Sphingomonas G10 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to a sphingomonas sp. Strain with a soil reclamation fertilizing effect, which is preserved in China General Microbiological Culture Collection Center on October 17, 2024 with a preservation number of CGMCC No.32244. The strain has the capabilities of nitrogen fixation, phosphate solubilization, potassium solubilization and growth promotion, can also significantly promote straw decomposition and nutrient release, and has the advantages of high efficiency, high yield, high yield and the like. When being used in reclamation soil in a coal mine area, the fertilizer can improve the crop yield, effectively and remarkably increase the number of soil microorganisms, change the community structure of the soil microorganisms, improve the diversity of the soil microorganisms and accelerate the recovery of soil microbial communities, and can be widely applied to improvement of the fertility of the reclamation soil in the coal mine area.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a sphingomonas capable of accelerating soil fertilization in coal mining areas and application thereof. Background Art

[0002] Coal is known as black gold, but while coal mining has made great contributions to the country's economic construction and development, it has also caused serious damage to land resources and the natural environment. Reclaiming the damaged cultivated land in the mining area into farmland is of great significance for adhering to my country's cultivated land red line, alleviating the contradiction between people and land, and maintaining food security and ecological security.

[0003] At present, the research on fertilization of reclaimed farmland in my country mainly focuses on the traditional application of chemical fertilizers, organic fertilizers and organic materials such as straw. However, these measures have problems such as low utilization rate of chemical fertilizers, slow release of nutrients from organic fertilizers and organic materials such as straw, and slow fertility improvement (Zhang Yunlong et al., Chinese Journal of Eco-Agriculture , 2020, 28(08): 1219-1229).

[0004] Microbial reclamation technology is a biotechnology measure that uses the inoculation advantages of microorganisms to comprehensively manage and improve the soil in the reclamation area. It establishes a mutually beneficial symbiotic system between functional strains and rhizosphere microorganisms by inoculating some beneficial and growth-promoting functional microorganisms, improves the microenvironment of the reclaimed soil, and accelerates the improvement of the reclaimed land soil, so as to achieve the purpose of fertilizing the soil, increasing production and increasing efficiency (Luo Zhengming, et al. Applied and Environmental Microbiology ,2020, 86: e00180-20; Xu Qicheng, et al. Molecular Ecology , 2022, 31: 161-173).

[0005] However, in production practice, the shortage of microbial strains with significant effects is a bottleneck problem in the development of microbial remediation technology. Summary of the invention

[0006] The purpose of the present invention is to provide a Sphingomonas G10 with the ability of nitrogen fixation, phosphorus solubilization, potassium solubilization and growth promotion in view of the deficiencies in the prior art. The Sphingomonas G10 can be used for the restoration of reclaimed soil in coal mining areas, which can significantly promote straw decomposition and soil nutrient activation, accelerate the process of soil fertilization in coal mining areas, and promote crop growth and development.

[0007] To achieve the above-mentioned purpose, the present invention provides a Sphingomonas G10, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 17, 2024, with the deposit number CGMCC No. 32244.

[0008] The Sphingomonas G10 was isolated from the reclaimed soil in the coal mining area of ​​Xishandi Village, Xiangyuan County, Changzhi City, Shanxi Province. It is a round, slightly protruding golden colony and is Gram-negative.

[0009] The strain was sequenced by 16S rDNA and compared with NCBI to identify it as belonging to Sphingomonas sp. Sphingomonas sp .).

[0010] Further strain performance testing revealed that the Sphingomonas G10 has the ability to fix nitrogen, solubilize phosphorus and potassium, and promote growth, and can significantly promote straw decomposition and nutrient release.

[0011] Therefore, the present invention also provides the use of the Sphingomonas G10 in fertilizing the reclaimed soil in coal mining areas.

[0012] Furthermore, the present invention also provides a Sphingomonas agent containing the Sphingomonas G10.

[0013] More specifically, the present invention also provides a method for preparing a sphingomonas agent, which specifically comprises: 1) Inoculating the Sphingomonas G10 into an activation medium and culturing to prepare a seed solution; 2) Mixing the seed liquid with sterilized bran and brown sugar in a mass ratio of 4:4:1 to obtain a microbial agent, and performing solid fermentation culture in an incubator at 25-28° C.; 3) The fermented culture is dried to a water content of less than 10%, and stirred to obtain a Sphingomonas G10 bacterial agent.

[0014] The activation culture medium uses a conventional LB culture medium, which can be prepared according to the LB culture medium preparation method in the "Molecular Cloning Experiment Guide".

[0015] Furthermore, the Sphingomonas G10 is inoculated into an activation culture medium, and cultured for 24 hours at 25-28° C. and 180 r / min to prepare a seed solution.

[0016] Furthermore, the solid fermentation culture time is preferably 3 to 7 days.

[0017] Furthermore, in the present invention, the fermented culture is preferably dried at a constant temperature of 30-35°C.

[0018] The Sphingomonas G10 bacterial agent prepared by the present invention has the ability of nitrogen fixation, phosphorus solubilization, potassium solubilization and growth promotion, can significantly promote straw decomposition and nutrient release, and is suitable for preparing a microbial bacterial agent for soil reclamation in coal mining areas.

[0019] The Sphingomonas G10 bacterial agent of the present invention is used as a microbial bacterial agent for reclaiming soil in coal mining areas, which can improve the fertility of the reclaimed soil in coal mining areas and promote crop yield increase.

[0020] The Sphingomonas G10 bacterial agent of the present invention is used as a microbial bacterial agent for reclamation of soil in coal mining areas, which can increase the number of microorganisms in the soil and improve the diversity of soil microorganisms.

[0021] The Sphingomonas G10 screened by the present invention shows extremely strong soil adaptability. As a highly efficient microorganism with the functions of nitrogen fixation, phosphorus solubilization, potassium solubilization and growth promotion, it can promote the decomposition of straw and the release of nutrients. At the same time, field test results show that the strain can improve the soil nutrient content in coal mining areas, and has a significant effect on improving the organic matter, total nitrogen, quick-acting potassium and effective phosphorus content of the reclaimed soil in the coal mining areas. Further experiments show that the strain can improve the yield of soybeans or corn.

[0022] More importantly, the Sphingomonas G10 of the present invention can also effectively increase the number of microorganisms in the soil, effectively and significantly change the structure of the soil microbial community, improve the diversity of soil microorganisms, and accelerate the recovery of the soil microbial community, and can be widely used in the restoration of reclaimed soil in coal mining areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a strain morphology of Sphingomonas.

[0024] Figure 2 This is a scanning electron microscope image of the Sphingomonas strain. Implementation

[0025] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solution of the present invention so that those skilled in the art can well understand and utilize the present invention, rather than limiting the scope of protection of the present invention.

[0026] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0027] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art. Example

[0028] Example 1

[0029] This example illustrates that Sphingomonas G10 ( Sphingomonas sp .) (Accession No. CGMCC No. 32244) screening and identification process.

[0030] 1. Sample Collection

[0031] The 0-20 cm surface layer samples of reclaimed soil in a typical coal mining area were collected in Xishandi Village, Xiangyuan County, Changzhi City, Shanxi Province (N 36°28′17.846″, E 113°00′50.383″). Six groups were collected according to different reclamation years and different fertilization measures and stored in groups at 4°C.

[0032] 2. Determination of functional strains for fertile soil and fields

[0033] The soil nutrients of the 6 groups of soil samples collected were measured. According to the nutrient classification standards of the second national soil survey, groups 1, 2, 4, and 5 of the reclaimed soils were classified as low-fertility reclaimed soils, and groups 3 and 6 were classified as high-fertility reclaimed soils. The microbial community composition of these two groups of soil samples was taken as the research object.

[0034] The test results showed that the bacterial abundance in the high-fertility reclaimed soil group was much higher than that in the low-fertility group. There were specific microorganisms with significantly increased abundance in the high-fertility group, and the number of specific microorganisms that increased significantly in the high-fertility group was positively correlated with its soil fertility level.

[0035] Therefore, these microorganisms were identified as key functional strains to promote the improvement of soil fertility in reclaimed soil, specifically: Proteobacteria ( Proteobacteria ), α-Proteobacteria ( Alphaproteobacteria )、Sphingomonasales( Sphingomonadales )、Sphingomonasaceae( Sphingomonadaceae ), Sphingomonas spp. Sphingomonas ).

[0036] Based on the above analysis, it was determined that the key target microbial strain for promoting soil fertility improvement is Sphingomonas. Subsequently, a targeted screening method was used to isolate and obtain fertile soil microbial strains that can promote soil fertility improvement in reclaimed soil in coal mining areas from the dominant Sphingomonas bacteria.

[0037] 3. Preliminary screening of bacterial strains

[0038] Based on the method of specific primer-PCR combined with streptomycin resistance plates for specific isolation of Sphingomonas, four strains of yellow colonies were initially obtained, marked as 1#, 3#, 13# and 21#, respectively. After three purifications, they were stored in 4°C and -80°C refrigerators using the slant streak method and glycerol preservation method, respectively, for subsequent experiments.

[0039] 4. Strain rescreening

[0040] The 16SrDNA was amplified by PCR on the four strains (1#, 3#, 13# and 21#) obtained in the initial screening, and the products were observed on a 3.6% agarose gel on a gel electrophoresis imager. The genomic DNA amplification result of strain 13# was consistent with the expected result, and strain 13# was determined to be the experimental target bacteria, named G10.

[0041] 5. Strain Identification

[0042] According to the experimental contents and methods recorded in the Manual of Common Bacterial Systematic Identification and Principles, Methods and Practices of Bacterial Systematics, the strain G10 obtained by screening was analyzed and identified by bacterial morphology, physiological and biochemical analysis and 16SrDNA.

[0043] The morphological characteristics of this strain are: the colonies are round, golden and opaque, with neat edges, smooth and convex surfaces, shiny, moist, and easy to pick up. The colony size is about 1 to 1.5 cm ( Figure 1 ). Scanning electron microscope revealed that the bacteria were short rod-shaped, with raised surfaces and smooth edges ( Figure 2 ).

[0044] The physiological and biochemical characteristics of the strains were further tested, as shown in Table 1.

[0045]

[0046] The results of physiological and biochemical tests of the strain were: Gram staining negative, methyl red (MR) positive, acetyl methyl alcohol (VP) negative, citrate growth positive, catalase positive, indole test negative, starch hydrolysis test positive, and the reactions were positive with lactose, mannitol, sucrose, glucose, xylose, maltose, starch and fructose as the only carbon sources, and positive with nitrate nitrogen and ammonium nitrogen as the only nitrogen sources.

[0047] The strain G10 was sequenced by 16S rDNA gene, and the BLAST homology comparison was performed by NCBI. Combined with the above morphological observations and physiological and biochemical test results, it was finally determined that the strain G10 belonged to Sphingomonas ( Sphingomonas sp .).

[0048] The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​October 17, 2024, with the deposit number CGMCC No. 32244.

[0049] Example 2

[0050] Sphingomonas G10 was cultured on Axubei medium, inorganic phosphorus medium, Montgina organic phosphorus medium and potassium-solubilizing bacteria selective medium respectively.

[0051] The preparation methods of each culture medium are as follows: Axubei medium: contains 0.2 g / L potassium dihydrogen phosphate, 10 g / L glucose, 0.2 g / L sodium chloride, 0.2 g / L magnesium sulfate, 0.3 g / L potassium sulfate, 5 g / L calcium carbonate and 15 g / L agar; Inorganic phosphorus medium: containing 5 g / L tricalcium phosphate, 10 g / L glucose, 0.2 g / L sodium chloride, 0.5 g / L ammonium sulfate, 0.3 g / L magnesium sulfate, 0.2 g / L potassium chloride, 0.03 g / L ferrous sulfate, 0.03 g / L manganese sulfate, 0.5 g / L yeast extract and 15 g / L agar; Montgina organophosphorus medium: contains 0.5 g / L ammonium sulfate, 10 g / L glucose, 0.3 g / L potassium chloride, 0.3 g / L sodium chloride, 0.03 g / L manganese sulfate, 0.03 g / L ferrous sulfate, 0.4 g / L yeast extract, 0.2 g / L lecithin, 5 g / L calcium carbonate and 15 g / L agar; Potassium-dissolving bacteria selective culture medium: contains 0.5 g / L yeast powder, 10 g / L sucrose, 2 g / L disodium hydrogen phosphate, 0.5 g / L magnesium sulfate, 1 g / L potassium feldspar powder, 1 g / L calcium carbonate, 1 g / L ammonium sulfate and 15 g / L agar.

[0052] The specific culture results are shown in Table 2. Sphingomonas G10 can grow on all the above culture media, but the colony morphology on different culture media is slightly different. This shows that Sphingomonas G10 has the ability to fix nitrogen, solubilize phosphorus and potassium.

[0053]

[0054] Furthermore, the round brown nitrogen-fixing bacteria (ACCC 10006), Bacillus megaterium (ACCC 11107), Bacillus sporogenes (ACCC 60254), silicate bacteria (ACCC 10013) and IAA-producing Pseudomonas sp. (ACCC 60210) purchased from the Culture Collection Management Center were used as control strains to compare the nitrogen fixation ability, organic phosphorus solubilization ability, inorganic phosphorus solubilization ability, potassium solubilization ability and growth promotion ability (IAA production) with Sphingomonas G10. The results showed that Sphingomonas G10 improved by 15.04%, 375%, 285.54%, 8.16% and 30.06% respectively compared with the control strains (see Table 3 for details), indicating that Sphingomonas G10 has strong abilities in nitrogen fixation, phosphorus solubilization, potassium solubilization and growth promotion, especially in terms of organic phosphorus solubilization and inorganic phosphorus solubilization.

[0055]

[0056] Note: The control strains used for each test index are: Azotobacter rotundus (ACCC 10006), Bacillus megaterium (ACCC 11107), Paenibacillus (ACCC 60254), silicate bacteria (ACCC 10013) and IAA-producing Pseudomonas (ACCC 60210).

[0057] The above results prove that the Sphingomonas G10 of the present invention has the characteristics of nitrogen fixation, phosphorus and potassium solubilization and growth promotion.

[0058] Example 3

[0059] This example verifies through decomposition experiments that the Sphingomonas G10 of the present invention has the characteristics of promoting straw decomposition and nutrient release.

[0060] Weigh 300 g of air-dried soil, adjust the humidity to 60% of the field water capacity, and place it in a plastic cup.

[0061] Weigh 7.5 g of crushed straw that has been sieved through a 2 mm sieve, and add 0.1 mL of Sphingomonas G10 fermentation broth (effective viable count 10) per gram of straw. 8 CFU / mL), add bacterial solution, stir evenly and put it into a nylon mesh bag, and bury it in the plastic cup filled with soil as shown in the example.

[0062] Except for replacing the Sphingomonas G10 fermentation broth with an equal volume of sterile water, other steps remained unchanged, and straw without adding bacterial agent was used as a control example.

[0063] The plastic cups of the embodiment and the comparative example were placed in a culture chamber at 25°C for culture. After 72 days, the weight change of the straw in the nylon mesh bag and the nutrient content of the straw were measured, and the decomposition rate and nutrient release rate of the straw were calculated. The results are listed in Table 4.

[0064] The specific calculation formula is as follows: Decomposition rate (%) = (initial cultivation straw mass (g) - straw mass at sampling (g)) / initial cultivation straw mass (g) × 100% Nutrient release rate (mg / d) = (nutrient content of straw in initial culture (mg) - nutrient content of straw at sampling (mg)) / culture days (d)

[0065]

[0066] The results showed that Sphingomonas G10 could effectively promote the decomposition and nutrient release of straw.

[0067] Example 4

[0068] The Sphingomonas G10 (CGMCC No. 32244) Sphingomonas sp .) was inoculated into LB medium (containing 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride), and cultured at 28°C and 180 r / min for 24 h to obtain the strain density OD 600 Seed solution with a value of 0.6 to 0.8.

[0069] The prepared seed liquid was mixed with sterilized bran and brown sugar in a mass ratio of 4:4:1, placed in an incubator, and solid fermented at 28°C. During the culture process, samples were taken at intervals and the number of viable bacteria of the inoculum was observed by the plate dilution counting method until the number of effective viable bacteria per gram of the sphingomonas agent was greater than 10 9 CFU.

[0070] The fermented culture is dried at a constant temperature of 35°C until the water content is less than 10%, then stirred evenly again, and stored at room temperature to obtain a powdered sphingomonas agent.

[0071] Example 5

[0072] A soybean planting test was carried out in a field on a newly reclaimed land in a coal mining subsidence area in Changzhi City, Shanxi Province using the Sphingomonas agent prepared in Example 4 to investigate the effect of the agent on improving crop yield and soil fertility in the coal mining subsidence area.

[0073] The experiment was based on returning straw to the field and applying inorganic fertilizer, and the microbial agent in Example 4 was added, and no microbial agent was added as a control. The amount of microbial agent used was 20 kg / mu, and soybeans were planted after ploughing into the soil with the base fertilizer.

[0074]

[0075] Note: Different lowercase letters in the same column indicate significant differences at p<0.05.

[0076] From the results in Table 5, it can be seen that the Sphingomonas agent provided in Example 4 of the present invention has significant effects in improving the crop yield of reclaimed soil in coal mining areas, and increasing soil organic matter, alkaline nitrogen, available phosphorus, and available potassium.

[0077] Example 6

[0078] In this example, a field corn planting experiment was conducted on newly reclaimed land filled with coal gangue in Gujiao City, Shanxi Province, to investigate the effect of Sphingomonas agent on crop yield, microbial quantity and community structure diversity of newly reclaimed land filled with coal gangue.

[0079] The experiment was based on the test field of returning straw to the field and applying inorganic fertilizer, and the microbial agent in Example 4 was added, and the control was not added with the microbial agent. The amount of microbial agent used was 20 kg / mu, and corn was planted after ploughing into the soil with the base fertilizer.

[0080]

[0081] From the results in Table 6, it can be seen that the Sphingomonas agent provided in Example 4 of the present invention has a significant effect on increasing the crop yield of reclaimed soil in coal mining areas, effectively and significantly increasing the number of soil microorganisms, changing the community structure of soil microorganisms, and improving the diversity of soil microorganisms.

[0082] The above embodiments of the present invention do not describe all the details in detail, nor limit the present invention to the above embodiments. Various changes, modifications, substitutions and variations made by ordinary technicians in this field without departing from the principles and purpose of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Sphingomonas ( Sphingomonas sp .) G10, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 32244.

2. Use of the Sphingomonas G10 described in claim 1 as a strain for fertilizing soil in coal mining areas.

3. Use of the Sphingomonas G10 described in claim 1 as a strain for nitrogen fixation, phosphorus solubilization, potassium solubilization and growth promotion.

4. Use of the Sphingomonas G10 described in claim 1 as a strain for promoting straw decomposition and nutrient release.

5. Use of the Sphingomonas G10 described in claim 1 as a strain for improving soil fertility in coal mining area reclamation and increasing crop yield.

6. Use of the Sphingomonas G10 described in claim 1 as a strain for increasing the number of microorganisms in reclaimed soil in coal mining areas and improving soil microbial diversity. 7 . A Sphingomonas agent, comprising the Sphingomonas G10 according to claim 1 .

8. The method for preparing the sphingomonas agent according to claim 7, comprising: 1) Inoculating the Sphingomonas G10 into an activation medium and culturing to prepare a seed solution; 2) Mixing the seed liquid with sterilized bran and brown sugar in a mass ratio of 4:4:1 to obtain a microbial agent, and performing solid fermentation culture in an incubator at 25-28° C.; 3) The fermented culture is dried to a water content of less than 10%, and stirred to obtain a Sphingomonas G10 bacterial agent.

9. The preparation method according to claim 8, characterized in that The Sphingomonas G10 was inoculated into an activation culture medium, and cultured at 25-28° C. and 180 r / min for 24 hours to prepare a seed solution.

10. The preparation method according to claim 8, characterized in that The solid fermentation culture time is 3 to 7 days.

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