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Aug 2020

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Measurements of Root Colonized Bacteria Species
根定植细菌种类的测量   

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Abstract

Root-associated bacteria are able to influence plant fitness and vigor. A key step in understanding the belowground plant-bacteria interactions is to quantify root colonization by the bacteria of interest. Probably, genetic engineering with fluorescence markers is the most powerful way to monitor bacterial strains in plant. However, this could have some collateral problems and some strains can be challenging to label. In this sense, bacterial inoculation under properly controlled conditions can enable reliable and reproducible quantification of natural bacterial strains. In this protocol, we describe a detailed procedure for quantification of root-associated bacteria. This method applies non-aggressive samples processed with morphological identification and PCR-based genetic fingerprinting. This easy-to-follow protocol is suitable for studying bacterial colonization of plants grown either in artificial medium or in soil.

Keywords: Plant-bacteria interaction (植物与细菌的相互作用), Rhizobacteria (根际细菌), PGPR (植物根际促生菌), Pathogen (病原体), Colonization (定植), Root (根), Soil (土)

Background

Plants naturally live with various soil bacteria in the rhizosphere, which refers to a thin layer of soil adhering to the roots. While some rhizobacteria have no observable effects on plants, others are either pathogens that cause detrimental effects or growth-promoting rhizobacteria (PGPR) that promote plant vigor (Mendes et al., 2013; Olanrewaju et al., 2019). The capacity of bacterial pathogens or PGPR to impact plant growth is tightly correlated with their level of bacterial root colonization. Therefore, the investigation of bacterial root colonization is an important stepping stone to understanding the belowground plant-bacteria interactions.


The abundance of bacteria strain can be assessed by visualization of fluorescence signals by modifying them to express a transgenic marker gene encoding the fluorescent protein such as GFP (Rochat et al., 2010; Krzyzanowska et al., 2012; Saad et al., 2018). The abundance of bacteria strain can also be measured by PCR-based amplification of the bacterial genomic DNA (Maciá-Vicente et al., 2009; Mendis et al., 2018). These two methods detect the bacterial strain of interest regardless of the presence or absence of other bacterial species; nevertheless, both methods have their potential limitations. The fluorescence-assisted detection may be limited by technical difficulties during genetic transformation, by the possibility that the introduction of the transgene may interfere with wild-type bacterial behavior, and by the risks posed to environmental safety. On the other side, PCR-based bacterial detection requires that levels of the target DNA templates be above the PCR detection limits, and that the target sequence be specific enough to represent the bacterial strain of interest only.


In addition to the fluorescence-based and the PCR-based methods, root-associated bacteria can also be quantified by in vitro culturing. This increases the detection limits, followed by counting the colony-forming units (CFUs), if the bacterial strain of interest is culturable with an artificial growth medium. With proper experimental setups, the method of counting CFUs is efficient, affordable, reliable, and reproducible for quantification of root-associated bacteria. By using Arabidopsis thaliana and B. megaterium YC4-R4 as a model system for studying plant-bacteria interactions, herein we describe a protocol for bacteria quantification based on counting CFUs from in vitro bacterial cultures. This protocol, which includes an optional genetic fingerprinting step by Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR, has been applied to different bacterial species in studies where plants were either grown in artificial medium or in soil, and under control or stress conditions (Vílchez et al., 2020). In addition to studying root-associated bacteria, this methodology can be applied to studies involving other types of plant organs or other types of bacteria-host systems under optimized conditions.


Materials and Reagents

  1. 1.5 ml Eppendorf tubes (ShangYu Yite Plastic Co., Ltd, catalog number: MCTB015, or similar)

  2. 15 ml tubes

  3. Surgical blade and scalpellum (Qingdao Sinoland International Trade Co., Ltd, catalog number: SS-002021, or similar)

  4. Circular-shape (9 × 1.5 cm) and square-shape (56 × 35 × 30 cm) Petri dishes (ShangYu Yite Plastic Co., Ltd, catalog number: PD0009, or similar; Haimen Laiboreike Experiment Instrument Manufacturing Co., Ltd, catalog number: LB077, or similar)

  5. Disposable tissue grinder plastic pestle for 1.5 ml tubes (Corning, Axygen®, catalog number: PES-15-B-SI, or similar)

  6. Inoculation Loops (Renon Laboratory Experiment Co., Ltd, catalog number; 52150000 or similar)

  7. Tape (3M MicroporeTM, 1530C-0, or similar)

  8. Seeds of Arabidopsis thaliana

  9. Murashige and Skoog Basal Salt Mixture (MS) (Sigma-Aldrich, catalog number: M5524)

  10. Tryptone (Sigma, catalog number: T7293)

  11. Yeast extract (Sigma, catalog number: Y1625)

  12. NaCl (Sangon Biotech, catalog number: A501218)

  13. NaOH or KOH (Sangon Biotech, catalog numbers: A100583 and A610441)

  14. Agar (Sigma, catalog number: L2897)

  15. Agarose (Sigma, catalog number: A9539)

  16. RedSafeTM Nucleic Acid Staining Solution (Invitrogen, catalog number: S33102)

  17. 2K Plus Ladder (Transgenbiotech, Trans2K® Plus DNA Marker, catalog number: BM121)

  18. Double-distilled sterilized H2O

  19. 100% Ethanol (Sangon Biotech, catalog number: A500737)

  20. Bleach (commercial 5% NaClO diluted 1:5 with water)

  21. DNA extraction kit (Qiagen DNA Blood&Tissue, Qiagen, catalog number: 69504 or similar)

  22. Master Mix solution for PCR (Transgenbiotech, 2× EasyTaq® PCR SuperMix, AS111-01)

  23. Enterobacterial Repetitive Intergenic Consensus fingerprinting and 16S rRNA PCR amplification primers:

    ERIC (5’-ATGTAAGCTCCTGGGGATTCAC-3’)

    27F (5’-AGAGTTTGATCMTGGCTCAG-3’)

    1492R (5’-TACGGYTACCTTGTTACGACTT-3’)

Equipment

  1. Stainless steel tweezer with long fine point (Labdirect, catalog number: SA-Q2090 or similar)

  2. Analytical balance (Sartorius, Practum224-1S or similar)

  3. Mechanical pipettes (Eppendorf Research Plus, P1000, P100 and P10, or similar)

  4. Benchtop vortex mixer (DragonLab MX-S, or similar)

  5. Benchtop centrifuge (Eppendorf, Centrifuge 5810R with A-4-62 Model Rotor, or similar; up to 4,000 rpm/3,220 × g)

  6. Clean bench (BCM-1000A Biological Clean Bench, Airtech, or similar)

  7. Shaker incubator (Eppendorf, New BrunswickTM I26 Stackable Incubator Shakers, or similar)

  8. Rolling incubator (Kylin-Bell Lab Instruments Co., Ltd., Qilinbeier QB-128, or similar)

  9. Autoclave

  10. Pasteur oven

  11. Plant growth chamber (Percival CU36L5 or similar) or growth room.

  12. Culture oven (Shanghai JingHong DHG-9038A, or similar)

  13. Electrophoresis devices (Bio-Rad, PowerPacTM Basic Power Supply + Wide Mini-Sub Cell GT Cell, or similar)

  14. A gel imaging system (Bio-Rad, ChemiDoc XRS+TM Gel Imaging System, or similar)

  15. Thermocycler (Bio-Rad, T100 Thermal Cycler, or similar)

Procedure

  1. Bacterial culture using B. megaterium YC4-R4 as an example

    1. Refresh YC4-R4 from glycerol stock on LB-Agar plate and incubate at 37 °C for 24 h*/**.

    2. Take a single colony to inoculate 5 ml LB medium (in vitro) (recommended 15 ml tubes). Incubate overnight in a shaker (220 rpm, 37 °C). For soil inoculation, scale up by using 1 ml of pre-culture per liter of fresh culture (final volume depends on soil initial moisture and pot volume)*.

    3. Prepare 0.45% NaCl solution by mixing 4.5 g per liter of ddH2O. Autoclave for 20 min at 121 °C. Cool to room temperature before use.

    4. Centrifuge the culture at 3,220 × g for 10 min. Resuspend the pellet in the same volume of 0.45% NaCl. OD600 ~0.8-1.1 (log growth phase; generally, equals to 106-108 CFU/ml) is recommended.

    Notes:

    1. *This step shall be done under sterile conditions.

    2. **For tests using antibiotic-resistant strains, LB plates may be prepared with the appropriate antibiotics.


  2. Prepare bacteria growth medium

    1. Mix 10 g of tryptone, 5 g of yeast extract, and 10 g NaCl per liter of ddH2O.

    2. For making LB agar plates, add 15 g agar per liter.

    3. Autoclave for 20 min at 121 °C. For LB agar plates, pour the liquid medium into Petri dishes and allow it to cool down and solidify.


  3. Prepare plant growth medium

    1. Mix MS powder in half strength (2.21 g) and agar (0.7% and 1% for circular and square plates, respectively, depending on test requirements) per liter of ddH2O.

    2. Adjust pH to 5.7 with NaOH or KOH.

    3. Autoclave for 20 min at 121 °C. Pour the medium into the Petri dishes and allow it to cool down and solidify.


  4. Seed sterilization and plant growth conditions

    1. Mix seeds with absolute ethanol in 1.5 ml tubes and keep in agitation for 1 min. For 100 seeds, 1 ml of ethanol is recommended.

    2. Discard ethanol and add 20% bleach solution (from commercial bleach stock). For 100 seeds, 1 ml of 20% bleach solution is recommended. Agitate in a spinner wheel for 10-15 min.

    3. Discard bleach and wash three times with sterile ddH2O in a clean bench*.

    4. Plant the seeds on MS medium one by one with the aid of pipet tips*.

    5. Stratify the seeds at 4 °C for 48 h. After stratification, place the plates vertically in the growth chamber for 14 days under the following conditions: 22 °C; 12 h light/12 h dark cycle; 40% relative humidity; and up to 155 µmol·m−2s−1 light intensity.

      Note: *This step shall be done under sterile conditions.

  5. In vitro colonization test

    1. Under sterile conditions, place 15-20 14-days-after-germination (DAG) seedlings grown in ½ MS 1% agar (in squared Petri plates) per replica in a 1.5 ml sterile tube (volume of sterile tube depends on plant size). Add 1 ml of bacteria in 0.45% NaCl solution.

    2. Incubate the tube overnight on a shaker (220 rpm, 26 °C*).

    3. Discard the bacterial solution. Place the seedlings on a sterile surface, such as sterilized aluminum foil or paper, or a Petri dish. Cut roots with sterile razor blades and keep them in 1.5 ml tubes.

    4. Sterilize the root surface by adding 1 ml of 75% ethanol, treating for 7-10 min with continuous agitation (spin wheel or equivalent).

    5. Discard the ethanol and wash the roots for three times with sterile ddH2O.

    6. Discard the ddH2O and grind roots with sterile pistils (for 1.5 ml tubes).

    7. Add 1 ml of 0.45% NaCl to the tube, homogenize by vortexing, and prepare serial dilutions (usually 5 times 10-fold dilution should be enough).

    8. Plate drop by drop (10 drops of 10 µl approx. up to a total volume of 100 µl) each dilution in LB-agar plates, let them dry for 2 min and incubate at 30 °C** for 24 h (Figure 1).



      Figure 1. Example of drop-by-drop seeding of a serial dilution in a quarter-divided growth plate. Drops are carefully placed on the dry growth medium plate to avoid interferences.


    9. Keep and centrifuge the original solution at the maximal speed. Once root debris is pelleted, discard the supernatant and fully dry at 60 °C in an oven for 48 h and record this weight as the dry weight (DW), by using Pasteur oven.

    10. Count bacteria colony numbers and calculate the colony forming units (CFUs) by taking into account the dilution and plated volume. Normalize the CFU values with root DW to obtain the colonization rates. Colonization rate = (CFUs × dilution factor)/mg of root DW.

    Notes:

    1. All work shall be done under sterile conditions.

    2. *This temperature has been tested to ensure non-stressing conditions for plants; at the same time, bacteria growth under the same temperature should also be considered to avoid imposing stress to the bacteria.

    3. **The temperature used herein is optimal for colony-counting of the example bacteria. It is highly recommended to optimize the temperature for different strains in order to avoid overgrowth/overlapped colonies.


  6. Determination of colonization ratio in soil-grown plants

    1. Grow Arabidopsis seedlings as described above.

    2. Prepare soil based on the conditions required. In our tests, soil is prepared by meshing a mix 1:3 (v:v) of vermiculite and soil substrate. To minimize unwanted microbes in the soil, the soil needs to be tyndalized (75 °C for 3 h + cooling interval for 10 h + 75 °C for another 3 h). Transfer 5 DAG seedlings to the soil.

    3. Inoculate with bacteria in 0.45% NaCl solution. As reference, in a regular 0.4 L pot, inoculate 50 ml of bacteria solution around planted seedlings.

    4. Allow the plants to grow under conditions as required by the desired tests (e.g., with or without certain abiotic stress).

    5. Harvest the roots (usually 20-40 mg of roots) at the time when colonization quantification is desired. For root harvesting, manually detach the soil around each root (with the help of tweezers) and then use a soft tissue to detach the smallest and most adhered pieces of soil. Fine soil particles must be removed by 3 × ddH2O washing*. Once roots are clean, follow indications for sterilizing, grinding, preparing dilutions, and counting CFUs in Part E.

    Note: *This step shall be done under sterile conditions.


  7. Strain isolation and identification

    1. Although the soil was tyndalized, typically Part F will yield a small portion of bacteria other than the inoculated strain. To confirm the identity of the inoculated strain and to calculate its proportion in the total bacterial population, first classify bacterial colonies by morphology (use https://microbeonline.com/colony-morphology-bacteria-describe-bacterial-colonies/, as reference), then use sterile toothpicks to pick single colonies with similar morphology and transfer them to liquid LB medium for culturing. Usually, 20-30 colonies should be randomly sampled to avoid bias (Figure 2).



      Figure 2. An example of colonies with different morphologies in a regular root colonization quantification process. Different morphologies were selected by color, size, or brightness of the colonies. The colour of the arrows indicates a pre-group of strains: according to the following criteria: red – intense yellow color; blue – white color, big size; orange – white color, small size; and green – beige color, matte surface.


    2. Culture the individual colonies in 5 ml of liquid LB on a shaker (220 rpm, 30 °C) for 24 h.

    3. Centrifuge the culture (suggestion in bench centrifuge: 3,220 × g, 10 min), discard supernatant, and extract DNA from pellets by using a high-performance method. Here, a DNA extraction kit is suggested (see methods).

    4. Amplify and sequence 16S rRNA genes with the 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) (or equivalent), and BLAST the sequence to identify isolated species.

    5. To further distinguish the inoculated strain from other strains of the same species, perform PCR with ERIC primer (5’-ATGTAAGCTCCTGGGGATTCAC-3’) to generate fingerprinting phenotype patterns for each strain. Use the stock strains employed in the test as the reference. Run the samples in a 2% agarose gel to maximize band separation. Use GelJ to compare the patterns with the reference and obtain similarity indexes. Figure 3 includes a representative gel image output.



      Figure 3. Example of a representative ERIC-PCR gel image output (modified from Vílchez et al., 2020). MWM: molecular weight marker; Strains 1 to 4 were recovered from roots in inoculated soil; Inoc. Strain: originally soil-inoculated strain.

Data analysis

Each test should be carried out with at least three replicates per condition prepared as indicated above (in vitro or in soil). Results will be considered statistically different when P-value 0.05 according to Student’s t-test. The relative colonization rates are calculated as: RC = (colony numbers × dilution factor)/mg of root DW.

Notes

  1. If applying this method to other plant species, scale the recipe to fit the plant size. For grinding samples, it is recommended to use 1.5 ml tubes and the matching pistils. Automatic grinding may be performed at low vibration frequencies. In the case of using mechanical disruption devices such as TissueLyser, use low frequencies of disruption in order to avoid overheating and mechanical stress that may cause disruption of bacterial membranes. Mechanical disruption at high frequencies is used to extract total DNA from soil, sludge, or other complex samples. Aggressive grinding/disruption methods increase the likelihood of introducing biases in the final counting and identification of bacterial strains.

  2. The ethanol-based sterilization process has been tested in roots of Arabidopsis and tomato as the reference plants. In the case of Arabidopsis (Figure 4), 7-10 min (about 5 min for longer and thicker roots, as in tomato) was assessed as optimal root surface sterilization time (these tests were performed with Gram+ bacteria; consider shorter time for Gram-). Longer incubation periods could interfere with the quantification of the colonizing bacteria.



    Figure 4. Colony counts after ethanol treatment in Arabidopsis roots. Colony forming units (CFUs) counted in the last wash solution after treatment with ethanol (blue columns) and respective root colonization counting (red columns). The X-axis indicates ethanol treatment time. n=3, data are represented as mean ± SE.


  3. Nevertheless, it should be noted that this set up is for Arabidopsis plants. Sterilization time depends on the type of plant and bacterial population tested, so it is highly recommended to optimize sterilization time depending on the actual experimental set up.

Acknowledgments

Research in the laboratory of H.Z. has been supported by the Chinese Academy of Sciences (CAS). J.I.V. was supported by the CAS President’s International Fellowship Initiative fellowship. We thank Prof. Rosa Lozano-Duran at PSC and the anonymous reviewers for critical reading of the protocol.

Competing interests

The authors declare no competing interests.

References

  1. Krzyzanowska, D., Obuchowski, M., Bikowski, M., Rychlowski, M., and Jafra, S. (2012). Colonization of potato rhizosphere by GFP-tagged Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44 shown on large sections of roots using enrichment sample preparation and confocal laser scanning microscopy. Sensors 12 (12):17608-17619.
  2. Maciá-Vicente, J. G., Jansson, H. B., Talbot, N. J., and Lopez-Llorca, L. V. (2009). Real-time PCR quantification and live-cell imaging of endophytic colonization of barley (Hordeum vulgare) roots by Fusarium equiseti and Pochonia chlamydosporia. New Phytol 182(1): 213-228.
  3. Mendes, R., Garbeva, P., and Raaijmakers, J. M. (2013). The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol Rev 37(5): 634-663.
  4. Mendis, H. C., Thomas, V. P., Schwientek, P., Salamzade, R., Chien, J. T., Waidyarathne, P., Kloepper, J. and De La Fuente, L. (2018). Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions. Plos One 13(2): e0193119.
  5. Olanrewaju, O. S., Ayangbenro, A. S., Glick, B. R. and Babalola, O. O. (2019). Plant health: feedback effect of root exudates-rhizobiome interactions. Appl Microbiol Biot 103(1): 1155-1166.
  6. Rochat, L., Péchy-Tarr, M., Baehler, E., Maurhofer, M. and Keel, C. (2010). Combination of fluorescent reporters for simultaneous monitoring of root colonization and antifungal gene expression by a biocontrol pseudomonad on cereals with flow cytometry. Mol Plant Microbe Interact 23(7): 949-961.
  7. Saad, M. M., De Zelicourt, A., Rolli, E., Synek, L. and Hirt, H. (2018). Quantification of Root Colonizing Bacteria. Bio-protocol 8: e2927.
  8. Vílchez, J. I., Yang, Y., He, D., Zi, H., Peng, L., Lv, S., Kaushal, R., Wang, W., Huang, W., Liu, R., Lang, Z., Miki, D., Tang, K., Paré, P. W., Song, C. P., Zhu, J. K. and Zhang, H. (2020). DNA demethylases are required for myo-inositol-mediated mutualism between plants and beneficial rhizobacteria. Nature Plants 6: 983-995.


简介

[摘要]根系相关细菌能够影响植物的健康和活力。理解地下植物与细菌相互作用的关键步骤是量化目标细菌的根定植。也许,用荧光标记基因工程是监测植物细菌菌株的最有力的方式,但是这可能有一些担保的问题,有些菌株可被有挑战性的标签。从这个意义上说,在适当控制的条件下接种细菌可以对天然细菌菌株进行可靠且可重复的定量。在此协议中,我们描述了用于定量根相关细菌的详细程序。此方法适用于非侵略性样本处理编 形态鉴定和基于PCR的遗传指纹图谱。这种易于遵循的方案适用于研究在人工培养基或土壤中生长的植物的细菌定植。

[背景] :植物中天然活与在根际,这是指土壤附着在根的薄层各种土壤细菌。虽然有些根瘤菌对植物没有可观察到的作用,但其他根瘤菌是引起有害作用的病原体或促进植物活力的生长性根瘤菌(PGPR)(Mendes等人,2013; Olanrewaju等人,2019)。细菌病原体或PGPR影响植物生长的能力与其细菌根定殖水平紧密相关。因此,细菌根定殖的研究是了解地下植物与细菌相互作用的重要踏脚石。

可以通过可视化荧光信号来评估细菌菌株的丰度,方法是对其进行修饰以表达编码诸如GFP的荧光蛋白的转基因标记基因(Rochat等,2010; Krzyzanowska等,2012; Saad等,2018)。 )。丰度的细菌菌株也可以由细菌基因组DNA的基于PCR的扩增测定(Maciá酒店-森特等人,2009;门迪斯等人,2018) 。这两种方法可以检测目标细菌菌株,而与其他细菌物种的存在与否无关。但是,这两种方法都有其潜在的局限性。荧光辅助检测可能会受到基因转化过程中的技术难题,转基因引入可能干扰野生型细菌行为以及环境安全风险的限制。另一方面,基于PCR的细菌检测要求目标DNA模板的水平高于PCR检测极限,并且目标序列必须具有足够的特异性以仅代表目标细菌菌株。

除了基于荧光和基于PCR的方法外,还可以通过体外培养来定量与根相关的细菌。如果目标细菌菌株可与人工生长培养基一起培养,则这将增加检测限,随后对菌落形成单位(CFU)进行计数。通过适当的实验设置,计算CFU的方法是高效,价格合理,可靠且可重现的,用于定量与根相关的细菌。通过使用拟南芥和巨大芽孢杆菌YC4-R4作为研究植物与细菌相互作用的模型系统,在此我们描述了一种基于对来自体外细菌培养物的CFU进行计数的细菌量化方案。该协议包括通过肠细菌重复基因间共识(ERIC)-PCR进行的可选遗传指纹识别步骤,已应用于研究在植物在人工培养基或土壤中以及在受控或胁迫条件下生长的研究中的不同细菌物种(Vílchez等人,2020)。除了研究与根相关的细菌外,该方法还可以应用于在优化条件下涉及其他类型的植物器官或其他类型的细菌宿主系统的研究。

关键字:植物与细菌的相互作用, 根际细菌, 植物根际促生菌, 病原体, 定植, 根, 土



材料和试剂


1.5 ml Eppendorf管(上虞市伊特塑胶有限公司,目录号:MCTB015或类似产品)
15毫升管
手术刀和scalpellum(青岛中兰国际贸易有限公司,目录号:SS-002021或类似产品)
圆形(9×1.5 cm)和方形(56 × 35 × 30 cm )培养皿(上虞市伊特塑胶有限公司,目录号:PD0009或类似的产品;海门莱伯瑞克实验仪器制造有限公司,目录号:LB077或类似的)
一次性组织研磨塑料杵1.5毫升管中(Corning,爱思进® ,目录号:PES-15-B-SI,或类似的)
接种环(Renon实验室实验有限公司,目录号;52150000或类似产品)
胶带(3M Micropore TM ,1530C-0或类似的胶带)
拟南芥种子
Murashige和Skoog基础盐混合物(MS)(Sigma-Aldrich,目录号:M5524)
胰蛋白((西格玛(Sigma),目录号:T7293)
酵母提取物(西格玛,目录号:Y1625)
NaCl(Sangon Biotech,目录编号:A501218)
NaOH或KOH(Sangon Biotech,目录号:A100583和A610441)
琼脂(西格玛(Sigma),目录号:L2897)
琼脂糖(Sigma,目录号:A9539)
RedSafe TM核酸染色溶液(Invitrogen,目录号:S33102)
2K加梯(Transgenbiotech,Trans2K ®加DNA标记,目录号:BM121)
二次蒸馏灭菌H 2 O
100%乙醇(Sangon Biotech,目录号:A500737)
漂白剂(商业5%NaClO用水以1:5稀释)
DNA提取试剂盒(Qiagen DNA Blood&Tissue,Qiagen,货号:69504或类似产品)
用于PCR主混合物溶液(Transgenbiotech,2 × EasyTaq ® PCR超混合液,AS111-01)
菌ERIC ˚F ingerprinting和的16S rRNA PCR扩增引物:
埃里克(5'-ATGTAAGCTCCTGGGGATTCAC-3')

27F(5'-AGAGTTTGATCMTGGCTCAG-3')

1492R(5'-TACGGYTACCTTGTTACGACTT-3')


设备


不锈钢长柄镊子(Labdirect,目录号:SA-Q2090或类似产品)
分析天平(Sartorius,Practum224-1S或类似产品)
机械移液器(Eppendorf Research Plus,P1000,P100和P10或类似产品)
台式涡旋混合器(DragonLab MX-S或类似产品)
台式离心机(Eppendorf,带有A-4-62型转子的离心机5810R或类似产品;最高4,000 rpm / 3,220 × g )
净化台(BCM-1000A生物净化台,Airtech或类似产品)
摇床培养箱(Eppendorf,New Brunswick TM I26可堆叠培养箱摇床或类似设备)
滚动培养箱(麒麟贝尔实验室仪器有限公司,麒麟贝尔QB-128或类似产品)
高压釜
巴斯德烤箱
植物生长室(Percival CU36L5或类似的植物)或生长室。
文化烤箱(上海景洪DHG-9038A或类似产品)
电泳设备(Bio-Rad,PowerPac TM基本电源+宽子微型子电池GT电池或类似设备)             
凝胶成像系统(Bio-Rad,ChemiDoc XRS + TM凝胶成像系统或类似产品)
热循环仪(Bio-Rad,T100热循环仪或类似产品)


程序


以巨大芽孢杆菌YC4-R4为例的细菌培养
1.从LB-琼脂平板上的甘油储备液中刷新YC4-R4,并在37°C下孵育24 h * / **。     

2.取一个菌落接种5 ml LB培养基(体外)(建议使用15 ml管)。在振荡器(220 rpm,37°C)中孵育过夜。对于土壤接种,每升新鲜培养物中应使用1 ml预培养物来扩大规模(最终量取决于土壤的初始水分和盆栽量)*。     

3.通过将4.5 g /升ddH 2 O混合,制备0.45%NaCl溶液。在121°C下高压灭菌20分钟。使用前冷却至室温。     

4.将培养物以3,220 × g离心10分钟。将沉淀重悬于相同体积的0.45%NaCl中。OD 600 〜0.8-1.1(数生长期;通常,等于10 6 -10 8 CFU / ml)的建议。     

ñ OTES:

*此步骤应在无菌条件下进行。
**对于使用抗生素抗性菌株的测试,可以使用适当的抗生素制备LB平板。


准备细菌生长培养基
1.每升ddH 2 O混合10 g胰蛋白p,5 g酵母提取物和10 g NaCl 。     

2.为了制作LB琼脂平板,每升添加15 g琼脂。     

3.在121°C下高压灭菌20分钟。对于LB琼脂板,将液体培养基倒入培养皿中,使其冷却并固化。     



准备植物生长培养基
1.将MS粉末分别以每升ddH 2 O的一半强度(2.21 g)和琼脂(分别根据测试要求,分别为圆形和方形平板分别为0.7%和1%)混合。     

2.用NaOH或KOH将pH调节至5.7。     

3.在121°C下高压灭菌20分钟。将培养基倒入培养皿中,使其冷却并固化。     



种子灭菌和植物生长条件
将种子与无水乙醇在1.5 ml试管中混合,并保持搅拌1分钟。对于100颗种子,建议使用1毫升乙醇。
丢弃乙醇,并添加20%的漂白剂溶液(来自商业漂白剂库存)。对于100粒种子,建议使用1毫升20%漂白剂溶液。在转盘中搅拌10-15分钟。
丢弃漂白剂,并在无菌工作台中用无菌ddH 2 O洗涤3次。
借助移液器吸头*将种子逐一种植在MS培养基上。
在4℃下将种子分层48小时。分层后,在以下条件下将板垂直放置在生长室中14天:22°C;光照12小时/黑暗12小时周期;相对湿度40%;以及高达155 µmol·m -2 s -1的光强度。
注意:*此步骤应在无菌条件下进行。


体外定植试验
在无菌条件下,放置15〜20 14 -天之后萌发在½MS 1%琼脂(在平方培养皿)每个副本在1.5ml无菌管中生长(DAG)秧苗(无菌管的体积取决于植物的大小) 。在0.45%NaCl溶液中加入1 ml细菌。
将管在摇床上(220 rpm,26°C *)孵育过夜。
丢弃细菌溶液。将幼苗放在无菌表面上,例如无菌铝箔或纸或培养皿中。用无菌剃刀刀片切开根部,并将其保存在1.5 ml的试管中。
加入1 ml的75%乙醇对根部表面进行消毒,并持续搅拌(转盘或类似装置)处理7-10分钟。
丢弃乙醇,并用无菌ddH 2 O清洗根部3次。
丢弃ddH 2 O,并用无菌雌蕊研磨根部(用于1.5 ml管)。
向试管中加入1 ml的0.45%NaCl,涡旋匀浆,并准备连续稀释液(通常5倍10倍稀释液就足够了)。
一滴一滴板(10滴10μl的AP的p ROX。多达100微升的总体积)在LB琼脂平板上的各稀释,让它们干燥为2分钟,并培育在30℃下** 24小时(图1)。





图1.在四分之一生长板中逐滴接种系列稀释液的实例。将液滴小心地放置在干燥的生长培养基板上,以避免干扰。


保持并以最大速度离心原始溶液。沉淀完根屑后,弃去上清液,并在60°C的烤箱中完全干燥48小时,并使用巴斯德烤箱将该重量记录为干重(DW)。
计算细菌菌落数,并考虑稀释度和接种量,计算菌落形成单位(CFU)。用根DW归一化CFU值以获得定植率。定植率=(CFUs ×稀释倍数)/根DW的mg。
ñ OTES:

所有工作应在无菌条件下进行。
*此温度已经过测试,可确保植物无压力条件;同时,还应考虑在相同温度下细菌的生长,以避免对细菌施加压力。
**本文使用的温度对于示例细菌的菌落计数是最佳的。强烈建议针对不同菌株优化温度,以避免菌落过度/重叠。


测定土壤中植物的定植率
1.如上所述生长拟南芥幼苗。   

2.根据所需条件准备土壤。在我们的测试中,土壤是通过将1:石与土壤基质的1:3(v:v)混合物混合而制成的。为了最大程度地减少土壤中不需要的微生物,需要对土壤进行定级化处理(75°C持续3小时+冷却间隔10小时+ 75°C持续3小时)。将5株DAG幼苗转移到土壤中。   

3.在0.45%NaCl溶液中接种细菌。作为参考,在普通的0.4 L锅中,在种植的幼苗周围接种50 ml细菌溶液。   

4.使植物在所需测试要求的条件下生长(例如,有或没有某些非生物胁迫)。   

5.在需要定植定量时,收获根(通常为20-40 mg根)。要进行根部收获,请使用镊子手动剥离每个根部周围的土壤,然后使用软组织剥离最小且粘附最紧密的土壤。必须通过3 × ddH 2 O洗涤*清除细小的土壤颗粒。根部清洁后,按照指示进行消毒,研磨,准备稀释液并计算E部分中的CFU。   

注意:*此步骤应在无菌条件下进行。


菌株隔离和鉴定
1.尽管土壤已被封土,但通常F部分将产生除接种菌株以外的一小部分细菌。要确认已接种菌株的身份并计算其在总细菌种群中的比例,请首先按形态对细菌菌落进行分类(使用https://microbeonline.com/colony-morphology-bacteria-describe-bacterial-colonies/,参考),然后使用无菌牙签挑出形态相似的单个菌落,并将其转移至液体LB培养基中进行培养。通常,应随机取样20-30个菌落,以免产生偏差(图2)。     










图2.在常规的根定殖量化过程中具有不同形态的菌落的示例。通过菌落的颜色,大小或亮度选择不同的形态。箭头的颜色表示预应变株:根据以下标准:红色–强烈的黄色;蓝色–白色,大号;橙色–白色,体积小;和绿色–米色,表面无光泽。


2.在摇床上(220 rpm,30°C)在5 ml液体LB中培养各个菌落24 h。     

3.离心培养物(建议在台式离心机中进行:3,220 × g ,10分钟),弃去上清液,并通过高效方法从沉淀物中提取DNA。在这里,建议使用DNA提取试剂盒(请参见方法)。     

4.用27F(5'-AGAGTTTGATCMTGGCTCAG-3')和1492R(5'-TACGGYTACCTTGTTACGACTT-3')(或同等物)扩增16S rRNA基因并测序,然后对其进行BLAST鉴定,以鉴定分离的物种。     

5.为了进一步区分已接种菌株与相同物种的其他菌株,请使用ERIC引物(5'-ATGTAAGCTCCTGGGGATTCAC-3')进行PCR,以为每个菌株生成指纹表型。使用测试中使用的储备菌株作为参考。在2%的琼脂糖凝胶中运行样品以最大程度地分离谱带。使用GelJ将模式与参考进行比较,并获得相似性指标。图3包括代表性的凝胶图像输出。     






图3.具有代表性的ERIC-PCR凝胶图像输出的实施例(从V改性í lchez等人,2020)。MWM:分子量标记;从接种土壤的根中回收1-4株。伊诺克 菌株:最初是土壤接种的菌株。


数据分析


每种测试均应按照上述指示的条件(体外或在土壤中)进行至少三个重复。根据学生的t检验,当P值˂0.05时,结果将被认为具有统计学差异。相对定植率的计算公式为:RC =(菌落数×稀释倍数)/根DW的mg。


笔记


如果将此方法应用于其他植物物种,则按配方缩放以适合植物大小。对于研磨样品,建议使用1.5 ml试管和配套的雌蕊。自动研磨可以在低振动频率下进行。在使用机械破坏设备(例如TissueLyser)的情况下,请使用低频率的破坏,以避免可能引起细菌膜破坏的过热和机械应力。高频机械破坏可用于从土壤,污泥或其他复杂样品中提取总DNA。积极的研磨/粉碎方法增加了在细菌菌株的最终计数和鉴定中引入偏差的可能性。
基于乙醇的灭菌过程已在拟南芥和番茄作为参照植物的根中进行了测试。在拟南芥中(图4),将7-10分钟(较长和较粗的根大约需要5分钟,如在番茄中)被认为是最佳的根部表面消毒时间(这些测试是用Gram +细菌进行的;考虑到较短的消毒时间)。公克-)。较长的孵育时间可能会干扰定殖细菌的定量。





图4.乙醇处理后的拟南芥根中的菌落数。在用乙醇处理后,最后洗涤溶液中的菌落形成单位(CFU)计数(蓝色柱)和相应的根定殖计数(红色柱)。X轴表示乙醇处理时间。n = 3,数据表示为平均值±SE。


但是,应该注意的是,该设置是针对拟南芥植物的。灭菌时间取决于所测试的植物和细菌种群的类型,因此强烈建议根据实际实验设置优化灭菌时间。


致谢


HZ实验室的研究得到了中国科学院(CAS)的支持。联谊会得到了CAS院长的国际研究金计划奖学金的支持。我们感谢PSC的Rosa Lozano-Duran教授和匿名审阅者对协议的严格阅读。


利益争夺


作者宣称没有利益冲突。


参考


D.Krzyzanowska,M.Obuchowski,M.Bikowski,M.Rychlowski和S.Jafra(2012)。GFP标记的枯草芽孢杆菌MB73 / 2,假单胞菌属菌种对马铃薯根际的定殖。P482和O骨属sp。使用富集样品制备和共聚焦激光扫描显微镜在根的大切片上显示了A44。传感器12(12):17608-17619。
Maciá-Vicente,JG,Jansson,HB,Talbot,NJ和Lopez-Llorca,LV(2009)。大麦(Fusarium equiseti)和衣原体衣原体(Pochonia chlamydosporia )对大麦(Hordeum vulgare )根的内生菌落的实时PCR定量和活细胞成像。新Phytol 182(1):213-228。
Mendes,R.,Garbeva,P.和Raaijmakers,JM(2013)。根际微生物组:植物有益,植物病原和人类病原微生物的意义。FEMS微生物修订版37(5):634-663。
Mendis,HC,Thomas,VP,Schwientek,P.,Salamzade,R.,Chien,JT,Waidyarathne,P.,Kloepper,J.和De La Fuente,L.(2018)。在非无菌土壤和田间条件下,通过植物生长促进根瘤菌牢固芽孢杆菌I-1582和解淀粉芽孢杆菌QST713进行根定殖的菌株特异性定量分析。Plos One 13(2):e0193119。
Olanrewaju,OS,Ayangbenro,AS,Glick,BR和Babalola,OO(2019)。植物健康:根系分泌物-根茎生物组相互作用的反馈作用。Appl Microbiol Biot 103(1):1155-1166。
Rochat,L.,Péchy-Tarr,M.,Baehler,E.,Maurhofer,M.和Keel,C.(2010)。结合荧光报告基因,通过流式细胞仪通过谷物上的生物防治假单胞菌同时监测根部定植和抗真菌基因的表达。摩尔植物微生物相互作用23(7):949-961。
萨阿德(Saad,MM),De Zelicourt,A.,Rolli,E.,Synek L.和Hirt,H.(2018)。根定殖细菌的定量。生物协议8:e2927。
Vílchez,JI,Yang,Y.,He,D.,Zi,H.,Peng,L.,Lv,S.,Kaushal,R.,Wang,W.,Huang,W.,Liu,R.,Lang ,Z.,Miki,D.,Tang,K.,Paré,PW,Song,CP,Zhu,JK and Zhang,H.(2020)。DNA脱甲基酶是肌醇介导的植物与有益根际细菌之间的相互作用所必需的。自然植物6:983-995。
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引用:Ignacio, V. J., Yang, Y., Yi, D. and Zhang, H. (2021). Measurements of Root Colonized Bacteria Species. Bio-protocol 11(7): e3976. DOI: 10.21769/BioProtoc.3976.
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