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Isolation of Rhizosphere Bacterial Communities from Soil
从土壤中分离根际细菌群落   

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Abstract

Rhizosphere bacterial communities have become a major focal point of research in recent years, especially regarding how they affect plants and vice versa (Philippot et al., 2013). Changes in microbial density and diversity within the rhizosphere occur in a spatial temporal manner. The soil zone closest to the plant roots has the most density and diversity of microbes (Clark, 1940). The lack of methods to consistently isolate rhizosphere samples in a spatially defined manner is a major bottleneck in rhizosphere microbiology. We hypothesized that microbes with increasing affinities to and distance from the plant root can be isolated using increasing strengths of physical disruption. Sonication is an excellent choice due to the ability to gently remove rhizosphere soil and bacterial biofilms without damaging plant roots (Doi T et al., 2007; Bulgarelli et al., 2012; Lundberg et al., 2012). In addition, simply increasing the time of sonication can increase the amount of physical force. We used such an approach to consistently isolate microbial communities with different affinities to the soybean roots (White et al., 2014). This article describes the use of successive sonication to isolate distal, middle, and proximal soil from the rhizosphere of soybean roots.

Materials and Reagents

  1. Soybean seedlings (Glycine max) in the vegetative stage (~ V3 to V5 period)
  2. Soil with a history of soybean cultivation
  3. dH2O
  4. K2HPO4 (VWR International, catalog number: BDH0266-500 g )
  5. KH2PO4 (VWR International, catalog number: BDH0268-500 g )
  6. NaCl (Sigma-Aldrich, catalog number: S7653-1 kg )
  7. Tween-20 (Sigma-Aldrich, catalog number: P9416-100 ml )
  8. Phosphate buffered saline Tween 20 (PBST) (see Recipes)

Equipment

  1. Razor blade
  2. Tweezers
  3. 15 ml conical-bottom polypropylene centrifuge tubes (3 per sample) (VWR International, catalog number: 89039-670 )
  4. 50 ml conical-bottom polypropylene centrifuge tubes (3 per sample) (VWR International, catalog number: 21008-940 )
    Note: Needed if plant roots are too large for 15 ml centrifuge tubes.
  5. Styrofoam raft to suspend centrifuge tubes in sonicator (homemade)
  6. Sonicator (Input: 117 V-50-60HZ 1ϕ, Output: 70 W 42KHZ +/-6%) (Thermo Fisher Scientific, model: FS20 )
  7. Centrifuge with a fixed angle rotor for 15 and 50 ml conical bottom tubes at 4 °C capable of at least 5,000 x g relative centrifugal force (120 V 12 A 60 Hz 1,300 W) (Example: Eppendorf, model: 5804R 15 amp version)

Procedure

  1. Either directly sow plant seeds or plant seedlings into soil of interest and allow seeds/seedlings to grow for desired amount of time (minimum of 1 week suggested for soybean plants).
    Notes:
    1. Although larger roots (ex. mature tree roots) are not recommended for this procedure, representative samples of the root system can be used depending on the research question.
    2. Amount of growth time depends on the research focus, for example the impact of a particular root exudate or the plant growth stage on the soil microbial community.
  2. Carefully remove plant seedlings by saturating the soil with dH2O or gently loosening the soil by hand to avoid damage to the roots.
    Note: Using an excessive amount of dH2O during saturation (i.e. resulting in a soil consistency thinner than mud) risks a loss of sample size and rhizosphere bacteria.
  3. Submerge the roots in a still pool of dH2O and gently shake the roots (as if painting a picture or dunking a teabag) to remove the larger soil particles. Skip this step if plant seedlings were removed by soil saturation in the previous step. See Figure 1 for example of soybean roots before and after the removal of large soil particles.


    Figure 1. Thirty-six day old soybean roots before (A) and after (B) submersion in a still pool of dH2O to remove large soil particles. The amount of soil clinging to the plant roots can vary depending on soil properties, the root architecture, and the size(s) of the plant roots.

  4. Use a razor blade to sever the plant roots (cutting near the plant stem).
  5. Place the severed roots into separate, labeled 15 ml centrifuge tubes filled with 10 ml of PBST, ensuring they are completely submerged (may use tweezers to gently push roots deeper into the tube).
    Notes:
    1. Roots should be placed into the centrifuge tube vertically.
    2. Ensure the centrifuge tube is not packed with the root sample. The number of roots placed into one tube depends on root size and/or the desire to keep root samples separate (ex. pooling all roots from one plant together, pooling multiple roots from several plants together, or keeping each root from one plant separate). Overly large roots, or too many roots in one tube, will lead to poor sample isolation whereas tiny roots, or too few roots in one tube, will yield a miniscule sample size.
    3. For seedlings with larger root systems, use a 50 ml centrifuge tube filled with 45 ml of PBST in this step and all subsequent steps. See Figure 2 for demonstrative sample of an acceptable amount of roots in a single tube.


    Figure 2. Soybean roots submerged in 10 ml of PBST within a 15 ml centrifuge tube

  6. Firmly secure the centrifuge tube lids, then place the tubes in a floating raft within a sonicator filled with dH2O.
    Note: Ensure the centrifuge tubes do not touch the bottom or sides of the sonicator (see Figure 3 for demonstrative diagram).


    Figure 3. Diagram demonstrating how to properly load samples and floating raft into the sonicator filled with dH2O. Centrifuge tubes should be submerged up to the 10 or 45 ml line (dependent on if a 15 or 50 ml centrifuge tube was used). Tubes should not touch the bottom or edges of the sonicator.

  7. Subject the centrifuge tubes to sonication for 60 sec, then turn off the sonicator (see Figure 4 for sonication summary).
    Notes:
    1. This sonication yields the rhizosphere soil furthest from the plant root or soil with least affinity to the plant root, noted as the “distal soil” sample.
    2. Use the same sonication time for both the 15 and 50 ml centrifuge tubes.


    Figure 4. Diagram of successive sonication procedure for isolation of distal, middle, and proximal soil samples from plant roots. Distal soil samples consist of the rhizosphere soil furthest from and with least affinity to the plant root. Middle soil samples consist of the rhizosphere soil that is closer to and with relatively less affinity the plant root. Proximal soil samples consist of the rhizosphere soil closest to and with highest affinity the plant root. Image adapted from a previous article (White et al., 2014).

  8. Using tweezers, gently remove the root(s) from the current centrifuge tube(s) and transfer into a new, labeled centrifuge tube (or tubes) containing 10 ml of fresh PBST.
    Note: Keep roots/samples separated in the same manner used for the first sonication. Do not pool roots/samples from different centrifuge tubes together.
  9. Firmly secure the centrifuge tube lids, place the tubes in the floating raft within the sonicator, and subject the tubes to sonication for 60 sec. Then turn off the sonicator.
    Note: This sonication yields the rhizosphere soil that is closer to the plant root, noted as the “middle soil” sample.
  10. Using tweezers, gently remove the root(s) from the current centrifuge tube(s) and transfer into a new, labeled 15 ml centrifuge tube (or tubes) containing 10 ml of fresh PBST.
    Note: Again, keep roots/samples separated in the same manner used for the first sonication. Do not pool roots/samples from different centrifuge tubes together.
  11. Firmly secure the centrifuge tube lids, place the tubes in the floating raft within the sonicator, and subject the tubes to sonication for 10 min. Then turn off the sonicator.
    Note: This sonication yields the rhizosphere soil closest to the plant root including any biofilms, noted as the “proximal soil” sample. At this point, soil should not be visible on the plant root.
  12. Using tweezers, gently remove the root(s) from the current centrifuge tube(s) and either discard the roots or place them into a new, labeled centrifuge tube (or tubes) filled with fresh PBST, then store the tubes at 4 °C until needed. Harvested samples may then be immediately used for bacterial cultivation or further processed for DNA or RNA isolation. If seeking to isolate DNA or RNA, complete the next 2 steps of the protocol. For bacterial cultivation, promptly subject the samples to a series of 6 to 10 fold dilutions using sterile dH2O and select several of these dilutions for plating (dilutions >10-3 recommended). When plating the chosen dilutions, ensure the appropriate nutrient medium (or media) is chosen. One hundred microliters of the chosen dilution should be dispensed onto the center of the petri dish and spread across the media using a flame-sterilized glass spreader. The petri dish should then be inverted and incubated under the ideal cultivating conditions (i.e. time and temperature). See Figure 5 for an example of bacterial cultivation via petri dish.
    Notes:
    1. Distal, middle, and proximal soil samples are all useful for bacterial cultivation. However, proximal soil samples are preferable as they contain the bacteria that most likely affect the plant directly and vice versa.
    2. Possible media for bacterial cultivation include a soil extract medium such as SESOM, DR2A + supplements, and R2A solidified with agar or gellan (Tamaki et al., 2005; Vilain et al., 2006).


    Figure 5. Bacterial cultivation of proximal soil samples from untransformed soybean roots on nutrient media solidified with agar (A-C) or gellan (D-F). Nutrient media consisted of DR2A+ (A, D), R2A (B, E), and SESOM (C, F). Bacterial samples acquired from a 10-5 dilution. Black dots and red circles indicate the presence of individual bacterial colonies.

  13. After securing the lids on all the centrifuge tubes, place them into a 4 °C centrifuge and subject them to centrifugation at 5,000 x g for 10 min or 4,500 x g for 15 min (depending on the limits of the centrifuge).
  14. Once centrifugation is complete, discard supernatant and either immediately use the pellets for DNA or RNA isolation or store them at -80 °C until needed.

Limitations of the method

  1. Sonication times may vary depending on the types of plant roots used as well as the properties of the soil in which they were grown.
  2. It is uncertain how useful this procedure is for soil fungi.
  3. Sample sizes will be small (likely < 0.3 g when using 15 ml centrifuge tubes) and decrease from sonication to sonication, with proximal soil samples being the smallest. This might be an issue for methods such as proteomics and metabolomics that generally require a larger sample size.
  4. Age of the plant makes a difference (root system is very large at later stages). This procedure is better suited for smaller root sizes. For perennial plants or older plants with large root systems, one can use a golf cup cutter (4” to 8” diameter) to obtain a soil core (6” to 12” deep) and obtain root segments from that by placing it in water and allowing the soil to separate from the roots. Obviously, this would depend on whether the representative samples of the root system would suffice to answer the research question.

Recipes

  1. Phosphate buffered saline Tween 20 (PBST) (500 ml, pH of 7.2)
    1. Add 0.605 g of K2HPO4 to 300 ml of dH2O, stir until K2HPO4 is completely dissolved
    2. Add 0.17 g of KH2PO4 to mixture, stir until KH2PO4 is completely dissolved
    3. Add 4.1 g of NaCl to mixture, stir until NaCl is completely dissolved
    4. Adjust pH with NaOH or HCl until final pH is 7.2
    5. Add dH2O to mixture until the final volume is 500 ml, stir to ensure even distribution
    6. Sterilize solution via autoclaving (liquid cycle, 121 °C for 30 min)
    7. Add 250 µl of Tween20 to mixture, gently swirl to ensure even distribution
      Note: Adding Tween20 before autoclaving will result in frothing overflow due to bubble formation.
    8. Store at room temperature (~20 °C)

Acknowledgments

This protocol was established in a previously published study (White et al., 2014). Funding for this research was provided by the South Dakota Agricultural Experiment Station and the South Dakota Soybean Research and Promotion Council. We would also like to thank R. Gelderman (SDSU) for providing soil samples, M. Hildreth (SDSU) for providing the sonicator used for this research, and Al Miron for providing the soybean plant depicted in Figure 1.

References

  1. Bulgarelli, D., Rott, M., Schlaeppi, K., Ver Loren van Themaat, E., Ahmadinejad, N., Assenza, F., Rauf, P., Huettel, B., Reinhardt, R., Schmelzer, E., Peplies, J., Gloeckner, F. O., Amann, R., Eickhorst, T. and Schulze-Lefert, P. (2012). Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature 488(7409): 91-95.
  2. Clark, F. E. (1940). Notes on types of Bacteria associated with plant roots. Transactions of the Kansas Academy of Science (1903-) 43:75-84.
  3. Doi T, H. Y., Abe, J. and Morita, S. (2007). Analysis of rhizosphere bacteria of rice cultivated in Andosol lowland and upland fields using molecular biological methods. Plant Root 1:66-74.
  4. Lundberg, D. S., Lebeis, S. L., Paredes, S. H., Yourstone, S., Gehring, J., Malfatti, S., Tremblay, J., Engelbrektson, A., Kunin, V., del Rio, T. G., Edgar, R. C., Eickhorst, T., Ley, R. E., Hugenholtz, P., Tringe, S. G. and Dangl, J. L. (2012). Defining the core Arabidopsis thaliana root microbiome. Nature 488(7409): 86-90.
  5. Philippot, L., Raaijmakers, J. M., Lemanceau, P. and van der Putten, W. H. (2013). Going back to the roots: the microbial ecology of the rhizosphere. Nat Rev Microbiol 11(11): 789-799.
  6. Tamaki, H., Sekiguchi, Y., Hanada, S., Nakamura, K., Nomura, N., Matsumura, M. and Kamagata, Y. (2005). Comparative analysis of bacterial diversity in freshwater sediment of a shallow eutrophic lake by molecular and improved cultivation-based techniques. Appl Environ Microbiol 71(4): 2162-2169.
  7. Vilain, S., Luo, Y., Hildreth, M. B. and Brozel, V. S. (2006). Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil. Appl Environ Microbiol 72(7): 4970-4977.
  8. White, L. J., Jothibasu, K., Reese, R. N., Brozel, V. S. and Subramanian, S. (2015). Spatio temporal influence of isoflavonoids on bacterial diversity in the soybean rhizosphere. Mol Plant Microbe Interact 28(1): 22-29.

简介

根际细菌群落已成为近年来研究的主要焦点,特别是关于它们如何影响植物或反之亦然(Philippot等人,2013)。根际微生物密度和多样性的变化以空间时间方式发生。最接近植物根部的土壤区具有最密集和多样性的微生物(Clark,1940)。缺乏以空间定义的方式一致地分离根际样品的方法是根际微生物学的主要瓶颈。我们假设可以使用增加的物理破坏的强度来分离具有对植物根部的亲和力和距植物根部的距离增加的微生物。超声是一个很好的选择,因为能够轻轻地去除根际土壤和细菌生物膜而不损害植物根(Doi T等人,2007; Bulgarelli等人,2012年; Lundberg等人,2012)。此外,简单地增加超声处理的时间可以增加物理力的量。我们使用这样的方法来一致地分离对大豆根具有不同亲和力的微生物群落(White等人,2014)。本文介绍了使用连续超声处理来隔离远端,中间和近端土壤与大豆根部的根际。

材料和试剂

  1. 在营养阶段(〜V3至V5期)的大豆幼苗(大豆)
  2. 具有大豆栽培史的土壤
  3. dH 2 2 O

  4. HPO <4>(VWR International,目录号:BDH0266-500g)

  5. (VWR International,目录号:BDH0268-500g)
  6. NaCl(Sigma-Aldrich,目录号:S7653-1kg)
  7. 吐温-20(Sigma-Aldrich,目录号:P9416-100ml)
  8. 磷酸盐缓冲盐水Tween 20(PBST)(参见配方)

设备

  1. 剃刀刀片
  2. 镊子
  3. 15ml锥形底聚丙烯离心管(每个样品3个)(VWR International,目录号:89039-670)
  4. 50ml锥形底聚丙烯离心管(每个样品3个)(VWR International,目录号:21008-940)
    注意:如果植物根部对于15ml离心管太大,则需要。
  5. 聚苯乙烯泡沫塑料筏在超声波仪(自制)中悬挂离心管
  6. 超声波仪(输入:117V-50-60HZ1φ,输出:70W 42KHZ +/- 6%)(Thermo Fisher Scientific,型号:FS20)
  7. 用固定角转子离心,在15℃和50ml锥形底部管中,在4℃,能够具有至少5,000×g相对离心力(120V 12 A 60Hz 1,300W)(实施例:Eppendorf,型号 :5804R 15 amp版本)

程序

  1. 将植物种子或植物幼苗直接播种到感兴趣的土壤中,并允许种子/幼苗生长所需的时间量(对于大豆植物推荐至少1周)。
    注意:
    1. 虽然不推荐使用更大的根(例如成熟的树根)   这个程序,可以使用根系统的代表性样品 取决于研究问题。
    2. 生长时间取决于   对研究重点,例如特定根的影响 渗出物或植物生长阶段对土壤微生物群落的影响
  2. 通过用dH 2 O饱和土壤或用手轻轻松开土壤以小心地除去植物幼苗,以避免损坏根。
    注意:在饱和期间使用过量的dH 2 O(即导致比泥土更稠的土壤稠度)可能会损失样品大小和根际细菌。
  3. 将根浸没在dH 2 O 2的静止池中,并轻轻摇动根(如同绘画或浸泡茶袋一样)以除去较大的土壤颗粒。如果在前一步骤中通过土壤饱和除去植物幼苗,则跳过此步骤。参见图1,例如在除去大的土壤颗粒之前和之后的大豆根

    图1.在dH 2 O的静止池中浸没之前(A)和之后(B)的三十六天大豆根以除去大的污垢颗粒。粘附到植物根部的土壤可以根据土壤性质,根构造和植物根部的尺寸而变化。

  4. 使用剃刀刀片切断植物根(切割植物茎附近)。
  5. 将切断的根部放置在装有10ml PBST的单独的,标记的15ml离心管中,确保它们完全浸没(可以使用镊子轻轻地将根更深地推入管中)。 注意:
    1. 根应垂直放入离心管中。
    2. 确保离心管没有装入根样品。 的 放置在一个管中的根的数量取决于根大小和/或 希望保持根部样本分开(例如,将所有根集合在一起 植物在一起,将多个植物的多个根汇集在一起,或 保持来自一个植物的每个根分开)。 过大的根,或太 许多根在一个管中,将导致较差的样品分离,而微小 根或在一个管中太少的根将产生微小的样品大小。
    3. 对于具有较大根系的幼苗,使用50ml离心机 在该步骤和所有后续步骤中用45ml PBST填充。 参见图2的可接受量的根的示范性样品 在单个管中。


    图2.在15ml离心管中浸没在10ml PBST中的大豆根

  6. 牢固固定离心管盖,然后将管放置在充满dH 2 O的超声波仪内的浮动筏中。
    注意:确保离心机管不会接触超声波仪的底部或侧面(见图3的说明图)。


    图3.显示如何将样品和浮动筏正确装载到装有dH 2 O的超声波仪中的图。离心管应浸没至10或45 ml管线取决于是否使用15或50ml离心管)。试管不应接触超声波仪底部或边缘。

  7. 使离心管超声处理60秒,然后关闭超声波仪(见图4的超声处理摘要)。
    注意:


图2.在15ml离心管中浸没在10ml PBST中的大豆根

  • 牢固固定离心管盖,然后将管放置在充满dH 2 O的超声波仪内的浮动筏中。
    注意:确保离心机管不会接触超声波仪的底部或侧面(见图3的说明图)。


    图3.显示如何将样品和浮动筏正确装载到装有dH 2 O的超声波仪中的图。离心管应浸没至10或45 ml管线取决于是否使用15或50ml离心管)。试管不应接触超声波仪底部或边缘。

  • 使离心管超声处理60秒,然后关闭超声波仪(见图4的超声处理摘要)。
    注意:
    ... Note: Keep roots/samples separated in the same manner used for the first sonication. Do not pool roots/samples from different centrifuge tubes together.
  • Firmly secure the centrifuge tube lids, place the tubes in the floating raft within the sonicator, and subject the tubes to sonication for 60 sec. Then turn off the sonicator.
    Note: This sonication yields the rhizosphere soil that is closer to the plant root, noted as the "middle soil" sample.
  • Using tweezers, gently remove the root(s) from the current centrifuge tube(s) and transfer into a new, labeled 15 ml centrifuge tube (or tubes) containing 10 ml of fresh PBST.
    Note: Again, keep roots/samples separated in the same manner used for the first sonication. Do not pool roots/samples from different centrifuge tubes together.
  • Firmly secure the centrifuge tube lids, place the tubes in the floating raft within the sonicator, and subject the tubes to sonication for 10 min. Then turn off the sonicator.
    Note: This sonication yields the rhizosphere soil closest to the plant root including any biofilms, noted as the "proximal soil" sample. At this point, soil should not be visible on the plant root.
  • Using tweezers, gently remove the root(s) from the current centrifuge tube(s) and either discard the roots or place them into a new, labeled centrifuge tube (or tubes) filled with fresh PBST, then store the tubes at 4 °C until needed. Harvested samples may then be immediately used for bacterial cultivation or further processed for DNA or RNA isolation. If seeking to isolate DNA or RNA, complete the next 2 steps of the protocol. For bacterial cultivation, promptly subject the samples to a series of 6 to 10 fold dilutions using sterile dH2O and select several of these dilutions for plating (dilutions >10-3 recommended). When plating the chosen dilutions, ensure the appropriate nutrient medium (or media) is chosen. One hundred microliters of the chosen dilution should be dispensed onto the center of the petri dish and spread across the media using a flame-sterilized glass spreader. The petri dish should then be inverted and incubated under the ideal cultivating conditions (i.e. time and temperature). See Figure 5 for an example of bacterial cultivation via petri dish.
    Notes:
    1. Distal, middle, and proximal soil samples are all useful for bacterial cultivation. However, proximal soil samples are preferable as they contain the bacteria that most likely affect the plant directly and vice versa.
    2. Possible media for bacterial cultivation include a soil extract medium such as SESOM, DR2A + supplements, and R2A solidified with agar or gellan (Tamaki et al., 2005; Vilain et al., 2006).


    Figure 5. Bacterial cultivation of proximal soil samples from untransformed soybean roots on nutrient media solidified with agar (A-C) or gellan (D-F). Nutrient media consisted of DR2A+ (A, D), R2A (B, E), and SESOM (C, F)。从10 -5稀释获得的细菌样品。 黑点和红色圆圈表示存在单个细菌菌落
  • 在将盖子固定在所有离心管上之后,将它们置于4℃离心机中并使其在5,000xg下离心10分钟或4,500xg离心15分钟( 取决于离心机的极限)。
  • 一旦离心完成,丢弃上清液,立即使用沉淀用于DNA或RNA分离或将其储存在-80℃直到需要。
  • 方法的限制

    1. 超声处理时间可以根据所使用的植物根类型以及它们生长的土壤的性质而变化。
    2. 不确定这个程序对土壤真菌有多有用
    3. 样品尺寸将很小(当使用15ml离心管时可能<0.3g),并且从超声处理降至超声处理,近端土壤样品最小。对于通常需要更大样本量的蛋白质组学和代谢组学等方法,这可能是一个问题
    4. 植物的年龄有所不同(根系在后期阶段非常大)。此过程更适合较小的根部尺寸。对于多年生植物或具有大根系的较老植物,可以使用高尔夫球杯切割器(直径4"至8")获得土壤芯(6"至12"深),并通过将其置于水中获得根段并允许土壤与根分离。显然,这取决于根系的代表性样本是否足以回答研究问题

    食谱

    1. 磷酸盐缓冲盐水Tween 20(PBST)(500ml,pH为7.2)
      1. 将0.605g的K 2 HPO 4加入到300ml的dH 2 O中,搅拌直到K 2 HPO 4为止, sub> 4 完全溶解
      2. 向混合物中加入0.17g的KH 2 PO 4,搅拌直到KH 2 PO 4完全溶解为止。
        />
      3. 向混合物中加入4.1g NaCl,搅拌至NaCl完全溶解
      4. 用NaOH或HCl调节pH,直到最终pH为7.2
      5. 向混合物中加入dH 2 O,直至最终体积为500ml,搅拌以确保均匀分布
      6. 通过高压灭菌(液体循环,121℃,30分钟)灭菌溶液
      7. 向混合物中加入250μlTween20,轻轻旋转以确保均匀分布 注意:在高压灭菌之前加入Tween20会由于气泡形成而导致起泡溢出。
      8. 储存于室温(〜20°C)

    致谢

    该协议在先前发表的研究中建立(White等人,2014)。 这项研究的资金由南达科他州农业实验站和南达科他州大豆研究和促进委员会提供。 我们还要感谢R. Gelderman(SDSU)提供土壤样品,M. Hildreth(SDSU)提供用于本研究的超声波仪,Al Miron提供图1所示的大豆植物。

    参考文献

    1. Bulgarelli,D.,Rott,M.,Schlaeppi,K.,Ver Loren van Themaat,E.,Ahmadinejad,N.,Assenza,F.,Rauf,P.,Huettel,B.,Reinhardt,R.,Schmelzer, E.,Peplies,J.,Gloeckner,FO,Amann,R.,Eickhorst,T。和Schulze-Lefert,P。 显示拟南芥根栖息的细菌性微生物群的结构和装配线索。 自然 488(7409):91-95。
    2. Clark,F.E。(1940)。 与植物根系相关的细菌类型的说明 堪萨斯科学院(1903-)43:75-84。
    3. Doi T,H.Y.,Abe,J。和Morita,S。(2007)。 在Andosol低地和高地田间种植的水稻根际细菌分析使用分子生物学方法。 植物根 1:66-74
    4. Lundberg,DS,Lebeis,SL,Paredes,SH,Yourstone,S.,Gehring,J.,Malfatti,S.,Tremblay,J.,Engelbrektson,A.,Kunin,V.,del Rio,TG,Edgar,RC ,Eickhorst,T.,Ley,RE,Hugenholtz,P.,Tringe,SGand Dangl,JL(2012)。 定义核心拟南芥根微生物组 自然 488(7409):86-90。
    5. Philippot,L.,Raaijmakers,J.M.,Lemanceau,P。和van der Putten,W.H。(2013)。 回到根源:微生物生态学 rhizosphere。 Nat Rev Microbiol 11(11):789-799。
    6. Tamaki,H.,Sekiguchi,Y.,Hanada,S.,Nakamura,K.,Nomura,N.,Matsumura,M.and Kamagata,Y。 通过分子和改良的栽培技术对浅层富营养湖的淡水沉积物的细菌多样性进行比较分析。 Appl Environ Microbiol 71(4):2162-2169。
    7. Vilain,S.,Luo,Y.,Hildreth,M.B.and Brozel,V.S.(2006)。 在液体土壤中分析土壤腐生菌的生命周期 提取物和在土壤中。 Appl Environ Microbiol 72(7):4970-4977。
    8. White,L.J.,Jothibasu,K.,Reese,R.N.,Brozel,V.S.and Subramanian,S。(2015)。 异黄酮对大豆根际细菌多样性的时空影响。植物微生物互动 28(1):22-29。
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    引用:White, L. J., Brözel, V. S. and Subramanian, S. (2015). Isolation of Rhizosphere Bacterial Communities from Soil. Bio-protocol 5(16): e1569. DOI: 10.21769/BioProtoc.1569.
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