Pseudomonas, saline-alkaline tolerant and / or growth-promoting microbial inoculum, preparation method and application of pseudomonas and saline-alkaline tolerant and / or growth-promoting microbial inoculum

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to pseudomonas, a saline-alkaline tolerant and / or growth-promoting microbial agent, a preparation method and application. The invention provides a strain of Pseudomonas brassicae E4, and the preservation number of the Pseudomonas brassicae E4 is CCTCC (China Center for Type Culture Collection) NO: M 20241638. The embodiment of the invention proves that the pseudomonas E4 disclosed by the invention can enhance the saline-alkaline tolerance of plants and has a growth promoting effect to a certain extent, so that the pseudomonas E4 can be deduced to be applied to the aspects of saline-alkaline land improvement, plant saline-alkaline tolerance and the like in China.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Pseudomonas, a salt-alkali resistant and / or growth-promoting bacterial agent, a preparation method and an application. Background Art

[0002] Soil salinization is a global ecological problem that leads to land degradation, reduced agricultural productivity and even food security issues. A large amount of Ca2+ is accumulated in saline-alkali soil. 2+ , K + 、Na + Salt ions such as salt ions are redistributed in the horizontal or vertical direction of the soil, causing salt to gradually accumulate on the soil surface, affecting the normal growth of plants; under this condition, saline-alkali soil becomes compacted, soil porosity becomes smaller, and air permeability and water permeability become worse and worse, affecting the growth, abundance, metabolism of functional microorganisms in the soil, as well as the activity of enzymes such as soil invertase, alkaline phosphatase, catalase and urease, and reducing the conversion rate of soil organic matter. Soil salinization will destroy soil structure, reduce soil fertility, change the structure of soil animal communities, and affect the absorption and metabolic functions of plants.

[0003] Rapeseed is one of the important crops in my country. It has strong salt-alkali tolerance and shows significant effects in improving saline-alkali land. Exploring microbial resources that promote rapeseed growth and salt-alkali tolerance and using plant and microbial resources to repair soil salinization are of great significance to the management of salinized land in my country.

[0004] It has been reported in the prior art that Pseudomonas has a disease prevention effect, as shown in Chinese patent CN202310311925.7_—A disease-preventing and growth-promoting Pseudomonas FP818 and the strain in its application. The strain is used to inhibit tomato bacterial canker, rice white leaf blight, crucifer black rot, rice leaf streak pathogen, watermelon acidophilus and solanacearum; there is no relevant report in the prior art that salt-alkali-tolerant Pseudomonas has a growth-promoting effect on rapeseed. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Pseudomonas, a salt-alkali resistant and / or growth-promoting bacterial agent, a preparation method and an application. The Pseudomonas (Pseudomonas brassicacearum) E4 provided by the present invention can improve the salt-alkali tolerance of plants and promote the growth of plants.

[0006] In order to solve the above technical problems, the following technical solutions are proposed:

[0007] The invention provides a strain of Pseudomonas brassicacearum E4, whose deposit number is CCTCC NO: M 20241638.

[0008] The present invention provides a salt-alkali resistant and / or growth-promoting bacterial agent, comprising the Pseudomonas E4 described in the above technical solution.

[0009] Preferably, the viable count of Pseudomonas E4 in the salt-alkali resistant and / or growth-promoting bacterial agent is ≥ 1×10 8 CFU / g.

[0010] Preferably, the bacterial agent includes Pseudomonas E4 bacterial solution.

[0011] The present invention provides a method for preparing the salt-alkali resistant and / or growth-promoting bacterial agent described in the above technical solution, and the method for preparing the bacterial agent comprises: inoculating Pseudomonas E4 in a culture medium for culturing to obtain the bacterial agent.

[0012] Preferably, the culture temperature is 26-35° C., and the culture time is 11-17 hours.

[0013] The present invention provides the use of the Pseudomonas E4 described in the above technical solution or the salt-alkali tolerant and / or growth-promoting bacteria described in the above technical solution or the salt-alkali tolerant and / or growth-promoting bacteria prepared by the preparation method described in the above technical solution in one or more of the following 1) to 6);

[0014] 1) Improving the salt-alkali tolerance of plants and / or improving saline-alkali land;

[0015] 2) Plant growth promotion;

[0016] 3) Increase the chlorophyll content of plants;

[0017] 4) Increase the content of plant amino acids;

[0018] 5) Increase the soluble sugar content of plants;

[0019] 6) Reduce the sodium ion content and / or sodium-potassium ratio of plants.

[0020] Preferably, the plant comprises rapeseed and / or Arabidopsis thaliana.

[0021] The present invention provides a method for improving plant salt-alkali tolerance and / or promoting plant growth, comprising the following steps: applying the Pseudomonas E4 described in the above technical scheme or the salt-alkali tolerant and / or growth-promoting bacterial agent described in the above technical scheme or the bacterial agent prepared by the preparation method described in the above technical scheme to plants.

[0022] Preferably, the application method includes at least one of soil mixing, root irrigation and spraying.

[0023] Beneficial effects of the present invention: The present invention provides a strain of Pseudomonas brassicacearum E4, with a deposit number of CCTCC NO: M 20241638. The Pseudomonas E4 provided by the present invention can promote plant growth under saline-alkali conditions and improve the salt-alkali tolerance of plants. The results of the embodiment show that under 100mM NaCl salt stress conditions, E4 treatment can increase the fresh weight of Arabidopsis thaliana by 241% and the root length by 17.5%; in a matrix soil with a mass content of NaCl of 1.2%, E4 treatment increases the fresh weight of the aboveground part of rapeseed by 56.65%, the aboveground dry weight by 115.3%, the soluble sugar content by 78.47%, the proline content by 36.37%, the chlorophyll content by 18.73%, the sodium ion content by 47.81%, and the sodium-potassium ratio by 40.17%. In a potted experiment on saline-alkali soil in Baicheng, Jilin, E4 treatment increased the aboveground fresh weight of rapeseed by 73.96%, the aboveground dry weight by 77.05%, reduced the sodium ion content by 2.64%, and reduced the sodium-potassium ratio by 11.34%. It can be seen that the Pseudomonas E4 of the present invention can be applied to saline-alkali land improvement and has the ability to enhance the salt-alkali tolerance of plants.

[0024] Biological Deposit Description

[0025] Pseudomonas brassicacearum E4 was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number CCTCCNO: M 20241638, and the deposit address is: Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The strain morphology of Pseudomonas E4 grown in 1 / 2TSB medium for 1 day;

[0027] Figure 2 is the phylogenetic tree of Pseudomonas E4;

[0028] Figure 3 The figure shows the results of the test on the salt-alkali tolerance of Arabidopsis thaliana enhanced by Pseudomonas E4 on 100 mM NaCl plates; A, B, and C are the phenotype of Arabidopsis thaliana cultured on 100 mM NaCl plates for 25 days, the fresh weight measurement results after 25 days of culture, and the root length measurement results after 25 days of culture, respectively;

[0029] Figure 4The figure is the result of enhancing the salt-alkali tolerance of rapeseed by Pseudomonas E4 under the condition of 1.2% NaCl matrix soil; A, B, C, D, E, F, G, H respectively represent the growth phenotype of rapeseed after 40 days of cultivation, the fresh weight of the aboveground part of rapeseed, the dry weight of the aboveground part of rapeseed, the chlorophyll content of rapeseed, the soluble sugar content of rapeseed, the proline content of rapeseed, the sodium ion content of rapeseed, and the sodium-potassium ratio of rapeseed;

[0030] Figure 5 Pseudomonas E4 enhances the salt-alkali tolerance of rapeseed in saline-alkali soil in Baicheng, Jilin; A and B represent the growth phenotype of rapeseed cultured in saline-alkali soil in Baicheng, Jilin for 40 days, C, D, E, and F represent the fresh weight of the aboveground part of rapeseed, the dry weight of the aboveground part of rapeseed, the sodium ion content of rapeseed, and the sodium-potassium ratio of rapeseed, respectively;

[0031] Figure 6 This is the result of improving saline-alkali soil with Pseudomonas E4. DETAILED DESCRIPTION

[0032] The present invention provides a strain of Pseudomonas brassicacearum E4, with a deposit number of CCTCC NO: M 20241638. The 16S rDNA gene sequence of the Pseudomonas brassicacearum E4 of the present invention is shown in SEQ ID NO.1.

[0033] The Pseudomonas E4 screening of the present invention is obtained by screening the rapeseed variety Huayouza 62 planted in saline-alkali soil in Baicheng, Jilin Province, and the root samples of the rapeseed variety Huayouza 62 are screened. Pseudomonas E4 can regulate the osmotic balance, ion absorption and transport of plants and reduce Na + The toxicity of salt-alkali stress is alleviated by stabilizing cell morphology and protein structure, maintaining hormone balance and cell metabolism level, and other physiological mechanisms to enhance the salt-alkali tolerance of plants and achieve plant growth promotion. Pseudomonas E4 appears as light green, round colonies on 1 / 2TSB medium plates, with raised colonies, moist and smooth surfaces, and neat edges.

[0034] The present invention extracts the 16S rDNA sequence of Pseudomonas E4 strain, performs BLAST homologous sequence search in the NCBI database, and constructs a phylogenetic tree using Mega 7 software. Figure 2 , indicating that strain E4 is Pseudomonas brassicacearum.

[0035] The present invention provides a salt-alkali resistant and / or growth-promoting bacterial agent, comprising the Pseudomonas E4 described in the above technical solution.

[0036] In the present invention, the viable count of Pseudomonas E4 in the salt-alkali resistant and / or growth-promoting bacterial agent may be ≥1×10 8 CFU / g, or 1×10 8 CFU / g.

[0037] In the present invention, the application form of Pseudomonas E4 in the salt-alkali resistant and / or growth-promoting bacteria preferably includes bacterial liquid. The preparation method of the bacterial liquid of the present invention preferably includes: inoculating the Pseudomonas E4 in a culture medium for culturing to obtain a fermentation liquid, and centrifuging and resuspending the fermentation liquid to obtain a bacterial liquid. The culture temperature of the present invention can be 26-35°C, or 28-30°C; as an embodiment, the culture temperature can be 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C; the culture time can be 11-17h, or 12-15h; as an embodiment, the culture time can be 11, 12, 13, 14, 15, 16 or 17h; the culture speed can be 160-230rpm, or 180-210rpm, or 200rpm; as an embodiment, the culture speed can be 160, 170, 180, 190, 200, 210, 220 or 230rpm. The fermentation liquid is obtained. The present invention preferably obtains the bacterial liquid after centrifugation and resuspending the fermentation liquid. The centrifugal speed of the present invention can be 6600-7400rpm, 6800-7300rpm, or 7000rpm; as an embodiment, the centrifugal speed can be 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300 or 7400rpm; the centrifugal time can be 8-12min, or 10min; after centrifugation, the bacterial cells are obtained and the bacterial solution is prepared by resuspension. The resuspension solvent is preferably a phosphate buffer; the number of live Pseudomonas E4 in the bacterial solution can be ≥1×10 8 CFU / mL, or 1×10 8 CFU / mL. The culture medium of the present invention preferably includes a liquid culture medium; the liquid culture medium of the present invention preferably includes a 1 / 2TSB liquid culture medium; the 1 / 2TSB liquid culture medium of the present invention may be composed of: 15 g of tryptone, 5 g of soy peptone, 5 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose and 1 L of ddH2O.

[0038] The present invention provides a method for preparing the salt-alkali resistant and / or growth-promoting bacterial agent described in the above technical solution, wherein the preparation method of the bacterial agent comprises: inoculating Pseudomonas E4 in a culture medium for culturing to obtain the bacterial agent. The number of live Pseudomonas E4 cells in the bacterial agent of the present invention can be ≥1×10 8 CFU / mL, or 1×108 CFU / mL. The bacterial agent of the present invention preferably includes at least one of a liquid agent, a powder agent, and a granule agent; the liquid agent preferably includes a bacterial solution, and the preparation method of the bacterial solution has been discussed above and will not be repeated here. The present invention has no special limitation on the preparation method of the powder agent, and a conventional method can be used. The present invention has no special limitation on the preparation method of the granule agent, and a conventional method can be used.

[0039] After applying the salt-alkali resistant and / or growth-promoting bacteria agent of the present invention, the growth of plants can be significantly promoted while the salt-alkali tolerance of plants can be improved.

[0040] The present invention provides the use of the Pseudomonas E4 described in the above technical solution or the salt-alkali tolerant and / or growth-promoting bacterial agent described in the above technical solution in one or more of the following 1) to 6):

[0041] 1) Improving the salt-alkali tolerance of plants and / or improving saline-alkali land;

[0042] 2) Plant growth promotion;

[0043] 3) Increase the chlorophyll content of plants;

[0044] 4) Increase the content of plant amino acids;

[0045] 5) Increase the soluble sugar content of plants;

[0046] 6) Reduce the sodium ion content and / or sodium-potassium ratio of plants.

[0047] The plants of the present invention may include rapeseed and / or Arabidopsis, or may be rapeseed and Arabidopsis. The plant growth promotion of the present invention preferably includes increasing at least one of the dry weight, fresh weight, root length and number of lateral roots of the plant, or may be increasing the fresh weight, root length and number of lateral roots of the plant. When the plant is Arabidopsis, the dry weight, fresh weight, root length and number of lateral roots of the Arabidopsis are increased; the fresh weight preferably includes the fresh weight of the aboveground part; the dry weight preferably includes the dry weight of the aboveground part; Pseudomonas E4 can improve the salt-alkali tolerance of Arabidopsis under 100mM NaCl salt stress conditions, and significantly increase the fresh weight and root length.

[0048] When the plant is rapeseed, at least one of the dry weight, fresh weight, chlorophyll content, soluble sugar content and amino acid content of the rapeseed can be increased, or at least one of the dry weight, fresh weight, chlorophyll content, soluble sugar content and amino acid content of the rapeseed can be increased. In the present invention, the amino acid can be proline. Increasing the proline content of a plant can enhance the plant's drought resistance, salt resistance and cold resistance, and the bacterial agent of the present invention increases the proline content in rapeseed, indicating that the stress resistance of rapeseed can be improved. When the plant is rapeseed, the sodium ion content and / or sodium-potassium ratio in the rapeseed can be reduced, or at least one of the sodium ion content and sodium-potassium ratio in the rapeseed can be reduced; the reduction in the sodium ion content and sodium-potassium ratio of the plant indicates that the plant has salt-alkali tolerance. As an embodiment, after culturing Pseudomonas E4, a bacterial solution is obtained. After the bacterial solution is used to irrigate the roots of rapeseed, Pseudomonas E4 can increase the fresh weight, dry weight, proline content, chlorophyll content and soluble sugar content of rapeseed, and reduce the sodium ion content and sodium-potassium ratio of the plant. This indicates that Pseudomonas E4 regulates the osmotic balance, ion absorption and transport of plants to reduce Na + It can alleviate the harm of salt-alkali stress to rapeseed by stabilizing cell morphology and protein structure, maintaining hormone balance and cell metabolism level and other physiological mechanisms, and enhance rapeseed's salt-alkali tolerance.

[0049] In the present invention, the application form preferably includes soil mixing and / or root irrigation, and more preferably root irrigation. In the present invention, when the Pseudomonas E4 agent is used for root irrigation in the laboratory stage, the application mass of the Pseudomonas E4 agent can be 9-11 mL per seedling, and can be 10 mL; the application time is preferably after the seedling grows two true leaves.

[0050] The present invention provides a method for improving plant salt-alkali tolerance and / or promoting plant growth, comprising the following steps: applying the Pseudomonas E4 described in the above technical scheme or the salt-alkali tolerant and / or growth-promoting bacterial agent described in the above technical scheme or the bacterial agent prepared by the preparation method described in the above technical scheme to plants.

[0051] In the present invention, the application method preferably includes at least one of soil mixing, root irrigation and spraying.

[0052] The salt-alkali tolerant and / or growth-promoting bacteria agent of the present invention has been discussed in detail in the aforementioned technical scheme, and will not be repeated here. The application amount of the present invention for soil mixing, root irrigation and spraying has been discussed in detail in the aforementioned technical scheme, and will not be repeated here. After applying the salt-alkali tolerant and / or growth-promoting bacteria agent of the present invention, it can significantly promote the growth of plants and improve the salt-alkali tolerance of plants.

[0053] Soil salinization is one of the main environmental factors that restrict plant growth and agricultural productivity. Improving crop yields in saline-alkali land through agricultural microbial technology is of great significance to the development of my country's agriculture. Rapeseed is one of the important crops in my country, with strong salt-alkali tolerance, and shows significant effects in the improvement of saline-alkali land. Exploring microbial resources that promote rapeseed growth and salt-alkali tolerance, and using microbial resources to repair soil salinization, not only helps to solve the problem of the lack of microbial resources for enhancing the salt-alkali tolerance of crops in my country, but also can clarify scientific issues such as the mechanism of action of soil microorganisms in enhancing plant salt-alkali tolerance. In this study, a strain of Pseudomonas brassicacearum E4 was isolated from the root part of the salt-alkali tolerant rapeseed variety Huayouza 62 in the saline-alkali soil of Baicheng, Jilin. The Pseudomonas E4 provided by the present invention can be applied to the improvement of saline-alkali land in my country, and has the effect of enhancing the salt-alkali tolerance of plants.

[0054] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] 1. Saline-alkali soil in Baicheng, Jilin Province. Saline-alkali soil in Baicheng, Jilin Province was sent to Wuhan Academy of Agricultural Sciences for determination of physical and chemical properties. The results of the determination of physical and chemical properties are as follows: the pH value of saline-alkali soil is 10, the organic matter content is 19.14 g / kg, the soil conductivity EC is 6.92 mS / cm, the effective phosphorus content is 7.88 mg / kg, the available potassium content is 230 mg / kg, and the sodium ion content is 2294 mg / kg. Among the physical and chemical properties of soil, soil conductivity and pH value reflect the degree of soil salinization, soil organic matter content and soil available phosphorus content are closely related to soil fertility and phosphorus cycle in soil, respectively, and the available potassium content in soil can reflect the supply of available potassium in soil.

[0056] 2. Test plants: The rapeseed variety is "Huayouza 62", which is preserved in this experiment. The soil for plant growth is matrix soil and vermiculite, and the volume ratio of matrix soil to vermiculite is 2:1. The rapeseed is cultured in a plant culture room under the conditions of temperature 22℃, humidity 75%, 12h / d light culture and 12h / d dark culture.

[0057] 3. Determination method

[0058] (1) Determination of fresh weight and dry weight of rapeseed

[0059] The rapeseed was cut at the junction of the root and stem, the surface moisture was absorbed with absorbent paper, and the above-ground part was measured and recorded as the fresh weight; the rapeseed material with fresh weight was placed in a 105°C oven for 30 minutes, and then dried in an 85°C oven to constant weight, and the dry weight of the rapeseed was weighed.

[0060] (2) Determination of chlorophyll content

[0061] The chlorophyll content was determined by the 95% ethanol colorimetric method: 0.5 g of fresh rapeseed leaves were weighed, cut into pieces, and 2 mL of 95% ethanol (hereinafter referred to as 95% ethanol) was added, ground, and filtered. The residue on the filter paper was rinsed with 2 mL of 95% ethanol until the residue faded, and the filtrate was collected and diluted to 5 mL with 95% ethanol to obtain the sample extract. 0.5 mL of the sample extract was diluted with 4 mL of 95% ethanol, and the absorbance was measured at wavelengths of 663 nm and 645 nm, respectively. 95% ethanol was used to adjust the absorbance to zero. Three parallel experiments were performed. Chlorophyll content C = [(12.7A 663 -2.69A 645 )+(22.9A 645 -4.68A 663 )]·V·N·W -1 , where C represents the chlorophyll content (μg·g -1 FW), V represents the volume of the extract (mL), N represents the dilution factor, W represents the fresh weight of the sample (g), A 663 and A 645 Represent the absorbance values ​​measured at 663nm and 645nm respectively.

[0062] (3) Determination of soluble sugar content

[0063] The content of soluble sugar was determined by thiobarbituric acid colorimetry: 0.5 g rapeseed leaves were weighed and cut into pieces, and a small amount of quartz sand and 4 mL of 10% trichloroacetic acid (TCA) solution were added to fully grind the obtained grinding solution, and the centrifugal speed was 4000 r / min, and the centrifugal time was 10 min. The supernatant obtained by centrifugation was the sample extract. 2 mL of sample extract was taken, and 2 mL of 0.6% thiobarbituric acid (TBA) solution was added thereto, mixed, boiled in a boiling water bath for 15 min, cooled to room temperature quickly, and the supernatant was taken after centrifugation. The absorbance was measured at a wavelength of 450 nm. 2 mL of distilled water was used instead of sample extract in the control group. Three parallel experiments were set up for each group. C = 11.71 × A 450 ×V×W -1 , C represents the soluble sugar content (μmol·g - 1 FW), V represents the volume of the extract (mL), W represents the fresh weight of the sample (g), A 450 Represents the absorbance value measured at a wavelength of 450nm.

[0064] (4) Determination of proline content

[0065] The standard curve solutions are proline solutions with different mass concentrations, and the proline concentrations in the proline solutions are 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL, respectively.

[0066] The proline content was determined by the acid ninhydrin colorimetric method: First, a standard curve of standard proline content was prepared. Then, 0.5 g of rapeseed leaves were weighed and cut into pieces, and 5 mL of 3% sulfosalicylic acid solution was added to the solution and boiled in a water bath for 10 min, then quenched and filtered. 2 mL of the filtrate was added with 2 mL of glacial acetic acid and 3 mL of colorimetric solution, and boiled in a water bath for 40 min together with the standard solution, and then quenched to room temperature, and 5 mL of toluene was added, shaken, and the supernatant was taken after standing for stratification. The absorbance was measured at a wavelength of 530 nm, and each group of experiments was repeated three times. The proline content was calculated using the formula: C = (C0·V T ·V S -1 )·W -1 Where C represents the proline content (μg·g -1 FW), W represents the fresh weight of the sample (g), C0 represents the proline content in the extract obtained from the standard curve (μg), V S Indicates the volume (mL) absorbed during measurement, V T Indicates the total volume of the extract (mL).

[0067] (5) Determination of sodium and potassium ion content

[0068] The dried rapeseed sample was ground and passed through a 40-mesh stainless steel sieve to obtain the dried rapeseed sample for use. The sodium and potassium ion content of the dried rapeseed sample was determined by flame photometry and commissioned to Wuhan Puness Biotechnology Co., Ltd. for testing.

[0069] 4.1 / 2TSB solid medium consists of 15 g tryptone, 5 g soy peptone, 5 g sodium chloride, 2.5 g potassium dihydrogen phosphate, 2.5 g glucose, 15 g agar and 1 L ddH2O;

[0070] The composition of 1 / 2TSB liquid culture medium is: 15 g tryptone, 5 g soy peptone, 5 g sodium chloride, 2.5 g potassium dihydrogen phosphate, 2.5 g glucose and 1 L ddH2O.

[0071] Example 1

[0072] 1. Screening of strains. The salt-alkali tolerant rapeseed variety Huayouza 62 was planted in saline-alkali soil in Baicheng, Jilin Province. Huayouza 62 was pulled out with its roots, and root samples of Huayouza 62 at a depth of 10 cm were collected; the collected root samples were placed in a 50 mL sterile centrifuge tube.

[0073] First, wash away the soil particles visible to the naked eye in the root sample with sterile water, then transfer the root to a new 50mL centrifuge tube, add 30mL of sterile phosphate buffer to the tube, and place it on a flat shaker for washing at room temperature. The shaker speed is 180rpm, and the washing time is 15min. Washing for 15min is counted as one wash, and the washing is repeated 3 times in total. After washing, use sterile filter paper to absorb the residual liquid in the root. Cut the washed and water-absorbed root tissue into 2mm small segments with sterile scissors, mix them, and put them into a 1.5mL sterile centrifuge tube. Add 200μL of 10mM magnesium chloride solution to the centrifuge tube. The magnesium chloride solution has been sterilized; then use a sterile grinding rod to grind the root until the root is ground into a homogenate to obtain a root homogenate. The root homogenate was transferred to a tube with a volume of 50 mL, containing 25 mL of magnesium chloride solution with a molar concentration of 10 mM. After transfer, it was mixed and allowed to stand at room temperature for 15 min. The resulting solution was diluted by a 10-fold gradient dilution method to 10, 100, 1000, and 10000 times gradient dilutions, which were all spread on a 1 / 2 TSB solid culture medium plate. The colony growth was observed every day, and a single colony was picked for identification and preservation. One of the salt-tolerant bacteria was named E4. The colony morphology of strain E4 grown on 1 / 2 TSB culture medium for 1 day was as follows: Figure 1 As shown, the colonies are light green, round, convex, with a moist and smooth surface and neat edges.

[0074] 2. Identification of strain E4

[0075] TA cloning: 1 μL of pMD18-TVector, 4 μL of DNA of strain E4 and 5 μL of Solution I (dissolved on ice) were mixed, and the mixed system was placed in a 16°C metal bath overnight for ligation to obtain a ligation product. The competent cells of Escherichia coli were taken out from -80°C and placed on ice. The competent cells were thawed for about 5 to 10 minutes. 60 μL of competent cells were added to 10 μL of ligation product, and the mixture was evenly obtained by pipetting and beating. The mixture was placed on ice for 30 minutes, and the mixture was quickly placed in a 42°C water bath for heat shock for 45 seconds. The mixture was then placed on ice for 5 minutes, and then transferred to 400 μL of SOC medium solution preheated at 37°C, and placed at 37°C, 220 r / min shaking for 45 minutes to obtain a bacterial solution; the bacterial solution was taken out and spread on a LB medium plate containing 250 μL / mL of ampicillin (Amp), blown dry and placed at 37°C overnight for culture, and a single colony was picked with a sterilized toothpick for colony PCR identification. PCR identification is as follows: the sequence of the vector primer M13F is selected as: 5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO.2) and the sequence of the universal primer 1492R is: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO.3), and the above two primers are used for PCR amplification. The PCR reaction program is: 95℃ pre-denaturation for 5min, 94℃ denaturation for 1min, 55℃ annealing for 1min, 72℃ extension for 90s, pre-denaturation, denaturation, annealing and extension for 30 cycles, and then extension at 72℃ for 10min, and then the target band is detected in agarose gel with a mass concentration of 1%. Fresh bacterial liquid with the target band is selected and sent to Qingke Biotechnology Co., Ltd. for testing.

[0076] Construction of phylogenetic tree: The complete 16S rDNA sequence of the strain was obtained by removing the primer sequences at both ends of the sequence obtained after sequencing, as shown in SEQ ID NO.1; the complete sequence was then BLASTed on NCBI to obtain several sequences with extremely high sequence similarity, and the evolutionary tree was constructed using MEGA7.0 software. The results are shown in Figure 2 As shown. Figure 2 It can be known that strain E4 is Pseudomonas brassicacearum, that is, Pseudomonas brassicacearum E4.

[0077] SEQ ID NO.1:GCGTACTGACTGCGGTCGACGATTAGAGTTTGATCCTG GCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGGTGCTTGCACCTCTTGAGAGCGGCGGACGGGTGAGTAAAGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGCTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACGTTAGTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTCCAAGCGTTAATCGGAATTACTGGGCGTAAAGCCCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGTCGAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTAAATTCGAAGCAACGCGAAAAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGGGGACGGTTACCACGGTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAAATCTCTAGAGGTCCCCGGGTACCGAGCTCGAGTTCC。

[0078] Example 2 Arabidopsis thaliana Plate Growth Promotion Test

[0079] Preparation of E4 bacterial agent: scrape a little E4 bacteria into a conical flask containing 200 ml TSB liquid culture medium, shake at 28°C, 200 rpm for 12 hours, then centrifuge at 4°C, 5000 rpm to collect the bacteria, remove the supernatant and resuspend the bacteria in PBS to obtain the bacterial agent.

[0080] Preparation of disinfectant: Mix sterile water and 84 disinfectant in a volume ratio of 1:1 to obtain disinfectant.

[0081] The surface of Arabidopsis seeds was disinfected with disinfectant for 5 minutes, then washed 7 times with sterile water, and placed in a 4°C refrigerator for vernalization for 2 to 3 days to obtain vernalized seeds.

[0082] Prepare 1 / 2MS solid culture medium plates. The culture dishes used are square dishes. 1 / 2MS solid culture medium does not contain sucrose, but contains 100mM NaCl. Treatment group (E4+100mM NaCl): Draw a 6×6 grid on the 1 / 2MS solid culture medium plate, sow the vernalized seeds on the second horizontal line above the plate, and Figure 2 A; then drop 5 μL of Pseudomonas E4 inoculum on the Arabidopsis seeds and seal the plate to prevent contamination. Control group (100 mM NaCl): Same as the treatment group, the only difference is that 5 μL PBS is dropped on the Arabidopsis seeds. OD of the inoculum 600 It is about 0.2.

[0083] Four parallel experiments were set up for each treatment group and control group. The square dishes of the treatment group and control group were placed in a plant culture room for cultivation at 22°C, 12h / d light and 12h / d dark. After 25 days of cultivation, the growth was statistically analyzed, the root length of Arabidopsis thaliana was measured, the fresh weight of Arabidopsis thaliana was weighed, and the promotion rate of root length and fresh weight of Arabidopsis thaliana by different strains was calculated and photographed and recorded.

[0084] The results are shown in Table 1 and Figure 3 , the results of anova analysis showed that Pseudomonas E4 promoted the better growth of Arabidopsis thaliana under salt stress conditions, and the number of lateral roots increased significantly (see Figure 3 The fresh weight and root length increased significantly (**** represents P < 0.0001, * represents P < 0.05), and the fresh weight increased by 159% compared with the control group (see Figure 3 B), root length increased by 7.14% (see Figure 3 Middle C). Figure 3 Middle B, Figure 3 n in C = 29-40 biological replicates.

[0085] Table 1 Results of various indicators of Arabidopsis thaliana

[0086]

[0087]

[0088] Example 3 Matrix soil potting test

[0089] Preparation of E4 inoculant: scrape a little E4 bacteria into a conical flask containing 200ml TSB liquid medium, shake at 28℃, 200rpm for 12 hours, then centrifuge at 4℃, 5000rpm to collect the bacteria, remove the supernatant and resuspend the bacteria in PBS to make the inoculant, which can be used for root irrigation. The number of E4 live bacteria in the inoculant is 1×10 8 CFU / mL.

[0090] Treatment group (E4): The sterilized rapeseed seeds were soaked in sterilized water at room temperature for 2 hours, and then placed on a culture dish covered with moist filter paper, and germinated at 25°C for 3-4 days. The rapeseed seedlings with the same growth were selected and transplanted into 10×10 plastic square pots. The cultivation medium was matrix soil and vermiculite, and the volume ratio of matrix soil to vermiculite was 2:1. Four rapeseed seedlings were planted in each pot. After the seedlings grew two true leaves, they were treated with 1.2% saline solution, and the amount of saline added was 2L. After the saline treatment for 2-3 days, the roots were irrigated with bacterial agents, and 10mL of bacterial agents were injected around the roots of each seedling.

[0091] Control group (Control): Same as the treatment group, the only difference is that it is treated with saline with a mass concentration of 1.2%, but no bacterial agent is added.

[0092] Treatment group and control group, each group was set up with 7 square pots, each pot has 4 rapeseed, cultured for 40 days and then various indicators were measured. The results are shown in Table 2-1, 2-2 and Figure 4 .

[0093] Table 2-1 Test results of various indicators of rapeseed

[0094]

[0095]

[0096] Table 2-2 Test results of various indicators of rapeseed

[0097]

[0098] Note: Table 2-2 shows the “mean ± standard deviation” results of the corresponding indicators in Table 2-1.

[0099] Figure 4 The error bars represent the mean ± standard deviation. The data were analyzed by t-test. * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0100] Salt water was added to the substrate soil to make the mass content of NaCl 1.2%. After 40 days of treatment with strain E4, the strains treated with the strain grew larger and better than those in the control group ( Figure 4 E4 can significantly increase the fresh weight and dry weight of the aboveground part of rapeseed. Compared with the control group, the aboveground fresh weight increased by 58.21% ( Figure 4 Middle B), aboveground dry weight increased by 91.55% ( Figure 4 Middle C), Figure 4 In A, n=24 biological replicates in B and C. Chlorophyll content, soluble sugar content, proline content, sodium ion content and sodium-potassium ratio were also measured. Compared with the control group, the chlorophyll content of rapeseed treated with E4 increased by 25.27%, the soluble sugar content increased by 77.14%, the proline content increased by 51.16%, the sodium ion content decreased by 33.30%, and the sodium-potassium ratio decreased by 27.39% ( Figure 4 (middle D~H). Figure 4 In D, n=4 biological replicates, in E, n=3 biological replicates, and in H, n=3 biological replicates.

[0101] Example 4 Potted Planting Test in Saline-Alkaline Soil

[0102] Treatment group (E4): The disinfected rapeseed seeds were soaked in sterilized water at room temperature for 2 hours, then placed on a culture dish covered with moist filter paper and germinated at 25°C for 3-4 days to obtain rapeseed seedlings with uniform growth for transplanting. The transplanting culture container was a 10×10 plastic square pot. The matrix in the square pot was a mixture of Jilin Baicheng saline-alkali soil and vermiculite. The volume ratio of Jilin Baicheng saline-alkali soil and vermiculite was 1:1 during mixing.

[0103] Each treatment had 7 pots, 4 seeds were planted in each pot, and the roots were regularly irrigated with bacterial agents. 10 mL of bacterial agent was injected around the root of each seedling, and the number of viable bacteria in the bacterial agent was 1×10 8 CFU / mL.

[0104] The control group was regularly irrigated with PBS buffer, and 10 ml of PBS buffer was injected around the root of each seedling. The treatment group and the control group were cultured for 40 days and various indicators were measured. The measurement method was the same as described above. The results are shown in Table 3 and Figure 5 .

[0105] Table 3 Test results of various indicators of rapeseed

[0106]

[0107]

[0108] Figure 5The error bars are mean ± standard deviation. The data were analyzed by t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Potted plant experiments were conducted using saline-alkali soil in Baicheng, Jilin Province. Figure 5 Middle A and Figure 5 The B in the figure shows the phenotype of rapeseed grown in saline-alkali soil in Baicheng, Jilin Province for 40 days. After being treated with strain E4, the fresh weight of rapeseed increased by 87.05% ( Figure 5 C), the dry weight increased by 72.57% ( Figure 5 D), where C and D are the results of n=24 biological replicates. The sodium and potassium ion contents of rapeseed were also measured. After strain E4 treatment, the sodium ion content and sodium-potassium ratio were significantly reduced compared with the control group, with the sodium ion content reduced by 1.96% and the sodium-potassium ratio reduced by 5.89% ( Figure 5 ZhongE and Figure 5 F), where E and F are the results of n=3 biological replicates.

[0109] Soil samples were taken from the treatment group and the control group after 40 days of cultivation to determine the pH value. Three biological replicates were set for each group. The results are shown in Tables 4 and Figure 6 It can be seen that Pseudomonas brassicacearum E4 can reduce the pH of saline-alkali soil and has the potential to be used in improving saline-alkali soil. Figure 6 The error bars are mean ± standard deviation, and the data were analyzed by t-test. *** indicates P < 0.001.

[0110] Table 4 Results of saline-alkali land improvement

[0111]

[0112] In summary, the present invention uses 1 / 2TSB medium, through limiting dilution and plate culture methods, to isolate a strain of Pseudomonas brassicacearum E4 from the roots of the salt-alkali tolerant rapeseed Huayouza 62 planted in saline-alkali soil in Baicheng, Jilin Province. The strain has a strong salt-alkali tolerance, and Pseudomonas brassicacearum E4. It can be seen that strain E4 can be used as a microbial agent for enhancing plant salt-alkali tolerance, and provides strain resources for the development of saline-alkali land microbial fertilizers.

[0113] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas brassicacearum E4, with a deposit number of CCTCC NO: M20241638.

2. A salt-alkali resistant and / or growth-promoting bacterial agent, characterized in that: It comprises the Pseudomonas E4 described in claim 1.

3. The salt-alkali resistant and / or growth-promoting bacterial agent according to claim 2, characterized in that: The number of live bacteria of Pseudomonas E4 in the salt-alkali resistant and / or growth-promoting bacterial agent is ≥1×10 8 CFU / g.

4. The salt-alkali resistant and / or growth-promoting bacterial agent according to claim 2 or 3, characterized in that: The bacterial agent includes Pseudomonas E4 bacterial liquid.

5. The method for preparing the salt-alkali resistant and / or growth-promoting bacterial agent according to any one of claims 2 to 4, characterized in that: The preparation method of the bacterial agent comprises: inoculating Pseudomonas E4 into a culture medium for culturing to obtain the bacterial agent.

6. The preparation method according to claim 5, characterized in that: The culture temperature is 26-35° C., and the culture time is 11-17 hours.

7. Use of the Pseudomonas E4 according to claim 1, the salt-alkali tolerant and / or growth-promoting bacteria according to any one of claims 2 to 4, or the salt-alkali tolerant and / or growth-promoting bacteria prepared by the preparation method according to claim 5 or 6 in one or more of the following 1) to 6); 1) Improving the salt-alkali tolerance of plants and / or improving saline-alkali land; 2) Plant growth promotion; 3) Increase the chlorophyll content of plants; 4) Increase the content of plant amino acids; 5) Increase the soluble sugar content of plants; 6) Reduce the sodium ion content and / or sodium-potassium ratio of plants.

8. The use according to claim 7, characterized in that: The plants include rapeseed and / or Arabidopsis thaliana.

9. A method for improving plant salt-alkali tolerance and / or promoting plant growth, characterized in that: The method comprises the following steps: applying the Pseudomonas E4 described in claim 1, the salt-alkali tolerant and / or growth-promoting bacterial agent described in any one of claims 2 to 4, or the bacterial agent prepared by the preparation method described in claim 5 or 6 to plants.

10. The method according to claim 9, characterized in that: The application method includes at least one of soil mixing, root irrigation and spraying.

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