by using distilled water, the pH value is adjusted to be 7.2 +/-0.2, and the tryptone, the soybean peptone and the sodium chloride are used after being sterilized by pressure steam at 121 ℃.

Preparing insoluble inorganic phosphorus solid culture medium, wherein the inorganic phosphorus is selected from calcium phosphate, and the formula is shown in table 1:

TABLE 1 formulation of insoluble inorganic phosphorus solid NBRIP Medium (1L)

Reagent	Company goods number	Dosage of
			Glucose	Sigma-Aldrich G8270	10g
MgCl₂·6H₂O	Sigma-Aldrich M2670	5g
			MgSO₄·7H₂O	Sigma-Aldrich M2643	0.25g
KCl	Sigma-Aldrich P3911	0.2g
			(NH₄)₂SO₄	Sigma-Aldrich A4418	0.1g
Calcium phosphate	Sigma-Aldrich 21218	1.00058g
			Agar	Solarbio A8190	12g

The insoluble inorganic phosphorus solid medium of Table 1 was added to 20ul of the bacterial solution of the above Klebsiella R1452. Adding the bacteria solution, covering with a cover, sealing with parafilm sealing film after the bacteria solution is completely dried, and culturing upside down. The bacteria liquid is added to the center of the area without splashing so as not to influence the observation of the effect of the phosphorus dissolving ring. 3 replicates were set. Culturing at 28 deg.C for about 3 days, and increasing the diameter of phosphorus-dissolving ring to increase the phosphorus-dissolving capacity. The results show that the Klebsiella R1452 can generate a phosphorus-solubilizing ring on the insoluble inorganic phosphorus solid medium (the left figure of FIG. 1), and the phosphorus-solubilizing ring is obvious.

Preparing an insoluble inorganic phosphorus liquid culture medium, wherein the inorganic phosphorus is selected from calcium phosphate, and the formula is shown in table 2:

TABLE 2 insoluble inorganic phosphorus liquid basal Medium formulation

The insoluble inorganic phosphorus liquid basic culture medium is filled into 6 conical flasks of 100ml, 30ml of each flask is respectively added with 0.03g of calcium phosphate, and the mixture is sterilized by steam under the pressure of 121 ℃ for standby. Adding 300ul of bacterial liquid with OD of 0.5 into 3 bottles of culture medium, using the other three bottles as blank control, placing the bottles in a shaking table at 28 ℃ and culturing at 180rpm, sampling the culture medium at 0h, 10h, 20h, 30h, 40h and 50h respectively, taking three samples in each bottle, wherein each sample is 200ul, filtering by using a membrane with the diameter of 0.22um, storing in a refrigerator at-20 ℃, and repeating the steps as the technology; biological replicates were used between the three flasks.

0 mg.L is prepared by using a phosphorus standard stock solution^-1、0.15mg·L^-1、0.25mg·L^-1、0.5mg·L^-1、1mg·L^-1、1.5mg·L^-1、2mg·L^-1、2.5mg·L^-1Adding the standard solution with concentration gradient into the ELISA plate, diluting the sample collected at each time point by 100 times, and sucking 100 mu L of the sample and adding the sample into the ELISA plate. Adding 100ul water and 50ul molybdenum antimony color development resisting agent into each hole of the ELISA plate, developing for 30min, performing colorimetry with 882nm microplate reader, calculating the result, and drawing a time line graph, wherein the result is shown in the right graph of FIG. 1, and the soluble phosphorus concentration reaches 135 mg.L after 20h^-1It is shown that the Klebsiella R1452 has high phosphorus-solubilizing properties for inorganic phosphorus.

Wherein, the molybdenum-antimony stock solution: 10g of ammonium molybdate [ (NH) are weighed₄)₆Mo₇O₂₄·4H₂O]Dissolved in 300ml of water at about 60 ℃ and cooled. 70ml of concentrated sulfuric acid is slowly injected into 500ml of water, stirred evenly and cooled. Dilute sulfuric acid is injected into ammonium molybdate solution and stirred evenly at any time. 100ml of 0.3% (m/v) potassium antimony tartrate [ K (SbO) C₄H₄O₆]The solution was finally diluted to 2L with water and stored in a brown bottle. The ammonium molybdate concentration in this stock solution was 0.5%.

Wherein, the molybdenum-antimony color-developing resisting agent: 0.1g of ascorbic acid is weighed and dissolved in 20ml of molybdenum-antimony stock solution, the validity period is 24h at room temperature, and the ascorbic acid can be stored for 7 days in a refrigerator at the temperature of 2-8 ℃.

Wherein, the phosphorus standard stock solution is 100mg/L]: weighing KH dried at 105 deg.C₂PO₄0.4394g, dissolving in 200ml water, adding 5ml concentrated sulfuric acid, and adding water to make volume to 1L, wherein the stock solution can be stored for a long time. Here, 100mg/L means the mass of phosphorus.

Wherein, the phosphorus standard working solution is [5mg/L ]]: the phosphorus standard stock solution is diluted with water (the bacterial solution is diluted with water, and the soil is 0.5M NaHCO)₃Solution dilution) is accurately diluted by 20 times, and the product can not be stored for a long time when being prepared.

Example 2 Klebsiella R1452 hydrolysis of organic phosphorus

The Klebsiella R1452 is streaked and activated on a 1/2TSB solid culture medium plate, sealed and cultured at 28 ℃ for about 3 days, then a single colony is selected to be placed in a 1/2TSB liquid culture medium, and cultured for about 3 days at 28 ℃ shaking table and 180rpm, thus obtaining the fermentation liquid. Centrifuging the fermentation liquid in 2900g high speed centrifuge for 10min, discarding supernatant in ultra clean bench, adding 25ml sterile water, and mixing by vortex. Repeating the above steps for 2 times, re-suspending with 15ml sterile water for the last time, vortex, taking out 1ml, and measuring bacterial OD with spectrophotometer_600nmValue, recorded and diluted to OD_600nmA bacterial solution of klebsiella R1452 was obtained at 0.5, and the content of klebsiella R1452 in the bacterial solution of klebsiella R1452 was 5 × 10⁸cfu/mL。

Preparing insoluble organophosphorus solid culture medium, wherein the formula is shown in table 3, the organophosphorus uses phytin, and the formula is shown in table 3:

TABLE 3 insoluble organophosphorus solid NBRIP Medium formulation (1L)

Reagent	Company goods number	Dosage of
			Glucose	Sigma-Aldrich G8270	10g
MgCl₂·6H₂O	Sigma-Aldrich M2670	5g
			MgSO₄·7H₂O	Sigma-Aldrich M2643	0.25g
KCl	Sigma-Aldrich P3911	0.2g
			(NH₄)₂SO₄	Sigma-Aldrich A4418	0.1g
Phytic acid calcium magnesium	TCI P0410	0.9467g
			Agar	Solarbio A8190	12g

20ul of the aforementioned Klebsiella R1452 bacterial liquid was added to the insoluble organophosphorus solid medium of Table 3. Adding the bacteria solution, covering with a cover, sealing with parafilm sealing film after the bacteria solution is completely dried, and culturing upside down. The bacteria liquid is added to the center of the area without splashing so as not to influence the observation of the effect of the phosphorus dissolving ring. 3 replicates were set. Culturing at 28 deg.C for about 3 days, and increasing the diameter of phosphorus-dissolving ring to increase the phosphorus-dissolving capacity. The results show that the Klebsiella R1452 can generate a phosphorus-solubilizing ring on the insoluble organophosphorus solid medium (the left panel of FIG. 2), and the phosphorus-solubilizing ring is obvious.

Preparing an insoluble organophosphorus liquid culture medium, wherein the organophosphorus is phytin, and the formula is shown in Table 4:

TABLE 4 basic culture medium formulation for insoluble organophosphorus liquid

The basic culture medium of insoluble organophosphorus liquid is filled into 6 conical flasks of 100ml, 30ml of each flask, 0.028g of phytin is added respectively, and the mixture is sterilized by steam under the pressure of 121 ℃ for standby. Adding 300ul of bacterial liquid with OD of 0.5 into 3 bottles of culture medium, using the other three bottles as blank control, placing the bottles in a shaking table at 28 ℃ and culturing at 180rpm, sampling the culture medium at 0h, 10h, 20h, 30h, 40h and 50h respectively, taking three samples in each bottle, wherein each sample is 200ul, filtering by using a membrane with the diameter of 0.22um, storing in a refrigerator at-20 ℃, and repeating the steps as the technology; biological replicates were used between the three flasks.

0 mg.L is prepared by using a phosphorus standard stock solution^-1、0.15mg·L^-1、0.25mg·L^-1、0.5mg·L^-1、1mg·L^-1、1.5mg·L^-1、2mg·L^-1、2.5mg·L^-1Adding the standard solution with concentration gradient into the ELISA plate, diluting the sample collected at each time point by 100 times, and sucking 100 mu L of the sample and adding the sample into the ELISA plate. Adding 100ul of water and 50ul of molybdenum-antimony color development resisting agent into each hole of the ELISA plate, developing for 30min, performing colorimetry with 882nm microplate reader, calculating the result, and drawing a time line diagram, wherein the result is shown in the right diagram of FIG. 2, and the concentration of soluble phosphorus reaches 82.2 mg.L after 50h^-1It is shown that the Klebsiella R1452 has high phosphorus-solubilizing properties for organophosphorus.

Wherein, the molybdenum-antimony stock solution: 10g of ammonium molybdate [ (NH) are weighed₄)₆Mo₇O₂₄·₄H₂O]Dissolved in 300ml of water at about 60 ℃ and cooled. 70ml of concentrated sulfuric acid is slowly injected into 500ml of water, stirred evenly and cooled. Dilute sulfuric acid is injected into ammonium molybdate solution and stirred evenly at any time. 100ml of 0.3% (m/v) potassium antimony tartrate [ K (SbO) C₄H₄O₆]The solution was finally diluted to 2L with water and stored in a brown bottle. The ammonium molybdate concentration in this stock solution was 0.5%.

In conclusion, the Klebsiella R1452 can hydrolyze inorganic insoluble phosphorus and organic phosphorus, a large amount of unavailable organic phosphorus exists in soil, and compared with bacteria only capable of dissolving inorganic insoluble phosphorus, the Klebsiella R1452 can assist plants to utilize more kinds of phosphorus sources in the soil, and is beneficial to reducing the use of chemical fertilizers. Compared with the phosphorus-dissolving bacteria separated from the soil, the phosphorus-dissolving bacteria separated from the plant root system are easier to colonize on the plant root system, form a long-term mutualistic symbiosis relationship with the plant, have influence on the growth and development of the plant, and are also beneficial to the long-term effect of the phosphorus-dissolving action in the soil.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of genetics and developmental biology of Chinese academy of sciences

<120> rice rhizosphere Klebsiella and application thereof

<130> GNCSY200475

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1418

<212> DNA

<213> Klebsiella sp (klebsiella sp.)

<400> 1

tacctgcaag tcgagcggta gcacagagag cttgctctcg ggtgacgagc ggcggacggg 60

tgagtaatgt ctgggaaact gcctgatgga gggggataac tactggaaac ggtagctaat 120

accgcataac gtcgcaagac caaagtgggg gaccttcggg cctcatgcca tcagatgtgc 180

ccagatggga ttagctggta ggtggggtaa cggctcacct aggcgacgat ccctagctgg 240

tctgagagga tgaccagcca cactggaact gagacacggt ccagactcct acgggaggca 300

gcagtgggga atattgcaca atgggcgcaa gcctgatgca gccatgccgc gtgtgtgaag 360

aaggccttcg ggttgtaaag cactttcagc ggggaggaag gcgntgaggt taataacctc 420

ancgattgac gttacccgca gaagaagcac cggctaactc cgtgccagca gccgcggtaa 480

tacggagggt gcaagcgtta atcggaatta ctgggcgtaa agcgcacgca ggcggtctgt 540

caagtcggat gtgaaatccc cgggctcaac ctgggaactg cattcgaaac tggcaggcta 600

gagtcttgta gaggggggta gaattccagg tgtagcggtg aaatgcgtag agatctggag 660

gaataccggt ggcgaaggcg gccccctgga caaagactga cgctcaggtg cgaaagcgtg 720

gggagcaaac aggattagat accctggtag tccacgctgt aaacgatgtc gatttggagg 780

ttgtgccctt gaggcgtggc ttccggagct aacgcgttaa atcgaccgcc tggggagtac 840

ggccgcaagg ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg 900

gtttaattcg atgcaacgcg aagaacctta cctggtcttg acatccacag aactttccag 960

agatggattg gtgccttcgg gaactgtgag acaggtgctg catggctgtc gtcagctcgt 1020

gttgtgaaat gttgggttaa gtcccgcaac gagcgcaacc cttatccttt gttgccagcg 1080

gtnnggccgg gaactcaaag gagactgcca gtgataaact ggaggaaggt ggggatgacg 1140

tcaagtcatc atggccctta cgaccagggc tacacacgtg ctacaatggc atatacaaag 1200

agaagcgacc tcgcgagagc aagcggacct cataaagtat gtcgtagtcc ggattggagt 1260

ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg tagatcagaa tgctacggtg 1320

aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtggg ttgcaaaaga 1380

agtaggtagc ttaaccttcg ggagggcgct taccactt 1418

Claims

1. Klebsiella, characterized by: the Klebsiella is Klebsiella sp with a strain number of R1452, and the registration number of the Klebsiella sp is CGMCC No.21199 in the common microorganism center of China Committee for culture Collection of microorganisms.

2. Use of a Klebsiella or/and a metabolite of Klebsiella according to claim 1, in any of the following applications:

3. A product characterized by: comprising the Klebsiella R1452 or/and the Klebsiella R1452 metabolite according to claim 1.

4. The product of claim 3, wherein: is any one of the following products:

v2, product for promoting phosphorus absorption of plants;

v3, product for improving soil.

5. Use of the product of claim 3 or 4 in any of the following applications:

x2, the use of said product for promoting phosphorus uptake in plants;

x3, and the application of the product in soil improvement.

6. A culture of klebsiella according to claim 1, characterized in that: the culture has at least one function of the following W1-W3:

w1, hydrolyzed inorganic insoluble phosphorus and organic phosphorus;

w2, promoting plant phosphorus absorption;

w3, improving soil.

7. Method for culturing the bacterium klebsiella R1452 according to claim 1, characterized in that: comprising the step of culturing said Klebsiella in a culture medium.

8. A process for preparing the product of any of claims 3 or 4, characterized in that: comprising the step of obtaining a product by using said Klebsiella R1452 and/or a metabolite of said Klebsiella R1452 as an ingredient of said product.

9. The method of claim 8, wherein: the product is a liquid microbial inoculum or a solid microbial inoculum.