Psychrophilic Pseudomonas 77S3, bacterial agent and application thereof

Abstract

The present invention provides a psychrophilic Pseudomonas 77S3, a bacterial agent and an application thereof. The psychrophilic Pseudomonas 77S3 is classified and named as psychrophilic Pseudomonas psychrotolerans, and was deposited in the China Center for Type Culture Collection on May 29, 2023, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC M 2023852. The use of the above-mentioned psychrophilic Pseudomonas 77S3 can solve the problem of soybean growth difficulties under salt stress in the prior art, and is applicable to the field of plant microbial technology.

Description

Technical Field

[0001] The present invention relates to the technical field of plant microorganisms, and in particular to a psychrophilic Pseudomonas 77S3, a bacterial agent and applications thereof. Background Art

[0002] Endophytes are microorganisms that live inside plant tissues without causing obvious disease symptoms in plant tissues. They are used to distinguish epiphytes that live on the surface of plants. Endophytes in soybeans can be isolated and identified from the roots, stems or nodules of soybeans. During the domestication and long-term cultivation of soybeans, the symbiotic endophytic microorganisms in soybeans are lost, the diversity gradually decreases, and the original excellent characteristics of wild soybeans in resisting biotic and abiotic stresses are lost.

[0003] In agricultural production, soil salinization is an important factor affecting crop production. Soil salinization can lead to reduced water and air permeability, osmotic potential, and imbalance of ion concentration, which can have an adverse effect on plant growth and development. Soybeans are susceptible to salt stress. The growth of soybeans is inhibited under high salt concentrations, affecting the agronomic traits of soybeans, resulting in soybean yield and quality.

[0004] In the prior art, soil properties are usually improved by flooding salinized soil with water or applying chemical fertilizers. However, large amounts of watering can easily lead to plant root corruption, poor soil property improvement effects, and waste of water resources. The use of chemical fertilizers can easily pollute the environment and have adverse effects on the health of animals, plants, and humans. Therefore, there is an urgent need for an effective and environmentally friendly substance to improve salinized soil so that soybeans can grow and develop into soybean plants with excellent traits under salt stress. Summary of the invention

[0005] The main purpose of the present invention is to provide a psychrophilic Pseudomonas 77S3, a bacterial agent and an application thereof, so as to solve the problem in the prior art that soybeans are difficult to grow under a salt stress environment.

[0006] In order to achieve the above-mentioned purpose, according to one aspect of the present invention, a psychrophilic Pseudomonas 77S3 is provided, which is classified and named as psychrophilic Pseudomonas psychrotolerans, and was deposited in the China Center for Type Culture Collection on May 29, 2023, with the deposit address of Wuhan University, Wuhan, China, and the deposit number of CCTCC M 2023852.

[0007] In order to achieve the above object, according to a second aspect of the present invention, a bacterial agent is provided, which includes psychrophilic Pseudomonas 77S3.

[0008] Furthermore, the bacterial agent includes a liquid bacterial agent or a solid bacterial agent.

[0009] Furthermore, the bacterial agent includes water and psychrophilic Pseudomonas 77S3, and the OD600 of the bacterial agent is 0.8-1.2.

[0010] Furthermore, the carrier of the bacterial agent includes a solid carrier, a liquid carrier or a gel carrier.

[0011] According to a third aspect of the present invention, there is provided a method for improving the salt-alkali tolerance of soybeans, the method comprising treating soybeans with Pseudomonas refractory 77S3 or a bacterial agent.

[0012] Furthermore, the treatment method includes any one or more of a seed soaking method, a bacteria-mixing method, a root irrigation method or a foliar spraying method.

[0013] Furthermore, the seed soaking method includes soaking soybean seeds in a liquid bacterial agent at 20 to 25° C. for 4 to 6 hours to obtain soaked soybeans.

[0014] Furthermore, the method of mixing bacteria into soil includes mixing the bacterial agent with cultivation soil to prepare mixed soil, and planting soybeans in the mixed soil; preferably, the soybeans are treated together by the seed soaking method and the method of mixing bacteria into soil, and the soybean seeds after the seed soaking treatment are planted in the mixed soil.

[0015] According to a fourth aspect of the present invention, there is provided a use of psychrophilic Pseudomonas 77S3, a bacterial agent or a method for improving the salt-alkali tolerance of soybean in soybean planting.

[0016] By applying the technical solution of the present invention, soybean endophytes were successfully isolated and screened, and psychrophilic Pseudomonas 77S3, which still has a growth-promoting effect under salt stress, was obtained. When psychrophilic Pseudomonas 77S3 is applied to soybean planting, it can significantly improve the salt-alkali tolerance of soybeans under salt stress, allowing soybeans to grow and develop under salt stress environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 The figure shows the growth state morphology of the endophyte separated and purified in Example 2 of the present invention on beef extract peptone solid culture medium containing different sodium chloride concentrations (200mM, 300mM, 500mM, respectively) and different pH values ​​(pH=7.0, pH=9.0, respectively).

[0019] Figure 2The figure shows the growth state morphology of endophyte 77S3 on beef extract peptone medium according to Example 2 of the present invention.

[0020] Figure 3 A standard curve diagram of phosphorus concentration (mg / L) and OD720 of endophyte 77S3 in Example 2 of the present invention is shown.

[0021] Figure 4 The figure shows the results of phosphate solubilization of endophytic bacteria 77S3 on NBRIP solid culture medium according to Example 2 of the present invention.

[0022] Figure 5 The graph shows the results of the effective phosphorus content of endophyte 77S3 at 2d, 4d and 6d according to Example 2 of the present invention.

[0023] Figure 6 A standard curve diagram of endophyte IAA concentration and OD530 in Example 2 of the present invention is shown.

[0024] Figure 7 The results of IAA production by endophyte 77S3 at 2d, 4d and 6d according to Example 2 of the present invention are shown.

[0025] Figure 8 The morphology of endophyte 77S3 observed under an optical microscope in Example 3 of the present invention is shown.

[0026] Fig. 9 A schematic diagram of the phylogenetic evolutionary tree of strain 77S3 based on the 16s rDNA gene sequence according to Example 4 of the present invention is shown.

[0027] Fig.10 The diagram shows the root phenotype results of Wm82 soybean seedlings treated with endophyte 77S3 inoculum and blank inoculum under salt stress according to Example 5 of the present invention.

[0028] Fig.11 The phenotypic result graphs of Wm82 soybean seedlings treated with endophyte 77S3 inoculum and blank inoculum under non-salt stress and salt stress in Example 5 of the present invention are shown, wherein CK-0mM is a seedling phenotypic result graph of soybean seeds irrigated with 0mM NaCl and treated with blank inoculum, 77S3-0mM is a seedling phenotypic result graph of soybean seeds irrigated with 0mM NaCl and treated with endophyte 77S3 inoculum, CK-150mM is a seedling phenotypic result graph of soybean seeds irrigated with 150mM NaCl and treated with blank inoculum, and 77S3-150mM is a seedling phenotypic result graph of soybean seeds irrigated with 150mM NaCl and treated with endophyte 77S3 inoculum.

[0029] Fig.12 The graph shows the comparative results of stem fresh weight, stem dry weight, root fresh weight, root dry weight, plant height and root length data of Wm82 soybean seedlings treated with endophyte 77S3 inoculum and blank inoculum under non-salt stress and salt stress in Example 5 of the present invention. DETAILED DESCRIPTION

[0030] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0031] Glossary:

[0032] Halodurans refers to microorganisms that can grow in both salty and non-salty environments. The highest salt tolerance of highly salt-tolerant bacteria can reach 20% (relative to the salt concentration of the environment in which they are located).

[0033] Salt stress environment: refers to an environment where the soil or water contains too much salt, exceeding the limit that organisms can tolerate within a certain range, thus causing adverse effects and pressure on the organisms in the environment. When the sodium chloride content in the soil exceeds 100mM, the growth and development of soybeans are affected by salt stress.

[0034] As mentioned in the background art, the prior art generally uses chemical fertilizers to enhance the stress resistance of plants and maintain the plant's demand for nutrients. However, the abuse of chemical fertilizers not only pollutes the environment, but also has adverse effects on plants, animals and humans.

[0035] The present invention screened and obtained a psychrophilic Pseudomonas 77S3 from soybean endophytes. After soybeans were inoculated with the strain, the strain 77S3 reproduced in the soybeans, which could improve the salt tolerance of soybeans, so that soybeans had a growth-promoting effect under salt stress, avoiding the use of chemical fertilizers to cause environmental pollution and other side effects on the plant body, and finally achieving the purpose of soybeans being able to grow and develop under a salt stress environment under the premise of protecting the environment. On this basis, the applicant proposed a series of protection schemes.

[0036] In the first typical embodiment of the present application, a psychrophilic Pseudomonas 77S3 is provided, which is classified and named as psychrophilic Pseudomonas psychrotolerans, and was deposited in the China Center for Type Culture Collection on May 29, 2023. The storage address is Wuhan University, Wuhan, China, and the storage number is CCTCC M 2023852.

[0037] This strain is an endophyte isolated from soybeans. When inoculated into soybeans, it can improve the salt-alkali tolerance of soybeans. This strain also has the ability to solubilize phosphate and secrete IAA (auxin, indole-3-acetic acid). During the growth process of soybeans, it can provide nutrients and auxin to soybeans and promote the growth of soybeans.

[0038] In a second typical embodiment of the present application, a bacterial agent for improving the salt-alkali tolerance of soybean is provided, and the bacterial agent includes the above-mentioned psychrophilic Pseudomonas 77S3.

[0039] The above-mentioned bacterial agent includes the psychrophilic Pseudomonas 77S3, an endophytic bacterium of soybean with salt-alkali tolerance. The above-mentioned bacterial agent is used to treat soybean seeds, roots, stems, leaves or soybean planting media, which can directly improve the salt-alkali tolerance of soybeans or improve the properties of soybean planting media during the germination and / or growth of soybeans, so that soybeans can germinate, grow and develop even in a salt stress environment. The above-mentioned planting medium can provide the nutrients and support required for plant growth. Plant planting media include natural media or artificial synthetic media, natural media include but are not limited to soil, peat or sand, and artificial synthetic media include but are not limited to organic fertilizers, soil conditioners or hydroponic nutrient solutions.

[0040] In a preferred embodiment, the above-mentioned bacterial agent includes a liquid bacterial agent or a solid bacterial agent.

[0041] Liquid microbial agents can act on the seeds, roots, stems, leaves or planting media of plants. The use methods of liquid microbial agents include but are not limited to one or more of the following methods: mixing microbial agents into the soil, soaking seeds, root irrigation and foliar spraying.

[0042] Solid microbial agents usually exist in the form of powder, granules or tablets, with a long storage life and high viability. Solid microbial agents can be directly applied to the planting medium of plants, or mixed with seeds and organic fertilizers to achieve the purpose of promoting plant growth.

[0043] In a preferred embodiment, the bacterial agent includes water and the psychrophilic Pseudomonas 77S3, and the OD600 of the bacterial agent is 0.8-1.2.

[0044] The preparation method of the above-mentioned bacterial agent includes activating psychrophilic Pseudomonas 77S3, culturing it overnight and then inoculating it into a liquid culture medium for proliferation and culture to obtain psychrophilic Pseudomonas 77S3 bacterial liquid; centrifuging the psychrophilic Pseudomonas 77S3 bacterial liquid, removing the supernatant, and obtaining psychrophilic Pseudomonas 77S3 bacterial bodies; mixing the psychrophilic Pseudomonas 77S3 bacterial bodies with water to obtain a bacterial agent; preferably, the above-mentioned liquid culture medium includes beef extract peptone liquid culture medium; preferably, the temperature of the above-mentioned proliferation and culture is 28-30°C, the shaking speed is 160-180rpm, and the culture time is 24-26h.

[0045] In a preferred embodiment, the bacterial agent carrier includes a solid carrier, a liquid carrier or a gel carrier.

[0046] Common microbial agent carriers include but are not limited to the following:

[0047] 1. Solid carrier: Solid carriers can provide a surface for microorganisms to grow and attach, such as powdered carriers (such as corn flour, wood fiber, etc.) or granular carriers. These solid carriers mainly provide the support required by microorganisms and protect them from the adverse effects of the external environment. Solid carriers also include granular carriers: granular carriers are tiny granular substances, such as talcum powder, sand, etc. Granular carriers can provide a surface and pore structure for microorganisms to attach, increasing the storage stability and release effect of microbial agents.

[0048] 2. Liquid carrier: Liquid carrier is usually a liquid medium, such as water, saline, infiltration fluid, etc. These liquid carriers can provide suitable environmental conditions for microorganisms, including moisture, dissolved oxygen, pH value, etc., to promote the growth and activity of microorganisms.

[0049] 3. Gel carrier: Gel carriers are often used in preparations to stabilize microbial agents, such as gelatin, agar and agar. Gel has the ability to retain water and maintain microbial activity, and can increase the adhesion and stability of microbial agents.

[0050] The composition of microbial agents usually consists of microorganisms, carriers and other auxiliary agents. In addition to the carrier that provides growth support, microbial agents may also contain protective agents (such as protective agents, antioxidants, etc.) to improve the storage stability of the agent, nutrients, surfactants and other additives that have a positive impact on the growth and adaptability of microorganisms.

[0051] The above-mentioned beef extract peptone liquid culture medium includes nutrients and inorganic salts; preferably, the above-mentioned beef extract peptone liquid culture medium includes nutrients and inorganic salts; the nutrients include beef extract and peptone; preferably, the inorganic salts include one or more of sodium chloride, potassium chloride or magnesium chloride; preferably, in the above-mentioned beef extract peptone liquid culture medium, the mass content of beef extract is 0.1-0.5wt%, the mass content of peptone is 0.4-3.0wt% and the mass content of sodium chloride is 0.2-1.0wt%.

[0052] The above-mentioned nutrients are the main components of the liquid culture medium for culturing bacteria, and their components can provide nutrients to maintain the activity of microorganisms, so that they can grow and reproduce normally in the culture medium. The above-mentioned nutrients for maintaining activity include carbon sources, nitrogen sources, phosphorus sources and vitamins. Carbon source refers to the source of carbon elements required by microorganisms during growth, which can provide the carbon skeleton required for cell structure composition and metabolite synthesis, including but not limited to one or more of glucose, fructose, maltose, starch or cellulose. Nitrogen source refers to the source of nitrogen elements required by microorganisms during growth, which can provide amino acids and base compounds containing nitrogen atoms in proteins and nucleic acids, including but not limited to one or more of glutamic acid, aspartic acid, urea or nitrate nitrogen compounds. Phosphorus source refers to the source of phosphorus elements required by microorganisms during growth, including but not limited to phosphates or phosphate esters. The above-mentioned phosphate refers to a compound composed of phosphate ions and metal ions or hydrogen ions, including potassium dihydrogen phosphate and ammonium dihydrogen phosphate. The above-mentioned phosphate ester refers to a compound containing a phosphate group and an alcohol group connected by an ester bond, including dimethyl butyrate diphosphate and trimethyl acetate triphosphate. The above vitamins can promote the growth activity of the strain, including but not limited to one or more of the B vitamins, vitamin C (ascorbic acid), vitamin E (tocopherol) or vitamin K. The above B vitamins include but are not limited to vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or nicotinic acid), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine or pyridoxone), vitamin B7 (biotin), vitamin B9 (folic acid) and vitamin B12 (choline). The role of the B vitamins includes promoting the energy metabolism of microorganisms or maintaining enzyme activity. The role of vitamin C includes improving microbial tolerance and activity, and can protect microorganisms from environmental stress and oxidative damage. The role of vitamin E includes protecting the cell membrane and cell structure of microorganisms, which helps to improve the survival rate and activity of microorganisms. The role of vitamin K includes promoting the growth and nutritional metabolism of microorganisms.

[0053] The functions of the above-mentioned inorganic salts in the beef extract peptone medium for culturing bacteria include maintaining the osmotic pressure balance of microbial cells and regulating the membrane potential of cells by providing sodium ions, chloride ions, calcium ions or potassium ions.

[0054] In a third typical embodiment of the present application, a method for improving the salt-alkali tolerance of soybeans is provided, comprising: treating soybeans with the above-mentioned psychrophilic Pseudomonas 77S3 or any of the above-mentioned bacterial agents.

[0055] In a preferred embodiment, the above-mentioned treatment method includes one or more of seed soaking, soil mixing with bacteria, root irrigation or foliar spraying.

[0056] The above-mentioned seed soaking method refers to soaking plant seeds in a microbial agent for a period of time to obtain plant seeds after soaking treatment; during the soaking process of the seeds in the microbial agent, a layer of colonies in the above-mentioned microbial agent can be formed on the surface of the seeds, which can be absorbed by the seeds to promote seed germination and growth. The above-mentioned soil mixing method refers to mixing the microbial agent with the cultivation soil of the plant before or during planting, and after obtaining the mixed soil, the plant grows in the mixed soil, and the colonies in the microbial agent can be formed in the mixed soil, and the soil structure and nutritional status are improved through the action of the colonies, and the stress resistance of the plant is enhanced. The above-mentioned root irrigation method refers to using liquid microbial agents to directly treat the roots of the plants, soak the roots of the plants, so that the plants can absorb the microorganisms in the microbial agent through the roots, and promote growth and development. The above-mentioned spraying method refers to placing the liquid microbial agent in a sprayer and spraying it evenly on the leaves of the plants, forming a layer of colonies in the above-mentioned microbial agent on the surface of the leaves, and absorbing it into the interior of the plants through the stomata covered on the leaves, so that the plants obtain the stress resistance of the microorganisms in the microbial agent and promote the growth of the plants.

[0057] In the above treatment methods, any one of the seed soaking method, the fungus mixing method, the root irrigation method or the foliar spraying method can be used to treat soybeans, which can improve the salt-alkali tolerance of soybeans. The plant height, rhizome length and weight of the treated soybeans planted in a saline-alkali environment are greater than those of untreated wild-type soybeans. The above-mentioned multiple treatment methods can also be used in combination, and soybeans can be treated using two or more methods, which can achieve the same or even better technical effect of improving the salt-alkali tolerance of soybeans compared to a single treatment method.

[0058] In a preferred embodiment, the seed soaking method comprises soaking the soybean seeds in the liquid bacterial agent at 20-25° C. for 4-6 hours to obtain soaked soybeans.

[0059] In a preferred embodiment, the above-mentioned bacteria-mixing soil method includes mixing any of the above-mentioned bacterial agents with cultivation soil to prepare mixed soil, and planting the above-mentioned soybeans in the mixed soil; preferably, the above-mentioned soybeans are treated together by the above-mentioned seed soaking method and the above-mentioned bacteria-mixing soil method, and the soybean seeds after the above-mentioned seed soaking treatment are planted in the above-mentioned mixed soil.

[0060] In a fourth preferred embodiment of the present application, the use of the psychrophilic Pseudomonas 77S3, any of the above-mentioned bacterial agents or any of the above-mentioned treatment methods in soybean planting is provided. Especially when applied in a salt stress environment, it has the beneficial effect of improving the salt-alkali tolerance of soybeans.

[0061] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0062] Example 1

[0063] A method for separating and activating soybean endophytes, comprising the following steps:

[0064] 1. Sample collection: Collect fresh salt-tolerant wild soybean plants and isolate endophytes from their roots, stems and leaves.

[0065] 2. Sample sterilization treatment: Weigh 2 g of sample and rinse with tap water for 30 minutes; soak in sterile water in a clean bench for 30 seconds, repeat 3 times; soak in 75% ethanol for 3-5 minutes; rinse with sterile water 4 times to obtain a sterile sample.

[0066] 3. Preparation of endophytic bacterial liquid: Use a sterile knife to decompose the sterile sample, place it in a mortar, add a small amount of quartz sand and 5 mL of normal saline, grind it fully into a paste, and dilute it with sterile water to obtain the endophytic bacterial liquid.

[0067] 4. Cultivation of endophytes: Use a pipette to take 200 μL of endophyte solution and spread it on beef extract peptone medium. Place it in the dark at 30°C for 3 to 5 days to obtain soybean endophytes.

[0068] 5. Isolation and activation of soybean endophytes: Soybean endophytes of different morphologies were separated and purified on beef extract peptone medium by using the plate streak method. All separated and purified endophytes were activated in beef extract peptone liquid medium (200 rpm, 30° C.) to obtain activated soybean endophytes.

[0069] Example 2

[0070] A method for screening salt-alkali tolerant endophytes.

[0071] 1. Preliminary screening of salt-alkali tolerant endophytes: The endophytes isolated and purified in the early stage were placed on beef extract peptone solid medium containing different sodium chloride concentrations (200mM, 300mM, 500mM) and different pH values ​​(pH=7.0, pH=9.0) to observe the bacterial growth status. Figure 1 As shown, salt-tolerant bacteria were preliminarily screened out.

[0072] Screening results: The endophyte isolated in the early stage of the experiment was numbered as 77S3. The endophyte 77S3 was able to survive on a beef extract peptone solid medium containing 500 mM NaCl and pH = 9.0. Figure 2 shown.

[0073] 2. Screening of salt-tolerant and alkali-tolerant endophytes with growth-promoting effects;

[0074] 1) Qualitative plate experiment, preliminary screening of phosphate-solubilizing endophytes:

[0075] The salt-alkali tolerant endophytes obtained earlier were inoculated on NBRIP solid medium and cultured at 30°C for 3 to 14 days under dark conditions. NBRIP solid medium contains insoluble Ca3(PO4)2. When it is converted into soluble phosphorus by endophytes, a transparent dissolution zone will appear around the strain. If a transparent zone is produced, it preliminarily indicates that the endophytes have the ability to dissolve phosphorus. Three parallels are set for each strain.

[0076] Phosphorus dissolution rate (D·d -1 ) is calculated as follows:

[0077] Phosphorus dissolution rate (D·d -1 ) = transparent zone diameter (D) / colony diameter (d)

[0078] Screening results: 77S3 was spotted on NBRIP solid medium and cultured for 3-7 days. An obvious transparent circle could be seen near the strain, such as Figure 4 As shown in Table 1, the solubility rate is 1.68.

[0079] Table 1

[0080] Strain number D / d(value) 77S3 1.68±0.06

[0081] 2) Shake flask quantitative experiment, determination of phosphate solubilization capacity:

[0082] Determine the phosphate solubilizing ability of endophytes with phosphate solubilizing ability in NBRIP liquid medium. Inoculate a single colony of endophytes into NBRIP liquid medium, set up three replicates, and culture under shaking conditions of 28°C and 200rpm. Determine the daily soluble phosphorus content in the shake flask by the molybdenum antimony method. The operation is as follows:

[0083] (1) Preparation of phosphorus standard curve: measure 0, 2, 4, 8, and 10 mL of phosphorus standard solution (5 mg / L) respectively into a 50 mL volumetric flask, dilute to 20 mL with water, add a few drops of 2,4-dinitrophenol as an indicator solution, adjust the pH of the solution so that it just turns slightly yellow, add 5 mL of molybdenum antimony anticolorimetric agent, shake well, and add water to make up to 50 mL. The mass concentration of phosphorus in this standard series of solutions is 0, 0.2, 0.4, 0.8, and 1.0 mg / L, respectively. Place at room temperature around 25°C for 30 minutes, use the 0 mg / L phosphorus standard solution as a control, and record the absorbance of other concentrations of phosphorus standard solutions at a wavelength of 720 nm. Use absorbance as the ordinate and phosphorus concentration (mg / L) as the abscissa to draw a phosphorus standard curve, as shown below: Figure 3 shown.

[0084] (2) Sample test: Centrifuge the bacterial solution at 6000 rpm for 10 min, take 2.5 mL of the supernatant into a 50 mL volumetric flask (the sample dilution factor is 20 times), and the color development steps and the steps for measuring the phosphorus content are the same as the phosphorus standard curve steps mentioned above. Finally, calculate the phosphorus concentration in the sample.

[0085] (3) A series of 0, 0.2, 0.4, 0.8, and 1.0 mg / L phosphorus standard solutions were compared with blank controls at a wavelength of 720 nm, and the OD values ​​were recorded. In order to quantitatively determine the phosphate solubilization ability of phosphate-solubilizing bacteria, the molybdenum antimony colorimetric method was used to determine the cumulative amount of phosphate solubilized by phosphate-solubilizing bacteria in the fermentation bottle every day. Ca3(PO4)2 was used as the only phosphorus source in the culture medium to determine the phosphate solubilization ability of phosphate-solubilizing bacteria and the pH value of the culture medium. The experiment detected the effective phosphorus content of the endophytic bacteria culture solution at 2d, 4d, and 6d, such as Figure 5 shown.

[0086] 3) Screening for the ability to produce IAA (auxin, indole-3-acetic acid):

[0087] The Salkowski colorimetric method was used to screen IAA-producing endophytes. The single colony of salt-alkali tolerant endophytes obtained earlier was inoculated into beef extract peptone liquid culture medium, set up three replicates, used a 50 mL conical flask with a liquid volume of 10 mL, and cultured overnight in dark conditions (200 rpm, 30°C), and finally adjusted to OD600 = 1.0.

[0088] Preparation of fermentation broth: Take 1 mL of the above culture broth and inoculate it into a medium containing L-tryptophan (200 mg L -1 ) in beef extract peptone liquid culture medium, using a 250mL conical flask with a liquid volume of 50mL, cultured in the dark at 30°C for 6 days.

[0089] Preparation of IAA standard curve: Prepare 0, 5, 10, 20, 40, 80 and 100 mg / L IAA standard solutions. Take 100 μL of the standard solution with different concentrations, mix it with an equal volume of Salkowski colorimetric solution, and place it at room temperature for 30 minutes in the dark to develop color. Finally, use an ELISA reader to compare the color at a wavelength of 530 nm and record the OD value. The standard curve of IAA concentration and OD530 is shown in the figure below. Figure 6 shown.

[0090] Filter results:

[0091] Endophyte 77S3 can produce IAA. The amount of IAA produced by endophyte 77S3 on the 2nd, 4th and 6th day after inoculation is as follows: Figure 7 shown.

[0092] The culture medium formulation used above includes:

[0093] Beef extract peptone medium: Take 3.0 g beef extract, 10.0 g peptone, 5.0 g sodium chloride, pH = 7.2 ± 0.1, and dilute to 1 L. Add 15%-20% agar powder to the solid medium.

[0094] Contains L-tryptophan (200mg·L -1 ) beef extract peptone liquid medium: weigh 2.0 g·L -1 Tryptophan was dissolved in 100 mL NaOH (100 mM), sterilized by membrane, added into sterilized beef extract peptone liquid culture medium, mixed and set aside.

[0095] NBRIP medium: glucose 10g, Ca3(PO4)2 5g, KCl 0.2g, MgCl2·6H2O 5g, MgSO4·7H2O 0.25g, (NH4)2SO4 0.1g, adjust pH to 7.0±0.2, and make up to 1 L. Add 15-20% (w / v) agar powder to the solid medium.

[0096] Example 3

[0097] A method for observing the morphology of soybean endophytes.

[0098] The endophytes screened above were stained using Gram staining, and the morphological characteristics of the bacteria were observed under an optical microscope under an oil immersion lens, and photographed and recorded. The operation steps are as follows:

[0099] 1. Smear: Take the bacteria to be tested and apply a thin layer in the center of the slide or add a little sterile water on the slide, mix the bacteria and water evenly and apply a thin layer.

[0100] 2. Drying: After smearing, let it dry naturally at room temperature, or slightly heat it over an alcohol lamp to make it dry quickly.

[0101] 3. Fixation: Hold one end of the slide with the specimen facing up and quickly move it back and forth outside the flame of the alcohol lamp 3 to 5 times, each time for 1 second. The temperature should not be too high to prevent bacterial protein denaturation. Let it cool before staining. It can also be fixed with methanol or ethanol.

[0102] 4. Initial staining: Add crystal violet staining solution and stain for 1 to 2 minutes, then rinse off the staining solution with clean water.

[0103] 5. Mordanting: Add Gram's iodine solution to cover the slide, leave it at room temperature for 1 to 2 minutes, and wash with water.

[0104] 6. Decolorization: Add decolorizing solution dropwise and shake for 10-30 seconds until the decolorizing solution no longer appears purple. Immediately rinse off the decolorizing solution with water to terminate the reaction.

[0105] 7. Re-staining: Add red re-staining solution and dye for 30-60 seconds, then wash with water.

[0106] 8. Drying and microscopic examination: Observe under an oil mirror.

[0107] The observations are as follows:

[0108] The bacterial morphology of endophyte 77S3 observed under an optical microscope is as follows Figure 8 The morphological characteristics are shown in Table 2.

[0109] Table 2

[0110]

[0111] Embodiment 4:

[0112] A method for DNA extraction and phylogenetic analysis of endophytes.

[0113] 1. Add the endophytic bacteria screened in the previous step to the beef extract peptone liquid culture medium, culture overnight in the dark (200 rpm, 30°C), and collect the bacteria by centrifugation.

[0114] 2. Extract the DNA of the whole bacterial genome using universal primers 27F (SEQ ID NO: 1: AGAGTTTGATCNTGGCTCAG, "N" represents adenine, guanine, cytosine or thymine) and 1492R (SEQ ID NO: 2: ACGGYTACCTTGTTACGACTT, "Y" represents cytosine or thymine).

[0115] To amplify the 16S rDNA of the bacterial whole genome, the PCR reaction system is shown in Table 3 and the reaction procedure is shown in Table 4.

[0116] 3. The PCR products were electrophoresed on 1.5% agarose gel. After electrophoresis, the PCR products were recovered from the gel, purified and sent to the sequencing department of Sangon Biotech Co., Ltd. for sequencing. The obtained 16SrDNA sequences were searched for homologous sequences in NCBI by Blast, and the sequences with higher scores were selected for comparison in MEGA7.0 software, and the phylogenetic tree was established by the neighbor-joining method.

[0117] Table 3

[0118]

[0119] Table 4

[0120]

[0121] The results are as follows:

[0122] The selected endophyte 77S3 was identified by 16S rDNA sequence analysis, and a phylogenetic tree was constructed using MEGA7.0. The schematic diagram is shown in the figure. Fig. 9 shown.

[0123] Example 5

[0124] Growth-promoting effect of endophytes on soybean under salt stress.

[0125] Plant materials: The plant materials used in the experiment were Williams 82 soybeans (Glycine max L. Merrill) stored in the laboratory, which were related crops of wild soybeans, the source of endophytes in the early stage of the experiment.

[0126] 1. Preparation of cultivation soil: The experimental potting medium is commercially available vermiculite. Distilled water is mixed into the vermiculite, and an equal amount of vermiculite is placed in a transparent tissue culture bottle with a diameter of 7 cm and a height of 10.5 cm, covered, and placed in a high-pressure steam sterilizer at 121°C for 1 hour. After sterilization, take it out and let it dry to room temperature. After secondary sterilization under the same conditions, let it dry to room temperature for use.

[0127] 2. Preparation of inoculation solution:

[0128] Blank inoculum: culture without inoculating bacteria in 100 mL of beef extract peptone liquid culture medium, centrifuge, discard the supernatant, and add sterile water for later use.

[0129] 77S3 inoculum: Inoculate 77S3 cells into 100 mL of beef extract peptone liquid culture medium. Collect the cells after cultivation, mix with sterile water, adjust OD600 to 1.0, and set aside.

[0130] 3. Soaking of soybean seeds: Use chlorine gas to sterilize the surface of Wm82 soybean seeds, and place them in blank inoculum and 77S3 inoculum respectively, and soak them at room temperature for 4 hours.

[0131] 4. Experimental grouping treatment method is shown in Table 5. The prepared inoculum solution was mixed into the tissue culture bottle containing the cultivation soil, 5 mL / pot, to obtain mixed soil. The soaked soybean seeds were planted in the tissue culture bottle containing the mixed soil, with 10 soybean seeds planted in each bottle.

[0132] When watering, T1, T2 and T3 were irrigated with distilled water, and T4, T5 and T6 were treated with an equal amount of 150mM NaCl solution. Six parallels were set up for each treatment group. All cultivations were carried out in a light incubator under consistent conditions. The parameters were set to 25℃ (16h light) and 25℃ (8h dark).

[0133] Table 5

[0134] Cultivation treatment Processing T1 Soybean seeds soaked in salt-free treatment + blank inoculant T2 Soybean seeds soaked in salt-free treatment and 77S3 inoculant T3 Soybean seeds treated with 150 mM NaCl + soaked in blank inoculum T4 150mM NaCl treatment + soybean seeds soaked in 77S3 inoculum

[0135] The results are as follows:

[0136] Soybean seeds were treated with salt. After 20 days of cultivation, soybean seedlings were sampled and photographed. The root morphology of soybeans under salt stress was as follows: Fig.10 The overall morphology of the seedlings is shown in Fig.11 shown.

[0137] exist Fig.10 In the figure, the three soybean roots on the left are the root phenotype results of soybean seedlings grown from soybean seeds treated with blank inoculum and irrigated with 150 mM NaCl, and the three soybean roots on the right are the root phenotype results of soybean seedlings grown from soybean seeds treated with endophyte 77S3 inoculum and irrigated with 150 mM NaCl.

[0138] The height, root length, stem fresh weight, stem dry weight, root fresh weight and root dry weight of soybean seedlings were measured. Fig.12 shown.

[0139] Under 150mM NaCl salt stress, 77S3 increased the plant height of Wm82 soybean seedlings by 16.29% compared with the control group; the root length increased by 15.73% compared with the control group; the stem fresh weight increased by 15.73% compared with the control group; the stem dry weight increased by 14.01% compared with the control group; the root fresh weight increased by 20.77% compared with the control group; and the root dry weight increased by 24.14% compared with the control group.

[0140] Therefore, the endophyte 77S3 isolated from salt-tolerant wild soybean had a significant growth-promoting effect on Wm82 seedlings under salt stress.

[0141] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the present invention successfully isolates and screens soybean endophytes from wild soybean plants, and obtains psychrophilic Pseudomonas 77S3 that still has a growth-promoting effect under stress. Inoculating psychrophilic Pseudomonas 77S3 into soybean seeds can significantly improve the salt-alkali tolerance of soybeans under salt stress; or applying psychrophilic Pseudomonas 77S3 to the cultivation soil, by changing the properties of the cultivation soil, ensures that soybean plants can continue to grow under salt stress and maintain excellent agronomic traits. This kind of psychrophilic Pseudomonas 77S3 can also solubilize phosphate and secrete IAA. The endophytic bacteria psychrophilic Pseudomonas 77S3 are used to treat soybean seeds or cultivation media, replacing the effects brought by chemical fertilizers, making up for the loss of important endophytic microorganisms in soybeans during long-term cultivation, and further reducing the dependence of crops on agricultural chemicals during planting.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A psychrophilic Pseudomonas 77S3, classified and named Pseudomonas psychrotolerans, was deposited in the China Center for Type Culture Collection on May 29, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC M 2023852.

2. A bacterial agent, characterized in that The bacterial agent includes the psychrophilic Pseudomonas 77S3 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent includes a liquid bacterial agent or a solid bacterial agent.

4. The bacterial agent according to claim 2, characterized in that The bacterial agent comprises water and the psychrophilic Pseudomonas 77S3, and the OD600 of the bacterial agent is 0.8-1.

2.

5. The bacterial agent according to claim 2, characterized in that The carrier of the bacterial agent includes a solid carrier, a liquid carrier or a gel carrier.

6. A method for improving the salt-alkali tolerance of soybeans, characterized in that: The method comprises: Treating soybeans using the psychrophilic Pseudomonas 77S3 of claim 1 or the bacterial agent of any one of claims 2 to 5; The treatment method is a seed soaking method.

7. The method according to claim 6, characterized in that The seed soaking method comprises soaking the soybean seeds in the liquid bacterial agent of claim 3 for 4 to 6 hours at 20 to 25° C. to obtain soaked soybeans.

8. The method according to claim 7, characterized in that planting the soybeans in the mixed soil; The soybeans are treated by using the seed soaking method and the bacteria-mixing soil method, wherein the bacteria-mixing soil method comprises: mixing the bacterial agent according to any one of claims 2 to 5 with cultivation soil to prepare the mixed soil; The method comprises: planting the soybean seeds treated by the seed soaking method in the mixed soil.

9. Use of the psychrophilic Pseudomonas 77S3 according to claim 1, the bacterial agent according to any one of claims 2 to 5, or the method according to claim 6 in soybean planting.

Images