Method for separating and culturing soil microorganisms based on high flux of membrane diffusion

Abstract

The invention discloses a method for high-throughput separation culture of soil microorganisms based on membrane diffusion. The method comprises the following steps: 1) dividing a soil sample into nutrient soil, enriched soil and culture medium soil, mixing the nutrient soil and a nutrient solution to obtain slurry, and diluting the enriched soil into sample diluents with different gradients; 2) adding the slurry into holes of a Transwell cell culture plate, adding a sample diluent into a small chamber, and carrying out enrichment culture; (3) diluting the culture in the small chamber in a gradient manner, respectively coating an R2A flat plate and a soil flat plate, and culturing to obtain R2A flat plate and soil flat plate single colonies; and 4) respectively inoculating the single colonies into a 96-deep-hole plate filled with an R2A liquid culture medium or a soil liquid culture medium for culturing through an automatic bacterium picking platform, and after the culture is completed, adding glycerol to preserve the culture so as to complete the separation and preservation of the soil microorganisms, thereby effectively improving the separation efficiency of the microorganisms and the richness of the culturable microorganisms.

Description

Technical Field

[0001] This invention relates to a method for high-throughput isolation and culture of soil microorganisms based on membrane diffusion in the field of microbial isolation and culture. Background Technology

[0002] Soil microorganisms, as the largest and most underutilized resource pool of Earth's biodiversity, play an irreplaceable role in agricultural microbial applications such as the development of novel biopesticides, plant disease prevention and growth promotion, microecological regulation, and soil pollution remediation. Soil contains tens of thousands of bacterial species, but due to the inefficiency of traditional isolation techniques, over 99% of soil microorganisms have yet to be successfully cultured. This microbial "dark matter" severely restricts the development of functional agricultural microbial agents, the discovery of novel antibiotics, and the in-depth analysis of soil ecological processes. Existing methods for isolating and culturing soil microorganisms face challenges in isolating and culturing oligotrophic strains and low-abundance microorganisms. Overcoming these technical bottlenecks and establishing an efficient, universal, high-throughput isolation system has become a core challenge in the field of agricultural biotechnology. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to obtain rare microorganisms and / or difficult-to-culture (uncultured) microorganisms from soil and / or increase the variety and / or abundance of soil microorganisms.

[0004] To address the above technical problems, this invention provides a method for isolating and culturing soil microorganisms.

[0005] The method for isolating and culturing soil microorganisms provided by this invention includes: 1) Divide the soil sample into three parts, namely nutrient soil, enriched soil and culture medium soil. Mix the nutrient soil and nutrient solution to obtain mud. Dilute the enriched soil with sterile water to obtain liquids of different gradients to obtain sample dilutions. 2) First, remove the small chamber with the permeable membrane at the bottom from the well of the Transwell cell culture plate. Then, add mud to the well, add sample diluent to the chamber, and then put the chamber back into the well. The amount of mud added should be sufficient to make the mud contact the permeable membrane. Then, carry out enrichment culture. 3) The cultures in the chamber were serially diluted with sterile water and spread onto R2A plates and soil plates, and cultured to obtain single colonies on R2A plates and soil plates, respectively. The R2A plates were made with R2A solid medium, which was made with R2A liquid medium and a solidifying agent. The soil plates were made with soil solid medium, which was made with soil liquid medium and a solidifying agent. The soil liquid medium was a liquid medium containing soil extract. 4) inoculate the R2A plate single colony into a culture plate containing the R2A liquid medium, inoculate the soil plate single colony into a culture plate containing the soil liquid medium, preserve the obtained culture, and complete the isolation culture.

[0006] In one embodiment of the present application, the step 2) comprises the step of providing the Transwell cell culture plate, wherein the Transwell cell culture plate comprises a multi-well cell culture plate, each well of which is equipped with a detachable chamber with a permeable membrane, and after placing one of the chambers into one well of the cell culture plate, the chamber separates the well into two independent culture spaces, and the culture space other than the chamber is referred to as the lower culture chamber.

[0007] In the above method, the multi-well is one or more wells.

[0008] In one embodiment of the present application, the multi-well is 12 wells.

[0009] In one embodiment of the present application, in the step 2), the enrichment culture is carried out at 28°C for 5 days with 180 rpm shaking.

[0010] In some embodiments of the present application, in the Transwell cell culture plate, the permeable membrane has micropores.

[0011] In one embodiment of the present application, the micropores have a pore size (diameter) of 0.1 µm.

[0012] In some embodiments of the present application, the nutrient solution comprises yeast extract, tryptone, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, sodium chloride, ferrous chloride, and water.

[0013] In some embodiments of the present application, the R2A liquid medium comprises yeast extract, peptone, casein hydrolysate, glucose, soluble starch, dipotassium hydrogen phosphate, anhydrous magnesium sulfate, sodium pyruvate, and water.

[0014] In some embodiments of the present application, the soil liquid medium is a liquid medium composed of the nutrient solution and soil extract.

[0015] In one embodiment of the present application, the nutrient solution comprises yeast extract 0.5 g / L, tryptone 0.5 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate heptahydrate 0.05 g / L, sodium chloride 0.1 g / L, and ferrous chloride 0.01 g / L, and the rest is water.

[0016] In one embodiment of the present application, the coagulant is agar.

[0017] In one embodiment of the present application, the R2A liquid medium is composed of yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium phosphate dibasic 0.3 g / L, magnesium sulfate anhydrous 0.024 g / L, and sodium pyruvate 0.3 g / L, and the rest is sterile water.

[0018] In one embodiment of the present application, the R2A solid medium is composed of yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium phosphate dibasic 0.3 g / L, magnesium sulfate anhydrous 0.024 g / L, and sodium pyruvate 0.3 g / L, and the rest is sterile water.

[0019] In some embodiments of the present application, the soil extract is a liquid (soil extract) extracted from the culture medium soil with sterile water.

[0020] In one embodiment of the present application, the ratio of the sterile water to the culture medium soil in the soil extract is 1 L of sterile water: 1 kg of the culture medium soil.

[0021] In one embodiment of the present application, the extraction is standing for 30 minutes at 20-30 °C.

[0022] In the above method, the ratio of the nutrient soil to the nutrient liquid in the slurry is 100 g of the nutrient soil: 200 mL of the nutrient liquid.

[0023] In one embodiment of the present application, the slurry is mixed by the nutrient soil and the nutrient liquid at a mass ratio of 1:2.

[0024] In one embodiment of the present application, the culture plate of step 4) is a deep-well culture plate, which is a culture plate with a volume of 1.8 mL per well.

[0025] In one embodiment of the present application, in step 4), an automatic bacterial picking instrument is used to inoculate the R2A flat plate single colony into a culture plate containing the R2A liquid medium, and an automatic bacterial picking instrument is used to inoculate the soil flat plate single colony into a culture plate containing the soil liquid medium.

[0026] The method for isolating and culturing soil microorganisms of the present application enhances the culturability of rare microorganisms, can obtain uncultured (uncultured) microorganisms, and improves the isolation efficiency of microorganisms and the richness of culturable microorganisms. The method for isolating and culturing soil microorganisms of the present application can obtain uncultured and / or uncultured bacteria, and can isolate and culture bacterial species with extremely low abundance in soil that cannot be isolated and cultured by traditional methods. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The flow chart of the method for isolating and culturing soil microorganisms of the present application.

[0028] Figure 2 The isolation and culture of soil microorganisms in the rhizosphere of wheat, wherein A is the genus level microbial richness; B is the species level microbial Wayne diagram.

[0029] Figure 3 The isolation and culture of soil microorganisms in the rhizosphere of watermelon, wherein A is the genus level microbial richness; B is the species level microbial Wayne diagram.

[0030] Figure 4 The 16S rDNA gene sequence alignment result of the FDXW-21 strain. DETAILED DESCRIPTION

[0031] The method for isolating and culturing soil microorganisms provided by the present application comprises the following steps: 1) The soil sample to be isolated is divided into three parts, which are respectively referred to as nutrient soil, enrichment soil and medium soil, the nutrient soil and nutrient solution are mixed to obtain mud; the enrichment soil is diluted with sterile water to obtain sample diluents with different gradients; 2) A Transwell cell culture plate is provided, the Transwell cell culture plate comprises a multi-well cell culture plate, each well is provided with a detachable chamber with a permeable membrane at the bottom, after one of the chambers is placed in one of the wells of the cell culture plate, the chamber divides the well into two independent culture spaces, and the other culture space except the chamber is referred to as a lower culture chamber; 3) The chamber with a permeable membrane at the bottom is first taken out from the well of the Transwell cell culture plate, then the mud is added to the well, the sample diluent is added to the chamber, and then the chamber is placed back into the well, the addition amount of the mud satisfies that the mud is in contact with the permeable membrane; and then enrichment culture is carried out; 4) The culture in the chamber obtained after the enrichment culture is diluted with sterile water respectively, and then coated on R2A flat plate and soil flat plate to form single colonies, and R2A flat plate single colonies and soil flat plate single colonies are obtained respectively; the R2A flat plate is a solid flat medium prepared by R2A solid medium, and the R2A solid medium is made of R2A liquid medium and coagulant; the soil flat plate is a solid flat medium prepared by soil solid medium, and the soil solid medium is made of soil liquid medium and coagulant; the soil liquid medium is a liquid medium containing soil extract; 5) The R2A flat plate single colonies are inoculated in a culture plate containing R2A liquid medium for culture, and the soil flat plate single colonies are inoculated in a deep hole culture plate containing soil liquid medium for culture, and the obtained culture is preserved to complete the separation and culture.

[0032] The step 4) is to enhance the culturability of rare microorganisms.

[0033] The application will be further described in detail in combination with specific embodiments, and the examples are only for illustrating the application, not for limiting the scope of the application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.

[0034] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0035] In the quantitative test in the following examples, three repeated experiments are set, and the average value is taken, unless otherwise specified.

[0036] Example 1, the method for separating and culturing soil microorganisms of the application The main process of the method for separating and culturing soil microorganisms of the application is as follows Figure 1 , which comprises the following steps: 1, sample collection 2-15 cm deep field crop rhizosphere soil 2 kg is taken by 5-point sampling method as the soil sample to be separated and cultured.

[0037] 2, preparation of mud, sample diluent The 1110 g of soil sample collected in step 1 is divided into three parts, which are respectively called nutrient soil, enrichment soil and medium soil. 100 g of nutrient soil, 10 g of enrichment soil and 1 kg of medium soil.

[0038] Mix 100 g of nutrient soil and 200 ml of nutrient solution to obtain a slurry. The nutrient solution is prepared as follows: take 0.5 g of yeast extract, 0.5 g of tryptone, 0.4 g of potassium phosphate dibasic, 0.05 g of magnesium sulfate heptahydrate, 0.1 g of sodium chloride, and 0.01 g of ferrous chloride, and make up to 1 L with sterile water, and sterilize at 121°C for 20 minutes.

[0039] Dilute 10 g of the enriched soil with sterile water to obtain liquid of different dilution gradients (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , and 10 -1 ).

[0040] The preparation method of the different gradient sample dilutions is as follows: take 10 g of soil, put it into a test tube containing 90 mL of sterile water, and shake at 30°C and 200 rpm for 30 minutes to obtain a soil suspension, which is 10 -1 sample dilution. Use a pipette to take 1 mL of the upper layer of the suspension into another test tube, and add 9 mL of sterile water. Shake for 1 minute in a vortex shaker (Scilogex MX-S vortex mixer, USA) to obtain a soil suspension, which is 10 -2 sample dilution. Repeat the above dilution process with the 10 -2 sample dilution to obtain 10 -2 , 10 -3 , and 10 -4 sample dilutions (10 -5 , 10 -3 , and 10 -4 sample dilutions, respectively). -5

[0041] 3. Enrichment culture 3.1 Provide a Transwell cell culture plate The Transwell cell culture plate in this example is a LABSELECT (Selected) 14201 cell culture chamber, which contains a Transwell cell plate with 12 wells (PC membrane, 12 mm, pore size 0.1 um). The cell culture plate includes 12 wells, each equipped with a detachable chamber with a permeable membrane (chamber in Figure 1 ), which divides the well into two independent culture spaces when a chamber is placed in a well. The other culture space, excluding the chamber, is called the lower culture chamber. The permeable membrane is a polycarbonate membrane with micropores.​Figure 1 The pore size (diameter) of the micropores is 0.1 pm.

[0042] Each of the three repeats was performed in triplicate, i.e. one 12-well Transwell cell culture plate was used for each repeat. The chamber was removed from the well of the Transwell cell culture plate, and the slurry was added to the well (1.2 ml / well) and the sample dilution was added to the chamber (0.5 ml / chamber). The chamber was then placed back into the well, and the slurry was added in an amount sufficient to contact the slurry with the permeable membrane of the chamber, and the liquid level of the slurry was higher than the permeable membrane. The entire Transwell cell culture plate was then sealed with Parafilm to prevent evaporation of water. This resulted in a sealed Transwell cell culture plate with 10 -1 sample dilution culture wells, 10 -2 sample dilution culture wells, 10 -3 sample dilution culture wells, 10 -4 sample dilution culture wells, and 10 -5 sample dilution culture wells. Two wells were used for each dilution of the sample dilution.

[0043] The sealed Transwell cell culture plate was placed in a shaker (ZWY-C2112B brand from Shanghai ZhiCheng Analytical Instruments Co., Ltd.) and incubated at 28°C and 180 rpm (rotation radius 35 mm) for 5 days. The incubation was completed. The culture obtained from the 10 -1 sample dilution was called 10 -1 enriched culture. The culture obtained from the 10 -2 sample dilution was called 10 -2 enriched culture. The culture obtained from the 10 -3 sample dilution was called 10 -3 enriched culture. The culture obtained from the 10 -4 sample dilution was called 10 -4 enriched culture. The culture obtained from the 10 -5 sample dilution was called 10 -5 enriched culture.

[0044] 4. Enhancing culturability of rare microorganisms 4.1. Preparation of R2A plates The R2A plate was prepared by pouring R2A solid medium at 55-60°C into a sterile petri dish with a diameter of 9 cm.

[0045] The preparation method of R2A solid medium is as follows: taking yeast extract 0.5 g, proteose peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, soluble starch 0.5 g, potassium phosphate dibasic 0.3 g, anhydrous magnesium sulfate 0.024 g, sodium pyruvate 0.3 g and agar 15 g, and then adding sterile water to 1 L, adjusting pH to 7.0, sterilizing at 121℃ for 20 minutes.

[0046] 4.2, preparation of soil plate The soil plate is a solid flat medium prepared by pouring soil solid medium at 55-60℃ into a sterile petri dish with a diameter of 9 cm.

[0047] The preparation method of soil solid medium is as follows: taking yeast extract 0.5 g, proteose peptone 0.5 g, potassium phosphate dibasic 0.4 g, magnesium sulfate heptahydrate 0.05 g, sodium chloride 0.1 g and ferrous chloride 0.01 g, and then adding soil extract to 1 L.

[0048] The soil extract is prepared as follows: mixing the medium soil of step 2 according to the ratio of 1 kg of soil and 1 L of sterile water, standing for 30 minutes at 20-30℃ for extraction, centrifuging at 5000 rpm for 20 minutes, and collecting the supernatant, which is the soil extract.

[0049] 4.3, plating The enrichment culture in each chamber obtained by step 3 is diluted by 10 1 , 10 2 , 10 3 , 10 4 , 10 5 and 10 6 times, respectively, and each enrichment culture obtains 10 -1 dilution enrichment culture, 10 -2 dilution enrichment culture, 10 -3 dilution enrichment culture, 10 -4 dilution enrichment culture, 10 -5 dilution enrichment culture and 10 -6 dilution enrichment culture, respectively. The following takes the 10 -1 enrichment culture of step 3 as an example to illustrate the method of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 and 10 6 times dilution: taking 0.1 ml of 10 -1 enrichment culture, and then adding it into a 2 mL Eppendorf tube containing 0.9 mL of sterile water, mixing well, to obtain 10-1 Dilution enrichment culture. Pipette 10 -1 Dilution enrichment culture. Pipette 100 microliters of the dilution enrichment culture into another 2 mL Eppendorf tube and add 900 microliters of sterile water, mix well to get 10 -2 Dilution enrichment culture, and so on, to get 10 -6 Dilution enrichment culture.

[0050] Each of the dilution enrichment cultures was plated on R2A plates and soil plates, 3 plates for each dilution enrichment culture, 0.1 ml per plate. Incubate at 30°C for 3 days.

[0051] According to the method, 10 -1 10 -1 Dilution enrichment culture, 10 -2 Dilution enrichment culture, 10 -3 Dilution enrichment culture, 10 -4 Dilution enrichment culture, 10 -5 Dilution enrichment culture, and 10 -6 Dilution enrichment culture were plated on R2A plates and incubated at 30°C for 3 days to get the cultures named as 10 -1 Enriched R2A solid culture-10 -1 , 10 -1 Enriched R2A solid culture-10 -2 , 10 -1 Enriched R2A solid culture-10 -3 , 10 -1 Enriched R2A solid culture-10 -4 , 10 -1 Enriched R2A solid culture-10 -5 , 10 -1 Enriched R2A solid culture-10 -6 ; 10 -1 10 -1 Dilution enrichment culture, 10 -2 Dilution enrichment culture, 10 -3 Dilution enrichment culture, 10 -4 Dilution enrichment culture, 10 -5 Dilution enrichment culture, and 10 -6 Dilution enrichment culture were plated on soil plates and incubated at 30°C for 3 days to get the cultures named as 10 -1 Enriched soil solid culture-10 -1 , 10 -1 Enriched soil solid culture-10-2 ,10 -1 Enrichment soil solid culture-10 -3 ,10 -1 Enrichment soil solid culture-10 -4 ,10 -1 Enrichment soil solid culture-10 -5 ,10 -1 Enrichment soil solid culture-10 -6 .

[0052] By analogy, the respective dilution enrichment cultures (10 -2 Dilution enrichment culture, 10 -3 Dilution enrichment culture, 10 -4 Dilution enrichment culture and 10 -5 Dilution enrichment culture) of each dilution enrichment culture (10 -1 Dilution enrichment culture, 10 -2 Dilution enrichment culture, 10 -3 Dilution enrichment culture, 10 -4 Dilution enrichment culture, 10 -5 Dilution enrichment culture and 10 -6 Dilution enrichment culture) of other enrichment cultures (10 -2 Enrichment culture, 10 -3 Enrichment culture, 10 -4 Enrichment culture and 10 -5 Enrichment culture) of step 3 were respectively inoculated into R2A plates and soil plates, and the cultures obtained by incubation at 30°C for 3 days were named respectively.

[0053] 5, Expansion culture All single colonies of R2A plates of step 4 were picked into step 96 deep well plates (Corning Corporation, item number P-DW-20-C-S) containing 1 ml R2A liquid medium per well using an automatic colony picker (ClonePicker-1 Cloning Colony Picker, Dehong Scientific Instruments Co., Ltd.), and incubated at 30°C for 3 days.

[0054] The R2A liquid medium was prepared as follows: 0.5 g of yeast extract, 0.5 g of peptone, 0.5 g of casein hydrolysate, 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of potassium phosphate dibasic, 0.024 g of anhydrous magnesium sulfate and 0.3 g of sodium pyruvate were taken, and sterilized water was added to make up to 1 L, the pH was adjusted to 7.0, and sterilized at 121°C for 20 minutes.

[0055] All single colonies of soil plates of step 4 were picked into step 96 deep well plates (Corning Corporation, item number P-DW-20-C-S) containing 1 ml soil liquid medium per well using an automatic colony picker (ClonePicker-1 Cloning Colony Picker, Dehong Scientific Instruments Co., Ltd.), and incubated at 30°C for 3 days.

[0056] The configuration method of the soil liquid medium is as follows: taking yeast extract 0.5 g, tryptone 0.5 g, potassium phosphate dibasic 0.4 g, magnesium sulfate heptahydrate 0.05 g, sodium chloride 0.1 g, and ferrous chloride 0.01 g, and using the soil extract solution of step 4.2 to make up to 1 L, sterilizing at 121°C for 20 minutes.

[0057] 6. Strain classification and identification From the culture obtained in step 5, a small amount of bacterial cells was lysed using a lysis buffer (Takara's Lysis Buffer for Microorganism to Direct PCR) to obtain bacterial genomic DNA. PCR amplification was performed in a PCR instrument (Bio-rad) using bacterial universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (sequence 1 in the sequence listing) and 1492R: 5'-CTACGGCTACCTTGTTACGA-3' (sequence 2 in the sequence listing). The PCR reaction system was ddH2O 8 μL, 2xM5 HiPer plus Taq HiFi 10 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, and genomic DNA 1 μL. After the reaction was completed, 3 μL was taken for 1% agarose gel electrophoresis detection to confirm the PCR amplification fragment. The PCR product was sent to a sequencing company for DNA sequencing. The obtained 16s rRNA gene sequence of the strain was analyzed by comparison in the rRNA / ITS databases in NCBI, and identified as the type of microorganism.

[0058] 7. Cryopreservation The remaining bacterial solution in the 96-well deep plate was added with an equal volume of 50% glycerol aqueous solution. The 96-well deep plate was sealed, numbered, and stored in a -80°C refrigerator.

[0059] According to the above method, the PC membrane with a pore size of 0.1 μm can ensure that the bacteria do not spread, and only small molecule substances can diffuse with each other to realize dynamic balance of nutrient supply and metabolite diffusion, simulating in-situ material exchange in soil. Combined with image recognition and mechanical arm picking bacteria system, the automation process of single colony picking is realized, and the efficiency of microbial separation and the richness of cultivable microorganisms are improved.

[0060] Example 2: High-throughput separation and culture of wheat rhizosphere soil microorganisms On March 27, 2025, in the wheat test field of Beijing Academy of Agriculture and Forestry Sciences in Haidian District, Beijing, 2-15 cm deep field wheat rhizosphere soil 2 kg was taken as the soil sample to be separated and cultured.

[0061] 1. The soil sample was subjected to isolation and cultivation of soil microorganisms by the method of the present application (Transwell treatment in Example 1). Figure 2

[0062] 2. Meanwhile, the soil sample was subjected to isolation and cultivation of soil microorganisms by the traditional LB separation method (LB treatment in Example 1). The specific method is as follows: Figure 2 2.1 Dilute 10 g of the soil sample into liquid of different gradient dilutions (10 -1 dilution liquid, 10 -2 dilution liquid, 10 -3 dilution liquid, 10 -4 dilution liquid, and 10 -5 dilution liquid) to obtain 5 kinds of sample dilution liquids of different dilutions.

[0063] The preparation method of different gradient sample dilution liquids is as follows: take 10 g of soil and put it into a test tube containing 90 mL of sterile water, shake at 30°C and 200 rpm for 30 minutes to prepare a soil suspension, which is the 10 -1 dilution liquid (10 -1 sample dilution liquid). Use a pipette to take 1 mL of the upper layer of the suspension into another test tube, add 9 mL of sterile water, and shake in a vortex shaker (Scilogex MX-S vortex mixer, USA) for 1 minute to prepare a soil suspension, which is the 10 -2 dilution liquid (10 -2 sample dilution liquid). Repeat the above dilution process with the 10 -2 dilution liquid to obtain 10 -3 , 10 -4 , and 10 -5 dilution liquids (10 -3 sample dilution liquid, 10 -4 sample dilution liquid, and 10 -5 sample dilution liquid), respectively.

[0064] 2.2 Coating LB plates The LB plate is a solid flat medium prepared by pouring LB solid medium at 55-60°C into a sterile culture dish with a diameter of 9 cm. The LB solid medium is prepared as follows: take 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar, and adjust the pH to 7.0 with sterile water to make 1 L.

[0065] Coat the 5 kinds of sample dilution liquids of different dilutions in 2.1 on LB plates and incubate at 30°C for 3 days. Coat 3 LB plates for each kind of sample dilution liquid. ​​

[0066] 2.3 The colonies in the 2.2 plate are manually inoculated into test tubes containing 2 mL of LB liquid medium, and cultured at 30°C for 3 days.

[0067] The LB liquid medium is prepared by the following method: taking 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, and then adding sterile water to make up to 1 L, and adjusting the pH to 7.0.

[0068] 2.4 Strain classification and identification The same as step 6 of Example 1.

[0069] 2.5 Cryopreservation The same as step 6 of Example 1.

[0070] 3. At the same time, the soil sample is subjected to soil microorganism separation and culture according to the traditional R2A separation method (R2A treatment in Figure 2 ).

[0071] The R2A separation method is different from the method of step 2 only in that the LB solid medium is replaced by the R2A solid medium of Example 1, the LB liquid medium is replaced by the R2A liquid medium of Example 1, and the other operations are the same as those of step 2.

[0072] 4. Results and analysis By systematically comparing the efficiency and richness of the separation and culture of soil microorganisms in the wheat rhizosphere soil by the separation and culture method of the present application with the LB separation method and the R2A separation method, the significant advantages of the method of the present application in the separation and culture of low-abundance microorganisms and rare microorganisms are revealed.

[0073] Each strain separated in steps 1, 2, and 3 is classified and identified according to the method of step 6 of Example 1, and the results are shown in Figure 2 , which shows that in the traditional culture method, the LB separation method separates and obtains a total of 134 microorganisms (belonging to 39 species of 13 genera), mainly from the Firmicutes phylum, Firmicutes , and the isolation rate of single species is only 29.10%. The R2A separation method separates and obtains a total of 132 microorganisms (belonging to 64 species of 27 genera), and the microbial flora contains the Firmicutes phylum, the Actinobacteria phylum, Actinobacteria , and the Proteobacteria phylum, Proteobacteria , which significantly improves the richness of the bacteria separated by the LB separation method, and the isolation rate of single species reaches 48.48%.

[0074] The method for isolating and culturing soil microorganisms of the present application isolates and obtains 178 strains of microorganisms, covering 111 species of 52 genera, and the isolation rate of single strain reaches 62.36%. Among them, 48.8% of the strains are microorganisms that cannot be isolated by traditional LB isolation method and R2A isolation method, including multiple bacteria of Bacteroidetes phylum. Bacteroidetes Brevundimonas pondensis , Psychrobacillus vulpis , Flavobacterium urocaniciphilum and 24 other bacteria, and 13 strains of bacteria with 16S rRNA gene sequence alignment similarity less than 97% are identified as new species. Thus, compared with the traditional LB isolation method and R2A isolation method, the method for isolating and culturing soil microorganisms of the present application can improve the richness of soil microbial isolation and effectively increase the isolation and culture of rare microorganisms, laying a foundation for the development of new germplasm resources of agricultural microorganisms.

[0075] In addition, the method for isolating and culturing soil microorganisms of the present application can realize accurate picking of small colonies by using an automatic colony picking instrument, increasing the isolation probability of rare species. For the same plate, manual picking can only identify 22 colonies, and the automatic colony picking instrument can accurately identify 44 colonies, and 38 strains of bacteria are cultured to be alive, significantly improving the picking and isolation of small colonies. In terms of technical efficiency, the traditional manual isolation method can only pick 30-50 colonies per hour, while the method for isolating and culturing soil microorganisms of the present application integrates the automatic colony picking instrument and the 96-well plate high-throughput culture technology, and the isolation efficiency is improved to 300-500 colonies / hour, which is 10 times higher than the manual operation efficiency. This breakthrough improvement not only significantly improves the amount of microbial isolation and culture per unit time, but also provides reliable technical support for the construction of macro-microbial resource library.

[0076] Example 3, High-throughput isolation and culture of watermelon rhizosphere soil microorganisms On April 29, 2025, in the watermelon planting base of Changziying Town, Daxing District, Beijing, 2-15 cm deep field watermelon rhizosphere soil 2 kg was taken as the soil sample to be isolated and cultured.

[0077] 1. According to the method of Example 1, the soil sample was subjected to soil microbial isolation and culture by the method for isolating and culturing soil microorganisms of the present application (Transwell treatment in Figure 3 ).

[0078] 2. At the same time, the soil sample was subjected to soil microbial isolation and culture by the traditional LB isolation method in Example 2 (LB treatment in Figure 3 ).

[0079] ​3. Simultaneously, soil microorganisms were isolated and cultured from the soil sample using the conventional R2A isolation method described in Example 2. Figure 3 (R2A processing in the middle).

[0080] 4. Results and Analysis The bacterial strains isolated in steps 1, 2, and 3 were identified according to the method in step 6 of Example 1. A total of 317 bacterial strains were identified and preserved. The results showed that... Figure 3 As shown.

[0081] like Figure 3 As shown, the bacterial abundance obtained by the method for isolating and culturing soil microorganisms of the present invention is much higher than that obtained by the LB isolation method and the R2A isolation method: the method for isolating and culturing soil microorganisms of the present invention isolated and cultured 52 unique bacterial species (38.8%), while the LB isolation method and the R2A isolation method isolated and cultured 37 (27.6%) and 30 (22.4%) unique bacterial species, respectively (the percentage of unique bacterial species in the total bacterial species, a total of 134 strains were identified), indicating that the bacteria isolated and cultured by the method for isolating and culturing soil microorganisms of the present invention have high uniqueness (e.g., Rheinheimera aquimaris , Fictibacillus phosphorivorans , Agrilutibacter solisilvae , Fictibacillus nanhaiensis These microbial strains are rarely reported to originate from the soil environment.

[0082] The results showed that the bacterial phylogenetic tree also indicated that the method for isolating and culturing soil microorganisms of the present invention produced a richer variety of bacteria. Rheinheimera , Brevundimonas , Ramlibacter The abundance of these bacteria in the soil is also extremely low, which indicates that the present invention can isolate and culture bacterial strains with extremely low abundance in the soil that cannot be isolated and cultured by traditional methods.

[0083] like Figure 4 The method for isolating and culturing soil microorganisms of the present invention isolated and cultured three strains that were identified as uncultured bacteria in NCBI, indicating that the method for isolating and culturing soil microorganisms of the present invention can obtain difficult-to-culture / uncultured bacteria.

[0084] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A method for isolating soil microorganisms in culture, characterized by, The method comprises the following steps: 1) a soil sample is divided into three parts, which are respectively called nutrient soil, enrichment soil and medium soil, the nutrient soil and nutrient solution are mixed to obtain mud, and the enrichment soil is diluted with sterile water to obtain sample diluent with different gradients; 2) first, a small chamber with a permeable membrane on the bottom is taken out from a hole of a Transwell cell culture plate, then the mud is added into the hole, the sample diluent is added into the small chamber, and the small chamber is put back into the hole, the amount of the mud added is enough to make the mud contact with the permeable membrane; then, enrichment culture is carried out; 3) the culture in the small chamber is diluted with sterile water with different gradients, and then R2A flat plates and soil flat plates are coated respectively, and then culture is carried out to obtain R2A flat plate single colonies and soil flat plate single colonies respectively; the R2A flat plate is prepared by using R2A solid culture medium, the R2A solid culture medium is prepared by using R2A liquid culture medium and a coagulant; the soil flat plate is prepared by using soil solid culture medium, the soil solid culture medium is prepared by using soil liquid culture medium and a coagulant, and the soil liquid culture medium is a liquid culture medium containing soil extract; 4) the R2A flat plate single colonies are inoculated into a culture plate containing the R2A liquid culture medium for culture, the soil flat plate single colonies are inoculated into a culture plate containing the soil liquid culture medium for culture, and the obtained culture is preserved, so that the separation and culture of soil microorganisms are completed.

2. The method of claim 1, wherein, The step 2) comprises the step of providing the Transwell cell culture plate, the Transwell cell culture plate comprises a multi-well cell culture plate, each hole of the cell culture plate is provided with a detachable small chamber with a permeable membrane on the bottom, and after the small chamber is put into a hole of the cell culture plate, the small chamber divides the hole into two independent culture spaces, and the other culture space except the small chamber is called a lower culture chamber.

3. The method according to claim 1 or 2, characterized in that, The permeable membrane has micropores, and the diameter of the micropores is 0.1 µm.

4. The method according to any one of claims 1 to 3, characterized in that, The nutrient solution comprises yeast extract, tryptone, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, sodium chloride, ferrous chloride and water; or / and, The R2A liquid culture medium comprises yeast extract, peptone, casein hydrolysate, glucose, soluble starch, dipotassium hydrogen phosphate, anhydrous magnesium sulfate, sodium pyruvate and water; Or / and, The soil liquid culture medium is a liquid culture medium composed of the nutrient solution and soil extract.

5. The method according to any one of claims 1 to 4, characterized in that, The R2A liquid culture medium comprises 0.5 g / L of yeast extract, 0.5 g / L of peptone, 0.5 g / L of casein hydrolysate, 0.5 g / L of glucose, 0.5 g / L of soluble starch, 0.3 g / L of dipotassium hydrogen phosphate, 0.024 g / L of anhydrous magnesium sulfate and 0.3 g / L of sodium pyruvate, and the rest is water; or / and, The nutrient solution comprises 0.5 g / L of yeast extract, 0.5 g / L of tryptone, 0.4 g / L of dipotassium hydrogen phosphate, 0.05 g / L of magnesium sulfate heptahydrate, 0.1 g / L of sodium chloride and 0.01 g / L of ferrous chloride, and the rest is water.

6. The method according to any one of claims 1 to 5, characterized in that, The soil extract is a liquid extracted from the medium soil by using sterile water.

7. The method of claim 6, wherein, The ratio of the sterile water to the culture medium soil in the soil extract is 1L of sterile water: 1kg of the culture medium soil.

8. The method according to any one of claims 1 to 7, characterized in that, The ratio of the nutrient soil to the nutrient liquid in the mud is 1 mass part of the nutrient soil: 2 mass parts of the nutrient liquid.

9. The method of any one of claims 1-8, wherein, The mud is mixed by the nutrient soil and the nutrient liquid according to the mass ratio of 1:

2.

10. The method of any one of claims 1-9, wherein, In step 4), the R2A plate single colony is inoculated into a culture plate containing the R2A liquid medium by using an automatic bacteria picking instrument, and the soil plate single colony is inoculated into a culture plate containing the soil liquid medium by using an automatic bacteria picking instrument.

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