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Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere
从植物叶际提取内生菌微生物群落DNA   

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Abstract

The plant phyllosphere, which represents all plant parts that are above the ground, is considered one of the most extensive ecosystems to be colonized by microorganisms, both at the surface as epiphytes or as endophytes within the plant. These plant-associated microbial communities are reservoirs of microbial diversity and they can be important for plant health. The characterization of microbial communities in diverse plants, such as Espeletia plants that are endemic to the Paramo ecosystem in the Andes Mountains, can shed light regarding possible interactions among microorganisms and microbial functional properties. Obtaining DNA from plant endophytic microbial communities involves various steps to ensure that samples are free of contamination from microorganisms present on the plant surface (epiphytes). Plant leaves are first surface sterilized, cut into pieces, homogenized using glass beads, and then used for DNA extraction using a commercially available kit. DNA samples are then quantified and analyzed using Qubit® 2.0 for use in PCR amplification of 16S rRNA genes.

Keywords: Endophyte(内生菌), Phyllosphere(叶际), Metagenomics(宏基因组学), Epiphyte(附生植物), 16S rRNA(16S rRNA)

Background

Extraction of endophytic DNA from plant samples has been done by several research groups and usually involves steps to minimize contamination from surface microbes. However, protocols must also be adapted to the plant material being studied and as such can incorporate different steps. Extraction of epiphyte DNA must also ensure that there is no contamination from endophytic microorganisms. The protocol described integrates elements from previous reports (Miles et al., 2012; Araujo et al., 2002), but was not identical given the characteristics of the plant material used. In this work we used leaves from Espeletia hartwegiana, a plant that is endemic to the Paramo ecosystem present in the Colombian Andean mountains. These leaves are characterized to be large and succulent with the presence of short hairs on the surface (pubescence) that require removal prior to leaf surface sterilization and DNA isolation. Here, although the pubescence is removed, the microorganisms associated with it are dislodged first to ensure a complete picture of the epiphyte community. In this case, sufficient DNA of good quality was recovered for PCR amplification and 16S rRNA gene analysis and for functional analysis using the GeoChip (Yan et al., 2015). Nonetheless, other downstream applications could require more DNA and hence more plant material.

Materials and Reagents

  1. Gloves
  2. 1.5 ml sterile microcentrifuge tubes
  3. 10, 100, 1,000 micropipette tips
  4. Sterile craft paper
  5. Ziploc bags (ethanol rinsed)
  6. Sterile swabs (preferably sponge top)
  7. Plant leaves (Espeletia sp.) ~10 g (Maybe more depending on the downstream application). Leaves were collected 1-2 days prior to processing, transported on dry ice and stored in sterile plastic bags at 4 °C
  8. Sterile dH2O
  9. 100% ethanol (Sigma-Aldrich, catalog number: E7023 )
  10. 5.25% sodium hypochlorite (Quidiscol Ltda, Bogotá, Colombia)
  11. Power Soil DNA Isolation Kit (MO BIO Laboratories, catalog number: 12888-50 )
  12. Sodium phosphate dibasic, Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
  13. Malt extract solid medium (OXOID, catalog number: CM0059 )
  14. Tris base (Promega, catalog number: H5133 )
  15. EDTA (Sigma-Aldrich, catalog number: E9884 )
  16. HCl (Mol Labs, catalog number: V4653 )
  17. 0.5 M NaH2PO4 (see Recipes)
  18. NAP buffer (see Recipes)
  19. Malt extract solid medium (see Recipes)
  20. TE buffer (see Recipes)

Note: All reagents used have a specific manufacturer and catalog number. Nonetheless, the user can use any molecular-grade reagent for the protocol.

Equipment

  1. Sterile 500-1,000 ml beakers, autoclaved
  2. 1-25 µl micropipette
  3. 10-100 µl micropipette
  4. 100-1,000 µl micropipette
  5. Sterile razors
  6. Vortex
  7. Sterile tweezers
  8. 25 °C incubator
  9. Sterile glass beads, autoclaved
  10. Laminar flow hood, preferably a class II BSC to avoid contamination
  11. Mini-Bead beater-96 (BioSpec Products)
  12. Qubit® 2.0 fluorometer (Thermo Fisher Scientific, USA)
  13. Autoclave

Procedure

  1. Leaves taken from Espeletia sp. plants (50 to 100 g) are transported to the laboratory inside sterile craft paper and Ziploc bags on dry ice. One set of leaves from each plant is then used for endophyte DNA extraction.
  2. Whole, intact leaves are first washed with sterile dH2O to remove dirt; this wash is performed by first submerging the leaves in sterile dH2O, sufficient to cover the entire leaf (about 500 ml in a 1 L beaker), and then rinsing with clean dH2O using a 100-1,000 µl micropipette.
  3. The intact leaves are placed in NAP buffer (500 ml) in a 1 L sterile beaker and vortexed for 1 min to dislodge epiphytic microbes residing on the plant surface. The fine short hairs, or surface pubescence, present on Espeletia plant leaves are removed by shaving using sterile razors since this facilitates the subsequent sterilization process.
  4. After shaving, leaves are rinsed by pipetting three times with sterile H2O (for large leaves this is approximately 100 ml) and then submerged in:
    1. 500 ml of 90% ethanol for 60 sec in a 1,000 ml beaker.
      Note: This depends on the size of the leaves; the beaker and the volume of ethanol can be smaller.
    2. 5.25% sodium hypochlorite solution in a similar beaker for 6 min.
    3. 70% ethanol for 30 sec.
    4. During this sterilization process it is important to completely submerge the leaf samples in each solution, solutions should be sterile and everything should be conducted preferably in a hood to avoid possible contamination from air. Leaf samples are transferred from one solution to another using sterile tweezers.
  5. The intact leaf samples are finally rinsed with sterile distilled H2O using the 1,000 µl micropipette.
  6. The sterilization is checked by making an imprint of each leaf on a plate of solid malt extract medium using sterile tweezers.
  7. Plates are incubated at 25 °C for up to three weeks, and checked daily for growth. Incubation is done at 25 °C because environmental isolates tend to prefer temperatures below 37 °C. The sterilization process can also be done with liquid medium.
  8. The leaf is cut into small pieces using the sterile razor and one gram is weighed out. These 1 g leaf fragments are further cut into 0.1 to 0.5 mm sections using sterile razors and placed in a 1.5 ml Eppendorf tube containing 1 g of sterile 0.1 mm diameter glass beads and 1 ml TE buffer. Alternatively, leaves can also be cut first into 0.1 to 0.5 mm sections, mixed and then weighed to obtain one gram of sample material. The plant material is processed immediately for extraction of DNA, but only those samples that proved to be sterile upon prolonged incubation on solid or in liquid media (step 7) are used for analysis of microbial communities.
  9. The plant material is then homogenized at room temperature (20-25 °C) in a Mini-Bead Beater for 5 min at 36 oscillations/sec.
  10. The DNA is extracted using the Power Soil DNA Isolation Kit, according to the manufacturer’s instructions, adding the recommended volume of the solution for a single extraction.
    Note: In some cases more than one individual extraction can be performed for a single sample.
  11. Extracted DNA is quantified using a Qubit® 2.0 fluorometer, following the manufacturer’s instructions. It is also possible to visualize the quality of the DNA by gel electrophoresis, but this depends on the amount of DNA obtained, which is usually low.

Notes

  1. During the sterilization, the leaves are processed intact and the amount of leaf material and water or NAP buffer depends on the size of the leaves.
  2. The sterilization of leave samples was checked using solid media, but liquid media could also be used. In this case, sterilized leaves can be submerged briefly in liquid medium (1-2 min), rinsed, and then the medium incubated for several days or weeks, checking for growth daily. Growth in any of the media indicates incomplete sterilization and therefore the respective samples must be discarded or re-sterilized.
  3. Depending on the amount of DNA required for downstream applications, more plant material can be used. If you have access to a bead beater that can accommodate larger volumes, you could add more leaf tissue and probably increase the DNA yields at the end.

Recipes

  1. 0.5 M NaH2PO4 (500 ml)
    34.50 g NaH2PO4 (F.W. 137.99)
    ~450 ml dH2O
    Dissolve, then bring up to volume with dH2O
    Sterilize by autoclaving at 121 °C for 15 min
    Note: A more concentrated solution (0.5 M) is prepared so it is easier to dilute to the desired concentration (124 mM).
  2. NAP buffer
    124 mM Na2HPO4
  3. Malt extract solid medium
    50 g MEA powder
    1 L of dH2O
    Sterilize by autoclaving at 115 °C for 10 min
  4. TE buffer
    10 mM Tris
    10 mM EDTA
    Adjust pH to 8.0

Acknowledgments

This work was financed by Colciencias (contract Nos. 573-2012 and 649-2013) and was performed under MADS contract No. 76-2013 for access to genetic resources and UAESPNN Research permit No. PIDB DTAO 021-10. The protocol was adapted from previously published work (Miles et al., 2012; Araujo et al., 2002). The authors declare that they have no conflict of interest that could impact on the design of implementation of this protocol.

References

  1. Araujo, W. L., Marcon, J., Maccheroni, W., Jr., Van Elsas, J. D., Van Vuurde, J. W. and Azevedo, J. L. (2002). Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl Environ Microbiol 68(10): 4906-4914.
  2. Miles, L. A., Lopera, C. A., González, S., de García, M. C., Franco, A. E. and Restrepo, S. (2012). Exploring the biocontrol potential of fungal endophytes from an Andean Colombian Paramo ecosystem. BioControl 57(5): 697-710.
  3. Yan, Q., Bi, Y., Deng, Y., He, Z., Wu, L., Van Nostrand, J. D., Shi, Z., Li, J., Wang, X., Hu, Z., Yu, Y. and Zhou, J. (2015). Impacts of the Three Gorges Dam on microbial structure and potential function. Sci Rep 5: 8605.

简介

代表地上所有植物部分的植物叶片被认为是被微生物定殖的最广泛的生态系统之一,在表面作为附生植物或植物内生植物。这些植物相关的微生物群落是微生物多样性的水库,它们对植物健康很重要。对安第斯山脉帕拉莫生态系统特有的不同植物,如埃斯佩雷西亚植物的微生物群落进行表征,可以揭示微生物与微生物功能特性之间的相互作用。从植物内生微生物群落中获取DNA涉及各种步骤,以确保样品没有植物表面(附生植物)上存在的微生物的污染。植物叶片首先被表面灭菌,切成块,用玻璃珠匀浆,然后用市售试剂盒进行DNA提取。然后使用Qubit 2.0对DNA样品进行定量和分析,用于16S rRNA基因的PCR扩增。

背景 从植物样品中提取内生DNA已经由几个研究小组进行,通常涉及到最小化表面微生物污染的步骤。然而,协议也必须适应于正在研究的植物材料,因此可以结合不同的步骤。附生植物DNA的提取也必须确保内生微生物没有污染。描述的方案将来自先前报告(Miles等人,2012; Araujo等人,2002)的元素综合起来,但是鉴于所使用的植物材料的特征, 。在这项工作中,我们使用了埃及埃斯特雷西亚hartwegiana 的叶子,这种植物是哥伦比亚安第斯山脉帕拉莫生态系统的特有植物。这些叶子的特征是大而多汁,在表面灭菌和DNA分离之前需要去除表面上存在短毛(柔毛)。在这里,尽管去除了柔毛,但与之相关的微生物首先被移除,以确保附生植物群落的完整图像。在这种情况下,回收足​​够的质量良好的DNA用于PCR扩增和16S rRNA基因分析,并使用GeoChip(Yan等人,2015)进行功能分析。然而,其他下游应用可能需要更多的DNA,因此需要更多的植物材料。

关键字:内生菌, 叶际, 宏基因组学, 附生植物, 16S rRNA

材料和试剂

  1. 手套
  2. 1.5 ml无菌微量离心管
  3. 10,100,1000个微量吸头尖端
  4. 无菌工艺纸
  5. Ziploc袋(乙醇漂洗)
  6. 无菌拭子(最好是海绵顶)
  7. 植物叶( Espeletia sp。)〜10 g(可能更多取决于下游应用)。在加工前1-2天收集叶子,在干冰上运输,并在4℃下储存在无菌塑料袋中
  8. 无菌dH 2 O
  9. 100%乙醇(Sigma-Aldrich,目录号:E7023)
  10. 5.25%次氯酸钠(Quidiscol Ltda,波哥大,哥伦比亚)
  11. 功率土壤DNA分离试剂盒(MO BIO Laboratories,目录号:12888-50)
  12. 磷酸氢二钠,Na 2 HPO 4(Sigma-Aldrich,目录号:S3264)
  13. 麦芽提取物固体培养基(OXOID,目录号:CM0059)
  14. Tris碱(Promega,目录号:H5133)
  15. EDTA(Sigma-Aldrich,目录号:E9884)
  16. HCl(Mol Labs,目录号:V4653)
  17. 0.5 M NaH 2 PO 4(参见食谱)
  18. NAP缓冲区(见配方)
  19. 麦芽提取物固体培养基(参见食谱)
  20. TE缓冲(见配方)

注意:所有使用的试剂都有特定的制造商和目录号。尽管如此,用户可以使用任何分子级试剂进行协议

设备

  1. 无菌500-1,000毫升烧杯,高压灭菌
  2. 1-25微升微量移液管
  3. 10-100微升微量移液管
  4. 100-1,000微升微量移液管
  5. 无菌剃须刀
  6. 涡流
  7. 无菌镊子
  8. 25℃孵化器
  9. 无菌玻璃珠,高压灭菌
  10. 层流罩,优选为II类BSC以避免污染
  11. 迷你珠打击机96(BioSpec产品)
  12. Qubit ® 2.0荧光计(Thermo Fisher Scientific,USA)
  13. 高压灭菌器

程序

  1. Espeletia sp。植物(50至100克)在无菌工艺纸和干冰上的Ziploc袋中运送到实验室。然后将来自每株植物的一组叶子用于内生真菌DNA提取。
  2. 首先用无菌dH 2 O O清洗整个完整的叶子以除去污垢;该洗涤是通过首先将叶子浸没在无菌dH 2 O 2中,足以覆盖整个叶子(在1L烧杯中约500ml),然后用清洁的dH 2 < / sub> O使用100-1,000微升微量移液管。
  3. 将完整的叶子放置在1L无菌烧杯中的NAP缓冲液(500ml)中,并振荡1分钟以去除驻留在植物表面上的附生微生物。通过使用无菌剃刀剃须来除去存在于埃斯佩雷西亚植物叶子上的细短毛或表面柔毛,因为这有助于随后的灭菌过程。
  4. 剃须后,通过用无菌H 2 O 2(对于大叶子约为100ml)移液三次冲洗叶子,然后浸没在:
    1. 500毫升90%乙醇,在1000毫升烧杯中放置60秒 注意:这取决于叶子的大小;烧杯和乙醇的体积可以更小。
    2. 5.25%次氯酸钠溶液在类似的烧杯中6分钟
    3. 70%乙醇30秒。
    4. 在这种灭菌过程中,重要的是要完全浸没在每个溶液中的叶片样品,溶液应该是无菌的,并且所有的物质都应优选在罩中进行,以避免可能的空气污染。叶片样品使用无菌镊子从一种溶液转移到另一种溶液。
  5. 使用1,000μl微量移液管,用无菌蒸馏的H 2 O 2终止完整的叶子样品。
  6. 通过使用无菌镊子在固体麦芽提取物培养基板上印刷每片叶片来检查灭菌。
  7. 将板在25℃下孵育长达三周,并每天检查生长。孵育在25°C进行,因为环境分离物往往倾向于低于37°C的温度。灭菌过程也可以用液体介质进行。
  8. 使用无菌剃刀将叶切成小块,称出一克。使用无菌剃刀将这些1g片段进一步切成0.1至0.5mm切片,并置于含有1g无菌0.1mm直径的玻璃珠和1ml TE缓冲液的1.5ml Eppendorf管中。或者,也可以将叶片首先切成0.1至0.5mm切片,混合,然后称重,得到1克样品。立即处理植物材料用于提取DNA,但只有在固体或液体培养基中长时间孵育后证明无菌的样品(步骤7)才用于微生物群落的分析。
  9. 然后将植物材料在室温(20-25℃)下在微型珠打浆机中以36次振荡/秒均化5分钟。
  10. 根据制造商的说明书,使用Power Soil DNA Isolation Kit提取DNA,为单次提取添加推荐的溶液体积。
    注意:在某些情况下,单个样本可以执行多次单独的提取。
  11. 按照制造商的说明书,使用Qubit 2.0荧光计量化提取的DNA。也可以通过凝胶电泳显示DNA的质量,但这取决于所获得的DNA的量,通常较低。

笔记

  1. 在灭菌期间,叶子被完整地处理,叶片材料和水或NAP缓冲液的量取决于叶子的大小。
  2. 使用固体培养基检查离开样品的灭菌,但也可以使用液体培养基。在这种情况下,灭菌的叶片可以短暂地浸没在液体培养基(1-2分钟)内,漂洗,然后将培养基孵育数天或数周,每天检查生长。任何一种培养基中的生长表明不完全消毒,因此各样品必须丢弃或重新消毒
  3. 根据下游应用所需的DNA量,可以使用更多的植物材料。如果您可以使用可以容纳更大体积的珠子搅拌器,您可以添加更多的叶子组织,并可能在最后增加DNA产量。

食谱

  1. 0.5M NaH 2 PO 4(500ml)
    34.50g NaH 2 PO 4(F.W.137.99)
    〜450ml dH 2 O O 溶解,然后与dH 2 O
    卷起 通过121℃高压灭菌15分钟来消毒
    注意:制备更浓缩的溶液(0.5M),因此更容易稀释至所需浓度(124mM)。
  2. NAP缓冲区
    124mM Na 2 HPO 4
  3. 麦芽提取物固体培养基
    50克MEA粉末
    1L的dH 2 O
    通过在115℃下高压灭菌10分钟来消毒
  4. TE缓冲区
    10 mM Tris
    10 mM EDTA
    将pH调节至8.0

致谢

这项工作由Colciencias(合同号573-2012和649-2013)提供资金,并根据MADS第76-2013号合同获取遗传资源和UAESPNN研究许可证编号PIDB DTAO 021-10。该协议改编自以前发表的作品(Miles等人,2012; Araujo等人,2002)。作者宣称他们没有利益冲突可能影响本协议实施的设计。

参考文献

  1. Araujo,WL,Marcon,J.,Maccheroni,W.,Jr.,Van Elsas,JD,Van Vuurde,JW and Azevedo,JL(2002)。&lt; a class ="ke-insertfile"href ="http: //www.ncbi.nlm.nih.gov/pubmed/12324338"target ="_ blank">内生细菌群体的多样性及其与柑橘类植物中的Xylella fastidiosa的相互作用。 Appl Environ Microbiol 68(10):4906-4914。
  2. Miles,LA,Lopera,CA,González,S.,deGarcía,MC,Franco,AE和Restrepo,S。(2012)。&nbsp; 探索安哥拉哥伦比亚Paramo生态系统的真菌内生真菌的生物防治潜力。 BioControl 57( 5):697-710。
  3. Yan,Q.,Bi,Y.,Deng,Y.,He,Z.,Wu,L.,Van Nostrand,JD,Shi,Z.,Li,J.,Wang,X.,Hu,Z., Yu,Y. and Zhou,J.(2015)。&nbsp; 三峡大坝对微生物结构和潜在功能的影响。 5:8605。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ruiz-Pérez, C. A. and Zambrano, M. M. (2017). Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere. Bio-protocol 7(4): e2142. DOI: 10.21769/BioProtoc.2142.
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Uma kumar
assistant professor
how to download this article
9/24/2017 11:41:29 PM Reply
María Zambrano
Molecular Genetics, Corporación Corpogen, Colombia

Hi, at the top of the online page (DOI: 10.21769/BioProtoc.2142), there is a link to download the pdf.

9/25/2017 7:26:37 AM