Pseudomonas capable of dissolving phosphorus and promoting growth under low-temperature condition and application of pseudomonas

Abstract

The invention relates to pseudomonas capable of solubilizing phosphorus and promoting growth under a low-temperature condition. Aiming at the problems of large temperature change amplitude and long low-temperature period in the growth period of coldness-loving crops such as oilseed rape and low effectiveness of conventional functional microorganisms under the low-temperature condition, the pseudomonas TWS23 which has the functions of dissolving inorganic phosphorus and promoting growth under the low-temperature condition is screened out, and the strain TWS23 can grow at 4-35 DEG C, is wide in temperature application range, and can be applied to the field of agricultural production. The organic phosphorus can be dissolved, the auxin indoleacetic acid is generated, and efficient planting of crops such as oilseed rape is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a pseudomonas capable of dissolving phosphorus and promoting growth under low temperature conditions and application thereof. Background Art

[0002] Rapeseed is the oil crop with the largest planting area and the widest regional distribution in my country. Rapeseed is a cool-loving crop that does not require much heat or soil. It can be divided into spring and winter rapeseed according to different sowing periods. The dividing line between the two major rapeseed areas of winter and spring is: starting from Shanhaiguan in the east, passing through the Great Wall and going south along the Taihang Mountains, from Wutai Mountain across the Yellow River to the eastern foot of the Helan Mountains, then passing through Liupan Mountain, through the upper reaches of the Bailong River to the lower reaches of the Yarlung Zangbo River. The area to the south and east is the winter rapeseed area, and the area to the north and west is the spring rapeseed area.

[0003] Rapeseed is a cool-loving crop. The temperature during its growth period fluctuates greatly, and the low-temperature period is long. The functional microorganisms required for its growth decrease in activity during the low-temperature season, and their functionality is greatly reduced. Pseudomonas is a common microorganism in high-altitude cold soils. Its growth temperature range is wide, which is more consistent with the temperature during the growth period of rapeseed, and it can play a role in most of the growth period of rapeseed. Most of the phosphate-solubilizing and growth-promoting microorganisms reported so far were screened under normal temperature conditions, and there are few for low-temperature crops. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention discloses a Pseudomonas that can solubilize phosphate and promote growth under low temperature conditions, named Pseudomonas rustica TWS23, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection on October 11, 2024, with the deposit number CGMCC No.32078.

[0005] A microbial agent can be prepared by using the Pseudomonas rustica TWS23.

[0006] A method for preparing a microbial agent comprises the following steps: Step 1, inoculating the Pseudomonas rustica TWS23 into LB liquid culture medium for liquid fermentation, the liquid fermentation conditions are: fermentation temperature of 28-30°C, rotation speed of 170-190r / min, fermentation time of 24-48h, and centrifuging to obtain Pseudomonas rustica TWS23 cells after the fermentation is completed; Step 2: resuspend the Pseudomonas rustica TWS23 bacteria in sterile water to prepare a bacterial suspension, which is a microbial agent.

[0007] As a preferred embodiment of the present invention, the effective viable count of the microbial agent is ≥ 0.2×10 8 cfu / ml.

[0008] As a preferred embodiment of the present invention, the microbial agent is prepared by adsorption, mixing, solid fermentation and the like to obtain a solid microbial agent, and the solid microbial agent can be used as a microbial organic fertilizer, that is, a bacterial fertilizer.

[0009] The above-mentioned Pseudomonas rustica TWS23 or microbial agent can promote the growth of cool-loving crops at 4-35°C, and can be used as a fertilizer for cool-loving crops such as rapeseed. By irrigating the roots or directly spraying Pseudomonas rustica TWS23 or microbial agent on the plants, leaves or soil, the growth of crops can be effectively promoted.

[0010] The above-mentioned Pseudomonas rustica TWS23 or microbial agent can dissolve inorganic phosphorus at 4-35°C and can be used to dissolve inorganic phosphorus.

[0011] The above-mentioned Pseudomonas rustica TWS23 or microbial agent can be used in the preparation of plant growth hormone indoleacetic acid (IAA).

[0012] Beneficial effects of the present invention: The present invention aims at the problem that the temperature of cool-loving crops such as rapeseed has a large fluctuation range during its growth period and a long low-temperature period, and conventional functional microorganisms have low effectiveness under low-temperature conditions. A Pseudomonas TWS23 with the function of dissolving inorganic phosphorus and promoting growth under low-temperature conditions is screened out. The strain TWS23 can grow at 4-35°C, has a wide temperature adaptability range, can dissolve organic phosphorus, and produce indoleacetic acid, a plant growth hormone, which is conducive to the efficient cultivation of crops such as rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The colony morphology of Pseudomonas sp. TWS23 on LB medium of the present invention;

[0014] Figure 2 This is a diagram showing the phosphorus solubilization effect of Pseudomonas sp. TWS23 of the present invention;

[0015] Figure 3 is a 16S rRNA phylogenetic tree of Pseudomonas sp. TWS23 of the present invention;

[0016] Figure 4 This is a diagram showing the effect of the IAA color development reaction of the Pseudomonas TWS23 bacteria of the present invention;

[0017] Figure 5 A statistical diagram of rapeseed plant height in an embodiment of the present invention;

[0018] Figure 6 This is a statistical diagram of the fresh weight of the aerial part of rapeseed in the embodiment of the present invention;

[0019] Figure 7 It is a statistical diagram of soil available phosphorus content in an embodiment of the present invention. DETAILED DESCRIPTION

[0020] Example 1

[0021] The present invention discloses a Pseudomonas that can dissolve phosphate and promote growth under low temperature conditions, named Pseudomonas rustica TWS23, which was deposited in the General Microbiology Center of China Culture Collection Committee on October 11, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 32078.

[0022] Isolation, screening and identification of Pseudomonas rustica TWS23 strain:

[0023] (1) Isolation of strains

[0024] Soil samples were collected from the high-cold 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.

[0025] (2) Screening of functional strains

[0026] 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 to obtain strain TWS23. After testing, strain TWS23 has the ability to dissolve inorganic phosphorus ( Figure 2 ).

[0027] 50ml LB plus tryptophan (concentration of 200mg / L) medium, inoculate 0.5ml seed liquid, 30℃, 180rpm shaker culture for 48h, 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 in a volume ratio of 50:1 and mix well), and add 2ml uninoculated LB liquid medium and an equal volume of colorimetric solution as a control. Those with pink color indicate that they can secrete indoleacetic acid IAA. After testing, strain TWS23 has the ability to produce indoleacetic acid IAA ( Figure 4 ).

[0028] (3) Strain identification

[0029] After culturing strain TWS23 on LB solid medium at 30°C for 48h, its colonies were smooth, beige, shiny, round, and 1-2mm in diameter. The results of phylogenetic tree comparison analysis constructed by 16S rRNA gene sequence showed that strain TWS23 had the highest homology with Pseudomonas rustica ( Figure 3 ), combined with the results of phylogenetic tree comparison analysis constructed with colony morphology and 16S rRNA gene sequences, strain TWS23 was identified as Pseudomonas rustica.

[0030] A microbial agent can be prepared by using the Pseudomonas rustica TWS23, and the preparation method thereof comprises the following steps: Step 1, inoculating the Pseudomonas rustica TWS23 into LB liquid culture medium for liquid fermentation, the liquid fermentation conditions are: fermentation temperature of 28° C., rotation speed of 170 r / min, fermentation time of 24 h, and centrifuging after the fermentation to obtain Pseudomonas rustica TWS23 cells; Step 2: resuspend the Pseudomonas rustica TWS23 cells in sterile water to obtain a bacterial suspension, which is a microbial agent; the effective viable count of the microbial agent is ≥ 0.2 × 10 8 cfu / ml.

[0031] The microbial agent is prepared by adsorption, mixing, solid fermentation and the like to obtain a solid microbial agent, and the solid microbial agent can be used as a microbial organic fertilizer, that is, a bacterial fertilizer.

[0032] Example 2

[0033] The content of this embodiment is basically the same as that of embodiment 1, except that the method for preparing the microbial agent by using the Pseudomonas rustica TWS23 comprises the following steps: Step 1, inoculating the Pseudomonas rustica TWS23 into LB liquid culture medium for liquid fermentation, the liquid fermentation conditions are: fermentation temperature of 30° C., rotation speed of 190 r / min, fermentation time of 48 h, and centrifugation after the fermentation to obtain Pseudomonas rustica TWS23 cells; Step 2: resuspend the Pseudomonas rustica TWS23 cells in sterile water to obtain a bacterial suspension, which is a microbial agent; the effective viable count of the microbial agent is ≥ 0.2 × 10 8 cfu / ml.

[0034] The microbial agent is prepared by adsorption, mixing, solid fermentation and the like to obtain a solid microbial agent, and the solid microbial agent can be used as a microbial organic fertilizer, that is, a bacterial fertilizer.

[0035] Example 3

[0036] The content of this embodiment is basically the same as that of embodiment 1, except that the method for preparing the microbial agent by using the Pseudomonas rustica TWS23 comprises the following steps: Step 1, inoculating the Pseudomonas rustica TWS23 into LB liquid culture medium for liquid fermentation, the liquid fermentation conditions are: fermentation temperature is 29° C., rotation speed is 180 r / min, fermentation time is 36 h, and after the fermentation is completed, centrifugation is performed to obtain Pseudomonas rustica TWS23 cells; Step 2: resuspend the Pseudomonas rustica TWS23 cells in sterile water to obtain a bacterial suspension, which is a microbial agent; the effective viable count of the microbial agent is ≥ 0.2 × 10 8 cfu / ml.

[0037] The microbial agent is prepared by adsorption, mixing, solid fermentation and the like to obtain a solid microbial agent, and the solid microbial agent can be used as a microbial organic fertilizer, that is, a bacterial fertilizer.

[0038] The culture medium used in the examples is specifically LB liquid culture medium (1 L) containing 10 g of tryptone, 5 g of yeast powder, and 10 g of NaCl, which is fixed to 1 L with deionized water and sterilized at 115° C. for 30 min.

[0039] LB solid medium (1 L) contained 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, 15 g of agar, and was diluted to 1 L with deionized water and sterilized at 115°C for 30 min.

[0040] The phosphate-dissolving solid culture medium (1 L) contained 10 g of glucose, 0.5 g of (NH4)2SO4, 0.5 g of yeast extract, 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·4H2O, 5 g of Ca3(PO4)2, and 20 g of agar powder. The volume was made up to 1 L with deionized water and sterilized at 115°C for 30 min.

[0041] Experiment on the Growth-Promoting Effect of Microbial Agents on Rapeseed

[0042] 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.

[0043] Sow rapeseed seeds in seedling cups. When the rapeseed seedlings grow 3 true leaves, select rapeseed seedlings with relatively consistent growth and transplant them into plastic pots filled with soil. Use 6 kg of soil per pot, mix with base fertilizer, and plant one rapeseed seedling in each pot. After transplanting, water the plastic pots and water them regularly afterwards.

[0044] The rapeseed seedlings transplanted into the plastic pots were provided with T1, which was watered with clean water as a control test, T2, which was watered with a solid bacterial agent of Bacillus megaterium sold on the market and diluted with sterile water to an effective viable count of 1.0×108 cfu / ml, and T3, which was watered with a microbial agent prepared by Pseudomonas rustica TWS23. There were three groups in total, and each group had 5 replicates. After the transplanted rapeseed seedlings grew stably for one week, the agent was irrigated with water to the roots, 6 ml per pot, and routine maintenance was performed after completion. 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.

[0045] The experiment showed that adding microbial agents can increase the effective phosphorus content in the soil and promote the growth of rapeseed. The effect of applying microbial agents prepared by Pseudomonas rustica TWS23 is better.

[0046] Combined with the test results of the above examples, a comprehensive analysis shows that Pseudomonas rustica TWS23 and the microbial agent prepared therefrom can effectively promote the growth of rapeseed, and have the function of solubilizing phosphorus and the ability to produce indoleacetic acid IAA, and can be used in promoting the growth of cool crops such as rapeseed, dissolving inorganic phosphorus, and preparing indoleacetic acid IAA.

[0047] The parts not described in detail herein are prior art. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer, and experimental steps not described in detail are referred to the prior art database.

[0048] 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 Pseudomonas bacterium that can dissolve phosphate and promote growth under low temperature conditions, characterized in that: Named Pseudomonas rustica TWS23, it was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 11, 2024, with the deposit number CGMCC No.32078.

2. A microbial agent, characterized in that: The product is prepared from the Pseudomonas sp. TWS23.

3. A microbial agent according to claim 2, characterized in that: The preparation method comprises the following steps: Step 1, inoculating the Pseudomonas rustica TWS23 into LB liquid culture medium for liquid fermentation, the liquid fermentation conditions are: fermentation temperature of 28-30°C, rotation speed of 170-190r / min, fermentation time of 24-48h, and centrifuging to obtain Pseudomonas rustica TWS23 cells after the fermentation is completed; Step 2: resuspend the Pseudomonas rustica TWS23 bacteria in sterile water to prepare a bacterial suspension, which is a microbial agent.

4. A microbial agent according to claim 3, characterized in that: The effective viable bacteria count of the microbial agent is ≥ 0.2×10 8 cfu / ml.

5. A microbial agent according to claim 3, characterized in that: The microbial agent is prepared through adsorption, mixing and solid fermentation to obtain a solid microbial agent.

6. Use of the Pseudomonas rustica TWS23 according to claim 1 or the microbial agent according to any one of claims 2 to 5 in promoting the growth of cool-loving crops under conditions of 4-35°C.

7. Use of the Pseudomonas rustica TWS23 according to claim 1 or the microbial agent according to any one of claims 2 to 5 in dissolving inorganic phosphorus at 4-35°C.

8. Use of the Pseudomonas rustica TWS23 according to claim 1 or the microbial agent according to any one of claims 2 to 5 in the preparation of indoleacetic acid (IAA).

Images