Lelliottia jeotgali and application thereof in saline-alkali soil improvement

Abstract

The invention relates to a salty seafood Lelliottijeotgali and application thereof in saline-alkali soil improvement, and further provides a soil conditioner, and the salty seafood Lelliottijeotgali has an obvious regulation effect on saline-alkali soil improvement and plant growth promotion.

Description

Technical Field

[0001] The invention belongs to the field of microbial engineering, and particularly relates to a saltwater seafood Lelliottia jeotgali and application thereof in improving saline-alkali land. Background Art

[0002] China has about 99.13 million hectares of saline-alkali land, accounting for 10% of the world's total saline-alkali land area, making China one of the countries with the most serious damage from salinization of cultivated land. In particular, in the oases and irrigation areas in the arid and semi-arid regions of the west, the problem of soil salinization is particularly prominent, seriously affecting agricultural production and ecological balance in these areas. Taking Inner Mongolia as an example, its salinized land is mainly concentrated in Bayannur City, Hohhot City, Baotou City, Ordos City in the western Hetao Irrigation Area, and Tongliao City and Chifeng City in the east. As of 2010, the area of ​​salinized land in Inner Mongolia has reached 3.16 million hectares, of which the salinized area of ​​cultivated land has reached more than 470,000 hectares, accounting for 40% of the irrigable area. What is more worrying is that the area of ​​secondary salinization of cultivated land is still increasing at a rate of 10,000 to 15,000 hectares per year. This phenomenon has aggravated the contradiction between people and land and posed a serious challenge to the sustainable development of local agriculture.

[0003] Irrational application of chemical phosphate fertilizers and soil salinization are the main causes of this problem. Long-term excessive use of chemical phosphate fertilizers has not only failed to effectively improve soil fertility, but has aggravated the degree of soil salinization, reduced soil biological activity and nutrient utilization. Therefore, it is particularly urgent to find a method that can both improve saline-alkali soil and increase crop yields. The application of salt-alkali tolerant phosphate-dissolving microorganisms came into being in this context. They reduce soil pH and increase the effective phosphorus content through metabolic acid-dissolving mechanisms, thereby improving the saline-alkali soil environment and promoting plant growth. In addition, these microorganisms can also work synergistically with other beneficial microorganisms to further enhance soil fertility and crop resistance.

[0004] However, although phosphate-solubilizing microorganisms exist in large quantities in the rhizosphere soil of plants, they have not yet been fully applied in agriculture as biological agents to improve saline-alkali soil or fix phosphorus in soil. The main reason is that the phosphate-solubilizing microorganisms currently screened out generally have low salt-alkali tolerance and are difficult to achieve optimal results in extreme environments. Therefore, it is urgent to strengthen research in enzymology, molecular biology, and colony ecology to screen out more efficient salt-alkali-tolerant phosphate-solubilizing microorganisms and optimize their performance through genetic engineering to meet the needs of practical applications. The current situation is that the application of phosphate-solubilizing microorganisms still faces many challenges, such as insufficient salt-alkali tolerance and lack of comprehensive agricultural applications. Therefore, it is particularly urgent to discover new soil-improving microorganisms at this stage. We need to increase investment in scientific research, strengthen the screening and optimization of phosphate-solubilizing microorganisms, and promote their widespread application in agricultural production to achieve the unity of economic, environmental, and ecological benefits, and make greater contributions to solving saline-alkali land problems and ensuring food security. Summary of the invention

[0005] In view of this situation, the inventors of the present invention have conducted in-depth research and successfully screened out a new type of salt-alkali tolerant phosphate-solubilizing microorganism, Lelliottia jeotgali, using genetic engineering technology. The strain was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 6, 2024, with a deposit number of CGMCC No.32940. Experimental verification shows that this bacterium has excellent salt-alkali tolerance and phosphate-solubilizing function, and exhibits a significant growth-promoting effect in a saline-alkali environment. It can not only effectively dissolve insoluble phosphorus, improve the effectiveness of phosphorus in the soil, improve the saline-alkali soil environment, and promote crop growth.

[0006] Another aspect of the present invention provides a soil conditioner, characterized in that it contains the Lelliottia jeotgali according to claim 1. The bacterial agent contained in the bacterial agent is solid, liquid or semi-solid, and the bacterial agent also includes an optional carrier and / or auxiliary material, and the optional carrier and / or auxiliary material is selected from at least one of a protective agent, an excipient, a binder, a disintegrant, a lubricant, a fragrance, a preservative, a stabilizer, a suspending agent, a dispersant and a diluent.

[0007] Another aspect of the present invention also provides the use of the above-mentioned saltwater seafood Lelliottia jeotgali in the improvement of saline-alkali land.

[0008] In one embodiment of the present invention, the application amount of the microbial agent to saline-alkali land is 5-40 kg / mu, and each gram of the microbial agent contains 50-500 million bacteria. A typical application amount is, for example, 10 kg / mu, and each gram of the microbial agent contains 200 million bacteria.

[0009] Another aspect of the present invention provides a plant growth promoter, characterized in that it comprises the Lelliottia jeotgali bacteria according to claim 1.

[0010] Another aspect of the present invention provides a soil improvement method, characterized in that the bacterial agent containing the saltwater seafood Lelliottia jeotgali according to claim 1 is applied to the land.

[0011] In one embodiment of the present invention, the application amount of the improved method of the present invention to the land is 5-40 kg / mu of microbial agent, and the bacterial content per gram of microbial agent is 0.5-500 million. The typical application amount is, for example, 10 kg / mu, and the bacterial content per gram of microbial agent is 200 million.

[0012] Compared with the prior art, the present invention has the following beneficial effects: from the perspective of biological improvement, the present invention performs functional evaluation and systematic identification of isolated halophilic microorganisms, growth-promoting microorganisms, and phosphate-solubilizing microorganisms; and organically combines them with organic fertilizers, and uses corn as a test crop in potted plants, in order to provide microbial agent products adapted to local conditions for the biological improvement of saline-alkali land. The more specific advantages of the present invention can be summarized as follows:

[0013] (1) Based on the above technical background, the present invention aims to provide a highly efficient saltwater seafood Lailiaote bacteria for reducing salinity and alkaline soil. In view of the above technical purpose, the present invention isolated a saltwater seafood Lailiaote bacteria from a saline-alkali soil sample in Inner Mongolia. It has been verified that the strain has the functions of solubilizing phosphate and promoting growth.

[0014] (2) The above strains are non-pathogenic bacteria and have a significant growth-promoting effect on corn.

[0015] (3) The present invention uses corn potted plants as an example to verify the effect of the strain on improving saline-alkali land. The verification results show that the above-mentioned bacterial agent can effectively increase crop yields in field experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a colony photo of the T4R87 strain of the present invention;

[0017] Figure 2 This is a hemolytic experiment of the T4R87 strain of the present invention. DETAILED DESCRIPTION

[0018] The exemplary embodiments, features and aspects of the present invention will be described in detail below based on specific examples, but the examples are only examples and do not limit the technical scope of the technical solution of the present invention.

[0019] Example 1

[0020] 1. Screening of strains for improving saline-alkali land

[0021] (1) Culture medium:

[0022] LB medium: NaCl 10g / L, peptone 10.0g / L, yeast extract 5.0g / L, agar 20.0g / L, water 1000mL; pH 7.2-7.4. Sterilization conditions 121℃, 20min.

[0023] Gao's medium No. 1: soluble starch 20.0g, KNO3 1.0g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, agar 20.0g, water 1000mL; pH 7.2-7.4. Sterilization conditions 121℃, 20min.

[0024] PDA medium: 200.0 g potato, 20.0 g sucrose, 20.0 g agar, 1000 mL water; pH 7.2-7.4. Sterilization conditions: 121°C, 20 min.

[0025] Halophilic bacteria medium (moderate): NaCl 100.0 g / L, NaHCO3 0.06 g / L, MgSO4·7H2O 1.0 g / L, KCl 2.0 g / L, CaCl2·2H2O 0.36 g / L, peptone 5.0 g / L, yeast extract 10.0 g / L, glucose 1.0 g / L, trace FeCl3, agar 20.0 g / L, water 1000 mL; pH 7.2-7.4. Sterilization conditions 115°C, 30 min.

[0026] NBRIP solid culture medium: MgCl2·6H2O 0.5g, MgSO4·7H2O 0.25g, Ca3(PO4)2 (or hydroxyapatite) 5g, (NH4)2SO4 0.1g, KCl 0.2g, glucose 10g, agar 20g, pH 7.0-7.5, sterilization at 115℃ for 30min.

[0027] (2) Sample collection

[0028] The soil drilling method was used to drill the 0-20 cm tillage layer of soil. Five points were randomly selected from each plot using an S-type drill. Five drilled soils were taken from each point, repeated three times, mixed and put into a ziplock bag. The retrieved soil needed to be sieved with a 2 mm sieve, put into a 50 ml centrifuge tube and stored in a -4 ° C incubator.

[0029] The soil around the roots was randomly selected and the crops of the current crop were dug out with roots. The soil attached to the plant roots was removed as much as possible, and then the plant roots were removed and placed in a 50 ml centrifuge tube filled with 0.85% sodium chloride solution (0.85 g / 100 ml water) and stored at low temperature.

[0030] (3) Separation and purification

[0031] Samples of different salinity in Inner Mongolia were collected from four locations: Runsheng Lake in Hangjinhou Banner, Bayannur City, Inner Mongolia Autonomous Region, the courtyard of the Academy of Agricultural Sciences of Bameng, Harimangnaoer Salt Lake in Xini Town, Ordos City, and Laodong Village in Tuoketuo County, and four types of arable soils: white saline soil, horse urine saline soil, horse urine saline soil, and salinized irrigation and silt soil. 10 ml of fresh samples were added to 100 ml of 5% sodium chloride LB liquid culture medium, and cultured at 37°C and 120 rpm for 2 days. 1 ml of bacterial solution was taken for gradient dilution, and 10 -4 , 10 -5 , 10 -6 0.1 ml of bacterial solution was inoculated onto LB solid selective medium plates, and each gradient was repeated three times, and inverted culture was carried out at 30°C for 2 days. A single colony was selected and repeatedly streaked for purification until no foreign bacteria were observed under a microscope to obtain a pure strain.

[0032] Results: A total of 116 bacterial strains were isolated.

[0033] (4) Screening of phosphate-solubilizing bacteria

[0034] Taking 116 strains as the routine, (1) the soil samples obtained from the field were placed in dry ice for storage and then stored in an ultra-low temperature freezer (-80°C) after being brought back.

[0035] (2) Take the soil out of the ultra-low temperature freezer (-80°C), then weigh 10 g of soil and put it into 90 mL of sterile water. Oscillate it at 180 rpm in a constant temperature oscillator at 37°C for 30 min.

[0036] (3) Pipette 1 mL of the original soil solution into a large test tube containing 9 mL of sterile water and mix thoroughly. Then, pipette 1 mL from this large test tube and add it into another test tube containing 9 mL of sterile water and mix thoroughly. Repeat this process to make 10 -1 -10 -6 For different gradient dilutions, pipette 100 μL of 10 -4 , 10 -5 , 10 -6 The dilutions were spread on LB plates, and each gradient was spread on two plate culture media, and cultured upside down in a 37°C constant temperature incubator for 2 days.

[0037] (4) Streak different single colonies onto LB plate culture medium according to their size, color, edge and other characteristics and incubate them upside down in a 37°C constant temperature incubator for 24 h.

[0038] (5) The isolated and purified cells were re-inoculated into NBRIP solid culture medium, and their growth was observed. The cells were incubated inverted at 37°C for 7 days.

[0039] (6) After 7 days of cultivation, the diameter of the phosphate solubilization zone (D) and the diameter of the bacterial colony (d) were observed and the ratio of the diameter of the phosphate solubilization zone to the diameter of the bacterial colony was calculated (when measuring the diameter with a vernier caliper, each circle was measured three times and the average value was calculated).

[0040] (7) Storage: Slant storage: Add 5 mL of LB solid culture medium to each test tube to make a slant. After solidification, streak the slant with purified bacteria. After the colonies grow at 37°C, replace the stopper with a rubber stopper and seal at 4°C. Activate once every 2-3 months.

[0041] Results: A total of 9 strains with high phosphate-solubilizing ability were obtained.

[0042] The phosphate solubilization performance of 9 strains was tested, and the results are as follows:

[0043] Table 1 Phosphate solubilization results of 9 bacterial strains

[0044]

[0045]

[0046] After initial screening, 9 strains among the 116 bacterial strains had phosphate solubilization contents higher than 200 μg / ml, so these 9 strains were used for subsequent experiments.

[0047] (4) Screening of growth-promoting bacteria

[0048] Corn seeds were soaked in 55-60℃ water for 20 minutes, the bacteria to be tested were cultured to OD600=3.0, and then the corn seeds were placed in a 50-fold diluted bacterial suspension and soaked for 6 hours. The soaked seeds were placed in a sterile culture dish covered with 2 layers of filter paper, 10 seeds were placed in each culture dish, and 5 ml of sterile water was added. Each treatment was repeated 3 times, and the seeds were soaked in LB culture medium with the same dilution concentration as a control, and placed in a 28℃ incubator. 2 ml of sterile water was added every day during the culture period, and the culture was cultured for 3 days.

[0049] Germination index = ∑ number of germinations at different times / number of germination days

[0050] Vitality index = germination index × length or mass of seedlings or young roots within a specified date

[0051] Germination index = ∑ number of germinations at different times / number of germination days.

[0052] Vitality index = germination index × length or mass of seedlings or young roots within the specified date.

[0053] After germination, corn seeds were sown in plug trays and transplanted into small pots when the second true leaf grew. When the seedlings grew to 4 leaves, they were watered with a 50-fold dilution of the test strain suspension (D600=3). The seedlings were watered once every 7 days, and the LB medium with the same concentration was used as a control. During the experiment, watering was carried out to keep the soil moisture of each treatment consistent. There were 3 treatments, 8 plants in each treatment, and watering was carried out twice. The stem diameter, plant height and fresh weight of the corn seedlings were measured.

[0054] Table 2 Results of corn seed germination

[0055]

[0056]

[0057] The strain T4R87 with the best germination and growth promotion effect was used for potted plant experiments. The results are shown in Table 3

[0058]

[0059] Table 3

[0060] Conclusion: T4R87 strain has obvious growth-promoting effect.

[0061] 3. Identification of strains

[0062] (1) Bacterial morphological characteristics: After culturing on LB plates at a constant temperature (37°C) for 24 h, the T4R87 strain was round, light yellow, opaque, and had smooth edges. Figure 1 As shown;

[0063] (2) Molecular identification: The fermentation broth of strain T4R87 was sent to Shanghai Shenggong Biotechnology Co., Ltd. for 16sRNA sequencing of the bacteria. The strain T4R87 was identified as Lelliottia jeotgali from salted seafood by BLAST comparison in NCBI.

[0064] Example 2 Strain Safety Evaluation

[0065] Hemolytic test: The selected microbial strains with the best phosphate-solubilizing and growth-promoting effects were streaked on blood agar plates, and Bacillus cereus was used as a positive control. The plates were cultured in a constant temperature incubator at 37°C for 24 hours, and whether transparent or translucent hemolytic rings appeared around the microbial strains was observed to determine whether the strains were hemolytic. The non-hemolytic strains were selected for the next test.

[0066] like Figure 2 No lytic ring appeared after the strain was cultured, proving that the strain was non-hemolytic and had high safety during production and use.

[0067] Example 3 Evaluation of the effect of strain application - field experiment

[0068] Test location: Zhonghexi Town, Dalate Banner, Ordos City

[0069] Test crop: Sunflower

[0070] Test treatment:

[0071] CK: blank control

[0072] T1: conventional fertilizer + soil conditioner No. 1 (bacterial agent 10 kg / mu, bacterial content 200 million / g)

[0073] T2: conventional fertilizers but no soil conditioners

[0074] Experimental plan: Each treatment was repeated 3 times, the experimental plot was 18m long, 6m wide and 108m in area. 2 The interval between plots was 1m, and 500 kg of soil conditioner was applied as base fertilizer per mu for field trials.

[0075] The average yield of all treatments was 4694.78 kg / hm2 for T1 treatment 2 , T2 without treatment was 4442.78kg / hm 2 , blank control: 2993.27kg / hm 2 The sunflower yield in the treatment with microbial agents was the highest, increasing by 5.67% compared with the treatment without microbial agents. Field data showed that the application of soil conditioners with microbial agents could affect the yield of sunflowers, and could achieve a small increase or stable yield compared with conventional fertilization.

[0076] Example 4 Evaluation of the effect of strain application - field experiment

[0077] Test location: Wuyuan County, Linhe City

[0078] Test crop: Oats

[0079] Test treatment:

[0080] CK: blank control

[0081] T1: conventional fertilizer + soil conditioner No. 1 (bacterial agent 10 kg / mu, bacterial content 200 million / g)

[0082] T2: conventional fertilizers but no soil conditioners

[0083] Experimental plan: Each treatment was repeated 3 times, the experimental plot was 18m long, 6m wide and 108m in area. 2 The interval between plots was 1m, and 500 kg of soil conditioner was applied as base fertilizer per mu for field trials.

[0084] The oat grass yield in T1 treatment was 9157.5 kg / hm2, and that in T2 treatment without application was 7180.31 kg / hm2. 2 , blank treatment: 4828.5kg / hm 2 The oat grass yield of T1 treatment increased by 27.53% compared with the treatment without microbial agent. The field data showed that the application of microbial agent soil conditioner can increase the oat grass yield.

[0085] Example 5 Evaluation of the effect of strain application - field experiment

[0086] Test location: Kezuozhong Banner, Tongliao City

[0087] Test crop: corn

[0088] Test treatment:

[0089] CK: blank control

[0090] T1: conventional fertilizer + soil conditioner No. 1 (bacterial agent 10 kg / mu, bacterial content 200 million / g)

[0091] T2: conventional fertilizers but no soil conditioners

[0092] Experimental plan: Each treatment was repeated 3 times, the experimental plot was 18m long, 6m wide and 108m in area. 2 The interval between plots was 1m, and 500 kg of soil conditioner was applied as base fertilizer per mu for field trials.

[0093] The average yield of corn in all soil conditioners was increased, with the average yields of T19873.45kg / hm2 and T29873.45kg / hm2, respectively. 2 , T2 without treatment was 7652.45kg / hm 2 , blank control: 7104.00kg / hm 2 The corn yield of T1 treatment increased by 16.18% compared with the treatment without microbial agent. Field data showed that the application of soil conditioner with microbial agent can increase corn yield to a certain extent.

[0094] The discovery and application of Lelliottia jeotgali from salted seafood of the present invention provides new ideas and technical support for solving the saline-alkali land problem in China and even the world. This microorganism optimized by genetic engineering has excellent salt-alkali tolerance and phosphorus solubility performance, can survive and function in extreme environments, and significantly improve soil fertility and crop yields.

[0095] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A salty seafood Lelliottia jeotgali, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 6, 2024, with the deposit number CGMCC No.32940.

2. A soil conditioner, characterized in that: It comprises the bacterial agent of Lelliottia jeotgali according to claim 1.

3. The soil conditioner according to claim 2, characterized in that The Lelliottiajeotgali bacterial agent is solid, liquid or semisolid, and the bacterial agent also includes optional carriers and / or auxiliary materials, and the optional carriers and / or auxiliary materials are selected from at least one of protective agents, excipients, adhesives, disintegrants, lubricants, fragrances, preservatives, stabilizers, suspending agents, dispersants and diluents.

4. Application of the saltwater seafood Lelliottiajeotgali according to claim 1 in improving saline-alkali land.

5. The use according to claim 3, characterized in that: The application amount for saline-alkali land is 5 to 40 kg / mu of bacterial agent, and the bacterial content per gram of bacterial agent is 50 to 500 million.

6. A plant growth promoter, characterized in that: It comprises the Lelliottia jeotgali bacteria according to claim 1.

7. A soil improvement method, characterized in that: The bacterial agent containing the saltwater seafood Lelliottia jeotgali of claim 1 is applied to the land.

8. The method according to claim 7, characterized in that The application amount for land is 5 to 40 kg / mu of fungal agent, and the bacterial content per gram of fungal agent is 50 to 500 million.

9. The method according to claim 7, characterized in that: The inoculum of Lelliottia jeotgali from salted seafood was activated and cultured at 30±0.5℃ before being applied to the land.

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