Enterobacter sphaeroides and application thereof in improving stress resistance of plants

Abstract

The invention discloses an enterobacter strain and application thereof in improving stress resistance of plants. The invention relates to the field of biological agriculture, and provides Enterobacter sphaeroides YJD-AD2-2, and the enrollment number of the Enterobacter sphaeroides YJD-AD2-2 in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms is CGMCC No.29524. The Enterobacter sphaeroides YJD-AD2-2 can be used for preparing the enterobacter sphaeroides YJD-AD2-2. The enterobacter sphaeroides YJD-AD2-2 provided by the invention can improve the resistance of arabidopsis thaliana, alfalfa, corn and rice to salt and alkali stress, specifically, the dry weight, fresh weight, plant length, leaf area and chlorophyll content are increased, the oxidation resistance is improved, wilt is reduced, and physiological and biochemical indexes are improved. The YJD-AD2-2 microbial inoculum improves the property and activity of saline alkali soil and improves the abundance and composition of microbial communities. Detection shows that the YJD-AD2-2 strain has the capabilities of secreting IAA (Indoleacetic Acid), ACC (Acetyl Cellulose) deaminase and siderophore, dissolving phosphorus and fixing nitrogen. The indexes show that the YJD-AD2-2 microbial inoculum has the potential of growth-promoting bacteria and has important application value in the field of agriculture.

Description

Technical Field

[0001] The invention relates to the field of biological agriculture, and in particular to a strain of Enterobacter kobeensis and application thereof in improving plant stress resistance. Background Art

[0002] Plants growing in natural environments are often subject to various biotic and abiotic stresses. Salt stress is one of the main factors threatening global sustainable agricultural production, threatening ecological security and food security. The lack of improvement measures for soda saline-alkali land is currently the bottleneck restricting the improvement and comprehensive utilization of soda saline-alkali land. Excessive salt concentration can cause soil degradation and change soil permeability and matrix potential, which is due to excessive exchangeable Na + and high soil pH can lead to clay swelling and dispersion, as well as a reduction in soil aggregates due to a decrease in soil permeability, available water capacity, and infiltration rate, leading to soil compaction and reduced vitality, thus making the soil unsuitable for crop growth. Salt stress can have various harmful effects on the physiological, biochemical, and molecular characteristics of plants and reduce crop productivity. Poor growth of plants under salt stress is caused by reduced nutrient mobilization, hormone imbalance, formation of reactive oxygen species (ROS), ion toxicity, and osmotic stress. In addition, the physicochemical properties of the soil caused by salinity will affect the activity of soil microorganisms, leading to the destruction of soil microbial diversity, thereby reducing the health of the soil.

[0003] Plant rhizosphere growth-promoting bacteria refer to a type of beneficial bacteria that live freely in the soil or are attached to the rhizosphere of plants and can promote plant growth, prevent and control diseases, and increase crop yields. Inoculation of plant rhizosphere growth-promoting bacteria can make ineffective nutrients in the soil effective, prevent and control crop diseases, and reduce the use of pesticides and fertilizers. It is a fundamental way to solve soil, water and food pollution and is generally considered to be an environmentally friendly and cost-effective way to increase crop yields. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to improve the normal growth of plants in saline-alkali land, increase the yield and biomass of plants, and improve the stress resistance of plants.

[0005] In order to solve the above technical problems, the present invention first screened a strain of Enterobacter kobeensis.

[0006] The Kobe Enterobacter provided by the present invention is Kobe Enterobacter (Enterobacter wuhouensis) CGMCC No. 29524, and its registration number in the General Microbiological Center of China National Microbiological Culture Collection Committee is CGMCC No. 29524.

[0007] The invention also provides a bacterial agent.

[0008] The bacterial agent provided by the present invention contains the above-mentioned Enterobacter kojiensis and / or metabolites of the Enterobacter kojiensis.

[0009] In the above-mentioned microbial agent, in addition to the active ingredient, the microbial agent also contains a carrier. The carrier may be a carrier commonly used in the field of pesticides and biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, soybean flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0010] The above-mentioned bacterial agents may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0011] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0012] In a specific embodiment of the present invention, the bacterial agent is a fermentation broth of the Enterobacter wuhouensis YJD-AD2-2 with a concentration of 2×108 CFU / mL. Further, the bacterial agent is prepared according to a method comprising the following steps: inoculating the Enterobacter wuhouensis YJD-AD2-2 into a bacterial culture medium and culturing the culture medium to obtain an OD 600 The bacterial solution (2×108 CFU / mL) with a value of 0.5-1.0 (such as 1.0) is the bacterial agent.

[0013] Furthermore, the bacterial culture medium is LB liquid culture medium.

[0014] In the preparation method of the bacterial agent, the culture conditions may be: 25-35°C (such as 25-30°C, 30-35°C, 25°C, 30°C or 35°C), 100-150r / min (such as 100-130r / min, 130-150r / min, 100r / min, 130r / min or 150r / min), culture for 30-60h (such as 30-48h, 48-60h, 30h, 48h or 60h).

[0015] The term "metabolite" refers to the primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process in which microorganisms absorb various nutrients from the outside world and generate substances and energy to maintain life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids and other monomers, as well as various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process in which microorganisms use primary metabolites as precursors to synthesize some substances that have no clear function in the life activities of microorganisms during a certain growth period. The products of secondary metabolism are secondary metabolites, most of which are compounds with relatively complex molecular structures. According to their functions, they can be divided into antibiotics, antioxidants, hormones, alkaloids, toxins and other types.

[0016] In the above, the metabolite can be obtained from the fermentation broth of the Kobe Enterobacter. The metabolite can be a sterile metabolite of the Kobe Enterobacter or a bacteria-containing metabolite of the Kobe Enterobacter. The sterile metabolite of the Kobe Enterobacter (sterile fermentation filtrate) can be prepared specifically according to the following method: the Kobe Enterobacter is cultured in a liquid culture medium, and the Kobe Enterobacter in the liquid culture (fermentation broth) is filtered out to obtain the sterile metabolite of the Kobe Enterobacter. The bacteria-containing metabolite of the Kobe Enterobacter can be prepared specifically according to the following method: the Kobe Enterobacter is cultured in a liquid fermentation medium, and the fermentation broth is collected, and the fermentation broth is the bacteria-containing metabolite of the Kobe Enterobacter.

[0017] The present invention also provides a culture of the above-mentioned Enterobacter kobeii, which is a substance obtained by culturing the above-mentioned Enterobacter kobeii in a bacterial culture medium.

[0018] The term "culture" refers to a general term for liquid or solid products (all materials in the culture container) that have a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing a microorganism, which can be a culture of a certain generation or a mixture of several generations.

[0019] The use of the above-mentioned Enterobacter kobe, Enterobacter kobe bacterial agent or Enterobacter kobe culture in at least one of the following also falls within the scope of protection claimed by the present invention.

[0020] The above application may specifically be any of the following:

[0021] (a1) Enhance the salt-alkali resistance of plants;

[0022] (a2) preparing products for enhancing the salt-alkali resistance of plants;

[0023] (a3) promoting plant growth;

[0024] (a4) preparing products for promoting plant growth;

[0025] (a5) Producing IAA;

[0026] (a6) preparing an IAA-producing product;

[0027] (a7) produces ACC deaminase;

[0028] (a8) preparing a product producing ACC deaminase;

[0029] (a9) siderophore production;

[0030] (a10) Preparation of siderophore-producing products

[0031] (a11) Phosphate dissolution;

[0032] (a12) preparing a phosphate-dissolving product;

[0033] (a13) nitrogen fixation;

[0034] (a14) preparing nitrogen-fixed products;

[0035] (a15) biofilm formation;

[0036] (a16) A product for preparing a biofilm.

[0037] In the above application, the plant growth promotion is embodied in whole or in part as follows:

[0038] (b1) promoting plant root elongation under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0039] (b2) promoting an increase in plant dry weight under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0040] (b3) promoting the increase of plant fresh weight under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0041] (b4) promoting plant growth under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0042] (b5) promoting the increase of plant leaf length under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0043] (b6) promoting the increase of plant leaf width under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0044] (b7) reducing plant leaf wilting under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0045] (b8) promoting the increase of chlorophyll content in plants under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0046] (b9) promoting the reduction of proline content in plants under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0047] (b10) promoting the reduction of malondialdehyde content in plants under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0048] (b11) promoting the enhancement of plant antioxidant enzyme activity under saline-alkali stress conditions or non-saline-alkali stress conditions;

[0049] The present invention also provides a method for preparing the bacterial agent, comprising the following steps: using the above-mentioned Enterobacter kojiensis as an active ingredient to obtain the bacterial agent.

[0050] The present invention also provides a bio-organic fertilizer, which contains the above-mentioned Enterobacter kojiensis or the bacterial agent or the above-mentioned culture.

[0051] The invention also provides a method for promoting plant growth.

[0052] The present invention also provides a method for promoting plant growth, which may include the following steps: applying the above-mentioned Enterobacter kojiensis or the above-mentioned bacterial agent or the above-mentioned culture to the test plant under saline-alkali stress conditions or non-saline-alkali stress conditions, thereby promoting plant growth.

[0053] The administration may be dripping.

[0054] The non-salt-alkali stress condition may be a normal growth condition (without any stress). In a specific embodiment of the present invention, water treatment is used as the non-salt-alkali stress condition.

[0055] In the above application or method, the plant may be a monocotyledonous plant or a dicotyledonous plant.

[0056] In the above application or method, the monocotyledonous plant may be a plant of the Gramineae family; the dicotyledonous plant may be a plant of the Leguminosae family or a plant of the Cruciferae family.

[0057] Furthermore, the grass plant may be corn or rice; the leguminous plant may be alfalfa, and the cruciferous plant may be Arabidopsis thaliana.

[0058] In one embodiment of the present invention, the plant is specifically a wild-type Arabidopsis Columbia-0 subtype. In another embodiment of the present invention, the plant is specifically a maize inbred line B73. In yet another embodiment of the present invention, the plant is specifically a rice variety Nipponbare.

[0059] Experiments have shown that the Enterobacter wuhouensis YJD-AD2-2 provided by the present invention can improve the resistance of Arabidopsis, alfalfa, corn and rice to salt-alkali stress, which is specifically manifested in a significant increase in dry weight, fresh weight and root length, an increase in the height of the aboveground part (i.e., an increase in plant height), an increase in leaf length and width, and changes in physiological and biochemical properties. Biochemical experiments using YJD-AD2-2 as a growth-promoting bacterium have shown that YJD-AD2-2 has strong salt-alkali stress tolerance and has the ability to dissolve phosphorus, fix nitrogen, secrete H + , siderophore, IAA and ACC deaminase and the ability to form biofilm are important evidences that YJD-AD2-2 has the potential to be a salt-tolerant and growth-promoting bacterium. The above experiments show that Kobe Enterobacter YJD-AD2-2 has important application value in the field of saline-alkali soil remediation.

[0060] Collection Instructions

[0061] Bacterial species name: Kobe Enterobacter

[0062] Latin name: Enterobacter wuhouensis

[0063] Strain ID: YJD-AD2-2

[0064] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0065] Abbreviation of depository institution: CGMCC

[0066] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0067] Deposit date: January 5, 2024

[0068] CGMCC Registration Number: CGMCC No.29524 BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The morphology and genus identification results of Kobe Enterobacter YJD-AD2-2 strain. A is the Gram staining result and YJD-AD2-2 bacterial cell morphology; B is the phylogenetic tree analysis.

[0070] Figure 2 The effect of YJD-AD2-2 bacterial agent on the growth of Arabidopsis seedlings under carbonate treatment conditions. The left side is alkaline solid culture medium; the right side is alkaline + bacterial agent solid culture medium.

[0071] Figure 3The effect of YJD-AD2-2 bacterial agent on the growth of alfalfa seedlings under alkaline stress conditions. A is the phenotype of alfalfa seedlings under various treatment conditions; B is the overall growth of alfalfa seedlings under various treatment conditions. 1 is the water control group; 2 is the water + bacterial agent group; 3 is the alkali solution control group; 4 is the alkali solution + bacterial agent group.

[0072] Figure 4 The effects of YJD-AD2-2 on the physiological and biochemical parameters of alfalfa seedlings under alkaline stress. A is plant height (above ground part); B is root length; C is fresh weight.

[0073] Figure 5 The effect of YJD-AD2-2 bacterial agent on the growth of corn seedlings under alkaline stress conditions. 1 is the water control group; 2 is the water + bacterial agent group; 3 is the alkali solution control group; 4 is the alkali solution + bacterial agent group.

[0074] Figure 6 The effects of YJD-AD2-2 on the antioxidant enzyme activities of corn seedlings under alkaline stress. A is SOD activity; B is POD activity; C is CAT activity; D is APX activity.

[0075] Figure 7 The effects of YJD-AD2-2 on the physiological and biochemical parameters of corn seedlings under alkaline stress. A is plant length (aboveground + underground parts); B is fresh weight; C is total chlorophyll content; D is MDA content; E is proline content.

[0076] Figure 8 The effect of YJD-AD2-2 bacterial agent on the growth of rice seedlings under alkaline stress conditions. 1 is the water control group; 2 is the water + bacterial agent group; 3 is the alkali solution control group; 4 is the alkali solution + bacterial agent group.

[0077] Fig. 9 The effects of YJD-AD2-2 on the physiological and biochemical parameters of rice seedlings under alkaline stress. A is plant length (aboveground + underground parts); B is fresh weight; C is total chlorophyll content; D is MDA content; E is proline content.

[0078] Fig.10 The effects of YJD-AD2-2 on the antioxidant enzyme activities of rice seedlings under alkaline stress. A is SOD activity; B is POD activity; C is CAT activity; D is APX activity.

[0079] Fig.11 pH suitable range and NaHCO tolerance of YJD-AD2-2 3 and NaCl capacity detection. A is the proliferation capacity detection of YJD-AD2-2 under different pH conditions; B is the detection of different concentrations of NaHCO 3C is the detection of the proliferation ability of YJD-AD2-2 under conditions of different NaCl concentrations.

[0080] Fig.12 YJD-AD2-2 strain secretes H + Ability testing.

[0081] Fig.13 The IAA secretion capacity of YJD-AD2-2 strain was tested. A is the qualitative result; B is the quantitative result.

[0082] Fig.14 The results are for the detection of ACC deaminase secretion ability of YJD-AD2-2 strain. A is the qualitative result; B is the quantitative result.

[0083] Fig.15 The siderophore secretion capacity of YJD-AD2-2 strain was tested. A is the qualitative result; B is the quantitative result.

[0084] Fig.16 This is the detection of the phosphorus solubilization ability of the YJD-AD2-2 strain. A is the qualitative result; B is the quantitative result.

[0085] Fig.17 This is a test of the nitrogen fixation ability of the YJD-AD2-2 strain (qualitative results).

[0086] Fig.18 The detection of the biofilm-forming ability of the YJD-AD2-2 strain. A is the qualitative result; B is the quantitative result. DETAILED DESCRIPTION

[0087] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0088] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0089] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0090] LB liquid culture medium: 10 g of tryptone, 5 g of yeast extract, and 30 g of sodium chloride, dissolve in distilled water, adjust the pH to 8.0, make up to 1 L, sterilize at 121°C for 15 min, and cool for later use.

[0091] LB solid medium: Add agar to LB liquid medium to a final concentration of 15 g / L and sterilize at 121°C for 15 min. Cool to about 55°C, pour into a culture dish, and cool until solidified before use.

[0092] The wild-type Arabidopsis thaliana Columbia-0 subtype is described in the following document: Kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J. Agenetic link between cold responses and flowering time through FVE in Arabidopsis thaliana. Nature Genetics. 2004, 36: 167-171. The wild-type Arabidopsis thaliana Columbia-0 subtype is hereinafter referred to as Arabidopsis thaliana.

[0093] Example 1. Isolation, Identification and Preservation of Enterobacter wuhouensis YJD-AD2-2 I. Isolation of YJD-AD2-2

[0094] 1. Add 5 g of plant rhizosphere soil sample to 45 mL of sterile distilled water, stir for 15 min, let stand for 10 min, then take 1 mL of the supernatant and add it to a sterile test tube containing 9 mL of sterile water and mix thoroughly (the dilution at this time is recorded as 10 -1 ), then draw 1mL from this test tube and add it to another sterile test tube containing 9mL of sterile water and mix well. Repeat this process to make 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Different dilutions of bacterial suspension. Take 0.1 mL of each dilution and evenly spread it on LB solid medium, and culture it at 30℃ for 2-3 days.

[0095] 2. After completing step 1, pick a single colony on the LB solid medium and purify it repeatedly for more than 3 times to obtain a single strain. One of the strains was named YJD-AD2-2.

[0096] 2. Identification of YJD-AD2-2

[0097] 1. Morphological identification

[0098] Gram stain:

[0099] ① Preparation: Take the bacterial culture for routine smear, dry and fix.

[0100] Gram staining should be performed using a culture of strain YJD-AD2-2 in its active growth phase. The smear should not be too thick to avoid false positives caused by incomplete decolorization. The flame fixation should not be too hot (the slide should not be too hot to touch).

[0101] ② Initial staining: Add crystal violet (just enough to cover the bacterial film) and stain for 1-2 minutes, then wash with water.

[0102] ③ Mordanting: Rinse away residual water with iodine solution, cover with iodine solution for about 1 minute, and wash with water.

[0103] ④ Decolorization: Use filter paper to absorb the residual water on the glass slide, tilt the glass slide, and use a dropper to add 95% ethanol for decolorization against a white background until the ethanol flowing out has no purple color, then wash with water immediately.

[0104] Ethanol decolorization is a key step in Gram staining: if the decolorization is insufficient, negative bacteria will be mistakenly stained as positive bacteria; if the decolorization is excessive, positive bacteria will be mistakenly stained as negative bacteria. Therefore, the decolorization time is generally 20-30s.

[0105] ⑤Re-dyeing: Re-dye with safranin solution for about 2 minutes and wash with water.

[0106] ⑥ Microscopic examination: After drying, observe with an oil microscope. Bacteria that are stained blue-purple are Gram-positive bacteria, and those that are stained red are Gram-negative bacteria.

[0107] The strain YJD-AD2-2 was stained with Gram stain. Figure 1 The bacteria shown in A were identified as Gram-negative bacteria. The bacteria were long rod-shaped and 3-10 μm in length.

[0108] 2. 16S rDNA sequence homology analysis

[0109] The specific analysis steps are as follows:

[0110] (1) Bacterial genome extraction

[0111] (2) Amplification of 16S rDNA sequence using specific primers

[0112] (3) Purification of PCR products

[0113] (4) DNA sequencing to obtain 16S rDNA sequence

[0114] (5) Compare the sample species information with known bacteria in the NCBI database

[0115] (6) Select similar bacterial species sequences and construct a phylogenetic tree

[0116] The 16S rDNA of strain YJD-AD2-2 is shown as SEQ ID No.1.

[0117] The double-stranded DNA molecule shown in SEQ ID No.1 was compared with the sequence in GenBank using Clustal X software, and a phylogenetic tree was constructed by combining the 16S rRNA sequences of similar species. Figure 1 B shows that strain YJD-AD2-2 has the highest homology with Enterobacter wuhouensis strain WCHEs120002, reaching 99.45%.

[0118] 3. Storage of Kobe Enterobacter YJD-AD2-2 strain

[0119] According to the above morphological, physiological and biochemical characteristics and 16S rDNA sequence homology analysis results, the bacteria YJD-AD2-2 isolated and purified in step 1 was identified as Enterobacter wuhouensis. It was deposited in the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​January 5, 2024, with a deposit number of CGMCC No. 29524. Hereinafter referred to as Enterobacter wuhouensis CGMCC No. 29524 or Enterobacter wuhouensis YJD-AD2-2.

[0120] Strain preservation: A single colony of Enterobacter wuhouensis YJD-AD2-2 was inoculated into LB liquid culture medium and cultured at 30°C for 16 hours to obtain a culture solution. One volume of the culture solution was mixed with one volume of 80% (v / v) glycerol aqueous solution and stored at -80°C.

[0121] Example 2: Preparation of Enterobacter kobe YJD-AD2-2 bacterial agent

[0122] The Enterobacter wuhouensis YJD-AD2-2 strain stored at -80°C in Example 1 was awakened on LB solid medium, and a single colony was picked and inoculated into a conical flask (specification: 500 mL) containing 100 mL LB liquid medium, and cultured at 30°C and 130 r / min for 24-36 h to obtain an OD 600 The bacterial solution with a value of about 1.0 (2×10 8 CFU / mL). The bacterial solution is the prepared YJD-AD2-2 bacterial agent.

[0123] Example 3: Application of Enterobacter kobeensis YJD-AD2-2 in improving the ability of Arabidopsis thaliana to resist salt-alkali stress

[0124] The size of the culture dish is 10cm×10cm.

[0125] 1. Preparation of culture medium

[0126] Alkaline solid medium: adjust the pH value of 1 / 2MS solid medium to 8.0, sterilize at 121℃ for 60min, and add NaHCO to the medium cooled to about 55℃. 3 The final concentration was 1.5 mM, and then the culture medium was poured into culture dishes (20-25 mL per culture dish) and cooled naturally to prepare an alkaline solid culture medium.

[0127] Alkali + bacterial agent (Kobe Escherichia coli YJD-AD2-2) solid culture medium: Take a culture dish containing alkaline solid culture medium, stand it upright, and use a toothpick to apply a trace amount of Kobe Escherichia coli YJD-AD2-2 bacterial agent prepared in Example 2 above on the surface of the culture medium below the lower quarter, and air-dry it naturally.

[0128] 2. Effect of Enterobacter kobe YJD-AD2-2 on the resistance of Arabidopsis to salt-alkali stress

[0129] The culture conditions were: 22°C, 12h light / 12h dark, and light intensity of 5000Lx.

[0130] 1) Arabidopsis thaliana (wild-type Arabidopsis thaliana Columbia-0 subtype) seeds were sterilized with a 2.6% (v / v) sodium hypochlorite aqueous solution for 10 min, and then washed three times with sterile water.

[0131] 2) After completing step 1), Arabidopsis seeds were sown on solid culture medium (alkaline solid culture medium, alkaline + bacterial agent solid culture medium) and vernalized at 4°C for three days. It should be noted that when the seeds were sown on the alkaline + bacterial agent solid culture medium, they were not in direct contact with the bacterial agent, that is, they were sown on the upper quarter of the culture dish of the alkaline + bacterial agent solid culture medium, and then cultured vertically. The root tips of Arabidopsis were observed and counted before contacting the bacterial agent (this experiment mainly studies the effect of the secretion of the bacterial agent on the growth of Arabidopsis).

[0132] 3) The solid culture medium obtained in step 2) is vertically cultured for 7 days or more to observe the growth and development phenotype of Arabidopsis thaliana.

[0133] The phenotype of Arabidopsis thaliana grown for 12 days is shown in Figure 2(The left side is alkaline solid medium, and the right side is alkaline + bacterial agent solid medium). The results showed that under saline-alkali stress conditions, the average taproot length of Arabidopsis seedlings on the solid medium coated with Kobe E. coli YJD-AD2-2 bacterial agent was significantly higher than that of the control alkaline solid medium (not coated with YJD-AD2-2 bacterial agent). Moreover, the number and length of lateral roots of seedlings treated with YJD-AD2-2 bacterial agent were also significantly higher than those of the control group ( Figure 2 ), the leaves of seedlings treated with the inoculant were larger and greener than those of the control group.

[0134] 4) Use ImageJ software to measure and count the taproot length of Arabidopsis seedlings in step 3 (n=80).

[0135] The statistical results are shown in Table 1. The significance was evaluated by one-way analysis of variance (***, P < 0.001).

[0136] Table 1. Statistics of main root length of Arabidopsis thaliana

[0137] Types of solid culture media Average taproot length (cm) Alkaline solid medium 1.852 Alkali + bacterial agent solid culture medium 3.735***

[0138] The results showed that under saline-alkali stress conditions, the average taproot length of Arabidopsis seedlings on the solid medium coated with Kobe E. coli YJD-AD2-2 was significantly higher than that on the control solid medium (not coated with YJD-AD2-2) (P<0.0001). This shows that Kobe E. coli YJD-AD2-2 can significantly promote the growth of Arabidopsis and improve its salt-alkali tolerance.

[0139] Example 4: Application of Enterobacter kobe YJD-AD2-2 bacterial agent in improving the ability of alfalfa to resist salt-alkali stress

[0140] Culture conditions: 22°C, 12h light / 12h dark, light intensity of 5000Lx.

[0141] Alkaline solution (30mM, Na 2 CO 3 :NaHCO 3 =1:9): 1.272g Na 2 CO 3 and 9.072 g NaHCO 3 Dissolve in 4000mL distilled water and set aside.

[0142] 1) Alfalfa seeds were sown in 16 medium-sized pots filled with nutrient soil (90 seeds in each pot). After 6 days of cultivation, a total of about 1,280 alfalfa seedlings were obtained, with about 80 seedlings in each pot (germination rate of about 89%).

[0143] 2) Divide the seedlings into two groups, each with 8 pots. One group is irrigated with 800 mL of 30 mM alkali solution (a large amount of liquid flows out from the bottom of the pot), and the other group is irrigated with 800 mL of distilled water. The treatment is then carried out once every 7 days, 500 mL each time, for a total of 3 times.

[0144] 3) The 8 pots of alfalfa seedlings irrigated with alkali solution were randomly divided into two groups: an alkali solution control group and an alkali solution + bacterial agent group, with 4 pots in each group. The 8 pots of alfalfa seedlings irrigated with distilled water were randomly divided into two groups: a water control group and a water + bacterial agent group, with 4 pots in each group. The experiment is as follows:

[0145] While the water + bacterial agent and alkali solution + bacterial agent groups were being treated for the first time, the YJD-AD2-2 bacterial agent (OD 2.0) prepared in Example 2 was evenly dripped onto the root of each alfalfa seedling. 600 =1), a total of 5 mL, cultured for 21 days; the water control group and the alkali solution control group were not treated with bacterial agents and cultured for 21 days under the same conditions.

[0146] 4) Observation of the growth and development phenotype of alfalfa seedlings in each group.

[0147] The phenotypes of alfalfa seedlings treated for 21 days are shown in Tables 2 and Figure 3 (1 is the water control group; 2 is the water + bacterial agent group; 3 is the alkali solution control group; 4 is the alkali solution + bacterial agent group). The statistical results showed that under the conditions of water and alkali solution treatment, the alfalfa seedlings in the group treated with YJD-AD2-2 bacterial agent showed a higher survival rate, higher plant height, longer taproot, more branches, and larger and greener leaves (Table 2, Figure 3 ), indicating that applying YJD-AD-2-2 inoculant under normal and alkaline treatment conditions can significantly promote the survival rate and growth of alfalfa seedlings.

[0148] Table 2. Statistics of plant height, root length and fresh weight of alfalfa seedlings

[0149] Average plant height (cm) Average root length (cm) Average fresh weight (g) Water control group 5.069 4.007 0.076 Water bacteria group 6.205 4.35 0.115 Alkaline solution control group 2.805 2.504 0.051 Alkaline solution group 6.965 5.011 0.075

[0150] 5) Effects of YJD-AD2-2 inoculant treatment for 21 days on the fresh weight and plant length of alfalfa seedlings under alkaline stress conditions.

[0151] Under alkaline stress conditions, the average fresh weight, plant height and root length of alfalfa seedlings in the alkali solution + bacterial agent group with YJD-AD2-2 bacterial agent were significantly higher than those in the alkali solution control group without YJD-AD2-2 bacterial agent ( Figure 4 AC). The significance was evaluated by one-way analysis of variance (*, P<0.05; ***, P<0.001).

[0152] It can be seen that under the conditions of water and alkali solution treatment, the application of Kobe Escherichia coli YJD-AD2-2 bacterial agent can significantly promote plant growth, and under alkaline stress, YJD-AD2-2 bacterial agent can significantly improve the alkaline stress resistance of alfalfa seedlings.

[0153] Example 5: Application of Enterobacter kobeensis YJD-AD2-2 in improving the ability of corn to resist salt-alkali stress

[0154] Alkaline solution (80 mM, Na 2 CO 3 :NaHCO 3 =1:9): 2.544 g Na 2 CO 3 and 18.144 g NaHCO 3 Dissolve in 3000mL distilled water and set aside.

[0155] Corn seedling culture conditions: 22℃; 12h light / 12h dark; light intensity of 5000Lx.

[0156] 1) 108 maize inbred line B73 seeds were taken and sown in 12 small pots filled with nutrient soil, with 9 seeds in each pot. Maize seedlings were obtained after culturing for 5 days (germination rate 100%).

[0157] 2) Divide the seedlings into two groups, each with 6 pots. One group is irrigated with 200 mL of 80 mM alkali solution, and the other group is irrigated with 200 mL of distilled water (a large amount of liquid flows out from the bottom). Then treat once every 5 days, 100 mL each time, for a total of 3 times.

[0158] 3) The 6 pots of corn seedlings irrigated with distilled water were randomly divided into two groups: a water control group and a water + bacterial agent group, with 3 pots in each group. The 6 pots of corn seedlings irrigated with alkali solution were randomly divided into two groups: an alkali solution control group and an alkali solution + bacterial agent group, with 3 pots in each group. While the water + bacterial agent group and the alkali solution + bacterial agent group were subjected to the first distilled water and alkali solution treatments, the YJD-AD2-2 bacterial agent (OD 200) prepared in Example 2 was evenly dripped onto the root of each corn seedling. 600 =1), 1 mL per pot, and then cultured for 15 days; no bacterial agent was applied to the water control group and the alkali solution control group, and they were cultured under the same conditions for 15 days.

[0159] 4) Observation of growth and development phenotype of corn seedlings in each group.

[0160] The phenotype of the whole seedling of corn seedlings grown for 15 days is shown in Figure 5(1 is the water control group, 2 is the water + bacterial agent group, 3 is the alkali solution control group, and 4 is the alkali solution + bacterial agent group). It can be observed that under water treatment conditions, the application of YJD-AD2-2 bacterial agent promoted the growth of corn seedlings. Under alkali treatment conditions, the growth of corn seedlings was severely inhibited, while the corn seedlings in the alkali + bacterial agent group with YJD-AD2-2 bacterial agent were taller, had larger and greener leaves, and had longer total root length.

[0161] 5) Effects of YJD-AD2-2 treatment for 15 days on plant length and fresh weight of corn seedlings under alkaline stress (80 mM).

[0162] The statistical results showed that the average plant length and fresh weight of corn seedlings treated with YJD-AD2-2 were significantly higher than those of the water control group without YJD-AD2-2. Under alkali treatment, the average plant length and fresh weight of corn seedlings decreased significantly compared with the water control group, while the alkali + bacterial agent group treated with YJD-AD2-2 increased significantly (Table 3, Figure 7 A, B), indicating that applying YJD-AD2-2 inoculant can significantly promote the growth of corn seedlings under normal and alkali treatment conditions. The significance was evaluated by one-way ANOVA (*, P<0.05; **, P<0.01; ***, P<0.001).

[0163] Table 3. Statistics of plant length and fresh weight of corn seedlings

[0164] Average plant length (cm) Average fresh weight (g) Water control group 1 90.027 3.025 Water + Bacteria Group 2 115.12 5.624 Alkaline solution control group 3 40.632 1.022 Alkali solution + bacterial agent group 4 70.197 3.982

[0165] 6) Effects of YJD-AD2-2 inoculant treatment on some physiological parameters of corn seedlings under alkaline stress for 15 days.

[0166] When plants are subjected to abiotic stress (such as alkaline stress), the level of reactive oxygen rises sharply. Reactive oxygen has a strong oxidative ability and has a destructive effect on the structure of macromolecular substances in cells. Therefore, excessive accumulation of reactive oxygen will inevitably cause damage to cells. For example, it causes peroxidation of cell membrane lipids to produce malondialdehyde (MDA), the content of which is an important parameter reflecting the body's antioxidant potential, which can reflect the body's lipid peroxidation rate and intensity, and can also indirectly reflect the degree of tissue peroxidation damage. Proline plays an important role in regulating the osmotic pressure and redox potential of cells, and its content reflects the degree of stress on cells. Chlorophyll content is an important evaluation indicator of plant photosynthesis capacity and growth status, and is often used to characterize plant growth status. Antioxidant enzymes can convert excessive reactive oxygen in plants into less toxic or harmless substances, balancing the level of reactive oxygen in the body. Oxidative stress is a state in which the free radicals produced in the body exceed the body's antioxidant clearance capacity, causing oxidative damage to cells and tissues. Defense against oxidative stress mainly relies on the body's antioxidant system. According to the different scavenging mechanisms, the antioxidant system can be roughly divided into two systems: enzyme antioxidant system and non-enzymatic antioxidant system. Each system has the total level of various antioxidant macromolecules, small molecules and enzymes in a variety of material systems, reflecting the total antioxidant capacity in the system.

[0167] The chlorophyll, proline, malondialdehyde (MDA) contents, peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities of corn seedlings were tested. Figure 6 and Figure 7 .

[0168] Under alkali treatment conditions, the total chlorophyll content of corn seedlings in the alkali control group was significantly lower than that in the water control group ( Figure 7 C); the proline and MDA contents were significantly higher than those in the water control group ( Figure 7 D, E), which seriously affected plant growth. However, the total chlorophyll content of corn seedlings in the alkali solution + bacterial agent group with YJD-AD2-2 bacterial agent was significantly higher than that in the alkali solution control group without YJD-AD2-2 bacterial agent ( Figure 7 C), while the proline and MDA contents were significantly lower than those in the alkali solution control group ( Figure 7 D, E). In addition, the antioxidant enzyme activity of corn seedlings in the alkali solution + bacterial agent group ( Figure 6) was also significantly higher than that of the alkali solution control group. The significance was evaluated by one-way analysis of variance (*, P<0.05; **, P<0.01; ***, P<0.001). The above results show that under alkali stress, YJD-AD2-2 can significantly promote plant growth and improve the alkali stress resistance of corn seedlings by adjusting the level of plant active oxygen and promoting the intensity of photosynthesis, greatly alleviating the cell damage caused by the increase in active oxygen levels, significantly improving the antioxidant capacity and osmotic pressure regulation ability of corn seedlings, and promoting the growth of corn seedlings.

[0169] Example 6: Application of Enterobacter kobe YJD-AD2-2 bacterial agent in improving the ability of rice to resist alkali stress

[0170] Culture conditions: 25°C; 12h light / 12h dark; light intensity of 5000Lx.

[0171] Alkali solution (40mM): 1.272g Na 2 CO 3 and 9.0731 g NaHCO 3 Dissolve in 3000mL distilled water and set aside.

[0172] 1) 150 seeds of the rice variety Nipponbare japonica rice were shelled and placed in a culture dish, a small amount of water was added, and then placed in a constant temperature incubator at 37° C. for 3-5 days until the seeds germinated and short roots appeared.

[0173] 2) Transplant the germinated rice seedlings into large milk tea pots filled with nutrient soil, 3 seedlings per pot, and obtain 40 pots of rice seedlings with a total of 120 seedlings.

[0174] 3) Take 20 pots of rice seedlings and water them with alkali solution once a week, 300 mL each time. Take another 20 pots of rice seedlings and water them with distilled water once a week, 300 mL each time. Water them 6 times in total.

[0175] 4) The 20 pots of rice seedlings watered with distilled water were randomly divided into two groups: a water control group and a water + bacterial agent group, with 10 pots in each group. The 20 pots of rice seedlings watered with alkali solution were randomly divided into two groups: an alkali solution control group and an alkali solution + bacterial agent group, with 10 pots in each group.

[0176] For the water + bacterial agent group and the alkali solution + bacterial agent group, 50 μL of the YJD-AD2-2 bacterial agent (OD 600 =1), cultured for 42 days; no bacterial agent was applied to the water control group and the alkali solution control group, and cultured for 42 days under the same conditions.

[0177] 5) Observation of the growth and development phenotype of rice seedlings in each group.

[0178] The phenotype of rice seedlings grown for 42 days is shown in Figure 8 (1 is the water control group, 2 is the water + bacterial agent group, 3 is the alkali solution control group, and 4 is the alkali solution + bacterial agent group). It can be observed that under water treatment conditions, the application of YJD-AD2-2 bacterial agent promoted the growth of rice. Under alkali treatment conditions, the growth of rice was severely inhibited, while the rice seedlings applied with YJD-AD2-2 bacterial agent had a higher survival rate, longer plant length, larger leaves, and longer total root length.

[0179] 6) Effects of YJD-AD2-2 treatment for 42 days on the fresh weight and plant length of rice seedlings under alkaline stress.

[0180] The statistical results are shown in Table 4. Under water treatment conditions, the average plant length and fresh weight of rice seedlings in the water + bacterial agent group with YJD-AD2-2 bacterial agent were significantly higher than those in the water control group without YJD-AD2-2 bacterial agent; under alkali solution treatment conditions, the average plant length and fresh weight of rice seedlings in the alkali solution + bacterial agent group with YJD-AD2-2 bacterial agent were significantly higher than those in the alkali solution control group without YJD-AD2-2 bacterial agent ( Fig. 9 A, B). The significance was evaluated by one-way analysis of variance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0181] Table 4. Statistics of plant length and fresh weight of rice seedlings

[0182] Average plant length (cm) Average fresh weight (g) Water control group 1 54.012 1.426 Water + Bacteria Group 2 61.914 1.924 Alkaline solution control group 3 40.043 0.857 Alkali solution + bacterial agent group 4 64.955 2.749

[0183] 7) Effects of YJD-AD2-2 inoculant treatment on some physiological parameters of rice seedlings under alkaline stress for 42 days.

[0184] Photosynthesis is a key metabolic process for the energy source of plant substances. Under salt stress, the structure of leaf cells changes significantly, photosynthesis is blocked, and the photosynthetic rate decreases. Chlorophyll content is an important evaluation index for plant photosynthesis capacity and growth status, and is often used to characterize plant growth status. When plants are subjected to abiotic stress (such as salt-alkali stress), the level of reactive oxygen rises sharply. Reactive oxygen has a strong oxidative ability and has a destructive effect on the structure of macromolecules in cells. Therefore, excessive accumulation of reactive oxygen will inevitably cause damage to cells. Antioxidant enzymes can convert excessive reactive oxygen in plants into less toxic or harmless substances to balance the level of reactive oxygen in the body. Excessive accumulation of reactive oxygen will inevitably cause damage to cells. For example, it causes peroxidation of cell membrane lipids to produce malondialdehyde (MDA), the content of which is an important parameter reflecting the body's antioxidant potential, which can reflect the body's lipid peroxidation rate and intensity, and can also indirectly reflect the degree of tissue peroxidation damage. Proline plays an important role in regulating the osmotic pressure and redox potential of cells, and its content reflects the degree of stress on cells.

[0185] The chlorophyll, proline, malondialdehyde (MDA) contents, peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities of rice seedlings were tested. Fig. 9 and Fig.10 .

[0186] Under alkali treatment conditions, the total chlorophyll content of rice seedlings in the alkali control group was significantly lower than that in the water control group ( Fig. 9 C); the proline and MDA contents were significantly higher than those in the water control group ( Fig. 9 D, E), which seriously affected plant growth. However, the total chlorophyll content of rice seedlings in the alkali solution + bacterial agent group with YJD-AD2-2 bacterial agent was significantly higher than that in the alkali solution control group without YJD-AD2-2 bacterial agent ( Fig. 9 C), while the MDA and proline contents were significantly lower than those in the alkali solution control group ( Fig. 9 D, E). In addition, the antioxidant enzyme activity of rice seedlings in the alkali solution + bacterial agent group ( Fig.10 ) was also significantly higher than that of the alkali solution control group. The significance was evaluated by one-way analysis of variance (*, P<0.05; **, P<0.01; ***, P<0.001). These results indicate that under alkaline stress, the YJD-AD2-2 agent significantly improved the antioxidant capacity of rice seedlings and greatly alleviated the cellular oxidative damage caused by the increase in reactive oxygen levels caused by alkaline stress; at the same time, it promoted photosynthesis, thereby promoting the growth of rice seedlings.

[0187] The above experimental results show that under normal and alkaline stress conditions, Kobe Enterobacter YJD-AD2-2 can improve the photosynthesis intensity, antioxidant capacity, and resistance to salt stress of plants, significantly promote plant growth, and improve the resistance of plants to alkaline stress.

[0188] Example 7: Detection of pH tolerance range of Kobe Enterobacter YJD-AD2-2 strain

[0189] Adjust the pH value of LB liquid medium to 3-10 with hydrochloric acid or sodium hydroxide, add 1 mL of YJD-AD2-2 bacterial solution (OD 600 =1) Culture and measure OD with a spectrophotometer every 2 hours 600 The results showed that the Kobe E. coli YJD-AD2-2 strain could not survive at pH 3, but could survive well at pH 4-10. It had the strongest proliferation ability at pH 4-7 and the lowest proliferation ability at pH 10 ( Fig.11 A) These results indicate that the YJD-AD2-2 strain has a wide pH adaptation range.

[0190] Example 8: Analysis of the tolerance of Kobe Enterobacter YJD-AD2-2 strain to alkaline and neutral salts

[0191] Add NaHCO to 100 mL of LB liquid medium. 3 Add 1 mL of YJD-AD2-2 bacterial solution (OD 600 =1) Culture and measure OD with a spectrophotometer every 2 hours 600 The results showed that the Kobe E. coli YJD-AD2-2 strain had a high activity in 60 mM NaHCO 3 There was no significant change in the growth in the solution, and then as the concentration increased, the proliferation ability decreased, but it was still able to maintain a certain degree of growth and reproduction, and it was able to survive even when it reached 120mM ( Fig.11 B), indicating that the YJD-AD2-2 strain has a higher ability to resist alkaline salt stress.

[0192] NaCl was added to 100 mL LB liquid medium to a final concentration of 0 M, 0.8 M, 1.2 M, 1.6 M, 2.0 M, and 2.2 M. 1 mL of YJD-AD2-2 bacterial culture (OD 600 =1) and cultured, and the OD was measured by spectrophotometer every 12 h. 600 The results showed that the Kobe E. coli YJD-AD2-2 strain could still survive in a 1.6M NaCl solution, but could not survive when the NaCl concentration reached a high concentration of 2.2M ( Fig.11 C). This indicates that the strain YJD-AD2-2 has a higher ability to resist neutral salt stress.

[0193] Example 9: Growth-promoting properties of Enterobacter kobe YJD-AD2-2 - secretion of H + Ability test

[0194] It is reported that most plant growth-promoting bacteria have the ability to secrete organic acids. + Capacity was measured.

[0195] The pH value of the LB liquid medium was adjusted to 8, and then 1 mL of YJD-AD2-2 bacterial solution (OD 600 =1) and cultured, and the OD was measured by spectrophotometer every 1.5 h. 600 The results showed that the pH value of the culture fluid dropped rapidly to 6.0 ( Fig.12), indicating that strain YJD-AD2-2 has a strong ability to produce hydrogen ions.

[0196] Example 10: Growth-promoting properties of Enterobacter kobe YJD-AD2-2 - detection of indoleacetic acid (IAA) secretion ability

[0197] It is reported that plant rhizosphere growth-promoting bacteria can generally secrete auxin (IAA) to promote plant growth. The ability of Enterobacter kobeensis YJD-AD2-2 to secrete IAA was determined.

[0198] Salkowski colorimetric solution (250 mL): concentrated H 2 SO 4 150 mL, 0.5 M FeCl 3 37.5mL, make up to volume with distilled water.

[0199] The Kobe Enterobacter YJD-AD2-2 strain prepared in Example 2 was inoculated into 1 mL of LB liquid medium containing 200 mg / L L-tryptophan (L-Try), and a strain that could not produce IAA was used as a negative control. After culturing at 30°C and 180 r / min for 36-48 h, 200 μL of the bacterial suspension was taken to a 48-well plate, 800 μL of Salkowski colorimetric solution was added, and the plate was placed at room temperature and away from light for 20 min before observation. The color turned red for positive, and the darker the color, the stronger the ability to secrete IAA.

[0200] The results of the Salkowski colorimetric reaction are shown in Fig.13 A (control group on the left, experimental group on the right). Judging from the color of the color developing solution, YJD-AD2-2 has a strong ability to secrete auxin. Taking IAA as the standard sample, the equation of the IAA standard curve is: y=0.0142x+0.0325 (R2=0.9925). The quantitative test results show that when the tryptophan concentration is 500ug / ml, the amount of IAA synthesis reaches 8.26mg / ml·OD 600 ( Fig.13 B). The significance was evaluated by one-way analysis of variance (***, P<0.001). The above results indicate that Kobe E. coli YJD-AD2-2 has a strong IAA secretion ability.

[0201] Example 11. Analysis of the Growth-Promoting Properties of Enterobacter kobeensis YJD-AD2-2—Ability to Secrete 1-Aminocyclopropane-1-carboxylic Acid (ACC) Deaminase

[0202] Plants produce a large amount of ethylene when they are under stress. High concentrations of ethylene can inhibit plant growth. Some microorganisms contain ACC deaminase, which can inhibit the production of ethylene precursor α-ketobutyric acid, thereby reducing the production of ethylene and reducing its inhibitory effect on plant growth. The ability of Enterobacter kojira YJD-AD2-2 to secrete ACC deaminase was determined.

[0203] DF medium (1 L): 0.1 mL of component a, 0.1 mL of component b, KH 2 PO 4 4.0g, Na 2 HPO 4 6.0 g, MgSO 4 7H 2 O0.2g, glucose 2.0g, 50% D-gluconic acid solution 4mL, citric acid 2.0g, (NH 4 ) 2 SO 4 2.0g, dilute to volume with deionized water, pH 7.0-7.2.

[0204] Component a (100 mL): H 3 BO 3 10mg, MnSO 4 ·H 2 O 11.19 mg, ZnSO 4 7H 2 O 124.6 mg, CuSO 4 ·5H 2 O 78.22 mg, MoO 3 10 mg, dilute to volume with deionized water.

[0205] Component b (10 mL): FeSO 4 7H 2 O 100 mg, dilute to volume with deionized water.

[0206] ADF medium (1 L): add (NH 4 ) 2 SO 4 Replaced with 0.5 M 1-aminocyclopropane-1-carboxylic acid (ACC).

[0207] A trace amount of Kobe Enterobacter YJD-AD2-2 strain was picked up and added to 10 μL of sterile water to prepare a bacterial solution, which was inoculated on DF solid medium (10% agar was added to the above DF liquid medium). After culturing for 3-4 days, the strain was transferred to ADF solid medium (10% agar was added to the above ADF liquid medium) with ACC as the only source of nitrogen. The plate was placed in an incubator at 28°C for inverted culture to observe whether the strain grew.

[0208] The experimental results are shown in Fig.14 , Kobe E. coli YJD-AD2-2 was cultured on ADF solid medium for 4 days and obvious colony growth was observed ( Fig.14 A), indicating that YJD-AD2-2 can produce ACC deaminase to produce nitrogen source from degrading ACC to maintain its own survival. In order to further detect the ability of YJD-AD2-2 to produce ACC deaminase, α-ketobutyric acid (Shanghai McLean Biochemical Technology Co., Ltd., catalog number: K835540-1g) was used as the standard sample, and the standard curve equation was measured as: y = 0.114x + 0.0584 (R2 = 0.9737). The quantitative test results showed that the ACC deaminase activity of YJD-AD2-2 was 0.119U / mg ( Fig.14 B). The significance was evaluated by one-way analysis of variance (**, P<0.01). The above results indicate that Enterobacter wuhouensis YJD-AD2-2 has a strong ability to secrete ACC deaminase.

[0209] Example 12: Analysis of the growth-promoting properties of Enterobacter kobe YJD-AD2-2 - ability to secrete siderophores

[0210] CAS medium (250 mL): Add 1 mM CaCl 2 0.2 mL, 1 mM MgSO 4 4mL, 20% glucose 2mL, 10% acid hydrolyzed casein 6mL, mix well, then add 37.8mL of pre-sterilized dye solution c, mix well to get CAS medium. Among them, solution a: 0.024g resazurin (CAS) dissolved in 20mL distilled water, then mixed with 4mL 1mM FeCl 3 Solution mixing; Solution b: 0.03g hexadecyltrimethylammonium bromide dissolved in 16mL distilled water. Slowly add 24mL solution a to 16mL solution b and mix thoroughly to obtain dye solution c. Culture medium d (200mL): 20mL 10×MM9 salt solution, dissolve 6.04g piperazine diethanol sulfonic acid in 150mL distilled water, mix the two and adjust the pH to 6.8 with 50% NaOH solution, and add 4g agar.

[0211] MKB medium (100 mL): 0.5 g tyrosine, 1.5 mL glycerol, KH 2 PO 4 0.25 g, MgSO 4 7H 2 O 0.25g, mix well, and adjust the pH to 7.2 with 50% NaOH solution.

[0212] Pick a small amount of Kobe E. coli YJD-AD2-2 strain and prepare a bacterial solution in 10 μL sterile water, inoculate it on a solid CAS plate, and place it in an inverted culture at 37°C. If the YJD-AD2-2 strain produces a clear orange-yellow transparent circle on the CAS plate, it means that the strain has the ability to secrete siderophore, and the size and color of the transparent circle indicate the ability of the strain to produce siderophore. The larger the transparent circle and the darker the color, the stronger the ability of the strain to produce siderophore. The experimental results are shown in Fig.15 , YJD-AD2-2 strain produced an orange-yellow transparent circle on the CAS plate ( Fig.15 A), indicating that the strain has the ability to secrete siderophore. A quantitative experiment was carried out. The strain was inoculated in 5mL MKB liquid culture medium and cultured at 150r / min and 30℃ for 48h. The culture solution was centrifuged at 5000rpm for 10min, and 1mL of supernatant was taken (reference value, Ar). Add 1mL of uninoculated MKB liquid culture medium and mix it with the CAS detection solution in a 1:1 ratio. After reacting at room temperature for 1h, deionized water was used as a control and the OD value (A) was measured at 630nm. The ratio of A / Ar represents the relative content of siderophore. The smaller the ratio, the stronger the ability of the strain to produce siderophore. The quantitative test results showed that the ability of YJD-AD2-2 to secrete siderophore was 1.25A / Ar( Fig.15 B). The significance was evaluated by one-way analysis of variance (***, P<0.001). The above results indicate that Enterobacter wuhouensis YJD-AD2-2 has a strong ability to secrete siderophore.

[0213] Example 13: Analysis of the growth-promoting properties of Kobe Enterobacter YJD-AD2-2 - phosphate solubilization ability

[0214] Organic and inorganic phosphorus in soil play a key role in plant growth. Inorganic phosphorus in alkaline soil is mainly Ca 3 (PO 4 ) 2, which cannot be directly absorbed and utilized by plants, limiting the growth and development of plants. Bacteria with phosphate-solubilizing function can secrete organic acids to dissolve the insoluble phosphorus-containing substances in the external environment of the bacteria, and convert them into phosphorus-containing substances that can be used by plants through different mechanisms. If the bacteria have the ability to solubilize phosphorus, a transparent circle will appear around the colony after being cultured on PKO medium (containing insoluble calcium phosphate) for a period of time.

[0215] PKO medium (1L): 10.0 g glucose, 5.0 g tricalcium phosphate, 5 g magnesium chloride, 0.1 g ammonium sulfate, 0.2 g potassium chloride, 0.25 g magnesium sulfate, 17 g agar, and the rest water, pH 7.0.

[0216] The experimental results are shown in Fig.16 After culturing E.coli YJD-AD2-2 on PKO medium for 72 hours, an obvious transparent circle appeared around the colony ( Fig.16 A), indicating that YJD-AD2-2 has the ability to dissolve phosphorus. The phosphorus dissolving ability of YJD-AD2-2 was quantitatively tested by molybdenum antimony colorimetry. Using phosphorus standard solution as the standard sample, a standard curve was obtained, and the calculation formula was: y = 0.183x + 0.2651 (R2 = 0.9907). The test results showed that the phosphorus dissolving ability of YJD-AD2-2 was 136.67 mg / L ( Fig.16 B). The significance was evaluated by one-way analysis of variance (***, P<0.001). The above results show that Enterobacter wuhouensis YJD-AD2-2 has a strong ability to dissolve phosphate.

[0217] Example 14: Analysis of the growth-promoting properties of Enterobacter kobe YJD-AD2-2 - nitrogen fixation ability

[0218] Ashby medium (1L): KH 2 PO 4 0.2g, NaCl 0.2g, MgSO 4 7H 2 O 0.2g, K 2 SO 4 ·2H 2 O0.2g, CaCO 3 5g, glucose 5g, mannitol 5g, agar 10g, pH 7.0, dilute to volume with deionized water.

[0219] If the strain has the ability to fix nitrogen, it can grow in Ashby medium without a nitrogen source. Using bacteria without the ability to fix nitrogen as a control, the control bacteria and Kobe Enterobacter YJD-AD2-2 strain were inoculated into Ashby solid medium and inverted at 30°C. The results are as follows Fig.17As shown, YJD-AD2-2 grew in Ashby medium, and the colonies were sticky, with neat edges and smooth surfaces, indicating that it had the ability to fix nitrogen.

[0220] Example 15: Analysis of the growth-promoting properties of Enterobacter kobe YJD-AD2-2 - the ability to form biofilm

[0221] Add 100 μL LB liquid culture medium to each test well of the 96-well plate, and inoculate 10 μL of overnight cultured YJD-AD2-2 bacterial solution. Incubate at 37°C for 36 hours. Aspirate the bacterial solution, add 200 μL of sterile PBS buffer to each well, and wash the wells 3 times. Add 100 μL of methanol to each well for fixation for 15 minutes, discard the methanol, and dry naturally. Add 100 μL of 1% crystal violet solution to each well, incubate at room temperature for 5 minutes, discard the unbound crystal violet staining solution, wash off the excess staining solution with distilled water, and dry at 37°C or room temperature. Add 100 μL of 33% glacial acetic acid solution to each well and let stand at 37°C for 30 minutes to dissolve the crystal violet ( Fig.18 A) Measure the OD of the solution in the culture well using an ELISA reader 590 The culture medium without inoculation was used as negative control, and twice the negative value was taken as the limit value. The significance was evaluated by one-way analysis of variance, ***, P<0.001. The quantitative experimental results showed that Kobe E. coli YJD-AD2-2 had a strong biofilm formation ability ( Fig.18 B) Biofilm is conducive to the colonization of E. kobeensis YJD-AD2-2 on the root surface, thereby stably exerting its role in promoting plant growth.

[0222] In summary, Kobe Enterobacter YJD-AD2-2 can survive in a wide pH range, has strong salt and alkali resistance, can produce organic acids, IAA, ACC deaminase, siderophore, has nitrogen fixation and phosphorus solubility, and can form biofilm. These characteristics make Kobe Enterobacter YJD-AD2-2 have a strong ability to promote plant growth.

[0223] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Enterobacter, characterized in that: The Enterobacter is Enterobacter wuhouensis, the strain number of which is YJD-AD2-2, and the registration number of which in the General Microbiological Center of China National Microbiological Culture Collection is CGMCC No.29524.

2. The culture of the Enterobacter according to claim 1, which is obtained by culturing the Enterobacter according to claim 1 in a microbial culture medium.

3. A microbial agent, characterized in that: The bacterial agent contains the Enterobacterium according to claim 1 or / and a metabolite of the Enterobacterium according to claim 1 or / and the culture according to claim 2.

4. A salt and / or alkali stress resistance preparation, characterized in that: The salt and / or alkali stress resistance preparation contains the Enterobacterium according to claim 1 or / and the metabolite of the Enterobacterium according to claim 1 or / and the culture according to claim 2 or / and the bacterial agent according to claim 3.

5. Any one of the following uses of the Enterobacterium according to claim 1, the metabolite of the Enterobacterium according to claim 1, the culture according to claim 2, the bacterial agent according to claim 3, and the salt and / or alkali stress resistance preparation according to claim 4: The above application may specifically be any of the following: (a1) Enhance the salt and alkalinity resistance of plants; (a2) preparing products for enhancing the salt and alkalinity resistance of plants; (a3) promoting plant growth; (a4) preparing products for promoting plant growth; (a5) Producing IAA; (a6) preparing an IAA-producing product; (a7) produces ACC deaminase; (a8) preparing a product producing ACC deaminase; (a9) siderophore production; (a10) Preparation of siderophore-producing products (a11) Phosphate dissolution; (a12) preparing a phosphate-dissolving product; (a13) nitrogen fixation; (a14) Preparing nitrogen-fixed products. (a15) Biofilm; (a16) A product for preparing a biofilm. In the above application, the plant growth promotion is embodied in whole or in part as follows: (b1) promoting the increase of plant fresh weight under saline-alkali stress conditions or non-saline-alkali stress conditions; (b2) promoting an increase in plant dry weight under saline-alkali stress conditions or non-saline-alkali stress conditions; (b3) promoting root elongation of plants under saline-alkali stress conditions or non-saline-alkali stress conditions; (b4) promoting plant growth under saline-alkali stress conditions or non-saline-alkali stress conditions; (b5) promoting the increase of plant leaf length under saline-alkali stress conditions or non-saline-alkali stress conditions; (b6) promoting the increase of plant leaf width under saline-alkali stress conditions or non-saline-alkali stress conditions; (b7) reducing plant leaf wilting under saline-alkali stress conditions or non-saline-alkali stress conditions; (b8) promoting the increase of chlorophyll content in plants under saline-alkali stress conditions or non-saline-alkali stress conditions; (b9) promoting the reduction of proline content in plants under saline-alkali stress conditions or non-saline-alkali stress conditions; (b10) promoting the reduction of malondialdehyde content in plants under saline-alkali stress conditions or non-saline-alkali stress conditions; (b11) promoting the enhancement of plant antioxidant enzyme activity under saline-alkali stress conditions or non-saline-alkali stress conditions; (b12) regulating plant secondary metabolites under saline-alkali stress conditions or non-saline-alkali stress conditions; (b13) Improve the vitality of saline-alkali soil and improve the physical and chemical properties of saline-alkali soil; (b14) Improve the abundance and composition of saline-alkali soil microbial communities.

6. The use according to claim 5, characterized in that The plant family is Cruciferae, Gramineae and Leguminosae. The plant is Cruciferae, and the Cruciferae plant is the following a1), a2) or a3): a1), Cruciferae; a2), Arabidopsis; a3), Arabidopsis thaliana. The grass plant may be the following b1), b2) or b3): b1), Poaceae; b2), Oryza or Zea; b3), Rice or Corn. The leguminous plant may be the following c1), c2) or c3): c1), Leguminosae; c2), Medicago; c3), Alfalfa.

7. A method for preparing the bacterial agent according to claim 3, comprising the following steps: using the Enterobacter as described in claim 1 as an active ingredient to obtain the bacterial agent.

8. Biological organic fertilizer, characterized by: The biological organic fertilizer contains the Enterobacter according to claim 1 or the culture according to claim 2 or the bacterial agent according to claim 3.

9. The use according to any one of claim 5 or the method according to claim 8, characterized in that: The plant is a monocotyledon or a dicotyledon.

10. A method for improving plant stress resistance and / or promoting plant growth, characterized in that: The method comprises treating a plant or a culture medium of a plant with the Enterobacterium according to claim 1, a metabolite of the Enterobacterium according to claim 1, a culture according to claim 2, or an anti-salt and / or alkali stress preparation according to claim 5.

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