Pseudomonas TWS2N1 and application thereof

Abstract

The invention discloses pseudomonas TWS2N1 and application thereof, and belongs to the technical field of agricultural microorganisms. The preservation number of the pseudomonas TWS2N1 is CGMCC (China General Microbiological Culture Collection Center) No.32348, and the pseudomonas TWS2N1 is preserved in the China General Microbiological Culture Collection Center on October 25, 2024. The strain is separated from high and cold soil in Branchiomega in the autonomous region of Tibet, can grow well at 4-35 DEG C, has high nitrogen fixation capacity, can produce growth-promoting substances such as IAA and promote growth and development of rape, can be used as a rhizosphere growth-promoting bacterium for cool-loving crops and promote growth and development of plants, and has high agricultural application prospects and economic values.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and in particular to a strain of Pseudomonas TWS2N1 and an application thereof. Background Art

[0002] Rapeseed is the oil crop with the largest planting area and the widest regional distribution in my country. my country is the country that produces the most rapeseed in the world. Rapeseed is a cool-loving crop that does not require much heat or soil. The temperature fluctuation range during its growth period is large, and the low temperature period is long. The activity of commonly used functional microorganisms decreases in the low temperature season, and their functionality is greatly reduced;

[0003] Pseudomonas is a common microorganism in alpine soil. It has a wide growth temperature range, which is consistent with the temperature of rapeseed's growth period, and can play a role in most of rapeseed's growth period. Most of the growth-promoting microorganisms reported so far are screened under normal temperature conditions, while there are fewer for low-temperature crops. The present invention isolates and screens a strain of Pseudomonas TWS2N1 from alpine soil that can grow at 4-35°C, has nitrogen fixation, and produces IAA, in order to help the efficient planting of crops such as rapeseed. Summary of the invention

[0004] The invention discloses a strain of Pseudomonas TWS2N1 and an application thereof, so as to solve the deficiencies of the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0006] A strain of Pseudomonas TWS2N1, the Pseudomonas TWS2N1 (Pseudomonas mohnii) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.32348.

[0007] Pseudomonas TWS2N1 bacteria prepared by the Pseudomonas TWS2N1 (Pseudomonas mohnii).

[0008] The preparation method of Pseudomonas TWS2N1 bacterial cells comprises inoculating Pseudomonas TWS2N1 (Pseudomonas mohnii) into LB liquid culture medium, and the liquid fermentation conditions are as follows: fermentation temperature is 28-30°C, rotation speed is 170-190r / min, and fermentation time is 24-48h. After the fermentation is completed, the Pseudomonas TWS2N1 bacterial cells are obtained by centrifugation.

[0009] Furthermore, the Pseudomonas sp. TWS2N1 cells are resuspended in sterile water to prepare a bacterial suspension, i.e., the microbial agent, with an effective viable count of ≥ 0.2×10 8 cfu / ml.

[0010] Furthermore, the microbial agent is a solid microbial agent or a biological organic fertilizer or a compound microbial fertilizer prepared by adsorption, mixing and solid fermentation, that is, the microbial fertilizer.

[0011] Furthermore, the microbial agent is used to promote the growth of cool-loving crops and increase the available nitrogen content in the soil under the condition of 4-35°C.

[0012] Furthermore, the Pseudomonas TWS2N1 (Pseudomonas mohnii) and / or the Pseudomonas TWS2N1 cells and / or the microbial agent and / or the microbial fertilizer are used to promote the growth of cool-loving crops, improve the nitrogen fixation capacity of the soil and produce IAA under conditions of 4-35°C.

[0013] Furthermore, the cool-loving crop is rapeseed.

[0014] Furthermore, the application method of Pseudomonas TWS2N1 (Pseudomonas mohnii) includes root irrigation and / or directly spraying and / or flushing the Pseudomonas TWS2N1 (Pseudomonas mohnii) and / or the Pseudomonas TWS2N1 cells and / or the microbial agent and / or the microbial fertilizer onto the plants / leaves and / or soil.

[0015] Beneficial effects of the present invention: The present invention solves the problem that the temperature of rapeseed varies greatly during its growth period and conventional functional microorganisms have low effectiveness under low temperature conditions, and screens out a strain of Pseudomonas TWS2N1 that has nitrogen fixation and IAA production functions in a large temperature range, in order to help the efficient cultivation of crops such as rapeseed. The screening test results of the present invention show that strain TWS2N1 can grow at 4-35℃ and has a wide temperature adaptability range. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The colony morphology of strain TWS2N1 on LB medium;

[0017] Figure 2 This is the IAA color reaction effect diagram of the fermentation liquid of strain TWS2N1;

[0018] Figure 3 is the 16S rRNA phylogenetic tree of strain TWS2N1;

[0019] Figure 4 The height of rapeseed plants under different treatments;

[0020] Figure 5 is the fresh weight of aboveground part of rapeseed in different treatments;

[0021] Figure 6 Available nitrogen content in soil under different treatments.

[0022] Biological deposit information

[0023] TWS2N1, classified and named Pseudomonas mohnii, is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 25, 2024, and the deposit number is CGMCC NO.32348. DETAILED DESCRIPTION

[0024] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] It should be noted that the details of the present invention are known to those skilled in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer, and the experimental steps not described in detail refer to pathophysiological experiments, online databases, etc.

[0026] Unless otherwise specified, the raw materials used in the following examples are as follows:

[0027] LB liquid medium (1 L): 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, dilute to 1 L with deionized water, and sterilize at 115 °C for 30 min.

[0028] LB solid medium (1 L): 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, 15-20 g of agar, dilute to 1 L with deionized water, and sterilize at 115°C for 30 min.

[0029] Ashby solid culture medium (1 L): KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, CaSO4·2H2O 0.2 g, NaCl 0.2 g, CaCO3 5 g, mannitol 10 g, agar 15-20 g, dilute to 1 L with deionized water, sterilize at 115℃ for 30 min.

[0030] Ashby solid culture medium (1 L): KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, CaSO4·2H2O 0.2 g, NaCl 0.2 g, CaCO3 5 g, mannitol 10 g, agar 15-20 g, dilute to 1 L with deionized water, sterilize at 115℃ for 30 min.

[0031] Example 1

[0032] A strain of Pseudomonas TWS2N1, the Pseudomonas TWS2N1 (Pseudomonas mohnii) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.32348.

[0033] Isolation, screening and identification of Pseudomonas mohnii TWS2N1:

[0034] (1) Isolation of strains

[0035] Soil samples were collected from the high-altitude mountainous areas of Qamdo, Tibet Autonomous Region. 10 g of soil sample was added to a conical flask containing 100 ml of sterile saline. After standing for 20 min, it was shaken on a shaker at 30 °C and 180 rpm for 30 min. 1 ml of the sample was added to 9 ml of sterile saline and then diluted 10 2 , 10 3 , 10 4 Times, take 100 μl of the above soil suspension and evenly spread it on the Ashur-Bai solid culture medium plate, culture it in a 10°C incubator for 48 h, pick a single bacterial colony with a sterile toothpick, streak it, purify it, and save it for later use.

[0036] (2) Screening of functional strains

[0037] 5 mL of semi-solid Aspergillus culture medium was placed in a 10 mm × 100 mm test tube, and the tube was sealed with a reverse rubber stopper after inoculation. After culturing for 12 hours, 1 / 10 volume of 10% acetylene gas was injected into the small test tube (to make the final concentration 1%), and the tube was cultured again for 24 hours under the same conditions. 0.5 mL of gas phase sample was drawn from the small test tube with a 1 mL disposable sterile syringe and injected into the sample injection port of the gas chromatograph to measure the peak area of ​​sample ethylene. The standard ethylene concentration is 508 μL / L. The working conditions of the gas chromatograph were set as follows: column temperature 70°C, injection port 120°C, detector temperature 180°C. The gas flow rates were: nitrogen 30 mL / L, hydrogen 30 mL / L, and dry air 60 mL / L. The nitrogenase activity was calculated according to the following formula, unit: μmol C2H4 / (mL·h).

[0038]

[0039] The strain TWS2N1 was tested to have the ability to fix nitrogen at 4-30°C, and its nitrogenase activity was 7.63-28.57 μmol C2H4 / (mL·h).

[0040] 50ml LB plus tryptophan (tryptophan concentration is 100mg / L) medium, inoculate 0.5ml seed liquid, and culture at 30℃, 180rpm shaking for 48h to obtain bacterial liquid. Take 2ml bacterial liquid in a test tube, add an equal volume of colorimetric solution (35% perchloric acid and 0.5mol / L ferric chloride solution are mixed at a volume ratio of 50:1), and add 2ml of uninoculated LB liquid medium and an equal volume of colorimetric solution as a control. Those with pink color indicate that they can secrete IAA. After testing, strain TWS2N1 has the ability to produce IAA ( Figure 2 ).

[0041] (3) Strain identification

[0042] After the strain TWS2N1 was cultured on LB solid medium at 30℃ for 48h, its colonies were smooth, white, shiny, round, and 3-5mm in diameter. The results of phylogenetic tree comparison analysis constructed by 16S rRNA gene sequence showed that the strain TWS2N1 had the highest homology with Pseudomonas mohnii ( Figure 3 ), combined with the results of phylogenetic tree comparison analysis constructed with colony morphology and 16S rRNA gene sequences, strain TWS2N1 was identified as Pseudomonas mohnii.

[0043] The Pseudomonas TWS2N1 bacterial cells prepared by Pseudomonas TWS2N1 (Pseudomonas mohnii) and the preparation method of Pseudomonas TWS2N1 bacterial cells are as follows: Pseudomonas TWS2N1 (Pseudomonas mohnii) is inoculated into LB liquid culture medium, and the liquid fermentation conditions are as follows: the fermentation temperature is 28°C, the rotation speed is 170r / min, and the fermentation time is 24h. After the fermentation is completed, the Pseudomonas TWS2N1 bacterial cells are obtained by centrifugation.

[0044] The bacterial suspension, i.e., the microbial agent, is prepared by resuspending the Pseudomonas TWS2N1 bacterial cells with sterile water, and the effective viable bacterial count is ≥ 0.2×10 8 cfu / ml.

[0045] The microbial fertilizer is a solid microbial agent or a biological organic fertilizer or a compound microbial fertilizer prepared by adsorption, mixing and solid fermentation of the microbial agent.

[0046] The microbial agent is used to promote the growth of cool-loving crops at 4°C and to increase the available nitrogen content in the soil. The cool-loving crop is rapeseed, and the agent is applied by root irrigation and / or directly spraying and / or flushing onto the plant / leaf surface and / or soil.

[0047] The Pseudomonas TWS2N1 (Pseudomonas mohnii) and / or the Pseudomonas TWS2N1 bacteria and / or the microbial agent and / or the microbial fertilizer are used to promote the growth of cool-loving crops, improve the nitrogen fixation capacity of the soil and produce IAA under 4°C conditions, and the cool-loving crop is rapeseed.

[0048] Example 2

[0049] The contents of this embodiment are basically the same as those of embodiment 1, except that a method for preparing Pseudomonas TWS2N1 bacterial cells is prepared by using Pseudomonas mohnii.

[0050] The Pseudomonas TWS2N1 bacterial cells prepared by Pseudomonas TWS2N1 (Pseudomonas mohnii) and the preparation method of Pseudomonas TWS2N1 bacterial cells are as follows: Pseudomonas TWS2N1 (Pseudomonas mohnii) is inoculated into LB liquid culture medium, and the liquid fermentation conditions are as follows: the fermentation temperature is 29°C, the rotation speed is 180r / min, and the fermentation time is 36h. After the fermentation is completed, the Pseudomonas TWS2N1 bacterial cells are obtained by centrifugation.

[0051] The bacterial suspension, i.e., the microbial agent, is prepared by resuspending the Pseudomonas TWS2N1 bacterial cells with sterile water, and the effective viable bacterial count is ≥ 0.2×10 8 cfu / ml.

[0052] The microbial fertilizer is a solid microbial agent or a biological organic fertilizer or a compound microbial fertilizer prepared by adsorbing, mixing and solid fermenting the microbial agent.

[0053] The microbial agent is used to promote the growth of cool-loving crops at 20° C. and to increase the available nitrogen content in the soil. The cool-loving crop is rapeseed, and the agent is applied by root irrigation and / or directly spraying and / or flushing onto the plant / leaf surface and / or soil.

[0054] The Pseudomonas TWS2N1 (Pseudomonas mohnii) and / or the Pseudomonas TWS2N1 bacteria and / or the microbial agent and / or the microbial fertilizer are used to promote the growth of cool-loving crops, improve the nitrogen fixation capacity of the soil and produce IAA at 20°C, and the cool-loving crop is rapeseed.

[0055] Example 3

[0056] The contents of this embodiment are basically the same as those of embodiment 1, except that a method for preparing Pseudomonas TWS2N1 bacterial cells is prepared by using Pseudomonas mohnii.

[0057] The Pseudomonas TWS2N1 bacterial cells prepared by Pseudomonas TWS2N1 (Pseudomonas mohnii) and the preparation method of Pseudomonas TWS2N1 bacterial cells are as follows: Pseudomonas TWS2N1 (Pseudomonas mohnii) is inoculated into LB liquid culture medium, and the liquid fermentation conditions are as follows: the fermentation temperature is 30°C, the rotation speed is 190r / min, and the fermentation time is 48h. After the fermentation is completed, the Pseudomonas TWS2N1 bacterial cells are obtained by centrifugation.

[0058] The bacterial suspension, i.e., the microbial agent, is prepared by resuspending the Pseudomonas TWS2N1 bacterial cells with sterile water, and the effective viable bacterial count is ≥ 0.2×10 8 cfu / ml.

[0059] The microbial fertilizer is a solid microbial agent or a biological organic fertilizer or a compound microbial fertilizer prepared by adsorbing, mixing and solid fermenting the microbial agent.

[0060] The microbial agent is used to promote the growth of cool-loving crops at 35° C. and to increase the available nitrogen content in the soil. The cool-loving crop is rapeseed, and the agent is applied by root irrigation and / or directly spraying and / or flushing onto the plant / leaf surface and / or soil.

[0061] The Pseudomonas TWS2N1 (Pseudomonas mohnii) and / or the Pseudomonas TWS2N1 bacteria and / or the microbial agent and / or the microbial fertilizer are used to promote the growth of cool-loving crops, improve the nitrogen fixation capacity of the soil and produce IAA at 35°C, and the cool-loving crop is rapeseed.

[0062] The following is a test of microbial agents on increasing soil effective nitrogen content and promoting rapeseed production:

[0063] The potted plant test site is the open-air platform of the factory area of ​​Hunan Tewos Ecological Co., Ltd. in Suining County, Hunan Province. The test time is from October 2023 to March 2024.

[0064] Sow rapeseed seeds in seedling cups. When the rapeseed seedlings grow 3 true leaves, select rapeseed seedlings with relatively consistent growth potential and transplant them into plastic pots (one rapeseed seedling per pot) filled with soil (6 kg of soil per pot, mixed with base fertilizer). After transplanting, water the plastic pots and water them regularly afterwards.

[0065] The rapeseed seedlings transplanted into the plastic pots were set up with three treatments, namely T1 (control treatment, clean water), T2 (commercially available round brown nitrogen-fixing bacteria agent), and T3 (TWS2N1 agent prepared in Example 2), with 5 replicates for each treatment. After the transplanted rapeseed seedlings grew stably for one week, the agent was irrigated with water to the roots, 6 ml per pot. After completion, routine maintenance was carried out. In March of the second year, rapeseed seedlings and rhizosphere soil samples were collected to measure the rapeseed plant height ( Figure 4 ), rapeseed aboveground fresh weight ( Figure 5 ), soil available nitrogen content ( Figure 6 ), and compare the rapeseed growth conditions and soil nutrient changes of each treatment.

[0066] The experiment showed that adding TWS2N1 microbial agent can increase the available nitrogen content in the soil and promote the growth of rapeseed. The effect of applying microbial agent prepared by Pseudomonas mohnii is better.

[0067] Components not described in detail herein are prior art.

[0068] Although the specific embodiments of the present invention are described in detail above, the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in this field without departing from the purpose of the present invention, and modifications or deformations without creative labor are still within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas sp. TWS2N1, characterized in that: The Pseudomonas TWS2N1 (Pseudomonas mohnii) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.32348.

2. The Pseudomonas TWS2N1 bacterial cell prepared according to the Pseudomonas TWS2N1 (Pseudomonas mohnii) according to claim 1.

3. The method for preparing Pseudomonas sp. TWS2N1 cells according to claim 2, characterized in that: Pseudomonas TWS2N1 (Pseudomonas mohnii) was inoculated into LB liquid culture medium. The liquid fermentation conditions were as follows: fermentation temperature was 28-30°C, rotation speed was 170-190 r / min, and fermentation time was 24-48 h. After the fermentation was completed, the Pseudomonas TWS2N1 cells were obtained by centrifugation.

4. A microbial agent containing the Pseudomonas sp. TWS2N1 bacterial cell according to claim 2 or 3, characterized in that: The Pseudomonas TWS2N1 bacteria are resuspended in sterile water to prepare a bacterial suspension, i.e., the microbial agent, with an effective viable bacterial count of ≥ 0.2×10 8 cfu / ml.

5. A microbial fertilizer containing the microbial agent according to claim 4, characterized in that: The microbial agent is a solid microbial agent or a biological organic fertilizer or a compound microbial fertilizer prepared by the microbial agent through adsorption, mixing and solid fermentation, that is, the microbial fertilizer.

6. Use of the microbial agent according to claim 4 in promoting the growth of cool-loving crops and increasing the available nitrogen content in the soil under the condition of 4-35°C.

7. Use of the Pseudomonas TWS2N1 (Pseudomonas mohnii) according to claim 1 and / or the Pseudomonas TWS2N1 bacteria according to claim 2 and / or the microbial agent according to claim 4 and / or the microbial fertilizer according to claim 5 in promoting the growth of cool-loving crops, improving the nitrogen fixation capacity of the soil and producing IAA under conditions of 4-35°C.

8. The use according to claim 6 or 7, characterized in that: The cool weather-loving crop is rapeseed.

9. A method for using Pseudomonas mohnii, characterized in that: The method comprises irrigating the roots and / or directly spraying and / or flushing the Pseudomonas TWS2N1 (Pseudomonas mohnii) described in claim 1 and / or the Pseudomonas TWS2N1 bacteria described in claim 2 and / or the microbial agent described in claim 4 and / or the microbial fertilizer described in claim 5 onto the plants / leaves and / or soil.

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