Salt-tolerant nitrogen-fixing bathrobacter pseudosucrose and application thereof

Abstract

The invention discloses a salt-tolerant nitrogen-fixing bathrobacter pseudosucrose and application thereof, and relates to the technical field of microorganisms, the salt-tolerant nitrogen-fixing bathrobacter pseudosucrose S12020 is obtained by separation, and the preservation number of the salt-tolerant nitrogen-fixing bathrobacter pseudosucrose S12020 is CGMCC (China General Microbiological Culture Collection Center) No.33020. The invention further discloses a preparation method of the salt-tolerant nitrogen-fixing bathrobacter pseudosucrose S12020. The K.pseudosacchari S12020 disclosed by the invention is a plant growth-promoting bacterium which is stable and efficient in nitrogen fixation and resistant to salt and alkali, the nitrogenase activity of the K.pseudosacchari S12020 is up to 1501.64 + / -81.12 nmol C2H4 (mg protein.h)-1, and the K.pseudosacchari S12020 can tolerate a culture medium environment with the NaCl concentration up to 8%. The strain has a good inhibition effect on pathogenic bacteria such as Xanthomonas oryzae pv. Oryzae, also has the functions of dissolving potassium, producing cellulase, producing siderophore, producing ammonia, producing IAA and the like, and can effectively improve the soil nutrition environment and promote plant growth. The strain is a plant probiotic which is high in nitrogen fixation activity, strong in salt resistance, good in growth promoting property, easy to culture, easy to ferment, easy to preserve, environment-friendly, harmless to people and livestock and the like, and has a good industrial production prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a salt-tolerant nitrogen-fixing Pseudosaccharosyringa bathyomarinus and its application. Background Art

[0002] Nitrogen is the most critical nutrient element during the growth process of plants and is an important component of amino acids and proteins. The available nitrogen in the soil is very limited and cannot meet the high-yield requirements of crops to the maximum extent. Artificially applying nitrogen fertilizers is one of the most important measures to achieve high crop yields. However, excessive application of nitrogen fertilizers will change the soil nitrogen cycle, significantly reduce the nitrogen in the microbial biomass, and lead to aggravated salinization. The excessive enrichment of salt ions and high pH value not only cause osmotic stress, water loss in plant cells and poisoning, but also affect the activities of microorganisms, restrict the transformation and supply of nitrogen and other nutrients, and result in a more lack of available nutrient elements in the soil.

[0003] In the agricultural ecosystem, about 24% of the nitrogen in the crop biomass comes from non-symbiotic N2 fixation. Nitrogen-fixing bacteria can convert the nitrogen in the air into ammonium salts that can be utilized by plants and provide essential nutrients for plant growth and development. According to the relationship between nitrogen-fixing bacteria and plants, biological nitrogen fixation is divided into three types: symbiotic nitrogen fixation, autotrophic nitrogen fixation, and associative nitrogen fixation. Among them, associative nitrogen-fixing bacteria form a loose association with plant roots and can fix the free nitrogen in the atmosphere. Such microorganisms are widely present in nature and play an important role in improving soil fertility and promoting plant growth. Compared with traditional symbiotic nitrogen-fixing bacteria, associative nitrogen-fixing bacteria have a wider host range and can form an associative nitrogen-fixing relationship with non-leguminous plants, thus expanding the applicable range of nitrogen fixation. The research on associative nitrogen-fixing bacteria and their related aspects has become a hot topic in current agricultural production applications and scientific research.

[0004] Applying nitrogen-fixing bacteria agents can reduce the harm caused by high salt stress to plants, promote plant growth, significantly improve the nitrogen supply capacity of the soil, reduce the soil pH and electrical conductivity, and thus improve the micro-ecological environment of plant roots. In addition, nitrogen-fixing bacteria can increase the effective nitrogen content and urease activity in saline-alkali soil, thereby increasing crop yields. However, the salinity of the soil directly affects the colonization and community structure of nitrogen-fixing bacteria. At present, the research and application of nitrogen-fixing bacteria mainly focus on soils with relatively low salinity, and there are few reports on nitrogen-fixing bacteria suitable for saline-alkali environments. The area of saline-alkali land in China is about 99.13 million hm 2 , accounting for about 70% of the total cultivated land area. Among them, the area of severely saline-alkali land with a pH higher than 9.0 and a salt content above 0.6% is increasing at a rate of 1.4% per year.

[0005] Kosakonia, as a type of beneficial microorganism, can provide nutrients and protection for plants through biological nitrogen fixation, biological control, etc., reduce the usage of chemical fertilizers and pesticides, and lower the environmental pollution caused by agricultural production. Existing research has shown that Kosakonia can produce plant hormones such as indole acetic acid (IAA), which stimulates the elongation and division of plant cells, thereby promoting plant growth. In addition, Kosakonia can also improve the soil environment, enhance soil fertility and air permeability, and provide better conditions for plant growth. However, there are few reports on efficient nitrogen-fixing and salt-tolerant Kosakonia pseudosacchari at home and abroad, and even fewer reports on its application in saline-alkali land improvement, agricultural production, and ecological restoration. Therefore, exploring the specific functions of Kosakonia pseudosacchari in efficient nitrogen fixation, salt tolerance, etc., and clarifying its growth-promoting effects on plants can provide an important resource basis for improving crop yield and quality and enhancing soil fertility in saline-alkali land. It can also promote the development of ecological agriculture and has broad application prospects.

[0006] For this reason, the present invention provides a salt-tolerant nitrogen-fixing Kosakonia pseudosacchari. By fully exploring the nitrogen-fixing microbial resources that can tolerate stress environments, this strain can convert nitrogen in the air into nutrients required by plants and establish a nutrient retention and relatively self-sustaining system for plant growth in saline-alkali habitats, thereby effectively reducing the usage of chemical fertilizers. Moreover, the development and application of the above salt-tolerant nitrogen-fixing strain are of great significance for increasing the crop yield in saline-alkali land. Summary of the Invention

[0007] Aiming at the above-mentioned defects existing in the prior art, the purpose of the present invention is to provide a salt-tolerant nitrogen-fixing Kosakonia pseudosacchari, which can form an associative nitrogen-fixing relationship with non-leguminous plants, convert nitrogen in the air into nutrients required by plants, and establish a nutrient retention and relatively self-sustaining system for plant growth in saline-alkali habitats, thereby effectively reducing the usage of chemical fertilizers.

[0008] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0009] The present invention provides a salt-tolerant nitrogen-fixing Kosakonia pseudosacchari strain, named Kosakonia pseudosacchari S12020, which was deposited at the General Microbiological Center of the China National Culture Collection of Microorganisms on December 11, 2024, and its deposit number is CGMCC No. 33020.

[0010] The present invention also provides a biological preparation, including the above-mentioned Kosakonia pseudosacchari S12020 and / or the metabolites of Kosakonia pseudosacchari S12020.

[0011] The present invention also provides the application of the above salt-tolerant nitrogen-fixing Kosakonia pseudosacchari strain or the above biological preparation in nitrogen fixation.

[0012] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in salt tolerance.

[0013] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in potassium solubilization.

[0014] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in cellulase production.

[0015] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in ammonia production.

[0016] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in siderophore production.

[0017] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in the secretion of the plant growth hormone IAA.

[0018] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in inhibiting Xanthomonas oryzae pv. oryzicola.

[0019] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in promoting the growth of rapeseed.

[0020] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in promoting the growth of wheat.

[0021] The present invention also provides the use of the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent in promoting the growth of maize.

[0022] The present invention also provides a biological fertilizer prepared using the above-mentioned salt-tolerant nitrogen-fixing Pseudosaccharibacter bathyalis strain or the above-mentioned biological agent.

[0023] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0024] The colonies of the strain K. pseudosacchari S12020 of the present invention are circular, with neat edges, opaque, milky white on the front, and smooth on the surface. And K. pseudosacchari S12020 of the present invention is a plant growth-promoting bacterium with stable and efficient nitrogen fixation and salt and alkali tolerance, and its nitrogenase activity is as high as 1501.64 ± 81.12 nmol C2H4 (mg protein·h) -1, it can tolerate a culture medium environment with a NaCl concentration as high as 8%. This strain has a good inhibitory effect on pathogenic bacteria such as Xanthomonas oryzae pv. oryzicola, and also has functions such as potassium solubilization, cellulase production, siderophore production, ammonia production, IAA production, etc., which can effectively improve the soil nutrient environment and promote plant growth. It is also a plant probiotic with high nitrogen fixation activity, strong salt tolerance, good growth-promoting characteristics, easy to culture, easy to ferment, easy to preserve, environmentally friendly and harmless to humans and livestock, etc., and has good industrial production prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a PCR gel electrophoresis diagram for the detection of the nitrogenase gene nifH of Kosakonia pseudosacchari S12020 in the present invention;

[0026] Figure 2 It is a phylogenetic tree constructed based on the 16S rRNA gene sequence in the present invention;

[0027] Figure 3 It is a bar chart for the detection of nitrogenase activity of Kosakonia pseudosacchari S12020 in the present invention;

[0028] Figure 4 It is the determination of the salt tolerance of Kosakonia pseudosacchari S12020 in the present invention (it can tolerate a culture medium with a Nacl concentration of 8%);

[0029] Figure 5 It is the determination of potassium solubilization of Kosakonia pseudosacchari S12020 in the present invention;

[0030] Figure 6 It is the determination of cellulose production of Kosakonia pseudosacchari S12020 in the present invention;

[0031] Figure 7 It is the determination of siderophore production of Kosakonia pseudosacchari S12020 in the present invention;

[0032] Figure 8 It is the determination of ammonia production of Kosakonia pseudosacchari S12020 in the present invention;

[0033] Figure 9 It is the determination of IAA secretion of Kosakonia pseudosacchari S12020 in the present invention;

[0034] Figure 10 Determination of the antagonistic ability of Kosakonia pseudosacchari S12020 against the pathogenic bacterium Xanthomonas oryzae pv. oryzicola in the present invention;

[0035] Figure 11 Determination of the growth-promoting ability of Kosakonia pseudosacchari S12020 on wheat in the present invention;

[0036] Figure 12 Determination of the growth-promoting ability of Kosakonia pseudosacchari S12020 on maize in the present invention;

[0037] Figure 13 Determination of the growth-promoting ability of Kosakonia pseudosacchari S12020 on rape in the present invention. Detailed implementation manners

[0038] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in combination with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] The reagents and materials used in the following embodiments are all conventional commercially available products unless otherwise specified; the experimental methods used in the following embodiments are all conventional methods in the art unless otherwise specified. The pathogenic bacteria used in the following embodiments can be collected in the wild or obtained from the Institute of Agricultural Resources and Agricultural Regional Planning, Chinese Academy of Agricultural Sciences for repeating the experiments of this application.

[0040] Explanation of the cited strains:

[0041] Xanthomonas oryzae pv. oryzicola that causes bacterial leaf streak of rice (Ye Wenxin, Chang Zheng, Liu Qianqian, etc. Mutation analysis of the xrvA gene encoding the virulence regulatory protein of Xanthomonas oryzae in Xanthomonas oryzae pv. oryzicola [J / OL]. Molecular Plant Breeding: 1-17 [2024-07-01]), hereinafter referred to as Xanthomonas oryzae pv. oryzicola.

[0042] Without conflict, the embodiments and features in the embodiments of the present invention may be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0043] The preparation methods of the culture media used in the following examples are as follows:

[0044] Luria - Bertani (LB) solid medium: 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl, 12 g of agar, made up to 1000 mL with distilled water, pH 7.0 - 7.2.

[0045] Luria - Bertani (LB) liquid medium: 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl, made up to 1000 mL with distilled water, pH 7.0 - 7.2.

[0046] Ashby solid medium: 0.2 g of KH2PO4, 0.2 g of MgSO4·7H2O, 0.2 g of NaCl, 5.0 g of CaCO3, 10 g of mannitol, 0.1 g of CaSO4·2H2O, 12 g of agar, made up to 1000 mL with distilled water.

[0047] Nitrogen - free medium: 10 g of sucrose, 0.12 g of NaCl, 0.5 g of K2HPO4·3H2O, 1 g of CaCO3, 0.2 g of MgSO4·7H2O, made up to 1000 mL with distilled water, pH 7.2.

[0048] Nutrient Agar (NA) medium: 3 g of beef extract, 5 g of peptone, 2.5 g of glucose, 12 g of agar, pH 7.0, made up to 1000 mL with distilled water.

[0049] Nutrient Broth (NB) medium: 3 g of beef extract, 5 g of peptone, 2.5 g of glucose, pH 7.0, made up to 1000 mL with distilled water.

[0050] Potassium - releasing medium: 5 g of sucrose, 0.5 g of MgSO4·7H2O, 0.005 g of FeCl3, 0.1 g of CaCO3, 2 g of sericite powder, 1.5% agar, made up to 1000 mL with distilled water.

[0051] Sodium carboxymethyl cellulose medium: 10 g of peptone, 10 g of yeast powder, 10 g of sodium carboxymethyl cellulose, 1 g of KH2PO4, 15 g of NaCl, 12 g of agar, made up to 1000 mL with distilled water;

[0052] Chrome azurol (CAS) blue qualitative detection medium:

[0053] Dissolve 0.012 g of chrome azurol sulphonate (CAS) in 10 mL of deionized water, and mix it evenly with 2 mL of 1 mmol / L FeCl3 solution to obtain solution a;

[0054] Dissolve 0.015 g of hexadecyl trimethyl ammonium bromide (HDTMA) in 8 mL of deionized water to obtain solution b;

[0055] Slowly add solution a to solution b and mix well to obtain dye solution c;

[0056] Add 10 mL of 0.1 mol / L phosphate solution (2.427 g of Na2HPO4·12H2O, 0.5905 g of NaH2PO4·2H2O, 0.075 g of KH2PO4, 0.125 g of NaCl, 0.25 g of NH4Cl, 100 mL of deionized water, diluted 10 times before use) and 6.04 g of piperazine diethanol sulfonic acid (pipes) into a clean Erlenmeyer flask containing 150 mL of distilled water, mix well and adjust the pH to 6.8 with 50% NaOH solution. Finally, add 4 g of agar powder to obtain culture medium d;

[0057] Sterilize dye solution c, culture medium d, 1 mmol / L CaCl2, 1 mmol / L MgSO4·7H2O, 20% glucose, and 10% casein amino acids (1215 °C, 20 min); respectively measure 0.2 mL of the above 1 mmol / L CaCl2, 4 mL of 1 mmol / L MgSO4·7H2O, 6 mL of 10% casein amino acids, and 2 mL of 20% glucose solution and add them to culture medium d; then slowly add dye solution c and shake well to obtain a blue qualitative detection medium.

[0058] Example 1 Isolation and Identification of Kosakonia pseudosacchari S12020

[0059] I. Isolation of Strain S12020

[0060] Clean the roots of corn plants of the same size and wash the soil completely clean. Cut the roots of the corn plants with sterilized scissors and cut them into several small sections about 1 cm long, put them into a sterile petri dish and weigh them. Each sample weighs 1 g and is repeated 3 times. The weighed samples are soaked in a sterile petri dish containing alcohol (75%, volume fraction) for 30 s, and then transferred to a sterile petri dish containing 25 mg·L -1 sodium hypochlorite and soak for 10 min. Finally, rinse the samples 3 times with sterile water.

[0061] The dilution separation method was used to isolate and purify microorganisms in the sample. 1 g of the sample was placed in a sterile test tube, appropriate sterile steel beads and sterile water were added, and it was ground by shaking. After grinding, the volume was fixed to 10 mL, and it was shaken for 30 min (120 r·min -1 ), and a sample suspension was obtained. Sample suspensions with different dilution gradients (1-10 -4 ) were prepared. 100 μL of the sample was taken from the dilution gradients of 10 -3 to 10 -4 respectively and smeared on the Ashby solid medium, and each concentration was repeated 3 times. The medium was inverted and cultured in an incubator at 30 °C for 3-7 d, and single colonies were picked from the plates with appropriate colony numbers. The parallel streaking method was used to purify the strain on the LB solid medium, and it was continuously streaked and separated 3 times to obtain the purified strain S12020.

[0062] II. Detection of the nitrogenase gene nifH of strain S12020

[0063] By streaking and inoculating strain S12020 on the LB solid medium, after culturing at 28 °C for 2 days, a small amount of monoclonal colonies were picked as the DNA template. The amplification primers for the nifH gene were nifH-F: '5-AAAGGYGGWATCGGYAARTCCACCAC-3'; nifH-R: '5-TTGTTSGCSGCRTACATSGCCATCAT-3', and the length of the amplification product was 500 bp ( Figure 1 , G was the positive control, and the amplification product of S12020 was the amplification product of this strain). The nifH gene was amplified according to the following PCR amplification system. The nifH PCR reaction system: The total PCR reaction system was 30 μL, including: 15 μL of 2×Taq PCR Master Mix; 1.5 μL of nifH_F; 1.5 μL of nifH_R; a small amount of DNA template; 12 μL of ddH2O; The PCR amplification program: The first step: pre-denaturation at 94 °C for 5 min; The second step: denaturation at 94 °C for 30 s; annealing at 55 °C for 30 s; extension at 72 °C for 1 min, for 35 cycles; The third step: sufficient extension at 72 °C for 5 min; store at 4 °C.

[0064] According to the blastx alignment analysis of the nifH (SEQ ID No.2) of S12020 amplified by PCR in NCBI, the results showed that the similarity between the amplified sequence and the nitrogenase gene sequence reached 99.12%, so strain S12020 has the nitrogenase gene nifH and is a nitrogen-fixing bacterium.

[0065] III. Identification of strain S12020

[0066] The strain S12020 was streaked on LB solid medium and cultured at 28 °C for 2 days. Then, a small amount of monoclonal colonies were picked as DNA templates, and the 16S rRNA gene was amplified according to the 16S PCR amplification system. 16S PCR reaction system: The total PCR reaction system was 30 μL, including: 15 μL of 2×Taq PCR Master Mix; 1.5 μL of 27F; 1.5 μL of 1492R; a small amount of DNA template; 12 μL of ddH2O. PCR amplification procedure: First step: Pre-denaturation at 94 °C for 5 min; Second step: Denaturation at 94 °C for 30 s; Annealing at 55 °C for 30 s; Extension at 72 °C for 1 min, for 35 cycles; Third step: Sufficient extension at 72 °C for 5 min; Store at 4 °C.

[0067] According to the alignment and analysis of the 16S rRNA (SEQ ID No.1) of S12020 obtained on EZBioCloud, the results showed that the similarity between S12020 and Kosakonia pseudosacchari JM-387 reached 99.34%. Based on the 16S rRNA sequence (SEQ ID No.1) of S12020 and the type strain of the genus Kosakonia, a phylogenetic tree Figure 2 ) was constructed, and it was found that the strain clustered with Kosakonia pseudosacchari DSM 27151 T into one branch, indicating a close genetic relationship. Therefore, the strain S12020 was identified as Kosakonia pseudosacchari.

[0068] Kosakonia pseudosacchari S12020 was deposited on December 11, 2024, at the China General Microbiological Culture Collection Center (abbreviated as CGMCC), and the deposit number was CGMCC No. 33020.

[0069] Kosakonia pseudosacchari S12020 is an aerobic Gram-negative bacterium, and the 16S rRNA and nifH gene sequences are shown in SEQ ID No.1 and SEQ ID No.2, respectively.

[0070] The preparation method of the metabolites of Kosakonia pseudosacchari S12020 is as follows: The strain S12020 was streaked on LB solid medium and cultured at 28 °C for 2 days. Then, monoclonal colonies were picked and inoculated into LB liquid medium, and cultured overnight at 28 °C under the condition of 220 rpm to obtain the mother liquor; then, it was transferred to LB liquid medium at a ratio of 1:1000 and cultured for 2 days under the same conditions to obtain the fermentation broth, which is the metabolite of the strain S12020.

[0071] In the present invention, Kosakonia pseudosacchari S12020 or / and the metabolites of Kosakonia pseudosacchari S12020, namely, the cells of Kosakonia pseudosacchari S12020 and the substances secreted by it into the culture medium.

[0072] Example 2 Determination of nitrogenase activity of Kosakonia pseudosacchari S12020

[0073] The acetylene reduction method was used to detect nitrogenase activity. The test strain S12020 was configured into a bacterial suspension with an OD 600nm = 0.1 and placed in a 25 mL headspace bottle. After culturing at 28 °C for 2 days, the cotton plug was replaced with a rubber plug for sealing. After extracting 1.5 mL of gas, 1.5 mL of C2H2 (final concentration of 1%) was added and cultured for 1 day. 0.2 mL of the mixed gas was extracted from the bottle and injected into a Shimadzu GC28A gas chromatograph to determine the production of C2H2 and C2H4. A headspace bottle filled with C2H2 without inoculating bacteria was used as a control. The chromatographic parameters were as follows: stainless steel chromatography column 2 mm × 1000 mm, column temperature 150 °C, hydrogen flame detector, temperature 20 °C, carrier gas N2, gas volume flow rate 30 mL·min -1 . The amount of ethylene generated in the reaction system was measured by gas chromatography and converted into acetylene reduction activity, expressed as ARA (nmol C2H4(mg protein h) -1 ). The nitrogenase activity was calculated using the following formula:

[0074] ARA (nmol C2H4(mg protein·h) -1 ) = (58.0 × Se × T × Pe) / (Sb × Te × P × t

[0075] × a × V)

[0076] In the formula, Se: ethylene peak area; T: Kelvin absolute temperature (T = 273.13 K); Pe: atmospheric pressure under test conditions (Pa); Sb: acetylene peak area; Te: temperature under test conditions (K); P: absolute atmospheric pressure (P = 101324.72 Pa); t: culture time (d); V: volume (mL); a: protein content (mg).

[0077] The results showed that the nitrogenase activity of strain S12020 was as high as 1501.64 ± 81.12 nmol C2H4(mgprotein·h) -1 ( Figure 3 ).

[0078] Example 3: Determination of the salt tolerance of Kosakonia pseudosacchari S12020

[0079] Prepare LB liquid media with 2%, 4%, 6%, 8%, and 10% NaCl concentrations in advance, using the LB medium with 1% NaCl concentration as a control. Pick a single colony of S12020 and inoculate it into the LB medium. After culturing at 28°C and 200 rpm for 24 h, transfer it to the LB media with different salt concentrations at an inoculation amount of 1%, and continue culturing at 28°C and 200 rpm for 24 h. Take out 1 ml from the culture flask every 12 h to measure its growth amount.

[0080] It can be seen from Figure 4 that the strain S12020 has strong salt tolerance. The strain S12020 grows better at 4% NaCl concentration compared to 2% NaCl concentration and has the potential of a halophilic bacterium. At 8% NaCl concentration, the strain S12020 shows a growth trend on the third day and continues to grow on the fourth day. The strain S12020 can tolerate and grow in a high-concentration salt environment.

[0081] Example 4: Kosakonia pseudosacchari S12020 has the ability to release potassium

[0082] Inoculate the activated strain S12020 into the LB liquid medium and shake-culture it overnight at 28°C and 220 rpm. Take 10 μL of the culture solution and spot-inoculate it on the potassium-releasing medium plate, using the spot-inoculation of 10 μL of LB liquid medium as a control treatment, with each treatment repeated 3 times. After air-drying, culture it in an inverted position in an incubator at 28°C for 7 d, and observe whether an oil-droplet-like liquid is formed.

[0083] Figure 5 The results show that obvious oil-droplet-like liquids appear around the inoculated colonies on the plate inoculated with the culture solution of strain S12020, indicating that the strain S12020 has the ability to release potassium.

[0084] Example 5: Kosakonia pseudosacchari S12020 has the ability to produce cellulase

[0085] Inoculate the activated strain S12020 into the LB liquid medium and shake-culture it overnight at 28°C and 220 rpm. Take 10 μL of the culture solution and spot-inoculate it on the sodium carboxymethyl cellulose medium plate, using the spot-inoculation of 10 μL of LB liquid as a control treatment, with each treatment repeated 3 times. After air-drying, culture it in an inverted position in an incubator at 28°C for 4 d, then pour 1 g / L Congo red staining solution to cover the plate, and then rinse it with 1 mol / L NaCl solution, and observe whether there is a clear zone.

[0086] Figure 6 The results showed that obvious clear zones appeared around the inoculated colonies on the detection plates inoculated with the culture solution of strain S12020, indicating that the cellulose in the plates was decomposed. This proved that strain S12020 had the ability to produce cellulase.

[0087] Example 6 Kosakonia pseudosacchari S12020 has the ability to produce siderophores

[0088] The activated strain S12020 was inoculated into LB liquid medium and cultured overnight with shaking at 28 °C and 220 rpm. 10 μL of the culture solution was spotted onto the plate of CAS blue qualitative detection medium and cultured in an inverted position in an incubator at 28 °C for 3 d. Whether a yellow halo appeared around the colonies was observed. Since siderophores compete for the iron ions chelated by EDTA in the medium, causing the medium to change from blue to yellow, the appearance of a yellow halo around the colonies indicated the production of siderophores.

[0089] Figure 7 The results showed that a yellow halo appeared around the colonies of strain S12020, proving that strain S12020 had the ability to produce siderophores.

[0090] Example 7 Quantitative determination of the ammonia production ability of Kosakonia pseudosacchari S12020

[0091] The freshly cultured S12020 was transferred to peptone ammonification medium and cultured with shaking at 28 °C and 200 rpm for 48 h. 200 μL of the culture solution was dropped onto a white ceramic plate, and the control was peptone ammonification medium without inoculation. 3 drops of Nessler's reagent were added to the culture solution and peptone ammonification medium. The appearance of a yellow or brownish-red precipitate indicated that the strain had the ability to produce NH3.

[0092] Figure 8 The results showed that obvious brownish-red precipitates appeared after adding Nessler's reagent to the culture solution of S12020, indicating that S12020 had significant ammonia production ability.

[0093] Example 8 Kosakonia pseudosacchari S12020 has the ability to produce IAA

[0094] The strain S12020 was inoculated into LB liquid medium containing 100 mg / L tryptophan and cultured with shaking at 28 °C and 200 rpm for 2 d. Then, it was centrifuged at 8000 r / min for 2 min, the supernatant was discarded, and the cells were resuspended with an equal volume of normal saline to obtain a cell suspension. Then, 100 μL of the cell suspension was dropped onto a white ceramic plate, and at the same time, 100 μL of Salkowski colorimetric solution was added. After standing in the dark at room temperature for 30 min, the plate was observed. If the color turned red, it indicated the ability to secrete IAA, and the darker the color, the stronger the IAA-producing ability; if the color did not change, it indicated the inability to secrete IAA.

[0095] Figure 9 The results showed that the strain S12020 showed a pink color in the dark, indicating that S12020 had the ability to secrete IAA, thus promoting the germination of plant seeds and the growth of seedlings.

[0096] Example 9 Determination of the antibacterial spectrum of Kosakonia pseudosacchari S12020

[0097] Information on the tested pathogenic bacteria, pathogenic fungi, and oomycetes is shown in Table 1. The antagonistic ability of the strain S12020 against 1 strain of pathogenic bacteria was detected.

[0098] Table 1 Information on the tested pathogenic bacteria and pathogenic fungi

[0099]

[0100] The plate antagonism test was used to detect the antagonistic ability of the strain S12020 against the above-mentioned pathogenic bacteria. The experimental method was as follows: The NA / LB medium with 1% agar content was melted and cooled to 45 °C at room temperature. The pathogenic bacteria that had been cultured overnight in the NA / LB liquid medium until the growth stationary phase were added to the cooled NA / LB medium at a ratio of 1:100, and then poured into plates. After drying, 10 μL of the culture solution of the strain S12020 (LB liquid shaking culture) was inoculated on the plates. After drying, the plates were inverted and cultured in an incubator at 28 °C. Plates inoculated only with the pathogenic bacteria were used as controls, and each treatment was set with 3 replicates. After culturing for 48 h, the diameter of the inhibition zone was observed and measured.

[0101] Figure 10 The results showed that the strain S12020 had a good antagonistic effect against Xanthomonas oryzae pv. oryzicola, indicating that the strain S12020 had the ability to resist plant pathogenic bacteria and had the potential to be developed into a biocontrol agent.

[0102] Example 10 Kosakonia pseudosacchari S12020 significantly promotes the growth of wheat

[0103] Mix nutrient soil, vermiculite, and natural soil (1:1:1) evenly and add them to an eight - row flower pot. After soaking with water, sow about 4 wheat seeds in each hole, cover them with a small amount of soil, and place them in a greenhouse at a temperature of 25 - 28 °C and a humidity of 65% - 75%. After about one week, wheat will grow. Select wheat seedlings with consistent growth and conduct a growth - promoting experiment.

[0104] Growth - promoting experiment: Pick a single colony of S12020 and inoculate it into LB medium. After culturing at 28 °C and 200 rpm for 24 h, transfer it to 50 mL of LB medium at an inoculation amount of 1% and continue culturing at 28 °C and 200 rpm for 24 h. After the culture is completed, use distilled water to prepare a bacterial suspension of S12020 with a concentration of 1×10 8 (OD 600 = 0.1), 1×10 7 (OD 600 = 0.01), 1×10 6 (OD 600 = 0.001) CFU·mL -1 . Irrigate 10 mL of the S12020 bacterial suspension to the roots of wheat. There are 40 plants in each treatment, with 3 replicates, and use distilled water irrigation as the control. Observe and record the growth situation every day. On the 21st day after irrigation, measure the physiological indexes of wheat, such as fresh weight, root length, and plant height.

[0105] Figure 11 The results show that after irrigation with S12020 bacterial suspensions at different concentrations, the wheat grows well. Compared with the control group, the plant height and fresh weight of wheat plants are significantly increased. Among them, after irrigation with the S12020 bacterial suspension at a concentration of OD 600 = 0.1, the average fresh weight of wheat reaches 0.62 ± 0.32 g, which is significantly 0.55 times higher than that of the control group, and the average plant height reaches 21.23 ± 4.51 cm, which is significantly 0.42 times longer than that of the control group. After irrigation with the S12020 bacterial suspension at a concentration of OD 600 = 0.01, the average fresh weight of wheat reaches 0.73 ± 0.24 g, which is significantly 0.82 times higher than that of the control group, and the average plant height reaches 18.73 ± 5.61 cm, which is significantly 0.25 times longer than that of the control group. After irrigation with the S12020 bacterial suspension at a concentration of OD 600 = 0.001, the average fresh weight of wheat reaches 0.60 ± 0.21 g, which is significantly 0.5 times higher than that of the control group, and the average plant height reaches 20.03 ± 4.51 cm, which is significantly 0.33 times longer than that of the control group.

[0106] Example 11 Kosakonia pseudosacchari S12020 significantly promotes the growth of maize

[0107] Mix nutrient soil, vermiculite, and natural soil (1:1:1) evenly and add them to flower pots. After soaking with water, sow 4 corn seeds in each pot, cover them with a small amount of soil, and place them in a greenhouse at a temperature of 25 - 28°C and a humidity of 65% - 75%. After about two weeks, when the seedlings grow out, select corn seedlings with consistent growth and conduct a growth promotion experiment.

[0108] Growth promotion experiment: Pick a single colony of S12020 and inoculate it into LB medium. After culturing at 28°C and 200 rpm for 24 h, transfer it to 50 mL of LB medium at an inoculation amount of 1%, and continue culturing at 28°C and 200 rpm for 24 h. After the culture is completed, use distilled water to prepare a bacterial suspension of S12020 with a concentration of 1×10 8 CFU·mL -1 . Irrigate 50 mL of the S12020 bacterial suspension at the roots of the corn. Treat 4 plants per pot, with 3 replicates, and use distilled water irrigation as the control. Observe and record the growth situation every day. On the 30th day after irrigation, measure the plant height, root length, leaf length, stem diameter, fresh weight, and weight of the underground part of the corn.

[0109] Figure 12 The results show that the corn after irrigation with the S12020 bacterial suspension grows vigorously. The average plant height reaches 67.29 ± 9.73 cm, which is significantly 0.15 times higher than that of the control group; the average stem length reaches 18.73 ± 4.35 cm, which is significantly 0.21 times higher than that of the control group; the average leaf length reaches 48.67 ± 6.87 cm, which is significantly 0.14 times higher than that of the control group.

[0110] Example 12 Kosakonia pseudosacchari S12020 significantly promotes the growth of rapeseed

[0111] Mix nutrient soil, vermiculite, and natural soil (1:1:1) evenly and add them to flower pots. After soaking with water, evenly scatter rapeseed seeds on the soil, cover them with a small amount of soil, and place them in a greenhouse at a temperature of 25 - 28°C and a humidity of 65% - 75%. After about one week, select rapeseed seedlings with consistent growth and conduct a growth promotion experiment.

[0112] Growth promotion experiment: Pick a single colony of S12020 and inoculate it into LB medium. After culturing at 28°C and 200 rpm for 24 h, transfer it to 50 mL of LB medium at an inoculation amount of 1%, and continue culturing at 28°C and 200 rpm for 24 h. After the culture is completed, use distilled water to prepare a bacterial suspension of S12020 with a concentration of 1×10 8 CFU·mL -1 . Irrigate 25 mL of the S12020 bacterial suspension at the roots of the rapeseed. Treat 4 plants per pot, with 3 replicates, and use distilled water irrigation as the control. Observe and record the growth situation every day. On the 30th day after irrigation, measure the chlorophyll content, plant height, fresh weight and other growth promotion indexes of the rapeseed.

[0113] Figure 13 The results showed that the rape plants treated by irrigating the roots with the suspension of S12020 bacteria grew vigorously, with an average plant height of 9.01±1.30 cm, which was significantly 0.34 times higher than that of the control group. Meanwhile, there was a tendency to promote chlorophyll synthesis and plant growth.

[0114] In summary, the culture of Pseudosaccharosyringa S12020 provided in Example 1 has at least one of the following functions B1 - B9: B1, nitrogen fixation; B2, salt tolerance (can tolerate a medium with a Nacl concentration of 8%); B3, potassium solubilization; B4, cellulase production; B5, siderophore production; B6, ammonia production; B7, secretion of auxin (IAA); B8, antagonism against Xanthomonas oryzae pv. oryzicola; B9, promotion of lateral root formation in plants; B10, increase in plant biomass.

[0115] The application of the metabolite (fermentation broth) of Pseudosaccharosyringa S12020 provided in Example 1 includes at least one of the applications of the fermentation broth in nitrogen fixation, salt tolerance (can tolerate a medium with a Nacl concentration of 8%), potassium solubilization, cellulase production, siderophore production, ammonia production, auxin (IAA) production, antagonism against Xanthomonas oryzae pv. oryzicola, promotion of lateral root formation in plants, or increase in plant biomass.

[0116] The above - described embodiments only represent the preferred embodiments of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A salt-tolerant and nitrogen-fixing strain of Pseudocercus pseudosucrose, characterized in that: The strain is named as Kosakonia pseudosacchari S12020, and its deposit number is CGMCC No.33020.

2. A biological agent, characterized in that: It includes the pseudosucrose microalgae S12020 and / or metabolites of pseudosucrose microalgae S12020 as described in claim 1.

3. Use of the salt-tolerant, nitrogen-fixing B. pseudosucrose strain of claim 1 or the biological preparation of claim 2 in nitrogen fixation.

4. Use of the salt-tolerant and nitrogen-fixing B. pseudosucrose strain according to claim 1 or the biological preparation according to claim 2 in salt tolerance and / or potassium solubilization.

5. Use of the salt-tolerant and nitrogen-fixing B. pseudosucrose strain according to claim 1 or the biological preparation according to claim 2 in producing cellulase and / or ammonia.

6. Use of the salt-tolerant and nitrogen-fixing B. pseudosucrose strain according to claim 1 or the biological preparation according to claim 2 in the production of siderophore.

7. Use of the salt-tolerant and nitrogen-fixing B. pseudosucrose strain according to claim 1 or the biological preparation according to claim 2 in secreting the plant growth hormone IAA.

8. Use of the salt-tolerant and nitrogen-fixing B. pseudosucrose strain of claim 1 or the biological agent of claim 2 in inhibiting bacterial leaf streak pathogen of rice.

9. Use of the salt-tolerant and nitrogen-fixing B. pseudosucrose strain of claim 1 or the biological agent of claim 2 in promoting the growth of any one or more of rapeseed, wheat and corn.

10. A biofertilizer prepared using the salt-tolerant and nitrogen-fixing Eubacterium pseudosucrose strain of claim 1 or the biological preparation of claim 2.

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