Pseudomonas TWS13 and application thereof

Abstract

The invention discloses pseudomonas TWS13 and application thereof, and belongs to the technical field of agricultural microorganisms. The preservation number of the pseudomonas TWS13 is CGMCC (China General Microbiological Culture Collection Center) No.32077, and the pseudomonas TWS13 is preserved in the China General Microbiological Culture Collection Center on September 25, 2024. The strain is separated from alpine soil in Branchiomega in the autonomous region of Tibet, can well grow under the condition of 4-35 DEG C, not only has strong inorganic phosphorus dissolving capacity, but also has an antagonistic effect on rice sheath blight pathogen, and has high agricultural application prospect and economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and in particular to a strain of Pseudomonas TWS13 and an application thereof. Background Art

[0002] Rice-oil rotation is a typical planting model in southern my country. It can not only make full use of the idle land in winter, but also play a certain inhibitory role in most diseases and insect pests in the soil, and significantly improve soil fertility. Since rice and rapeseed are planted in different seasons, the temperature difference between the two during their growth period is very large. The activity of commonly used functional microorganisms decreases in low temperature seasons, and their functionality is greatly reduced, making it difficult to meet the growth promotion needs of both crops at the same time. Pseudomonas is a common microorganism in high-altitude cold soils, and its growth temperature range is wide. It is the preferred microorganism for rice-oil rotation.

[0003] Most of the functional microorganisms reported so far were screened under normal temperature conditions, but few have a wide growth temperature range. The present invention isolated and screened a Pseudomonas TWS13 from high-cold soil that can grow at 4-35°C and has the function of degrading inorganic phosphorus and antagonizing rice sheath blight pathogens, in order to facilitate the efficient cultivation of rice-oil rotation. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Pseudomonas TWS13 and applications 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 TWS13, wherein the Pseudomonas TWS13 (Pseudomonas germanica) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of September 25, 2024 and a deposit number of CGMCC No.32077.

[0007] Pseudomonas TWS13 cells prepared by the Pseudomonas TWS13.

[0008] The preparation method of Pseudomonas TWS13 bacteria is as follows: inoculating Pseudomonas TWS13 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, centrifugation is performed to obtain Pseudomonas TWS13 bacteria;

[0009] Furthermore, the Pseudomonas TWS13 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 rapeseed growth and increase the effective phosphorus content in the soil under the condition of 4-35°C.

[0012] Furthermore, the Pseudomonas TWS13 and / or the Pseudomonas TWS13 cells and / or the microbial agent and / or the microbial fertilizer are used to promote rapeseed growth, dissolve inorganic phosphorus and antagonize rice sheath blight pathogens under 4-35°C conditions.

[0013] Furthermore, the application method of Pseudomonas TWS13 includes root irrigation and / or directly spraying and / or flushing the Pseudomonas TWS13 and / or the Pseudomonas TWS13 cells and / or the microbial agent and / or the microbial fertilizer onto the plants / leaves and / or soil.

[0014] Furthermore, the application scenario is rice-oil rotation.

[0015] Beneficial effects of the present invention: In view of the large temperature variation during the growth period of the two crops in the rice-oil rotation planting mode and the low effectiveness of conventional functional microorganisms under low temperature conditions, the present invention screened out a strain of Pseudomonas TWS13 that has the function of solubilizing phosphate and antagonizing rice sheath blight pathogens in a large temperature range, in order to help the efficient planting of rice-oil rotation. The screening test results of the present invention show that strain TWS13 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 TWS13 on LB medium;

[0017] Figure 2 This is a diagram showing the phosphorus solubilization effect of strain TWS13;

[0018] Figure 3 is the 16S rRNA phylogenetic tree of strain TWS13;

[0019] Figure 4 The strain TWS13 is used to antagonize rice sheath blight pathogen;

[0020] Figure 5 The height of rapeseed plants under different treatments;

[0021] Figure 6 is the fresh weight of aboveground part of rapeseed in different treatments;

[0022] Figure 7 Available phosphorus content in soil under different treatments.

[0023] Biological deposit information

[0024] TWS13, classified and named Pseudomonas germanica, is deposited in the General Microbiology Center of China Microbiological 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 September 25, 2024, and the deposit number is CGMCC NO.32077. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] Unless otherwise specified, the raw materials used in the following examples are as follows:

[0028] 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.

[0029] LB solid medium (1 L): 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, 15 g of agar, dilute to 1 L with deionized water, and sterilize at 115 °C for 30 min.

[0030] Phosphate-dissolving solid culture medium (1 L): glucose 10 g, (NH4)2SO4 0.5 g, yeast extract 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, FeSO4·7H2O 0.03 g, MnSO4·4H2O 0.03 g, Ca3(PO4)2 5 g, agar powder 20 g, dilute to 1 L with deionized water, sterilize at 115°C for 30 min.

[0031] Example 1

[0032] A Pseudomonas TWS13 (Pseudomonas germanica), which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of September 25, 2024 and a deposit number of CGMCC No.32077.

[0033] Isolation, screening and identification of Pseudomonas germanica TWS13:

[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 spread it evenly on LB medium (yeast powder 5g / L, peptone 10g / L, NaCl 10g / L, agar 15g / L) plates, culture in a 10℃ incubator for 48h, pick a single bacterial colony with a sterile toothpick, streak and purify it, and save it for later use.

[0036] (2) Screening of functional strains

[0037] The strain stored at 4°C was inoculated into LB liquid medium and cultured overnight at 30°C and 160r / min. The cultured bacterial solution was diluted and applied to the phosphate-dissolving solid medium and cultured upside down in a 30°C incubator. After 7 days of culture, colonies with phosphate-dissolving rings and different morphologies were selected. After testing, strain TWS13 has the ability to dissolve inorganic phosphorus ( Figure 2 ).

[0038] (3) Strain identification

[0039] After culturing strain TWS13 on LB solid medium at 30°C for 48h, its colonies were smooth, beige, shiny, round, and 1-3mm in diameter. The results of phylogenetic tree comparison analysis constructed by 16S rRNA gene sequence showed that strain TWS13 had the highest homology with Pseudomonas germanica ( Figure 3 ), combined with the results of phylogenetic tree comparison analysis constructed with colony morphology and 16S rRNA gene sequences, strain TWS13 was identified as Pseudomonas germanica.

[0040] The Pseudomonas TWS13 cells are prepared by Pseudomonas germanica. The preparation method of Pseudomonas TWS13 cells is as follows: Pseudomonas TWS13 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 TWS13 cells are obtained by centrifugation.

[0041] The bacterial suspension, i.e., the microbial agent, is prepared by resuspending the Pseudomonas TWS13 bacterial cells with sterile water, and the effective viable bacterial count is ≥ 0.2×10 8 cfu / ml.

[0042] 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.

[0043] The microbial agent is used to promote rapeseed growth and increase the effective phosphorus content in the soil at 4°C, and its application scenario is rice-rapeseed rotation. The agent is applied by root irrigation and / or directly sprayed and / or flushed onto the plants / leaves and / or soil.

[0044] The application of the Pseudomonas TWS13 and / or the Pseudomonas TWS13 cells and / or the microbial agent and / or the microbial fertilizer in promoting rapeseed growth, dissolving inorganic phosphorus and antagonizing rice sheath blight pathogens at 4°C is in rice-oil rotation.

[0045] Example 2

[0046] The contents of this embodiment are basically the same as those of embodiment 1, except that the method for preparing Pseudomonas TWS13 cells is prepared by using Pseudomonas germanica.

[0047] The Pseudomonas TWS13 cells are prepared by Pseudomonas Germanica. The preparation method of Pseudomonas TWS13 cells is as follows: Pseudomonas TWS13 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 TWS13 cells are obtained by centrifugation.

[0048] The bacterial suspension, i.e., the microbial agent, is prepared by resuspending the Pseudomonas TWS13 bacterial cells with sterile water, and the effective viable bacterial count is ≥ 0.2×10 8 cfu / ml.

[0049] 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.

[0050] The microbial agent is used to promote rapeseed growth and increase the effective phosphorus content in the soil at 20° C., and its application scenario is rice-rapeseed rotation. The agent is applied by root irrigation and / or directly sprayed and / or flushed onto the plants / leaves and / or soil.

[0051] The application of the Pseudomonas TWS13 and / or the Pseudomonas TWS13 cells and / or the microbial agent and / or the microbial fertilizer in promoting rapeseed growth, dissolving inorganic phosphorus and antagonizing rice sheath blight pathogens at 21° C. is in rice-oil rotation.

[0052] Example 3

[0053] The contents of this embodiment are basically the same as those of embodiment 1, except that the method for preparing Pseudomonas TWS13 cells is prepared by using Pseudomonas germanica.

[0054] The Pseudomonas TWS13 cells are prepared by Pseudomonas Germanica. The preparation method of Pseudomonas TWS13 cells is as follows: Pseudomonas TWS13 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 TWS13 cells are obtained by centrifugation.

[0055] The bacterial suspension, i.e., the microbial agent, is prepared by resuspending the Pseudomonas TWS13 bacterial cells with sterile water, and the effective viable bacterial count is ≥ 0.2×10 8 cfu / ml.

[0056] 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.

[0057] The microbial agent is used to promote rapeseed growth and increase the effective phosphorus content in the soil at 35° C., and its application scenario is rice-rapeseed rotation. The agent is applied by root irrigation and / or directly sprayed and / or flushed onto the plants / leaves and / or soil.

[0058] The application of the Pseudomonas TWS13 and / or the Pseudomonas TWS13 cells and / or the microbial agent and / or the microbial fertilizer in promoting rapeseed growth, dissolving inorganic phosphorus and antagonizing rice sheath blight pathogens at 35° C. is in rice-oil rotation.

[0059] The following is an experiment on the effect of microbial agents on increasing the effective phosphorus content in the soil and promoting the growth of rapeseed:

[0060] 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.

[0061] 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.

[0062] The rapeseed seedlings transplanted into plastic pots were set up in T1 (control treatment, clean water), T2 (commercially available Bacillus megaterium solid inoculant diluted with sterile water to an effective viable count of 1.0×10 8 cfu / ml), T3 (TWS13 bacterial agent prepared in Example 2), and 5 replicates were set for each treatment. After the transplanted rapeseed seedlings grew stably for one week, the bacterial agent was irrigated with water to irrigate 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 5 ), rapeseed aboveground fresh weight ( Figure 6 ), soil available phosphorus content ( Figure 7 ), and compare the rapeseed growth conditions and soil nutrient changes of each treatment.

[0063] Experiments have shown that adding microbial agents can increase the effective phosphorus content in the soil and promote rapeseed growth. The effect of applying microbial agents prepared by Pseudomonas germanica TWS13 is better.

[0064] The following is the identification of the antagonistic function of Pseudomonas germanica TWS13 against rice sheath blight pathogen:

[0065] Use a sterile puncher with a diameter of 2 mm to take a mycelium block of rice sheath blight pathogen and place it in the center of the LB solid culture medium plate, and place it in a 28°C incubator for culture. After 1 day, inoculate TWS13 at 4 points 2 cm away from the center of the pathogen, and place it in a 28°C incubator for 5-7 days to observe the antibacterial effect of the test strain. Figure 4 As shown, the results showed that strain TWS13 had a significant antagonistic effect against rice sheath blight pathogen.

[0066] Combined with the comprehensive analysis of the above test results, the strain TWS13 can effectively promote the growth of rapeseed, and has the function of solubilizing phosphate and antagonizing rice sheath blight pathogen.

[0067] 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. TWS13, characterized in that: The Pseudomonas TWS13 (Pseudomonas germanica) is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of September 25, 2024 and a deposit number of CGMCC No.32077.

2. The Pseudomonas TWS13 cell prepared according to claim 1.

3. The method for preparing Pseudomonas sp. TWS13 according to claim 2, characterized in that: Pseudomonas TWS13 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 TWS13 cells were obtained by centrifugation.

4. A microbial agent containing the Pseudomonas TWS13 bacterial cell according to claim 2 or 3, characterized in that: The Pseudomonas TWS13 bacteria 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.

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 rapeseed growth at 4-35°C and increasing the effective phosphorus content in the soil.

7. Use of the Pseudomonas TWS13 according to claim 1 and / or the Pseudomonas TWS13 cells according to claim 2 and / or the microbial agent according to claim 4 and / or the microbial fertilizer according to claim 5 in promoting rapeseed growth, dissolving inorganic phosphorus and antagonizing rice sheath blight pathogens under 4-35°C.

8. The application method of Pseudomonas sp. TWS13 is characterized by , including root irrigation and / or direct spraying and / or flushing onto the plants / leaves and / or soil with the Pseudomonas TWS13 described in claim 1 and / or the Pseudomonas TWS13 cells described in claim 2 and / or the microbial agent described in claim 4 and / or the microbial fertilizer described in claim 5.

9. The use according to claim 6 or 7, characterized in that: The application scenario is rice-oil rotation.

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