Coxsackie cocori strain and microbial inoculum and application of coxsackie cocori strain

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a coxakella cocori strain as well as a microbial agent and an application of the coxakella cocori strain. The invention discloses a strain of coxakella coraldii T6-2, which is preserved in the China General Microbiological Culture Collection Center on July 10, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.31262. The invention further discloses a preparation method of the coxakella coraldii T6-2. The strain has the characteristics of salt resistance, alkali resistance and high ammonium resistance; the strain has biological activities of dissolving phosphorus, fixing nitrogen and secreting IAA, and has an obvious promotion effect on soybean growth under salt stress; the strain has extremely strong ammonia nitrogen degradation activity, the initial concentration of ammonia nitrogen is in a range of 50-100 mg / L, and after treatment is performed for 36 h, the ammonia nitrogen degradation rate reaches 90% or above. The strain has extremely high application value in plant growth promotion and ammonia nitrogen waste biodegradation in saline-alkali and high-ammonium environments.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and specifically relates to a Cossackia coli strain and a bacterial agent and application thereof. Background Art

[0002] In agricultural production, excessive soil salt content will interfere with the normal growth of crops, resulting in reduced yields and even crop death. Ammonium nitrogen is one of the important forms of nitrogen directly absorbed and utilized by crop roots, but the long-term application of ammonium nitrogen fertilizers in agricultural production has led to excessive accumulation of NH in the soil in some areas. 4 + , far exceeding the optimum content (0.1-0.5 mmol / L) required for crop growth, causing ammonium poisoning. High salt and high ammonium stress has become an important environmental problem in agricultural production, seriously restricting the development of agriculture and animal husbandry.

[0003] Microbial strains are important resources for promoting plant growth and development. Growth-promoting bacteria can improve plant tolerance to abiotic stresses such as high salt and high ammonium to a certain extent. Microbial growth promotion is mainly achieved by producing a large number of secondary metabolites, which release slow-release nitrogen, phosphorus, and potassium in the soil. It promotes plant growth by fixing nitrogen, dissolving potassium, and dissolving phosphorus; at the same time, it produces iron carriers and IAA to enhance plant stress resistance. Exploring plant growth-promoting bacteria can alleviate the high-salt and high-ammonium environmental stress on crops, and open up new paths for the extensive development and utilization of microbial resources.

[0004] At present, the bacteria that can be used to promote growth found in the soil environment or inside plants are mainly Azospirillum, Pseudomonas, Bacillus, Microbacterium and Rhizobium, while Kosakonia cowanii, which is salt-tolerant and high-ammonium-tolerant and can secrete IAA, has not been reported yet. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a strain of Cossackia coli and its bacterial agent and application.

[0006] In one aspect, the present invention provides a strain of Kosakonia cowanii, wherein the strain is designated as Kosakonia cowanii T6-2.

[0007] Specifically, the Kosakonia cowanii T6-2 was screened and isolated from the root soil sample of wheat "Tongmai No. 6", and the deposit information of the Kosakonia cowanii T6-2 is as follows:

[0008] Strain name: T6-2;

[0009] Classification name: Kosakonia cowanii;

[0010] Date of receipt by the depository: July 10, 2024;

[0011] Time of issuance of the preservation certificate: July 18, 2024;

[0012] Depository: China General Microbiology Center (CGMCC);

[0013] Deposit number: CGMCC No.31262.

[0014] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] Furthermore, the 16S rDNA sequence of Coxsackie is shown in SEQ ID NO:1.

[0016] In another aspect, the present invention provides a bacterial agent containing Kosakonia cowanii T6-2, wherein the bacterial agent contains Kosakonia cowanii T6-2 bacterial cells and / or Kosakonia cowanii T6-2 fermentation broth.

[0017] Furthermore, the deposit number of the Cossackia coli in the bacterial agent is CGMCC No.31262.

[0018] In a third aspect, the bacterial agent can be prepared into any industrially acceptable dosage form.

[0019] It is easy for a person skilled in the art to know that in order to give full play to the efficacy of Kosakonia cowanii T6-2, it can be prepared into a preparation form that is easy to apply according to the needs of use. Based on this, further, the present invention provides a bacterial agent formulation containing Kosakonia cowanii T6-2. The bacterial agent is processed into a formulation formulation accepted in industry and agriculture, and the preparation contains an auxiliary agent that assists Kosakonia cowanii T6-2 in improving plant growth promotion. The bacterial agent can be formulated into a variety of formulations such as soluble liquid, water-dispersible granules, wettable powder, dispersible oil suspension, suspension, microemulsion, granule, and microcapsule with a certain sustained release effect. When preparing the above-mentioned different formulations, for a person skilled in the art, in addition to using the optional Kosakonia cowanii T6-2, it is also necessary to select a variety of auxiliary agents, and different formulation auxiliary components (auxiliaries) can be selected and used as needed. The auxiliary component may be one or more of a dispersion medium, a dispersant, an emulsifier, a wetting agent, a thickener, a defoaming agent, an antifreeze agent, a disintegrant, a binder, a filler, a carrier, and the like.

[0020] Furthermore, the Kosakonia cowanii or the bacterial agent containing the Kosakonia cowanii T6-2 bacterial body and / or the fermentation liquid of Kosakonia cowanii T6-2 is used in plant growth promotion.

[0021] Furthermore, the Kosakonia cowanii or the bacterial agent containing Kosakonia cowanii T6-2 bacteria and / or Kosakonia cowanii T6-2 fermentation liquid is used to promote plant growth under saline-alkali and high ammonium stress.

[0022] Furthermore, the Kosakonia cowanii T6-2 or the bacterial agent containing the Kosakonia cowanii T6-2 bacterial body and / or the fermentation liquid of Kosakonia cowanii T6-2 promotes plant growth through nitrogen fixation, phosphorus solubilization or IAA secretion by Kosakonia cowanii T6-2.

[0023] Furthermore, the Kosakonia cowanii or the bacterial agent containing the Kosakonia cowanii T6-2 bacterial body and / or the fermentation liquid of Kosakonia cowanii T6-2 is used in soybean growth promotion.

[0024] In a fourth aspect, the present invention also provides the use of Cossackia coelicolor in the biodegradation of ammonia nitrogen waste.

[0025] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0026] The present invention screens and separates a strain of Kosakonia cowanii T6-2 from a root soil sample of wheat "Tongmai No. 6", and the strain is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit number is: CGMCC No. 31262. It is verified that the strain of Kosakonia cowanii T6-2 has the characteristics of salt resistance, alkali resistance, and high ammonium resistance. The strain can survive in both salt and non-salt environments, and the maximum NaCl concentration it tolerates is 10% (w / v), and the maximum alkali resistance pH value is 11. The strain can grow normally in the range of ammonium nitrogen concentration of 0 to 2000 mg / L.

[0027] It has been verified that Kosakonia cowanii T6-2 has nitrogen fixation and phosphorus solubilization activities, and has strong IAA secretion activity, and the amount of IAA secreted is 25.3694±0.0030 mg / L. In the soybean hydroponic experiment, Kosakonia cowanii T6-2 has a significant promoting effect on the growth of soybean seedlings under salt stress.

[0028] The ammonia nitrogen degradation test showed that under the condition of initial ammonia nitrogen concentration of 50-100 mg / L, the ammonia nitrogen degradation rate of Kosakonia cowanii T6-2 reached more than 90% after treatment for 36 hours, indicating that Kosakonia cowanii T6-2 has extremely strong ammonia nitrogen degradation ability and the strain can be used in the biodegradation of ammonia nitrogen waste.

[0029] Kosakonia cowanii T6-2 can be used in plant growth promotion in saline-alkali and high-ammonium environments and can be used in ammonia nitrogen waste biodegradation. The invention provides a new biological resource for ammonia nitrogen waste biodegradation and plant growth promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a single colony of Kosakonia cowanii T6-2 on LB medium.

[0031] Figure 2 This is the growth curve of Kosakonia cowanii T6-2 in LB medium with different salt concentrations.

[0032] Figure 3 The growth of Kosakonia cowanii T6-2 was compared at different pH values ​​for 12 hours.

[0033] Figure 4 This is a comparison of the growth of Kosakonia cowanii T6-2 cultured under different ammonia nitrogen concentrations for 12 hours.

[0034] Figure 5 These are the test results of ammonia nitrogen degradation rate of Kosakonia cowanii T6-2 under different ammonia nitrogen concentrations.

[0035] Figure 6 These are the test results of the nitrogen fixation performance of Kosakonia cowanii T6-2.

[0036] Figure 7 These are the test results of the phosphate solubilization performance of Kosakonia cowanii T6-2.

[0037] Figure 8 These are the test results for IAA production by strain Kosakonia cowanii T6-2.

[0038] Fig. 9 Schematic diagram of the agronomic traits of Kosakoniacowanii T6-2 bacterial suspension on soybean seedling growth promotion under normal conditions and 100 mmol / L NaCl salt stress conditions.

[0039] Fig.10 The statistical results of Kosakonia cowanii T6-2 bacterial suspension on promoting soybean seedling growth under normal and salt treatment conditions. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is described below in conjunction with embodiments; however, the present invention is not limited to the following embodiments.

[0041] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0042] The experimental methods and detection methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0043] Example 1

[0044] This example is the separation and purification of strain T6-2.

[0045] Sample source: Root soil samples of the wheat variety “Tongmai No. 6” produced by Shaanxi Datang Seed Co., Ltd. in Yangling District, Xianyang City, Shaanxi Province.

[0046] Prepare high-salt and high-ammonium screening medium: LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) with NaCl and NH 4 Cl, prepare NaCl to a final concentration of 85mmol / L, NH 4 The liquid screening medium with a final Cl concentration of 10 mmol / L is adjusted to a pH value of 7.4-7.6 and sterilized at 121°C for 30 minutes to obtain a high-salt and high-ammonium liquid screening medium. The high-salt and high-ammonium solid screening medium is prepared by adding 1.5 wt% agar powder to the above formula.

[0047] Strain screening: Use a sterile brush to brush the rhizosphere soil of "Tongmai No. 6" onto sterile weighing paper on a clean bench, weigh 1 g of the rhizosphere soil sample and add it to 50 mL of high-salt and high-ammonium screening liquid culture medium, and culture in a shaking flask at 37°C and 120 r / min for 24 hours.

[0048] Strain separation and purification: The above soil high-salt and high-ammonium mixed solution was allowed to stand for 30 minutes, and 100 μL and 200 μL of the supernatant were respectively applied on two high-salt and high-ammonium solid screening culture media, and cultured at 37°C for 18 to 20 hours; after the colonies grew, the colonies with fast growth and large diameter were picked and inoculated in 3mL liquid screening culture media; after shaking and culturing at 37°C and 180 r / min for 16 hours, they were streaked on the high-salt and high-ammonium solid screening culture media, and cultured at 37°C to obtain single colonies. Pick a single colony, inoculate it in liquid LB culture media, and after shaking and culturing at 37°C and 180 r / min for 16 hours, prepare it into glycerol bacteria (bacterial liquid volume: 30% glycerol volume = 1:1), mark it as T6-2 strain, and store it at -80°C.

[0049] Cultivation of T6-2 strain: Take out the strain of T6-2 stored at -80℃, use an inoculation loop to streak the bacterial solution on the surface of the LB plate, incubate it upside down at 37℃ for 12h, pick a single colony and put it in 3mL of LB medium (tryptone 10g / L, yeast extract powder 5g / L, NaCl 10g / L), and incubate it at 37℃, 180rpm, with shaking for 12h.

[0050] Example 2

[0051] This example is to identify strain T6-2.

[0052] Morphological identification:

[0053] The strain T6-2 was cultured on LB solid medium plates at 37°C for 12 h. The single colonies were light yellow to orange-yellow, round, with neat edges and a smooth and moist surface. Figure 1 On the LB solid plate containing salt, white colonies appear.

[0054] Systematic identification:

[0055] Pipette 1.2 mL of fresh T6-2 bacterial suspension and transfer it to a 1.5 mL centrifuge tube with a pipette. Centrifuge at 10,000 rpm for 5 min at 4°C and discard the supernatant. Add 0.8 mL of DNA extraction buffer (100 mmol / L EDTA-Na 2 , 1mol / LNaCl, 100mmol / L Tris-HCl, 1% CTAB, pH 8.0), fully suspend the cells, mix well, and place in a water bath at 37°C for 1h. During this period, mix the centrifuge tube twice; add 250μL of V:V = 1:1 to the centrifuge tube Tris-saturated phenol and chloroform were fully mixed and extracted for 10 minutes, allowed to stand at room temperature for 2 minutes, and centrifuged at 10,000 rpm at 4°C for 15 minutes; 600 μL of the supernatant was aspirated into a new 1.5 mL centrifuge tube, and then 250 μL of chloroform was added to the centrifuge tube, and after sufficient mixing, allowed to stand at room temperature for 2 minutes, and centrifuged at 10,000 r / min at 4°C for 10 minutes; 500 μL of the supernatant was transferred to a new centrifuge tube with a pipette, and 0.6 times the volume of pre-cooled isopropanol was added, and allowed to stand at 4°C for 1 hour to precipitate DNA; centrifuged at 10,000 rpm at 4°C for 10 minutes, the supernatant was discarded, and the precipitate was washed twice with 70% ethanol pre-cooled at 4°C, placed on a clean bench to be blown dry, and 30 μL of distilled water was added to dissolve the DNA precipitate to obtain T6-2 genomic DNA.

[0056] The 16S rDNA gene of T6-2 was amplified using the universal primer pair 27F and 1492R. The sequences of the primer pair used are as follows:

[0057] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0058] 1492R:5'-GGCTACCTTGTTACGACTT-3'

[0059] PCR reaction system: 22 μL Gold Mixing (Beijing Qingke Biotechnology Co., Ltd.), 0.5 μL of each primer, 1-2 μL of DNA template. Amplification conditions: 94°C 4 min, 94°C 30 s, 55°C 30 s, 72°C 1 min (32 cycles), 72°C 5 min, 3 μL of PCR product was drawn and detected by 1% agarose gel electrophoresis, and then sent to Aoke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of the T6-2 strain was obtained by sequencing, and its full length is 1443 bp. The specific sequence is shown in SEQ ID NO: 1.

[0060] The 16S rDNA sequence of the T6-2 strain shown in SEQ ID NO:1 was compared with the nucleic acid data in GenBank (National Center for Biotechnology Information [http: / / www.ncbi.nlm / nih.gov]) by BLAST homology comparison. The results showed that the 16S rDNA of the T6-2 strain was 99% identical to the 16S rDNA sequence of Kosakonia cowanii (GenBank No.: OP910014.1), indicating that the T6-2 strain was Kosakonia cowanii and was named Kosakonia cowanii T6-2 (sequence submitted to GenBank, Accession No.: PP837988.1).

[0061] Example 3

[0062] This example is a test to evaluate the salt and alkali resistance of Kosakonia cowanii T6-2.

[0063] Evaluation of salt tolerance of Kosakonia cowanii T6-2:

[0064] In LB liquid medium containing 0%, 2%, 4%, 6%, 8%, and 10% (w / v) NaCl, Kosakonia cowanii T6-2 seed culture was inoculated at a 1% inoculation amount (1 mL of seed culture was inoculated in 100 mL of liquid culture medium), and cultured with shaking at 37°C and 180 rpm for 12 h. The OD of Kosakonia cowanii T6-2 was measured. 600 The growth curve was drawn using LB liquid culture medium containing 0%, 2%, 4%, 6%, 8%, and 10% (w / v) NaCl without inoculation of bacterial solution as blank.

[0065] The results are as follows Figure 2 As shown, Kosakonia cowanii T6-2 can grow in liquid LB culture medium with NaCl concentration of 0% to 10% (w / v), and its growth in LB culture medium containing 2% (w / v) and 4% (w / v) salt is better than that in salt-free LB culture medium.

[0066] Evaluation of alkali resistance of Kosakonia cowanii T6-2:

[0067] The pH value of LB liquid medium was adjusted to 5, 7, 9, and 11 with 1 mol / L NaOH or 1 mol / L HCl, and the seed culture of Kosakonia cowanii T6-2 was inoculated at 1% inoculation volume (1 mL of seed culture was inoculated in 100 mL of liquid culture medium), and the culture was shaken at 37°C for 12 h. The OD of Kosakonia cowanii T6-2 culture was measured. 600 The growth curve was drawn according to the value; the culture medium with pH values ​​of 5, 7, 9, and 11 without inoculation of bacterial solution was used as blank.

[0068] The results are as follows Figure 3 As shown, the results showed that Kosakonia cowanii T6-2 strain can grow in the pH range of 5-11.

[0069] Example 4

[0070] This example is an evaluation of the ammonium tolerance and ammonia nitrogen degradation performance of Kosakonia cowanii T6-2.

[0071] Ammonium tolerance test of Kosakonia cowanii T6-2:

[0072] Kosakonia cowanii T6-2 was inoculated into NM medium and cultured with different concentrations of (NH 4 ) 2 SO 4 The initial ammonium nitrogen concentration of NM medium was set to 50, 100, 200, 500, 1000, and 2000 mg / L. The culture conditions were 37°C, 180 rpm, and 5% (V / V) inoculation. The culture was continued for 12 h, and the bacterial OD was measured. 600 The preparation method of the NM medium is as follows: weigh 10.0 g CH 3 COONa, 7.0g K 2 HPO 4 , 3.0g KH 2 PO4 、0.1g MgSO 4 7H 2 O, 0.05 g FeSO 4 7H 2 O was dissolved in distilled water, and then the volume was made up to 1 L with distilled water, and the pH value was adjusted to 7.2.

[0073] The results of the ammonium tolerance test of Kosakonia cowanii T6-2 are as follows Figure 4 As shown, the results showed that Kosakonia cowanii T6-2 could grow in the range of ammonium nitrogen concentration from 0 to 2000 mg / L.

[0074] Ammonia nitrogen degradation performance test of Kosakonia cowanii T6-2:

[0075] The Kosakonia cowanii T6-2 bacterial suspension grown to the logarithmic growth phase was centrifuged at 8,000 rpm for 5 min at room temperature to collect the bacterial cells, which were resuspended in sterile water and adjusted to OD 600 The value was set to 1.0 as the inoculum; the above inoculum was inoculated into NM medium with initial ammonia nitrogen concentration of 50, 100, and 200 mg / L at a 5% (v / v) inoculum; at the same time, NM medium with initial ammonia nitrogen concentration of 50, 100, and 200 mg / L without inoculum was set as blank. Cultured in a constant temperature shaker at 37°C and 180 rpm, sampling was performed every 2 hours to measure the ammonia nitrogen concentration and bacterial OD 600 value.

[0076] Ammonia nitrogen concentration was detected using Nessler's reagent method:

[0077] First, prepare Nessler's reagent and potassium sodium tartrate solution. The preparation method of Nessler's reagent is: weigh 16g of sodium hydroxide, dissolve it in 50mL of ammonia-free distilled water and fully cool it to room temperature; weigh 7g of potassium iodide and 10g of mercuric iodide and dissolve them in distilled water, then slowly inject this solution into the sodium hydroxide solution under stirring, dilute it with distilled water to 100mL, store it in a polyethylene bottle, and seal it for storage. The preparation method of potassium sodium tartrate solution is: weigh 50g of potassium sodium tartrate and dissolve it in 100mL of distilled water, heat and boil to remove ammonia, cool it, and dilute it to 100mL with distilled water.

[0078] Then collect and culture Kosakonia cowanii T6-2 bacterial solution, centrifuge, take 500 μL of supernatant, add 1.0 mL Nessler's reagent and 1.0 mL potassium sodium tartrate solution, mix well and let stand for 10 minutes, measure the absorbance at a wavelength of 425 nm, and the result is as follows: Figure 5 shown.

[0079] The results showed that under the condition of initial ammonia nitrogen concentration of 50-100 mg / L, the ammonia nitrogen degradation rate reached more than 90% after treatment with Kosakoniacowanii T6-2 for 36 hours.

[0080] Example 5

[0081] This example is a growth promotion test of Kosakonia cowanii T6-2.

[0082] (1) Nitrogen fixation performance test of Kosakonia cowanii T6-2

[0083] The streak method was used to inoculate Kosakonia cowanii T6-2 on Ashby medium, and the culture was inverted at 37°C for 2-3 days to observe whether the strain could grow stably. If the strain could grow stably, it was considered to have nitrogen fixation function, and if the strain could not grow stably, it was considered to have no nitrogen fixation function. The preparation method of the Ashby medium is: weigh 5g mannitol, 0.2g MgSO 4 7H 2 O, 0.2 g KH 2 PO 4 , 0.2g NaCl, 0.1g CaSO 4 ·2H 2 O, 5g CaCO 3 , dissolve 15g agar powder in distilled water, then make up to 1L with distilled water and adjust the pH value to 7.0.

[0084] Test results such as Figure 6 As shown, Kosakonia cowanii T6-2 grew well on Ashby medium, indicating that it has nitrogen fixation function.

[0085] (2) Phosphate solubilization performance test of Kosakonia cowanii T6-2

[0086] Use a pipette to take 2 μL of activated Kosakonia cowanii T6-2 and inoculate it on the organophosphorus bacteria culture medium. Incubate in a 28°C constant temperature incubator for 3 days to observe whether a transparent circle appears. If a transparent circle appears, it indicates that the strain has the ability to dissolve phosphate. Otherwise, it indicates that the strain does not have the ability to dissolve phosphate.

[0087] The cross method was used to measure d (colony diameter) and D (transparent zone diameter), and D / d was calculated. The phosphorus solubilization ability of the strain was determined according to the ratio of transparent zone diameter to colony diameter (denoted as Mp). The preparation method of the organophosphorus bacteria culture medium is as follows: 10 g glucose, 0.2 g lecithin, 0.03 g MgSO 4 7H 2 O, 0.3 g NaCl, 0.5 g (NH 4 ) 2 SO 4 , 0.03 g FeSO 4 7H 2 O, 0.3 g NaCl, 0.4 g yeast extract, 5 g CaCO 3 , 0.03g MnSO 4 ·4H 2 O. Dissolve 20 g agar in distilled water, then dilute to 1 L with distilled water and adjust the pH to 7.0.

[0088] Test results such as Figure 7 As shown, after Kosakonia cowanii T6-2 was inoculated into the organophosphate culture medium and cultured, a transparent zone was produced, indicating that Kosakonia cowanii T6-2 had the function of dissolving organic phosphorus; the Mp value of Kosakonia cowanii T6-2 for dissolving organic phosphorus was calculated, and the Mp value was 1.87±0.06, indicating that Kosakonia cowanii T6-2 had a strong phosphorus dissolving performance.

[0089] (3) IAA production performance test of Kosakonia cowanii T6-2

[0090] IAA qualitative determination:

[0091] Using the colorimetric method, Kosakonia cowanii T6-2 was inoculated into LB liquid culture medium containing L-tryptophan (200 mg / L), and cultured on a shaker at 37°C 180 rpm for 4 days; 50 μL of bacterial suspension and 50 μL of Salkowski's reagent were added dropwise to a 96-well plate culture plate, and a positive control and a negative control were set up. The positive control was added with 50 μL of 50 mg / L indoleacetic acid, and the negative control was added with LB liquid culture medium containing L-tryptophan; the 96-well plate culture plate was protected from light for 30 minutes and its color change was observed. If it turned pink, it could produce IAA. The preparation method of the Salkowski's colorimetric solution is as follows: take 50 mL of 35% HClO 4 With 1mL 0.5mol / L FeCl3 Mix the solution evenly and prepare it before use.

[0092] The qualitative test results are as follows Figure 8 As shown by Figure 8 It was found that Kosakonia cowanii T6-2 has the ability to produce IAA.

[0093] IAA quantitative determination:

[0094] Prepare IAA standard solutions with mass concentrations of 0μg / mL, 0.5μg / mL, 1.0μg / mL, 5.0μg / mL, 10.0μg / mL, 15.0μg / mL, 20.0μg / mL, and 25.0μg / mL, add equal amounts of Salkowski colorimetric solution to test tubes of IAA standard solutions of different concentrations, place the test tubes in a 28℃ constant temperature incubator, and allow color development for 30 minutes in the dark. After the color development reaction is completed, perform colorimetry at a wavelength of 530nm, and draw a standard curve with absorbance as the horizontal axis and IAA concentration as the vertical axis.

[0095] After culturing Kosakonia cowanii T6-2 in a shake flask for 4 days, take 2 mL of bacterial solution in a centrifuge tube, centrifuge at 10,000 rpm for 10 minutes, take the supernatant and add it to a test tube, add an equal volume of Salkowski colorimetric solution, and place it in a 28°C constant temperature incubator away from light for 30 minutes for color development reaction. After the reaction is completed, the absorbance value of the sample is measured at a wavelength of 530 nm. Calculate the amount of IAA produced by Kosakonia cowanii T6-2.

[0096] According to calculation, the IAA production of Kosakonia cowanii T6-2 was 25.3694±0.0030 mg / L.

[0097] The qualitative and quantitative tests of IAA production by Kosakonia cowanii T6-2 showed that Kosakonia cowanii T6-2 could secrete IAA. The quantitative test results showed that the IAA production was 25.3694±0.0030 mg / L, indicating that Kosakonia cowanii T6-2 had strong IAA secretion activity.

[0098] Example 6

[0099] This example provides a test on the growth promotion of soybean seedlings by Kosakonia cowanii T6-2.

[0100] (1) Preparation of Kosakonia cowanii T6-2 bacterial suspension

[0101] Kosakonia cowanii T6-2 strain was inoculated into LB liquid medium and cultured at 37°C for 12 h. The fermentation filtrate and bacterial precipitate were obtained by centrifugation at 8,000 rpm for 5 min at room temperature. The bacterial precipitate was suspended in sterile water to prepare OD 600 The bacterial suspension is 1.0 for later use.

[0102] (2) Test on the effect of Kosakonia cowanii T6-2 bacterial suspension on soybean seedling growth

[0103] Four experimental groups were set up in the test experiment on the growth promotion of soybean seedlings by Kosakonia cowanii T6-2, namely: clean water + nutrient solution group (clean water soaking + Hoagland nutrient solution culture), bacterial suspension + nutrient solution group (bacterial suspension soaking + Hoagland nutrient solution culture), clean water + saline nutrient solution group (clean water soaking + Hoagland nutrient solution containing 100mmol / L NaCl culture), bacterial suspension + saline nutrient solution group (bacterial suspension soaking + Hoagland nutrient solution containing 100mmol / L NaCl culture).

[0104] Select soybean seeds of similar size and full grains, disinfect them with 75% ethanol solution for 1 minute, rinse them with tap water once, soak them in 5% sodium hypochlorite solution for 20 minutes, and rinse them with clean water 5 times.

[0105] The seeds were placed in a conical flask, and the soybean seeds were soaked in clean water and bacterial suspension for 12 hours, respectively. Then, the seeds were placed on filter paper soaked in clean water in a culture dish and germinated at 25°C for 3 days. Soybean seedlings with similar growth conditions and plant height of about 3 cm were selected and transplanted into a hydroponic device (the hydroponic device consists of a conical flask and a hydroponic planting basket). The culture solutions were Hoagland nutrient solution and Hoagland nutrient solution containing 100 mmol / L NaCl, respectively. Three replicates were set for each treatment group. On the 14th day of seedling growth, plant height, root length and fresh weight were measured. The preparation method of the Hoagland nutrient solution is as follows: weigh 0.945 g Ca(NO 3 ) 2 ·4H 2 O, 0.505g KNO 3 、0.080g NH 4 NO 3 , 0.136g KH 2 PO 4 、0.493g MgSO 47H 2 O was dissolved in distilled water, and then 2.5 mL of 400× iron salt solution and 5 mL of 200× trace element solution were added to the above solution. Finally, distilled water was added to make the volume to 1 L and the pH value was adjusted to 6.0.

[0106] The results of the growth promotion test are as follows Fig. 9 As shown, the bacterial suspension of Kosakonia cowanii T6-2 can promote the growth of soybean seedlings under salt stress conditions. Under normal and salt treatment conditions, Kosakonia cowanii T6-2 has a significant growth-promoting effect on soybean plant height and root length. Fig.10 The results showed that soybean seedlings treated with Kosakonia cowanii T6-2 suspension for 12 hours had thicker stems and more developed root systems than the control, further indicating that Kosakonia cowanii T6-2 can promote soybean growth.

[0107] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred embodiments of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A strain of Cossackia coli, characterized in that The Kosakonia cowanii T6-2 was named as Kosakonia cowanii T6-2, and was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31262.

2. The Cossackia coli according to claim 1, characterized in that The 16S rDNA sequence of Cossackia coli is shown in SEQ ID NO:

1.

3. The bacterial agent containing Cossackia coli according to claim 1, characterized in that The bacterial agent contains Kosakonia cowanii T6-2 bacterial cells and / or Kosakonia cowanii T6-2 fermentation liquid.

4. The bacterial agent according to claim 3, characterized in that The deposit number of the Coxsackievirus is CGMCC No.31262.

5. The bacterial agent according to claim 3 or 4, characterized in that The bacterial agent can be prepared into any industrially acceptable dosage form.

6. Use of the Cossackia coli according to claim 1 or 2 or the bacterial agent according to claim 4 or 5 in promoting plant growth.

7. The use according to claim 6, characterized in that: The plant growth promotion is to promote plant growth in a saline-alkali and high-ammonium environment.

8. The use according to claim 6, characterized in that: The plant growth promotion is achieved by Kosakonia cowanii T6-2 through nitrogen fixation, phosphorus solubilization or IAA secretion.

9. The use according to claim 6, characterized in that: The plant is a herb, and the herb is soybean.

10. Use of Cossackia coli according to claim 1 or 2 in biodegradation of ammonia nitrogen waste.

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