Salt-tolerant pseudomonas synxantha and application thereof

Abstract

The application discloses a salt-tolerant and nitrogen-fixing Pseudomonas taetrolens strain and application thereof, relates to the technical field of microorganisms, and discloses a salt-tolerant and nitrogen-fixing Pseudomonas taetrolens strain (S12020) with a preservation number of CGMCC No. K. pseudosacchari 33020, which is isolated and obtained. K. pseudosacchari The S12020 of the application is a plant growth-promoting bacterium which is stable, efficient in nitrogen fixation and salt-tolerant, and can tolerate a culture medium environment with a NaCl concentration of up to 8%. The strain has the functions of potassium solubilization, cellulase production, siderophore production, ammonia production, IAA production and the like, and can effectively improve the soil nutrient environment and promote plant growth. The strain has the advantages of high nitrogen fixation activity, strong salt tolerance, good growth promotion characteristics, easy cultivation, easy fermentation, good preservation, environmental friendliness, harmlessness to humans and animals and the like, and has a good industrial production prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a salt-tolerant Pseudomonas taetrolens and its application. BACKGROUND

[0002] Nitrogen is the most critical nutrient element in the process of plant growth, and is an important component of amino acids and proteins. The available nitrogen in soil is very limited and cannot meet the maximum demand of crop production. Artificial application of nitrogen fertilizer is one of the most important measures to achieve high crop yield, but excessive nitrogen fertilizer will change the soil nitrogen cycle, significantly reduce the nitrogen content in microbial biomass, and lead to the aggravation of salinization. In addition to causing osmotic stress, water loss and toxicity of plant cells, excessive accumulation of salt ions and high pH value also affect the activities of microorganisms, restrict the transformation and supply of nitrogen and other nutrients, and make the available nutrients in the soil even more scarce.

[0003] In the agricultural ecosystem, about 24% of the nitrogen in crop biomass comes from non-symbiotic N2O fixation. Nitrogen-fixing bacteria can convert atmospheric nitrogen into ammonium salt available to plants, providing 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, self-nitrogen fixation, and associative nitrogen fixation. Among them, associative nitrogen-fixing bacteria form a loose association with plant roots and can fix free nitrogen in the atmosphere. This type of microorganism widely exists in nature and plays 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 associative nitrogen-fixing relationships with non-legume plants, thereby increasing the scope of nitrogen fixation. Associative nitrogen-fixing bacteria and related research have become a hot topic in current agricultural production and scientific research.

[0004] The application of nitrogen-fixing bacteria can reduce the harm of high salt stress to plants, promote plant growth, and significantly improve the nitrogen supply capacity of soil, reduce soil pH and electrical conductivity, and thus improve the microecological environment of plant roots. In addition, nitrogen-fixing bacteria can increase the effective nitrogen content and urease activity of saline-alkali soil, thereby increasing crop yield. However, the degree of salinity-alkalinity of soil directly affects the colonization and community structure of nitrogen-fixing bacteria. Currently, research and application of nitrogen-fixing bacteria are mostly focused on soils with low salinity-alkalinity, and there are few reports on nitrogen-fixing bacteria suitable for saline-alkaline environments. The area of saline-alkali land in China is about 991.3 million hm 2 , accounting for about 70% of the total arable land. Among them, the severe saline-alkali land with pH higher than 9.0 and salt content more than 0.6% grows at a rate of 1.4% per year.

[0005] As a kind of beneficial microorganism, Kosakonia can provide nutrients and protection for plants through biological nitrogen fixation and biological control, reduce the use of chemical fertilizers and pesticides, and reduce the environmental pollution of agricultural production. Previous studies have shown that Kosakonia can produce plant hormones such as indole acetic acid (IAA), stimulate the elongation and division of plant cells, and thus promote the growth of plants. In addition, Kosakonia can also improve the soil environment, increase the soil fertility and air permeability, and provide better conditions for the growth of plants. However, there are few reports on high-efficiency nitrogen-fixing and salt-tolerant Pseudosaccharobacteria at home and abroad, and even fewer reports on its application in saline-alkali soil improvement, agricultural production and ecological restoration. Therefore, exploring the specific functions of Pseudosaccharobacteria, such as high-efficiency nitrogen fixation and salt tolerance, and clarifying its plant growth-promoting effect can provide important resource basis for improving crop yield and quality, improving soil fertility in saline-alkali soil, and promoting the development of ecological agriculture, which has broad application prospects.

[0006] Therefore, the present application provides a salt-tolerant nitrogen-fixing Pseudosaccharobacterium, which can convert nitrogen in the air into nutrients needed by plants and establish a nutrient retention and relative self-maintenance system for plant growth in saline-alkali habitats, thereby effectively reducing the use of chemical fertilizers. The development and application of the above salt-tolerant nitrogen-fixing strain are of great significance for improving crop yield in saline-alkali soil. SUMMARY

[0007] In view of the above defects in the prior art, the present application aims to provide a salt-tolerant nitrogen-fixing Pseudosaccharobacterium that can form a symbiotic nitrogen fixation relationship with non-leguminous plants, convert nitrogen in the air into nutrients needed by plants, and establish a nutrient retention and relative self-maintenance system for plant growth in saline-alkali habitats, thereby effectively reducing the use of chemical fertilizers.

[0008] To achieve the above-mentioned purpose, the technical solution of the present application is as follows:

[0009] The present application provides a salt-tolerant nitrogen-fixing Pseudosaccharobacterium strain, which is named Pseudosaccharobacterium (Kosakonia pseudosacchari, abbreviated as K. pseudosacchari) S12020, and was preserved in the China General Microbiological Culture Collection Center on December 11, 2024, with the preservation number CGMCC No. 33020 and the preservation address No. 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The present application also provides a biological agent comprising the above-mentioned Pseudosaccharobacterium S12020 and / or metabolites of Pseudosaccharobacterium S12020.

[0011] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in nitrogen fixation.

[0012] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in salt tolerance.

[0013] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in potassium release.

[0014] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in cellulase production.

[0015] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in ammonia production.

[0016] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in siderophore production.

[0017] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in secreting auxin IAA.

[0018] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in inhibiting Xanthomonas oryzae pv.oryzicola.

[0019] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in promoting growth of rape.

[0020] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in promoting growth of wheat.

[0021] The application further provides application of the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation in promoting growth of corn.

[0022] The application further provides a biological fertilizer prepared by using the salt-tolerant nitrogen-fixing Pseudosaccharophillus bathycala strain or the biological preparation.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The strain K.pseudosacchari S12020 of the application is round, with neat edges, opaque, white on the front, and smooth surface. -1, can tolerate NaCl concentration up to 8% of the medium environment. The strain has good inhibitory effect on rice bacterial leaf streak and other pathogenic bacteria, also has the functions of potassium release, cellulase production, siderophore production, ammonia production, IAA production, etc., 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 promotion characteristics, easy to culture, easy to ferment, good preservation, environmentally friendly and harmless to humans and animals, etc., and has good industrial production prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a PCR gel electrophoresis map for detecting nifH of nitrogenase gene of Kosakonia pseudosacchari S12020 in the application;

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

[0027] Figure 3 It is a column chart for detecting nitrogenase activity of Kosakonia pseudosacchari S12020 in the application;

[0028] Figure 4 It is a determination of salt tolerance (Nacl concentration of 8% of the medium) of Kosakonia pseudosacchari S12020 in the application;

[0029] Figure 5 It is a determination of potassium release of Kosakonia pseudosacchari S12020 in the application;

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

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

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

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

[0034] Figure 10 Determination of the growth promoting ability of Kosakonia pseudosacchari S12020 on wheat in the present application;

[0035] Figure 11 Determination of the growth promoting ability of Kosakonia pseudosacchari S12020 on corn in the present application;

[0036] Figure 12 Determination of the growth promoting ability of Kosakonia pseudosacchari S12020 on rape in the present application. DETAILED DESCRIPTION

[0037] In order to enable persons skilled in the art to better understand the present application, the technical solutions of the present application will be further described in detail below in combination with the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by persons skilled in the art without creative labor should belong to the protection scope of the present application.

[0038] The reagents and materials used in the following examples, etc., are all conventional commercially available products unless otherwise specified; the experimental methods used in the following examples are all conventional methods in the art unless otherwise specified. The pathogenic bacteria used in the following examples can be collected from the wild or obtained from the Institute of Agricultural Resources and Agricultural Regionalization, Chinese Academy of Agricultural Sciences, to repeat the experiments of the present application.

[0039] Reference strain explanation:

[0040] Xanthomonas oryzae pv. oryzicola (Ye Wenxin, Chang Yue, Liu Qianqian, et al. Mutation analysis of xrvA gene encoding virulence regulation protein of Xanthomonas oryzae pv. oryzicola [J / OL]. Molecular Plant Breeding: 1-17 [2024-07-01]) causes bacterial leaf streak of rice, hereinafter referred to as bacterial leaf streak of rice.

[0041] In the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present application will be described in detail below in combination with the embodiments.

[0042] The following is the preparation method of the culture medium used in the examples:

[0043] Luria-Bertani (LB) solid medium: yeast extract 5 g, tryptone 10 g, NaCl 10 g, agar 12 g, distilled water to 1000 mL, pH 7.0-7.2.

[0044] Luria-Bertani (LB) liquid medium: yeast extract 5 g, tryptone 10 g, NaCl 10 g, distilled water to 1000 mL, pH 7.0-7.2.

[0045] Ashby solid medium: KH2PO40.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaCO35.0 g, mannitol 10 g, CaSO4·2H2O 0.1 g, agar 12 g, distilled water to 1000 mL.

[0046] Nitrogen-free medium: sucrose 10 g, NaCl 0.12 g, K2HPO4·3H2O 0.5 g, CaCO31 g, MgSO4·7H2O 0.2 g, distilled water to 1000 mL, pH 7.2.

[0047] Nutrient Agar (NA) medium: beef extract 3 g, peptone 5 g, glucose 2.5 g, agar 12 g, pH 7.0, distilled water to 1000 mL.

[0048] Nutrient Broth (NB) medium: beef extract 3 g, peptone 5 g, glucose 2.5 g, pH 7.0, distilled water to 1000 mL.

[0049] Potassium-dissolving medium: sucrose 5 g, MgSO4·7H2O 0.5 g, FeCl30.005 g, CaCO30.1 g, sericite powder 2 g, 1.5% agar, distilled water to 1000 mL.

[0050] Sodium carboxymethyl cellulose medium: peptone 10 g, yeast powder 10 g, sodium carboxymethyl cellulose 10 g, KH2PO41 g, NaCl 15 g, agar 12 g, distilled water to 1000 mL.

[0051] Chromazurol (CAS) blue qualitative detection medium:

[0052] Take 0.012 g of chrome azurol (chrome azurol sulphonate, CAS) and dissolve it in 10 mL of deionized water, and mix it with 2 mL of 1 mmol / L FeCl3 solution to get solution a;

[0053] Take 0.015 g hexadecyl trimethyl ammonium bromide (HDTMA) and dissolve it in 8 mL of deionized water to obtain solution b;

[0054] Slowly add solution a to solution b and mix well to obtain dye solution c;

[0055] Take 10 mL of 0.1 mol / L phosphate solution (2.427 g Na2HPO4·12H2O, 0.5905 g NaH2PO4·2H2O, 0.075 g KH2PO4, 0.125 g NaCl, 0.25 g NH4Cl, 100 mL of deionized water, 10 times dilution when used), 6.04 g of piperazine diethanol sulfonic acid (pipes), and add them to a clean triangular 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;

[0056] Sterilize dye solution c, culture medium d, and 1 mmol / L CaCl2, 1 mmol / L MgSO4·7H2O, 20% glucose, and 10% casein amino acid (115°C, 20 min); respectively take 0.2 mL of 1 mmol / L CaCl2, 4 mL of 1 mmol / L MgSO4·7H2O, 6 mL of 10% casein amino acid, and 2 mL of 20% glucose solution and add them to culture medium d; then slowly add dye solution c and mix well to obtain a blue qualitative detection medium.

[0057] Example 1 Isolation and identification of Kosakonia pseudosacchari S12020

[0058] I. Isolation of strain S12020

[0059] The roots of corn plants of uniform size were cleaned, and the soil was completely washed clean. The roots of the corn plants were cut off with sterilized scissors, and the roots were cut into small pieces about 1 cm long, which were placed in a sterile petri dish and weighed, 1 g per sample, repeated 3 times. The weighed samples were 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 NaClO for 10 min. Finally, the samples were rinsed with sterile water 3 times.

[0060] The microorganisms in the samples were isolated and purified by dilution separation method. 1 g of sample was placed in a sterile test tube, and an appropriate amount of sterile steel beads and sterile water were added for shaking and grinding. After grinding, the volume was adjusted to 10 mL, and the mixture was shaken for 30 min (120 r·min -1), and a sample suspension was obtained. The sample suspension was prepared in different dilution gradients (1-10 -4 ) and 100 μL of each sample was taken from the 10 -3 -10 -4 dilution gradient and smeared on Ashby solid medium. Each concentration was repeated 3 times. The medium was incubated in an inverted 30°C incubator for 3-7 days, and single colonies were selected from the plates with appropriate colony numbers. The parallel streak method was used to purify the strains on LB solid medium, and the strains were separated by streaking 3 times to obtain the purified strain S12020.

[0061] II. Detection of the nitrogenase gene nifH of the strain S12020

[0062] The strain S12020 was inoculated on LB solid medium by streaking, and after 2 days of incubation at 28°C, a small amount of single colony was selected as a DNA template. The nifH gene amplification primers were nifH-F: '5-AA AGGYGGWATCGGYAARTCCACCAC-3'; nifH-R: '5-TTGTTSGCSGCRT ACATSGCCATCAT-3', and the length of the amplification product was 500 bp (G is the positive control, and S12020 is the amplification product of the strain). Figure 1 The nifH gene was amplified according to the following PCR amplification system. The nifH PCR reaction system: 30 μL of total PCR reaction system, including: 2x Taq PCR Master Mix 15 μL; nifH_F 1.5 μL; nifH_R 1.5 μL; DNA template in small amount; dd H2O 12 μL; PCR amplification program, first step: 94°C pre-denaturation for 5 min; second step: 94°C denaturation for 30 s; 55°C annealing for 30 s; 72°C extension for 1 min, 35 cycles; third step: 72°C full extension for 5 min; 4°C storage.

[0063] According to the blastx alignment analysis of the nifH (SEQ ID No. 2) of S12020 amplified by PCR, the results showed that the similarity of the amplified sequence to the nitrogenase gene sequence was 99.12%, and therefore the strain S12020 had the nitrogenase gene nifH and was a nitrogen-fixing bacterium.

[0064] III. Identification of the strain S12020

[0065] The strain S12020 was inoculated on LB solid medium by streaking, and after 2 days of culture at 28°C, a small amount of monoclonal colonies were picked as DNA templates for 16S rRNA gene amplification according to the 16S PCR amplification system. The 16S PCR reaction system: total PCR reaction system 30 μL, including: 2 × Taq PCR Master Mix 15 μL; 27F 1.5 μL; 1492R 1.5 μL; DNA template trace; ddH2O 12 μL. The PCR amplification program, first step: 94°C pre-denaturation for 5 min; second step: 94°C denaturation for 30 s; 55°C annealing for 30 s; 72°C extension for 1 min, 35 cycles; third step: 72°C full extension for 5 min; 4°C storage.

[0066] According to the alignment analysis of the obtained 16S rRNA of S12020 (SEQ ID No. 1) in EZBioCloud, the results showed that the similarity of S12020 with Kosakonia pseudosacchari JM-387 reached 99.34%. Based on the 16S rRNA sequence of S12020 (SEQ ID No. 1) and the Kosakonia type strain, a phylogenetic tree was constructed Figure 2 ), and it was found that the strain was clustered with Kosakonia pseudosacchari DSM 27151 T and had close genetic relationship, so the strain S12020 was identified as Kosakonia pseudosacchari.

[0067] Kosakonia pseudosacchari S12020 was preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 11, 2024, and the preservation number was CGMCC No. 33020.

[0068] 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.

[0069] The preparation method of the metabolites of Kosakonia pseudosacchari S12020 is as follows: the strain S12020 was inoculated on LB solid medium by streaking, and after 2 days of culture at 28°C, a small amount of monoclonal colonies were picked as DNA templates for 16S rRNA gene amplification according to the 16S PCR amplification system. The 16S PCR reaction system: total PCR reaction system 30 μL, including: 2 × Taq PCR Master Mix 15 μL; 27F 1.5 μL; 1492R 1.5 μL; DNA template trace; ddH2O 12 μL. The PCR amplification program, first step: 94°C pre-denaturation for 5 min; second step: 94°C denaturation for 30 s; 55°C annealing for 30 s; 72°C extension for 1 min, 35 cycles; third step: 72°C full extension for 5 min; 4°C storage.

[0070] In the present application, the Kosakonia pseudosacchari S12020 or / and metabolites of the Kosakonia pseudosacchari S12020, i.e. the Kosakonia pseudosacchari S12020 cell body and substances secreted by the Kosakonia pseudosacchari S12020 into the culture medium.

[0071] Example 2 Determination of nitrogenase activity of Kosakonia pseudosacchari S12020

[0072] The acetylene reduction method was used to detect nitrogenase activity. The strain S12020 to be tested was prepared into an OD 600nm = 0.1 bacterial suspension with nitrogen-free medium and placed in a 25 mL headspace bottle. After 2 d of culture at 28°C, the cotton plug was replaced with a rubber plug for sealing, 1.5 mL of gas was extracted, and then 1.5 mL of C2H2 (final concentration of 1%) was added, and the culture was continued for 1 d. 0.2 mL of 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 inoculated with bacteria and injected with C2H2 was used as a control. The chromatographic parameters were as follows: stainless steel chromatographic column 2 mm x 1000 mm, column temperature 150°C, hydrogen ion flame detector, temperature 20°C, carrier gas N 2， 2, gas volume flow rate 30 mL·min -1 . The amount of ethylene generated in the reaction system was determined by gas chromatography and converted into acetylene reduction activity, which was expressed as ARA (nmol C2H4(mg protein h) -1 ). The nitrogenase activity was calculated using the following formula:

[0073] ARA (nmol C2H4(mg protein·h) -1 ) = (58.0 x Se x T x Pe) / (Sb x Te x P x t x a x V)

[0074] In the formula, Se: ethylene peak area; T: absolute temperature in Kelvin (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).

[0075] The results showed that the nitrogenase activity of strain S12020 was as high as 1501.64 ± 81.12 nmol C2H4(mg protein·h) -1 ( Figure 3 ).

[0076] Example 3 Determination of salt tolerance of Kosakonia pseudosacchari S12020

[0077] LB liquid medium with 2%, 4%, 6%, 8%, 10% NaCl concentration was prepared in advance, and 1% NaCl concentration LB medium was used as a control; S12020 single colony was inoculated in LB medium, and after 24h culture at 28℃, 200rpm, 1% inoculation amount was transferred to S12020 single colony inoculated in LB medium with different salt concentrations, and continued to culture at 28℃, 200rpm for 24h. Every 12h, 1ml was taken from the culture bottle to measure the growth amount.

[0078] From the results shown in Table 1, it can be seen that the strain S12020 has a strong salt tolerance, and the strain S12020 grows better at 4% NaCl concentration than at 2% NaCl concentration, and has the potential of halophilic bacteria. At 8% NaCl concentration, the strain S12020 shows growth trend at the third day and continues to grow at the fourth day, and the strain S12020 can tolerate high concentration of salt environment and grow. Figure 4 Example 4 Kosakonia pseudosacchari S12020 has the ability to release potassium

[0079] Activated strain S12020 was inoculated in LB liquid medium and cultured overnight at 28℃, 220rpm. 10μL of the culture was spotted on the potassium release medium plate, and 10μL of LB liquid medium was spotted as a control treatment, and each treatment was repeated 3 times. After air-drying, the plates were inverted and cultured in a 28℃ incubator for 7d, and whether oil droplet-like liquid was formed was observed.

[0080]

[0081] The results shown in Table 2 show that the plate inoculated with strain S12020 culture liquid has obvious oil droplet-like liquid around the inoculated colony, indicating that the strain S12020 has the ability to release potassium. Figure 5 Example 5 Kosakonia pseudosacchari S12020 has the ability to produce cellulase

[0082] Activated strain S12020 was inoculated in LB liquid medium and cultured overnight at 28℃, 220rpm. 10μL of the culture was spotted on the sodium carboxymethyl cellulose medium plate, and 10μL of LB liquid was spotted as a control treatment, and each treatment was repeated 3 times. After air-drying, the plates were inverted and cultured in a 28℃ incubator for 4d, then 1g / L Congo red dye solution was poured to cover the plates, and then rinsed with 1mol / L NaCl solution, and whether transparent circle was observed.

[0083]

[0084] The results shown in Table 3 show that the plate inoculated with strain S12020 culture liquid has obvious oil droplet-like liquid around the inoculated colony, indicating that the strain S12020 has the ability to release potassium. Figure 6The results show that a clear transparent zone appears around the inoculated colony on the detection plate inoculated with the culture solution of strain S12020, indicating that the cellulose in the plate is decomposed. It is proved that strain S12020 has the ability to produce cellulase.

[0085] Example 6 Kosakonia pseudosacchari S12020 has the ability to produce siderophore

[0086] The activated strain S12020 was inoculated in LB liquid medium and shaken overnight at 28°C, 220 rpm. 10 μL of the culture solution was spotted on the CAS blue qualitative detection medium plate and incubated in an inverted incubator at 28°C for 3 days. Whether a yellow halo appeared around the colony was observed. Since the EDTA chelated iron ions in the siderophore competition medium make the medium change from blue to yellow, the appearance of a yellow halo around the colony indicates the production of siderophore.

[0087] Figure 7 The results show that a yellow halo appears around the colony of strain S12020, proving that strain S12020 has the ability to produce siderophore.

[0088] Example 7 Quantitative determination of the ammonia-producing ability of Kosakonia pseudosacchari S12020

[0089] Freshly cultured S12020 was transferred to a proteose peptone ammonification medium and shaken at 28°C, 200 rpm for 48 h. 200 μL of the culture solution was dropped on a white ceramic plate, and the control was the proteose peptone ammonification medium without inoculation. 3 drops of Nessler's reagent were added to the culture solution and the proteose peptone ammonification medium. The appearance of yellow or brown-red precipitate indicates that the strain has the ability to produce NH3.

[0090] Figure 8 The results show that a clear brown-red precipitate appears after Nessler's reagent is added to the culture solution of S12020, indicating that S12020 has a significant ammonia-producing ability.

[0091] Example 8 Kosakonia pseudosacchari S12020 has the ability to produce IAA

[0092] Strain S12020 was inoculated in LB liquid medium containing 100 mg / L tryptophan, and cultured at 28°C with 200 rpm for 2 d, then centrifuged at 8000 r / min for 2 min, the supernatant was discarded, and the same amount of normal saline was used to resuspend the bacteria to obtain a bacterial suspension. Then, 100 μL of the bacterial suspension was dropped on a white ceramic plate, and 100 μL of Salkowski colorimetric solution was added, and the color change was observed after the plate was placed at room temperature for 30 min in the dark. If the color turns red, it indicates that IAA can be secreted, and the deeper the color, the stronger the ability to produce IAA; if the color does not change, it indicates that IAA cannot be secreted.

[0093] Figure 9 The results show that strain S12020 shows pink color in the dark, indicating that S12020 has the ability to secrete IAA, thereby promoting the germination of plant seeds and the growth of seedlings.

[0094] Example 9 Determination of the antibacterial spectrum of Kosakonia pseudosacchari S12020

[0095] The information of the test pathogenic bacteria is shown in Table 1. The antagonistic ability of strain S12020 against 1 strain of pathogenic bacteria was detected.

[0096] Table 1 Information of the test pathogenic bacteria

[0097]

[0098] The antagonistic ability of strain S12020 against the above pathogenic bacteria was detected by plate antagonism test. The experimental method is as follows: after the NA / LB medium containing 1% agar was melted, it was cooled to 45°C at room temperature. The pathogenic bacteria that were cultured in NA / LB liquid medium overnight to the stable growth 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 strain S12020 culture solution (LB liquid shake culture) was inoculated on the plates. After drying, the plates were incubated in an incubator at 28°C, and the plates inoculated with pathogenic bacteria only were used as controls, and each treatment was set in triplicate. After 48 h of culture, the inhibition zone diameter was observed and measured.

[0099] The results show that strain S12020 has good antagonistic effect on Xanthomonas oryzae, indicating that strain S12020 has the ability to resist plant pathogenic bacteria and has the potential to be developed as a biocontrol agent.

[0100] Example 10 Kosakonia pseudosacchari S12020 significantly promotes the growth of wheat

[0101] The nutrient soil, vermiculite, natural soil (1:1:1) were mixed and added to the eight continuous row flower pots, and then soaked with water. About 4 seeds of wheat were sown in each hole, and then covered with a small amount of soil. The pots were placed in a greenhouse with a temperature of 25-28°C and a humidity of 65%-75%. After about a week, the wheat grew, and the wheat seedlings with consistent growth were selected for the growth promotion experiment.

[0102] Growth promotion experiment: S12020 single colonies were inoculated in LB medium, and cultured at 28°C, 200 rpm for 24h. Then, 1% of the culture was inoculated into 50 mL of LB medium, and cultured at 28°C, 200 rpm for another 24h. After the culture, S12020 was diluted with distilled water to obtain a bacterial suspension with a concentration of 1x10 8 (OD 600 =0.1), 1x10 7 (OD 600 =0.01), and 1x10 6 (OD 600 =0.001) CFU·mL -1 . 10 mL of the S12020 bacterial suspension was used to irrigate the roots of the wheat. There were 40 plants in each treatment, and the experiment was repeated three times. Distilled water was used as a control. The growth of the plants was observed and recorded every day. On the 21st day after irrigation, the fresh weight, root length, and plant height of the wheat were measured.

[0103] Figure 10 The results showed that the wheat plants grew well after irrigation with different concentrations of S12020 bacterial suspension. Compared with the control group, the plant height and fresh weight of the wheat plants were significantly increased. The average fresh weight of the wheat plants irrigated with the S12020 bacterial suspension with OD 600 =0.1 was 0.62±0.32g, which was 0.55 times higher than that of the control group. The average plant height was 21.23±4.51cm, which was 0.42 times higher than that of the control group. The average fresh weight of the wheat plants irrigated with the S12020 bacterial suspension with OD 600 =0.01 was 0.73±0.24g, which was 0.82 times higher than that of the control group. The average plant height was 18.73±5.61cm, which was 0.25 times higher than that of the control group. The average fresh weight of the wheat plants irrigated with the S12020 bacterial suspension with OD 600 =0.001 was 0.60±0.21g, which was 0.5 times higher than that of the control group. The average plant height was 20.03±4.51cm, which was 0.33 times higher than that of the control group.

[0104] Example 11 Kosakonia pseudosacchari S12020 significantly promotes the growth of corn

[0105] The nutrient soil, vermiculite, natural soil (1:1:1) were mixed and added to the flowerpot, and then soaked with water. Four corn seeds were sown in each pot, and then covered with a small amount of soil. The pots were placed in a greenhouse with a temperature of 25-28°C and a humidity of 65%-75%. After about two weeks, the seedlings were selected and used for the growth promotion experiment.

[0106] The S12020 single colony was inoculated in LB medium and cultured at 28°C and 200 rpm for 24 hours. Then, the culture was transferred to 50 mL of LB medium at an inoculation amount of 1%. The culture was further incubated at 28°C and 200 rpm for 24 hours. After the incubation, the S12020 was prepared into a bacterial suspension with a concentration of 1 x 10 8 CFU·mL -1 The S12020 bacterial suspension was used to irrigate the roots of the corn with 50 mL per pot. Four plants were treated in each pot, and the experiment was repeated three times. Distilled water was used as a control. The growth of the corn was observed and recorded every day. On the 30th day after irrigation, the plant height, root length, leaf length, stem diameter, fresh weight, and underground weight of the corn were measured.

[0107] Figure 11 The results showed that the corn treated with the S12020 bacterial suspension grew vigorously, with an average plant height of 67.29±9.73 cm, which was 0.15 times higher than the control group. The average stem length was 18.73±4.35 cm, which was 0.21 times higher than the control group. The average leaf length was 48.67±6.87 cm, which was 0.14 times higher than the control group.

[0108] Example 12: Kosakonia pseudosacchari S12020 significantly promotes the growth of rape

[0109] The nutrient soil, vermiculite, natural soil (1:1:1) were mixed and added to the flowerpot, and then soaked with water. The rape seeds were evenly spread on the soil, and then covered with a small amount of soil. The pots were placed in a greenhouse with a temperature of 25-28°C and a humidity of 65%-75%. After about one week, the seedlings were selected and used for the growth promotion experiment.

[0110] The S12020 single colony was inoculated in LB medium and cultured at 28°C and 200 rpm for 24 hours. Then, the culture was transferred to 50 mL of LB medium at an inoculation amount of 1%. The culture was further incubated at 28°C and 200 rpm for 24 hours. After the incubation, the S12020 was prepared into a bacterial suspension with a concentration of 1 x 10 8 CFU·mL -1 The S12020 bacterial suspension was used to irrigate the roots of the rape with 25 mL per pot. Four plants were treated in each pot, and the experiment was repeated three times. Distilled water was used as a control. The growth of the rape was observed and recorded every day. On the 30th day after irrigation, the chlorophyll content, plant height, and fresh weight of the rape were measured.

[0111] Figure 12 The results show that the rape treated by S12020 bacterial suspension by root irrigation has vigorous growth, the average plant height is 9.01±1.30 cm, which is 0.34 times higher than that of the control group, and has the trend of promoting chlorophyll synthesis and plant growth.

[0112] In summary, the S12020 culture provided by example 1 has at least one of the following functions B1-B9: B1, nitrogen fixation; B2, salt tolerance (can tolerate Nacl concentration of 8% medium); B3, potassium solution; B4, cellulase production; B5, ferritin production; B6, ammonia production; B7, plant growth hormone (IAA) secretion; B8, antagonism of rice stripe disease; B9, promoting plant lateral root formation; B10, improving plant biomass.

[0113] The application of the metabolite (fermentation broth) of S12020 provided by example 1 at least includes one of the following applications: nitrogen fixation, salt tolerance (can tolerate Nacl concentration of 8% medium), potassium solution, cellulase production, ferritin production, ammonia production, plant growth hormone (IAA) production, antagonism of rice stripe disease, promotion of plant lateral root formation or improvement of plant biomass.

[0114] The above examples only express the preferred embodiments of the present application, which are described in detail and specifically, but cannot be understood as the limitation of the scope of the present application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A salt-tolerant Pseudomonas cesiica strain, characterized in that, The strain was named *Pseudomonas sucrose* (…). Kosakonia pseudosacchari S12020, its accession number is CGMCC No.33020.

2. A biological agent, characterized in that, The Kosakonia pseudosacchari S12020 of claim 1.

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

4. Use of the salt-tolerant, nitrogen-fixing Kosakonia pseudosacchari strain of claim 1 or the biological preparation of claim 2 in potassium solubilization.

5. Use of the salt-tolerant, nitrogen-fixing Kosakonia pseudosacchari strain of claim 1 or the biological preparation of claim 2 in cellulase production.

6. Use of the salt-tolerant, nitrogen-fixing Kosakonia pseudosacchari strain of claim 1 or the biological preparation of claim 2 in ammonia production.

7. Use of the salt-tolerant, nitrogen-fixing Kosakonia pseudosacchari strain of claim 1 or the biological preparation of claim 2 in siderophore production.

8. Use of the salt-tolerant, nitrogen-fixing Kosakonia pseudosacchari strain of claim 1 or the biological preparation of claim 2 in plant growth hormone IAA secretion.

9. Use of the salt-tolerant, nitrogen-fixing Kosakonia pseudosacchari strain of claim 1 or the biological preparation of claim 2 in promoting the growth of rape, wheat and / or corn.

10. A biofertilizer prepared using the salt-tolerant, nitrogen-fixing Kosakonia pseudosacchari strain of claim 1 or the biological preparation of claim 2.

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