Culture medium for high-throughput separation of crop rhizobacteria, and preparation method therefor and use thereof

Abstract

A culture medium for high-throughput separation of crop rhizobacteria, and a preparation method therefor and the use thereof. The culture medium comprises a 1 / 10 TSB culture medium and an additive solution of the culture medium, and the additive solution of the culture medium is prepared by means of mixing a crop plant with a TSB culture medium, and pulverizing, fermenting, filtering and sterilizing the mixture. With regard to the culture medium, on the basis of a multispecies interaction theory in rhizosphere microbiome and by means of using the interaction network among multiple microorganisms, crop-derived nutrient components are fermented with in-situ rhizosphere microbiome, and the fermentation product is used as the carbon source and nitrogen source for the separation of rhizosphere microorganisms, thereby simulating the in-situ environment of rhizosphere microorganisms to the maximum, and obtaining more microbial species. High-throughput sequencing reveals that the culture medium can obtain more than 60% of bacterial species compared with an existing culture medium.

Description

Culture medium for high-throughput separation of crop rhizosphere bacteria, preparation method, and application thereof Technical Field

[0001] The present invention relates to the technical field of environmental microbiology, and in particular to a culture medium for high-throughput separation of crop rhizosphere bacteria, and a preparation method and application thereof. Background Art

[0002] The crop rhizosphere provides an important habitat for a large number of microorganisms. In particular, the secretions produced by the roots can serve as carbon and nitrogen sources for a variety of microorganisms. Therefore, a large number of microorganisms, such as filamentous fungi, yeasts, actinomycetes and bacteria, are enriched in the crop rhizosphere. These microorganisms are collectively referred to as the rhizosphere microbiome. They play an important role in promoting crop nutrient absorption, improving crop stress resistance, and promoting yield increases.

[0003] In recent years, with the development of high-throughput sequencing technology, researchers have gained a deeper understanding of rhizosphere microbial diversity. In particular, metagenomics has enabled a deeper understanding of the functions of the rhizosphere microbiome. As a vital resource for crop probiotic strains, a growing number of researchers are using high-throughput sequencing data to isolate rhizosphere bacteria, hoping to identify beneficial strains suitable for field cultivation, thereby alleviating pests and diseases and increasing crop yields. Currently, the most widely used high-throughput method is the limiting dilution-based method for isolating rhizosphere bacteria developed by Liu Yongxin et al. (Zhang, J., Liu, YX., Guo, X. et al. High-throughput cultivation and identification of bacteria from the plant root microbiota. Nat Protoc 16, 988–1012 (2021)). Furthermore, Chinese patent CN111518729A discloses a high-throughput isolation and cultivation method for crop root microbiomes. This method can isolate and cultivate large numbers of bacteria in a short period of time with minimal labor, rapidly identify bacterial species through a one-click sequence analysis process, and assemble crop root microbiomes, providing technical support for obtaining a wider range of crop root microbial resources. However, the rice root bacteria isolated and cultured using this method only account for approximately 60% of the total rice root bacterial species, and the Arabidopsis root bacteria isolated and cultured account for approximately 58% of the Arabidopsis root bacterial species. To increase the number of isolated rhizosphere bacterial species, the development of new culture media to meet the requirements of high-throughput isolation of rhizosphere microorganisms is urgently needed. Summary of the Invention

[0004] In order to increase the number of crop rhizosphere microorganism species obtained using a high-throughput separation and culture method, the present invention provides a culture medium for high-throughput separation of crop rhizosphere bacteria, a preparation method thereof, and an application thereof.

[0005] In a first aspect, the present invention provides a culture medium for high-throughput isolation of crop rhizosphere bacteria, comprising a 1 / 10 TSB culture medium and a culture medium additive; the culture medium additive is prepared by mixing crop plants with TSB culture medium, crushing, fermenting, filtering and sterilizing, and the crop is cucumber.

[0006] Furthermore, the ratio of 1 / 10 TSB medium to medium supplement was 1:1.

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned culture medium, comprising: preparing an agar aqueous solution and sterilizing it at high temperature; mixing 1 / 10 TSB culture medium, culture medium additive and agar aqueous solution to a final agar concentration of 1.2%, and preparing a culture medium plate after condensation.

[0008] Furthermore, the preparation method of the culture medium supplement specifically includes:

[0009] The whole plant of the crop to be separated is mixed with 1 / 10 TSB medium at a mass ratio of 1:1-10 and crushed, and then fermented at 4-25°C for 12 hours. The fermented product is filtered and sterilized to obtain the product.

[0010] Furthermore, different types of antibiotics are added to the culture medium according to conventional methods to inhibit the growth of non-target microbial groups. Commonly used antibiotics include nystatin, cycloheximide, penicillin, tetracycline, streptomycin sulfate, ampicillin, etc.

[0011] In a third aspect, the present invention provides an application of the above-mentioned culture medium in high-throughput separation and cultivation of crop rhizosphere microorganisms.

[0012] Furthermore, under sterile conditions, the crop root samples were ground to obtain a homogenate, which was fully diluted with the above-mentioned culture medium and then transferred to a 96-well plate for culture until the number of turbid wells in the 96-well plate no longer increased. The genomic DNA of the microorganisms in the turbid wells was extracted and subjected to high-throughput sequencing analysis to obtain the crop root microbiome.

[0013] Furthermore, the dilution factor was such that the number of turbid wells in the 96-well plate was ultimately 30-35.

[0014] Furthermore, the specific operation of the homogenate is as follows: collect crop root samples, rinse the roots with sterile water, transfer the roots to a new centrifuge tube, and add 30 mL of 10 mM magnesium chloride solution; place the centrifuge tube on a shaker, wash it repeatedly 3 times, take it out and absorb the liquid on the root with sterile filter paper; cut the roots into pieces with sterile scissors and mix them evenly, weigh 0.02 g of root tissue and put it into a 1.5 mL centrifuge tube; add 1 mL of sterile magnesium chloride solution to the 1.5 mL centrifuge tube, use 500 μL of magnesium chloride solution to grind the roots, use 500 μL of magnesium chloride solution to rinse the grinding rod, and grind it into a homogenate; transfer the homogenate to a 50 mL centrifuge tube containing 25 mL of 10 mM magnesium chloride, mix well and set aside.

[0015] Furthermore, the culture supplement was prepared by mixing 100 mL of 1 / 10 TSB medium with 65 g of cucumber seedlings with roots, which were fully crushed, fermented at 10° C. for 12 h, and filtered for sterilization.

[0016] The beneficial effects of the present invention are:

[0017] The culture medium for high-throughput isolation of crop rhizosphere bacteria provided by the present invention is based on the theory of multi-species interactions in the rhizosphere microbiome, utilizes the interaction network between multiple microorganisms, ferments crop-source nutrients using the in situ microbiome in the rhizosphere, and uses the fermentation products as the carbon and nitrogen sources for the isolation of rhizosphere microorganisms, maximally simulating the in situ environment of rhizosphere microorganisms, thereby obtaining more microbial species.

[0018] Compared with existing culture media, the culture medium provided by the present invention can obtain more types of bacterial phyla. Combined with high-throughput separation methods, it can obtain more than 60% of the bacterial species obtained by high-throughput sequencing, while conventional isolation and culture methods can only obtain 10% to 15% of the bacterial species. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0020] FIG1 is a photograph of the colony growth of cucumber rhizosphere bacteria after being isolated using different culture medium plates in Test Example 1 of the present invention.

[0021] Figure 2 is a graph showing the colony count and species number of cucumber rhizosphere bacteria isolated using different culture medium plates in Test Example 1 of the present invention; Figure A shows the colony count results of cucumber rhizosphere bacteria isolated using different culture medium plates; Figure B shows the species number results of cucumber rhizosphere bacteria isolated using the same culture medium plates; in the box plots, those with the same marked letters indicate no significant difference, and those with different marked letters indicate significant difference.

[0022] FIG3 is a community composition diagram obtained by collecting plate colonies and performing high-throughput sequencing in Test Example 1 of the present invention.

[0023] FIG4 is a graph showing the genus-level quantitative results of cucumber rhizospheric bacteria obtained by high-throughput separation and culture in Test Example 2 of the present invention. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention. Example

[0025] 1. Preparation of culture medium supplement

[0026] Collect the cucumber seedlings with roots, shake off the soil on the roots, and rinse the roots with sterile water to wash off the attached soil; mix 50 g of the washed cucumber seedlings with roots with 100 mL of 1 / 10 TSB medium, fully crush, and ferment at 10°C for 12 hours. Filter the fermentation liquid through 7-layer gauze folded back, then filter three times through a single-layer filter paper, and finally filter the filtrate through a 0.22 μm sterile filter to obtain the culture medium supplement A.

[0027] 2. Preparation of culture medium

[0028] Prepare an agar aqueous solution and sterilize at high temperature; mix 1 / 10 TSB medium, culture medium supplement A, and the agar aqueous solution so that the ratio of 1 / 10 TSB medium to culture medium supplement A is 1:1 and the final agar concentration is 1.2%. After condensation, prepare culture medium plate A.

[0029] The difference from Example 1 is that the weight of the cucumber seedlings with roots, fermentation conditions and fermentation time used in Examples 2-6 are shown in Table 1. Other treatments are the same as in Example 1. The culture medium supplement BF is prepared, and then the culture medium plate BF is prepared.

[0030] Table 1 Raw material dosage and fermentation conditions of culture medium additives in Examples 1-6

[0031] Examples Name of culture medium additive 1 / 10 TSB medium (mL) Weight of cucumber seedlings with roots (g) Fermentation temperature (°C) Fermentation time (h) 1A 10050 10122 B 10050 4123 C 10050 25 124 D 10065 10125 E 100120 10126 F 10025 1012

[0032] Test Example 1: Isolation and Culture of Cucumber Rhizosphere Bacteria

[0033] (1) The experiment requires a total of 8 culture medium plates: one is a 1 / 10 LBA culture medium plate, one is a 1 / 10 TSA culture medium plate with an agar concentration of 1.2%; the remaining six culture media are culture medium plates A to F prepared in Examples 1-6, respectively.

[0034] (2) Sampling: Cucumber plants that had been colonized for 60 days were collected in a greenhouse. After collection, the above-ground parts of the plants were cut off, and the soil attached to the roots was shaken off. 10 g of main roots, lateral roots, and adventitious roots were cut and placed in a sterile 50 mL centrifuge tube. After each plant was collected, the scissors were disinfected with 75% ethanol and rinsed three times with sterile water before use. After collection, the centrifuge tubes containing the samples were placed in an ice box and transported to the laboratory. 35 mL of sterile 1× PBS buffer was added to each sample tube, and the tubes were placed in a shaker at 180 r / min for 20 minutes. The surface moisture of the roots was then absorbed with sterile filter paper. This was repeated three times.

[0035] (3) Isolation and culture: Accurately weigh 0.02 g of the cleaned root sample, add 1 mL of sterilized 1× PBS buffer and grind thoroughly, then use 1× PBS buffer to dilute 10 times to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 25 μL of each concentration was pipetted with a pipette and dropped onto 8 culture medium plates, and immediately spread evenly with a sterile triangular glass coating rod. After the bacterial solution was fully absorbed, the plates were sealed with a sealing film, inverted and cultured at 28°C for 36 hours, and the number of colonies on each plate was counted. Six plates were coated with each culture medium for each dilution concentration. Among them, under the condition of 1000-fold dilution, the colony growth status on 8 culture medium plates is shown in Figure 1. The number of colonies grown on culture medium plate AF was significantly more than that on 1 / 10 LBA culture medium and 1 / 10 TSA culture medium. The plate counting results of 1000-fold dilution are shown in Table 2 and Figure 2A. It can be seen that the number of colonies grown on different culture media is significantly different (F 7.16 =15.247, p<0.001), the number of colonies grown on culture plate D was the largest.

[0036] Table 2 Number of colonies grown on different culture medium plates

[0037] Culture medium ABCDEF1 / 10 LBA1 / 10 TSA Colony count 96.6±7.1105.3±3.597.0±1.7127.3±4.5113.0±13.084.6±0.579.0±9.074.3±12.8

[0038] (4) Species identification: Select colonies with different colors, sizes, thickness, transparency and textures, and purify and culture them on TSA medium using the three-zone streak method with an inoculation loop. After obtaining the strain, streak it three times to obtain a pure strain. Group the strains according to colony morphology. Select representative strains and inoculate them into test tubes containing 2 mL of TSB liquid medium. Incubate at 28°C and 180 rpm for 12 hours. Then transfer 1 mL of bacterial liquid to a 2 mL centrifuge tube, centrifuge at 13,000 rpm for 2 minutes, collect the bacteria, extract DNA using a bacterial genome extraction kit, and then amplify the bacterial 16S rRNA gene using 27F and 1492R. PCR reaction system (25 μL): 1 μL DNA (20 ng), 0.5 μL of each primer, 12.5 μL 2× Taq PCR Buffer, 10.5 μL ddH2O. PCR amplification protocol: 95°C for 5 min; 94°C for 30 s, 52°C for 30 s, 72°C for 90 s, 35 cycles; 72°C for 10 min. PCR products were analyzed by 1.2% agarose gel electrophoresis, and samples with fragments approximately 1500 bp were sequenced at Sangon Biotech (Shanghai) Co., Ltd. using the Roche 454 platform. The resulting sequences were compared with closely related strains from GenBank for bacterial species identification. The bacterial species isolated from each plate were counted, as shown in Table 3 and Figure 2B. Plate D showed the highest number of bacterial species.

[0039] Table 3 Number of bacterial species grown on different culture medium plates

[0040] Culture medium ABCDEF1 / 10 LBA1 / 10 TSA Number of bacterial species 17.6±2.113.0±2.024.0±2.028.0±1.723.0±1.017.6±3.28.6±0.57.0±1.7

[0041] (5) High-throughput amplicon sequencing detection

[0042] All dilution plates containing colonies (D) and 1 / 10 TSA plates were rinsed thoroughly with 3 mL of sterile water. Bacteria on the surface were collected and mixed. Total DNA was extracted and amplified using specific primers 799F and 1193R to amplify the V5-V7 region of the rhizospheric bacterial 16S rRNA. A barcode sequence (provided by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.) was added to the 5' end of the PCR product. The PCR reaction system (50 μL) consisted of 5 μL (10 ng) DNA template, 1.5 μL of each primer, 5 μL of 2 mmol / L dNTPs, 2 μL of MgSO₄, 5 μL of 10× KOD Buffer, 1 μL of KOD Plus, and ddH₂O to make up to 50 μL. The PCR amplification program was as follows: 94°C for 2 min, 30 cycles of 94°C for 30 s, 63°C for 30 s, and 68°C for 30 s, and 68°C for 5 min.

[0043] Each sample was run in triplicate. The PCR products from the three replicates were mixed and analyzed by 2% agarose gel electrophoresis. The PCR products were excised and recovered using the AxyPrep DNA Gel Extraction Kit and sent to Shanghai Major Biopharmaceutical Technology Co., Ltd. for library construction and sequencing on an Illumina Miseq PE250 platform. Sequencing data were analyzed using the Shanghai Major Biopharmaceutical Technology Co., Ltd. Bioinformatics Cloud platform (www.majorbio.com). Quality control and data analysis were performed on the cloud platform using the QIIME v.1.9.1 (Quantitative Insights Into Microbial Ecology) software package. Sequences shorter than 200 bp, containing ambiguous bases, and containing primer mismatches of more than two bases were removed from the sequencing data before data processing. After screening, OTU clustering was performed using UPARS in the USEARCH software package at a 97% similarity threshold. The most abundant sequences were selected as representative OTUs and taxonomic analysis was performed using RDP classifier v.2.2. Taxonomic annotation was performed using the SILVA database (http: / / www.arb-silva.de) with a confidence threshold of 70%. OTU composition and read counts were calculated for each sample at different taxonomic levels. Due to limitations in the sequencing platform and annotation database, some sequences could not be annotated to species in this experiment. Therefore, a maximum likelihood tree of species composition was constructed at the genus level using FastTree 2.1.3 based on the sequence information. The read counts at the genus level were used as species abundance. The final results are shown in Figure 3. A total of 29 bacterial genera were detected on medium plate D, and 18 bacterial genera were detected on the 1 / 10 TSA medium plate.

[0044] Test Example 2 High-throughput Isolation, Cultivation, and Identification of Cucumber Root Bacteria

[0045] Collect cucumber root samples, rinse the roots with sterile water, transfer the roots to a new centrifuge tube, and add 30 mL of 10 mM magnesium chloride solution; place the centrifuge tube on a shaker and wash it repeatedly three times. After taking it out, use sterile filter paper to absorb the liquid on the roots; cut the roots into pieces with sterile scissors and mix them evenly. Weigh 0.02 g of root tissue and place it in a 1.5 mL centrifuge tube; add 1 mL of sterile magnesium chloride solution to the 1.5 mL centrifuge tube, use 500 μL of magnesium chloride solution to grind the roots, and use 500 μL of magnesium chloride solution to rinse the grinding rod and grind until it becomes a homogenous slurry; transfer the homogenate to a 50 mL centrifuge tube containing 25 mL of 10 mM magnesium chloride and mix well for later use.

[0046] This experiment set up three groups, namely control group 1, control group 2 and experimental group, with 3 replicates in each group and the average value was taken. The treatment methods for each group are as follows:

[0047] ① Control group 1: extract the total DNA from the homogenate, and then detect the number of bacterial species in the cucumber rhizosphere sample according to the method of "(5) High-throughput amplicon sequencing detection" in Test Example 1, which serves as control group 1 (i.e., Amplicon sequencing in Figure 4);

[0048] ② Control group 2: The homogenate was fully diluted with 1 / 10 TSB medium and then transferred to a 96-well plate for incubation until the number of turbid wells in the 96-well plate no longer increased. The dilution factor was such that the number of turbid wells in the 96-well plate finally reached 30-35. Genomic DNA of the microorganisms in the turbid wells was extracted, and the 16S rRNA gene of the isolated and cultured bacteria was amplified using double-flanked PCR with high throughput identification to obtain the cucumber root microbiome (i.e., 1 / 10 TSB in Figure 4).

[0049] ③ Experimental group: The homogenate was fully diluted using medium plate D prepared in Example 4 and then transferred to a 96-well plate for culture until the number of turbid wells in the 96-well plate no longer increased. The dilution factor was such that the final number of turbid wells in the 96-well plate was 30-35. Genomic DNA of the microorganisms in the turbid wells was extracted, and the 16S rRNA gene of the isolated and cultured bacteria was identified by high-throughput double-sided tag PCR amplification to obtain the cucumber root microbiome (i.e., D in Figure 4).

[0050] The high-throughput isolation and testing methods of cucumber rhizospheric bacteria in the control group 2 and the experimental group were specifically referred to the method of Liu Yongxin et al. (Zhang, J., Liu, Y.-X., Guo, X., Qin, Y., Garrido-Oter, R., Schulze-Lefert, P. and Bai, Y. (2021). High-throughput cultivation and identification of bacteria from the plant root microbiota. Nat Protoc 16(2): 988-1012.).

[0051] The results of the above three groups are shown in FIG4 . At the genus level, a total of 100.3 bacterial genera were detected using the high-throughput amplicon sequencing method, a total of 39.0 bacterial genera were isolated using the 1 / 10 TSB medium, and a total of 62.3 bacterial genera were isolated using the medium plate D prepared in Example 4. The number of bacterial species detected by the three methods was significantly different (F 2,6 =79.34, p<0.001). The high-throughput amplicon sequencing method can detect almost all bacterial species in the sample, but cannot obtain pure strains. The number of species isolated using the gradient dilution method combined with 1 / 10 TSB medium was significantly lower than that of the technical solution of the present invention, and the number of bacterial species isolated by the technical solution of the present invention was 59.7% higher.

[0052] Although the present invention has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A culture medium for high-throughput separation of crop rhizosphere bacteria, characterized in that: The method comprises 1 / 10 TSB culture medium and culture medium additive; the culture additive is prepared by mixing 100 mL 1 / 10 TSB culture medium with 65 g cucumber seedlings with roots, which are fully crushed, fermenting at 10° C. for 12 hours, and filtering and sterilizing; and the crop is cucumber.

2. The culture medium according to claim 1, characterized in that The mass ratio of 1 / 10 TSB medium to medium supplement is 1:

1.

3. A method for preparing the culture medium as claimed in claim 1, characterized in that: include: Prepare an agar aqueous solution and sterilize it at high temperature; mix 1 / 10 TSB culture medium, culture medium additive and agar aqueous solution to obtain a final agar concentration of 1.2%; and prepare a culture medium plate after condensation.

4. The preparation method according to claim 3, characterized in that: Antibiotics are added to the culture medium to inhibit the growth of non-target microbial species.

5. A use of the culture medium as claimed in claim 1 in high-throughput separation and cultivation of crop rhizosphere microorganisms, characterized in that: The crop is cucumber.

6. The use according to claim 5, characterized in that Under sterile conditions, the crop root sample is ground to obtain a homogenate, the homogenate is fully diluted with the culture medium as described in claim 1 and then transferred to a 96-well plate for culture until the number of turbid wells in the 96-well plate no longer increases, the genomic DNA of the microorganisms in the turbid wells is extracted, and high-throughput sequencing analysis is performed to obtain the crop root microbiome.

7. The use according to claim 6, characterized in that The dilution factor is such that the number of turbid wells in the 96-well plate will eventually be 30-35.

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