Lelliottia jeotgali and its application in saline-alkali soil improvement

Abstract

The present application relates to a kind of salty seafood Lelliottia jeotgali and its application in saline-alkali soil improvement, also provide soil conditioner, the present application has obvious regulating effect for saline-alkali soil improvement and plant growth promotion.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial engineering, and particularly relates to a Lelliottia jeotgali and application thereof in saline-alkali soil improvement. BACKGROUND

[0002] China has about 9913 million hectares of saline-alkali land, accounting for 10% of the total area of saline-alkali land in the world, which makes China one of the countries most seriously affected by the salinization of arable land. In particular, in the oases and irrigation areas of the western arid and semi-arid regions, the problem of soil salinization is particularly prominent, which seriously affects the agricultural production and ecological balance in these regions. Take Inner Mongolia as an example, its salinized land is mainly concentrated in the western Hetao Irrigation Area of Baicheng City, Hohhot City, Baotou City, Ordos City, and the eastern Tongliao City, Chifeng City, etc. As of 2010, the area of salinized land in Inner Mongolia reached 3.16 million hectares, of which the area of salinized arable land reached more than 470,000 hectares, accounting for 40% of the irrigable area. More worrying is that the area of secondary salinized arable land is still increasing by 10,000 to 15,000 hectares per year, which exacerbates the contradiction between man and land and poses a serious challenge to the sustainable development of local agriculture.

[0003] Unreasonable chemical phosphorus fertilizer application and soil salinization are the main causes of this problem. Long-term overuse of chemical phosphorus fertilizer not only fails to effectively improve soil fertility, but also aggravates the degree of soil salinization and reduces the biological activity and nutrient utilization rate of the soil. Therefore, it is particularly urgent to find a method that can both improve saline-alkali soil and increase crop yield. The application of salt-tolerant and phosphorus-solubilizing microorganisms emerges as the times require in this context. They can reduce the soil pH value and increase the effective phosphorus content through the mechanism of metabolic acid production and phosphorus solubilization, thereby improving the saline-alkali soil environment and promoting plant growth. In addition, these microorganisms can also synergize with other beneficial microorganisms to further enhance the soil fertility and the stress resistance of crops.

[0004] However, despite the abundance of phosphorus-solubilizing microorganisms in the rhizosphere soil of plants, the use of these microorganisms as bio-inoculants to improve saline-alkali soil or fix phosphorus in soil has not been fully utilized in agriculture. The main reason is that the salt-tolerant ability of the currently screened phosphorus-solubilizing microorganisms is generally not high, and it is difficult for them to perform optimally in extreme environments. Therefore, it is urgent to strengthen research from the aspects of enzymology, molecular biology, and colony ecology, to screen more efficient salt-tolerant phosphorus-solubilizing microorganisms, and to optimize their performance through genetic engineering methods to meet the needs of practical application. At present, the application of phosphorus-solubilizing microorganisms still faces many challenges, such as insufficient salt-tolerant ability, lack of comprehensive agricultural application, etc. Therefore, it is particularly urgent to find new soil improvement microbial bacteria at this stage. We need to increase scientific research investment, strengthen the screening and optimization of phosphorus-solubilizing microorganisms, and promote their wide application in agricultural production, so as to realize the unity of economic, environmental and ecological benefits, and make greater contributions to solving the problem of saline-alkali land and ensuring food security. SUMMARY

[0005] In view of the above situation, the inventors of the present application have successfully screened a new salt-tolerant phosphorus-solubilizing microorganism, Lelliottia jeotgali, using genetic engineering technology. The strain was deposited at the China General Microbiological Culture Collection Center on December 06, 2024, and the deposit number is CGMCC No. 32940. Experimental verification shows that this bacterium has excellent salt-tolerant ability and phosphorus-solubilizing function, and exhibits a significant growth-promoting effect in saline-alkali environments. It not only can effectively dissolve insoluble phosphorus, improve the availability of phosphorus in soil, improve the saline-alkali soil environment, and promote crop growth.

[0006] Another aspect of the present application also provides a soil conditioner, characterized in that it comprises Lelliottia jeotgali. The bacterial inoculant comprising the bacteria is solid, liquid or semi-solid, and the bacterial inoculant further comprises optional carriers and / or auxiliary materials selected from at least one of protective agents, excipients, binders, disintegrants, lubricants, flavors, preservatives, stabilizers, suspending agents, dispersing agents and diluents.

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

[0008] In one embodiment of the present application, the application amount for saline-alkali land is 5-40 kg / mu of bacterial inoculant, and the bacterial content per gram of bacterial inoculant is 0.5-500 million. A typical application amount is, for example, 10 kg / mu, and the bacterial content per gram of bacterial inoculant is 200 million.

[0009] Another aspect of the present application also provides a plant growth promoter, characterized in that it comprises Lelliottia jeotgali bacteria.

[0010] Another aspect of the present application also provides a soil improvement method, characterized in that: a bacterial agent comprising Lelliottia jeotgali is applied to the land.

[0011] In one embodiment of the present application, the application improvement method is applied to the land at a dose of 5-40 kg / mu of bacterial agent, and the bacterial content per gram of bacterial agent is 0.5-5 billion. A typical application dose is, for example, 10 kg / mu, and the bacterial content per gram of bacterial agent is 200 million.

[0012] Compared with the prior art, the present application has the following beneficial effects: the present application evaluates and identifies the isolated halophilic microorganism, growth-promoting microorganism, and phosphorus-solubilizing microorganism from the perspective of biological improvement, and combines the microorganism with organic fertilizer. Corn is used as a test crop for potting, so as to provide a microbial agent product suitable for biological improvement of saline-alkali land. The more specific advantages of the present application can be summarized as follows:

[0013] (1) Based on the above technical background, the present application aims to provide a high-efficiency Lelliottia jeotgali salt-alkali land improvement microbial agent. In view of the above technical purpose, the present application isolates a Lelliottia jeotgali strain from a sample of saline-alkali land in Inner Mongolia. The strain has been verified to have phosphorus-solubilizing and growth-promoting functions.

[0014] (2) The above strain is a non-pathogenic bacterium and has a significant growth-promoting effect on corn.

[0015] (3) The present application takes corn potting as an example to verify the saline-alkali land improvement effect of the strain. The verification results show that the above bacterial agent can effectively increase crop yield in field experiments. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a colony photograph of the T4R87 strain of the present application;

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

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

[0019] Example 1

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

[0021] (1) Culture medium:

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

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

[0024] PDA medium: potato 200.0 g, sucrose 20.0 g, agar 20.0 g, water 1000 mL; pH 7.2-7.4. Sterilization condition 121℃, 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, FeCl3 trace amount, agar 20.0 g / L, water 1000 mL; pH 7.2-7.4. Sterilization condition 115℃, 30 min.

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

[0027] (2) Sample collection

[0028] The soil in the plough layer of 0-20 cm was collected by soil drilling method. Five points were randomly selected in each sample plot, and five soil drills were taken from each point, with three repetitions. The soil was mixed and then placed in a self-sealing bag. The returned soil was sieved through a 2 mm sieve and stored in a 50 ml centrifuge tube at -4℃.

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

[0030] (3) Isolation and purification

[0031] Respectively, collect samples from different saline-alkali blocks in Inner Mongolia (from the Hunsheng Lake in Hanggin Rear Banner of Bayannur City and the Inner Mongolia Baicheng Institute of Agriculture, the Haligemangnaoer Salt Lake in Xini Town of Ordos City, the Labor Village in Tuoketuo County, 4 types of cultivated layer soil, i.e. sticky white saline soil, sticky urine salt soil, sticky urine salt soil, and salinized irrigated alluvial soil), take 10 ml of fresh sample into 100 ml 5% sodium chloride LB liquid medium, and culture at 37℃, 120 rpm for 2 days. Take 1 ml of bacterial liquid for gradient dilution, and inoculate 10 -4 ,10 -5 ,10 -6 μl of bacterial liquid 0.1 ml into LB solid selective medium plates, repeat three times for each gradient, and culture at 30℃ for 2 days. Select single colonies for repeated streaking and purification until no impurities are observed under a microscope to obtain pure strains.

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

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

[0034] With 116 strains as the routine, (1) the soil samples taken from the field are stored in dry ice and preserved in an ultra-low temperature freezer (-80℃) after being brought back.

[0035] (2) Take the soil from the ultra-low temperature freezer (-80℃), then weigh 10 g of soil into 90 mL of sterile water, and shake at 37℃ in a constant temperature shaker for 30 min.

[0036] (3) Take 1 mL of soil stock solution and add it to a large test tube containing 9 mL of sterile water, mix well, then take 1 mL from this large test tube and add it to another test tube containing 9 mL of sterile water, mix well, and so on to prepare 10 -1 -10 -6 different gradient dilutions, and take 100 μl of 10 -4 ,10 -5 ,10 -6 dilutions and spread them on LB plates, with each gradient spread on two plate media, and culture at 37℃ in a constant temperature incubator for 2 d.

[0037] (4) According to the size, color, and edge characteristics of the colonies, different single colonies are streaked on LB plate media, and cultured at 37℃ in a constant temperature incubator for 24 h.

[0038] (5) After isolation and purification, inoculate the re-inoculated NBRIP solid medium, observe the growth, and culture at 37℃ in a constant temperature incubator for 7 d.

[0039] (6) After 7 days of culture, the diameter of the phosphorus solubilization ring (D) and the diameter of the bacterial colony (d) were observed and the ratio of the diameter of the phosphorus solubilization ring to the diameter of the bacterial colony was calculated (when the diameter was measured by a vernier caliper, the diameter was measured three times for each ring and the average value was calculated).

[0040] (7) Preservation: slope preservation: 5 mL of LB solid medium was added to each test tube to make a slope, and after solidification, the purified bacteria were streaked on the slope. After the colonies were grown at 37°C, the stopper was replaced and stored at 4°C. Activation was performed once every 2-3 months.

[0041] Results: A total of 9 strains with high phosphorus solubilization ability were obtained.

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

[0043] Table 1: Results of phosphorus solubilization determination of 9 strains

[0044]

[0045]

[0046] After preliminary screening, 9 strains with phosphorus solubilization content higher than 200 μg / ml were obtained from 116 strains, and therefore 9 strains were subjected to subsequent experiments.

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

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

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

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

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

[0052] Vigor index = germination index × length or mass of seedlings or young roots within a specified period.

[0053] Corn seeds were sowed in hole tray after germination, and transplanted to small pots when the second true leaf emerged. When the seedlings grew to 4 leaves, the seedlings were irrigated with the test strain suspension (D600 = 3) diluted 50 times, irrigated once every 7 days, and the same concentration of LB medium was used as a control. The soil moisture of each treatment was kept consistent by irrigating with clean water during the test period. There were 3 treatments, each with 8 replicates, irrigated twice, and the stem diameter, plant height, and fresh weight of corn seedlings were measured.

[0054] Table 2 Determination results of corn seed germination

[0055]

[0056]

[0057] The T4R87 strain with the best germination and growth promotion effect was used for pot experiment, and the results are shown in Table 3

[0058]

[0059] Table 3

[0060] Conclusion: The T4R87 strain has obvious growth promotion effect.

[0061] 3. Strain identification

[0062] (1) Bacterial morphological characteristics: After incubation at 37°C for 24 hours on LB plates, the T4R87 strain showed a round, light yellow, and opaque shape with smooth edges, as shown in Figure 1 .

[0063] (2) Molecular identification: The T4R87 strain fermentation broth was sent to Shanghai Shenguo Biological Engineering Co., Ltd. for 16sRNA sequence determination of bacteria. Through BLAST comparison in NCBI, the T4R87 strain is Lelliottia jeotgali.

[0064] Example 2 Safety evaluation of strain

[0065] Hemolytic test: The screened phosphorus-dissolving and growth-promoting microbial strain was streaked on blood agar plates with Bacillus cereus as a positive control, and incubated at 37°C in an incubator for 24 hours. Whether a transparent or translucent hemolytic ring appeared around the microbial strain was observed to determine whether the strain had hemolytic activity. Strains without hemolytic activity were selected for further testing.

[0066] As Figure 2 shown, no hemolytic ring appeared after the strain was cultured, indicating that the strain had no hemolytic activity and was highly safe in production and use.

[0067] Example 3 Strain Application Effect Evaluation - Field Experiment

[0068] Test site: Zhonghe Xizhen, 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 fertilizer but not soil conditioner

[0074] Test scheme: 3 repetitions for each treatment, test plot 18 m long, 6 m wide, area 108 m 2 , 1 m apart between plots, 500 kg of soil conditioner was applied per mu of base fertilizer for field test.

[0075] The average yield of all treatments was T1 4694.78 kg / hm 2 , T2 without treatment 4442.78 kg / hm 2 , blank control 2993.27 kg / hm 2 , the yield of sunflower treated with microbial inoculant was the highest, increased by 5.67% compared with the treatment without microbial inoculant. Field data showed that the application of soil conditioner with microbial inoculant could affect the yield of sunflower. Compared with conventional fertilization treatment, it could achieve small increase or stable yield.

[0076] Example 4 Strain Application Effect Evaluation - Field Experiment

[0077] Test site: Wuyuan County, Linhe City

[0078] Test crop: oat

[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 fertilizer but not soil conditioner

[0083] Test scheme: 3 repetitions for each treatment, test plot 18 m long, 6 m wide, area 108 m 2 , 1 m apart between plots, 500 kg of soil conditioner was applied per mu of base fertilizer for field test.

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

[0085] Example 5: Effect evaluation of strain application in field experiment

[0086] Test site: Keyouzhongqi in Tongliao

[0087] Test crop: corn

[0088] Test treatment:

[0089] CK: blank control

[0090] T1: conventional fertilizer + soil conditioner No. 1 (10 kg / mu of microbial agent, 2 billion / g of microbial content)

[0091] T2: conventional fertilizer but without adding the soil conditioner

[0092] Test scheme: each treatment is repeated for 3 times, the test plot is 18 m long, 6 m wide, and the area is 108 m 2 , the interval between plots is 1 m, and 500 kg of soil conditioner is applied as base fertilizer per mu of land for field test.

[0093] The average yield of all the corns with the soil conditioner is increased, and the average yields are 9734.45 kg / hm2 of T1, 7652.45 kg / hm2 of T2 without adding the microbial agent and 7104.00 kg / hm2 of the blank control 2 2 2 . The corn yield of the T1 treatment is increased by 16.18% compared with the treatment without adding the microbial agent. The field data show that the application of the soil conditioner with the microbial agent can increase the corn yield.

[0094] The discovery and application of the Lelliottia jeotgali in the application provide a new idea and technical support for solving the problem of saline-alkali soil in China and even the whole world. The microorganism optimized by the genetic engineering method has excellent salt-tolerant and phosphorus-solubilizing properties, can survive and play a role in an extreme environment, and significantly improves soil fertility and crop yield.

[0095] ​​The above described embodiments are only to illustrate the preferred embodiments of the present application, and are not intended to limit the scope of the present application. Any modification and improvement of the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application.

Claims

1. A type of salted seafood Leroyella Lelliottia jeotgali Its characteristics are, It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 6, 2024, with accession number CGMCC No. 32940.

2. A soil conditioner, characterized in that, It comprises the features described in claim 1 Lelliottia jeotgali Bacterial agents.

3. The soil conditioner according to claim 2, characterized in that, The Lelliottia jeotgali The microbial agent is solid, liquid, or semi-solid, and the microbial agent further includes a carrier and / or auxiliary materials, wherein the carrier and / or auxiliary materials are selected from at least one of protective agents, excipients, binders, disintegrants, lubricants, fragrances, preservatives, stabilizers, suspending agents, dispersants, and diluents.

4. The salted seafood Leroyella as described in claim 1 Lelliottia jeotgali Applications in saline-alkali soils include salt and alkali tolerance, phosphorus solubilization, and promotion of the growth of sunflowers, oat grass, and corn.

5. The application according to claim 4, characterized in that: For saline-alkali land, the application rate of microbial agent is 5-40 kg / mu, and the microbial agent contains 0.5-500 million bacteria per gram.

6. A growth promoter for sunflower, oat grass, and corn in saline-alkali land, characterized in that, It comprises the features described in claim 1 Lelliottia jeotgali bacteria.

7. A soil improvement method for producing acid to solubilize phosphorus, lowering soil pH, and improving the saline-alkali environment in saline-alkali land, characterized in that, The salted seafood Leroyella as described in claim 1 Lelliottia jeotgali The microbial agent is applied to the land.

8. The method according to claim 7, characterized in that, The application rate for the soil is 5-40 kg / mu of microbial agent, and each gram of microbial agent contains 0.5-5 billion microorganisms.

9. The method according to claim 7, characterized in that, Salted seafood Leroyite Lelliottia jeotgali After the microbial agent is activated and cultured, it is cultured at 30±0.5℃ before being applied to the land.

Images