I Plate-based Assay for Studying How Fungal Volatile Compounds (VCs) Affect Plant Growth and Development and the Identification of VCs via SPME-GC-MS
用于研究真菌挥发性化合物(VCs)如何影响植物生长发育并通过SPME-GC-MS技术鉴定VCs的I板检测法   

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Molecular Plant Microbe Interactions
Oct 2018

 

Abstract

Biogenic volatile compounds (VCs) mediate various types of crucial intra- and inter-species interactions in plants, animals, and microorganisms owing to their ability to travel through air, liquid, and porous soils. To study how VCs produced by Verticillium dahliae, a soilborne fungal pathogen, affect plant growth and development, we slightly modified a method previously used to study the effect of bacterial VCs on plant growth. The method involves culturing microbial cells and plants in I plate to allow only VC-mediated interaction. The modified protocol is simple to set up and produces reproducible results, facilitating studies on this poorly explored form of plant-fungal interactions. We also optimized conditions for extracting and identifying fungal VCs using solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS).

Keywords: Arabidopsis thaliana (拟南芥), GC-MS (GC-MS), I plate (I板), Plant-fungal interaction (植物真菌互作), SPME (SPME), Verticillium dahliae (大丽轮枝菌), Volatile compounds (挥发性化合物)

Background

Volatile compounds (VCs) have been shown or suggested to play varied and crucial roles in mediating organismal interactions within and across kingdoms. Plants rely on VCs to attract pollinators, seed dispersers, and parasitoids (Baldwin, 2010; Herrmann, 2010). Animals have evolved sophisticated olfactory systems to detect and respond to foods, threats, and mates through volatile cues (Buck, 2004). Similarly, microbial VCs seem to perform diverse functions such as suppressing competitors, regulating their population density, and controlling morphological transitions (Bailly and Weisskopf, 2012; Bennett et al., 2012; Bitas et al., 2013). Roles of microbial VCs in plant growth, development, and stress response have been investigated using several experimental setups that physically separate microbial cells from plants so that only VC-mediated interaction can occur (Ryu et al., 2003; Kai and Piechulla, 2009; Xie et al., 2009; Hung et al., 2013; Vaishnav et al., 2015). Among them, a method employing bipartite Petri plate, also called I plate, has been most frequently used. We adopted I plate to study the effect of VCs produced by soilborne fungal pathogens on plant growth, development, and responses to biotic and abiotic stresses (Bitas et al., 2015; Li and Kang, 2018; Li et al., 2018b). In addition, we optimized a scheme for VC extraction and analysis to help identify fungal VCs that are responsible for modulating plant growth and development (Li et al., 2018b).

Here, we provide a detailed protocol for setting up an I plate assay used for evaluating the effect of VCs produced by Verticillium dahliae, a devastating soilborne fungal pathogen that infects hundreds of plant species, on Arabidopsis thaliana. This protocol enables rapid and straightforward determination of if and how fungal VCs affect plants. We also describe a protocol for capturing VCs through solid phase microextraction (SPME) and analyzing extracted VCs via gas chromatography-mass spectrometry (GC-MS). In combination, these protocols will help explore how VCs produced by diverse fungi affect plants and can also be applied to study VC-mediated interactions between microbes.

Materials and Reagents

  1. Surgical blades #10 and #11
  2. Parafilm (Bemis, catalog number: PM-99)
  3. Paper towel
  4. 10 µl and 1,000 µl micropipette tips
  5. 100 x 15 mm I plate (VWR, catalog number: 25384-310)
  6. 100 x 100 mm square plate (VWR, catalog number: 10799-140)
  7. 100 x 15 mm (VWR, catalog number: 25384-302) and 60 x 15 mm (VWR, catalog number: 25384-092) Petri plates
  8. 1.7 ml microcentrifuge tube (VWR, catalog number: 87003-294)
  9. 25 ml serological pipette (VWR, catalog number: 89130-900) 
  10. Filter unit with 0.2 µM cellulose membrane (Nalgene, catalog number: 121-0020)
  11. 1.5 ml sample vial (Shimadzu, catalog number: 221-34274-91), white cap with septum
  12. V. dahliae strains PD322 and PD413 (conidial suspension in 20% glycerol and stored at -80 °C)
  13. A. thaliana ecotype Col-0 seeds (Lehle Seed Co.) 
  14. Sterile MilliQ water
  15. Murashige and Skoog (MS) basal medium (Sigma-Aldrich, catalog number: M0404-10L)
  16. Sucrose (Alfa Aesar, catalog number: A15583)
  17. Granulated agar (Difco, catalog number: 214530)
  18. Potato dextrose agar (PDA) (Difco, catalog number: 213400)
  19. 200 proof ethanol (KOPTEC, catalog number: 64-17-5)
  20. 6% sodium hypochlorite (CLOROX) 
  21. n-Hexane (EMSURE, catalog number: 1043744000)
  22. C7-C30 saturated alkanes (Sigma-Aldrich, catalog number: 49451-U) 
  23. 99.99% pure helium gas 
  24. 0.5x PDA medium (see Recipes)
  25. MS agar medium (see Recipes)

Equipment

  1. Pyrex glass bottle
  2. 10 µl and 1,000 µl micropipettes
  3. Scalpel
  4. Forceps
  5. Cork borer (5 mm in diameter)
  6. SPME fiber holder (Supelco, catalog number: 57330-U)
  7. 15 ml clear glass vial (Supelco, catalog number: 27159), screw cap with PTFE/silicone septum 
  8. SPME fiber assembly with 50/30 µm DVB/CAR/PDMS fiber coating (Supelco, catalog number: 57328-U)
  9. Electric pipette controller (Drummond Scientific Co., catalog number: 4-000-110-TC)
  10. Vortex (VWR, model: Genie 2)
  11. Table-top centrifuge (Eppendorf, model: 5417C) 
  12. Table-top shaker (VWR, catalog number: 57018-754)
  13. Analytical balance (Mettler Toledo, model: AE-100)
  14. Dissecting microscope (Zeiss, model: Stemi 2000-C)
  15. Incubator (Sheldon Manufacturing, model: 1510E)
  16. Plant growth chamber (Conviron, model: CMP5090)
  17. Flexible-arm electrode holder (Mettler Toledo, catalog number: 30266628)
  18. GC-MS system (Shimadzu, model: GCMS-QP2010 ultra) equipped with AOC-20i auto injector (Shimadzu, catalog number: 221-72315-48) 
  19. Rtx-Wax capillary (60 m, 0.25 mm ID and 0.25 μm df) column (Restek, catalog number: 12426)
  20. 4 °C refrigerator
  21. Autoclave

Software

  1. ImageJ (version 1.52a) 
  2. GC-MS Solution (Shimadzu, version 2.72), a package supporting GC-MS real-time and post-run analyses
  3. National Institute of Standards and Technology (NIST) Mass spectral library (Shimadzu, version 11)

Procedure

  1. I plate assay (Figure 1)
    1. Use a sterile 10 µl pipette tip to streak V. dahliae stock on 0.5x PDA (Recipe 1) plate in a zigzag pattern and incubate at 22 °C for 10 days. 
    2. Surface sterilize A. thaliana seeds as follows:
      1. Add 1 ml 95% ethanol to a 1.7 ml microcentrifuge tube containing seeds, vortex, and incubate for 1 min.
      2. After removing ethanol, wash once with sterile MilliQ water and discard the water.
      3. Add 1 ml 6% sodium hypochlorite solution, vortex, and incubate for 15 min by shaking at 100 rpm.
      4. Wash twice with sterile MilliQ water after removing the sodium hypochlorite solution.
      5. Incubate seeds in 1 ml sterile MilliQ water at 4 °C in darkness for 3 days. 
    3. Prepare square plates with MS agar (Recipe 2) and slice the medium into 10 x 10 mm pieces using a sterilized scalpel with blade #10.
    4. Hold one seed at the tip of a 10 µl micropipette using suction and then release the seed onto each agar piece. Seal the plate with two layers of Parafilm and place in a plant growth chamber set at 22 °C, 12 h light (4,500 lux, 60 μmol photons m-2 s-1), and 60% relative humidity for 7 days.
    5. Prepare I plate by adding 8 ml MS agar to one compartment and 8 ml 0.5x PDA to the other compartment. 
    6. Transfer five A. thaliana seedlings (similar in size and growth stage) along with attached agar piece to the MS side of I plate using a sterilized scalpel with blade #10.
    7. Use a heat-sterilized cork borer to generate culture plugs along the actively growing margin of V. dahliae culture and place one plug (upside down) to the far end of the PDA side of I plate using a sterilized scalpel with blade #11.
    8. Seal the inoculated I plate with two layers of Parafilm and place in a plant growth chamber for a designated amount of time for each experiment (see Li et al., 2018b for specific examples).


    Figure 1. Workflow of the I plate-based assay

  2. Extraction of VCs produced by V. dahliae (Figure 2)
    1. Inoculate a plug of V. dahliae culture on 8 ml PDA slant in a 15 ml clear glass vial. Seal the vial with Parafilm. 
    2. Incubate at 22 °C until the culture fully covers the surface of PDA slant, which typically takes 8 days.
    3. Replace Parafilm with a screw cap containing PTFE/silicone septum and incubate for one day.
    4. Condition the SPME fiber before VC extraction by placing the SPME needle into the GC injection port set at 230 °C for 1 h.
    5. Insert the conditioned SPME fiber in the injection port and starting the GC temperature program shown in Table 1. Desorb the fiber for 5 min in the injection port. Retract the fiber and remove the needle after 5 min and check resulting chromatograms when the program is completed. The intensity of background peaks (= blank sample) should be very low. Otherwise, repeat the desorption of the fiber. 
    6. Extract VCs for 1 h by leaving the SPME fiber in the headspace of sampling vial. Use a flexible-arm electrode holder to lock the position of the SPME fiber holder so that the insertion depth of fiber is uniform for all extractions. 


    Figure 2. Workflow of volatile compound extraction

    Table 1. Conditions for gas chromatography-mass spectrometry analysis


  3. GC-MS analysis of extracted VCs (Figure 3)
    1. Analyze the extracted VCs using a GC-MS system (manual injection mode, Figure 3A) by following the conditions described in Table 1. Insert the SPME needle into the GC injection port immediately after retracting it from each sampling vial. Desorb VCs bound to the SPME fiber for 5 min. An Rtx-Wax capillary column is used as it exhibits better retention and separation of polar VCs. 
    2. Place the fiber in the GC injection port for 20 min for desorption before extracting another sample of VCs.
    3. Analyze C7-C30 saturated alkane standards using the automatic sample injection mode (Figure 3B). Connect the AOC-20i auto injector to the GC injection port. Place a 1.5 ml sample vial containing 0.5 ml solution of standards (1:50 dilution with n-hexane) on the rack of the auto injector. Inject 1 μl solution into the GC-MS system and analyze using the same column and conditions (Table 1).


    Figure 3. Gas chromatography-mass spectrometry analysis. A. Extracted volatile compounds (VCs) from V. dahliae were analyzed via the manual injection mode. B. Alkane standards were analyzed using the automatic sample injection mode.

Data analysis

  1. Assessment of root growth and development after individual VC treatments
    1. After co-cultivation, use forceps to gently pull A. thaliana seedlings from the medium without damaging their roots.
    2. Remove excess moisture on the roots using a paper towel.
    3. Detaching roots from the shoot using forceps, weigh the roots immediately using an analytical balance. 
    4. Mount the roots on a flat surface (e.g., 100 x 15 mm Petri plate) and add 3 ml water on them. 
    5. Gently spread the roots using forceps so that the primary and lateral roots do not overlap.
    6. Use the bottom of 60 x 15 mm Petri plate to flatten the roots (Figure 4A) for subsequent measurements: 
      1. Count the number of lateral roots, including all branches, under a dissecting microscope. 
      2. Take pictures of the roots and import them to ImageJ. Follow the instruction in ImageJ to measure the primary root length (Figure 4B).
    7. Calculate the lateral root density of each sample by dividing the number of lateral roots by the primary root length.


      Figure 4. Analysis of A. thaliana roots after treating with volatile compounds produced by V. dahliae. A. Flattened A. thaliana roots by pressing with a 60 x 15 mm Petri plate. B. Snapshot of ImageJ data used to measure the length of the primary root (indicated by the yellow line) after VC treatment. The start and end of the primary root were indicated using red arrows.

  2. Identification of individual compounds
    1. Peak finding, peak integration, and retention time correction were performed using the post-run analysis software in the GC-MS Solution package.
    2. The putative identity of each compound (peak) was determined using the following methods.
      1. Compare resulting mass spectral profiles with reference data archived in the NIST mass spectral library (version 11). The top hit(s) with match factors ≥ 90% were put on a “positive list” of tentatively identified compounds.
      2. Calculate the retention index (RI) of each compound using the following equation (for temperature programmed chromatography) (Kováts, 1958).


        where,
        x = Unknown compound in the sample
        n = The number of carbons in the alkane preceding the unknown compound
        N = The number of carbons in the alkane following the unknown compound
        Tx = The retention time of the unknown compound
        Tn = The retention time of the preceding alkane
        TN = The retention time of the following alkane

      The experimentally obtained RI of each peak was compared to those in the NIST Chemistry WebBook (https://webbook.nist.gov, using polar columns). For positive confirmation of identity, a maximum relative deviation of ± 2% from published values was used (Stoppacher et al., 2010).

Notes

  1. The I plate assay may not be suitable for studying the effect of VCs produced by fungi that grow rapidly, as fungal mycelia may grow over the central divider of I plate and contaminate the MS medium. We removed agar strips from both sides of the divider of I plate to prevent this kind of contamination when we studied fungi that grow faster than V. dahliae (Bitas et al., 2015). However, for fungi like Trichoderma, this measure was not sufficient (Li et al., 2018a).
  2. Due to the limited space of I plate, only small plants (e.g., A. thaliana and Nicotiana benthamiana) in their early growth stage are suitable for this assay. 
  3. Under the growth conditions used, growth promoting effect on A. thaliana becomes noticeable as early as after 7 days of co-cultivation with V. dahliae (Li et al., 2018b). 
  4. Because A. thaliana starts to initiate inflorescence development after 14 days of co-cultivation, we recommend that the duration of co-cultivation should not exceed 14 days. 
  5. Here, we only described how to analyze the root growth and development after VC exposure. Other traits of VC-exposed plants, such as shoot weight, chlorophyll content, physiological and molecular changes, can also be analyzed (e.g., Zhang et al., 2007 and 2008; Li et al., 2018b). 
  6. Several SPME fibers and GC columns may need to be evaluated to optimize the scheme for VC extraction and analysis. In our research, the DVB/CAR/PDMS 50/30 µm fiber coating extracted the largest number of VCs, and the Rtx-Wax capillary column enabled better separation of extracted VCs than the DB-5 column.
  7. Analysis of VCs extracted from uninoculated PDA slant is necessary to exclude VCs derived from the medium and the environment.

Recipes

  1. 0.5x PDA medium (1 L)
    19.5 g PDA
    Pour the autoclaved medium after cooling it down to 50 °C 
  2. MS agar medium (1 L)
    4.5 g MS and 7.5 g agar
    Add 5 ml 50% (w/v) sucrose (filter sterilized) after cooling down the autoclaved medium to 50 °C
    Mix well before pouring

Acknowledgments

Our research on fungal VCs has been supported by a grant to SK from the USDA-Specialty Crop Multi-State Program (AM170200XXXXG006), a fellowship to NL from the Storkan-Hanes-McCaslin Foundation, and the Huck Dissertation Research Award to NL. The I plate assay and the method for VC extraction were adopted from Ryu et al. (2003) and Stoppacher et al. (2010), respectively.

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Bailly, A. and Weisskopf, L. (2012). The modulating effect of bacterial volatiles on plant growth: current knowledge and future challenges. Plant Signal Behav 7(1): 79-85.
  2. Baldwin, I. T. (2010). Plant volatiles. Curr Biol 20(9): R392-397.
  3. Bennett, J. W., Hung, R., Lee, S. and Padhi, S. (2012). 18 Fungal and bacterial volatile organic compounds: An overview and their role as ecological signaling agents. In: Hock, B. (Ed.). Fungal Associations. Springer, Berlin, Heidelberg, 373-393.
  4. Bitas, V., Kim, H. S., Bennett, J. W. and Kang, S. (2013). Sniffing on microbes: diverse roles of microbial volatile organic compounds in plant health. Mol Plant Microbe Interact 26(8): 835-843.
  5. Bitas, V., McCartney, N., Li, N., Demers, J., Kim, J. E., Kim, H. S., Brown, K. M. and Kang, S. (2015). Fusarium oxysporum volatiles enhance plant growth via affecting auxin transport and signaling. Front Microbiol 6: 1248.
  6. Buck, L. B. (2004). Olfactory receptors and odor coding in mammals. Nutr Rev 62(11 Pt 2): S184-188; discussion S224-141.
  7. Herrmann, A. (2010). The chemistry and biology of volatiles. In: Herrmann, A. (Ed.). Chichester, John Wiley & Sons, Ltd.
  8. Hung, R., Lee, S. and Bennett, J. W. (2013). Arabidopsis thaliana as a model system for testing the effect of Trichoderma volatile organic compounds. Fungal Ecology 6(1): 19-26.
  9. Kai, M. and Piechulla, B. (2009). Plant growth promotion due to rhizobacterial volatiles--an effect of CO2? FEBS Lett 583(21): 3473-3477.
  10. Kováts, E. (1958). Gas-chromatographische charakterisierung organischer verbindungen. Teil 1: Retentionsindices aliphatischer halogenide, alkohole, aldehyde und ketone. Helvetica Chimica Acta 41(7): 1915-1932.
  11. Li, N., Alfiky, A., Wang, W., Islam, M., Nourollahi, K., Liu, X. and Kang, S. (2018a). Volatile compound-mediated recognition and inhibition between Trichoderma biocontrol agents and Fusarium oxysporum. Front Microbiol 9: 2614.
  12. Li, N. and Kang, S. (2018). Do volatile compounds produced by Fusarium oxysporum and Verticillium dahliae affect stress tolerance in plants? Mycology 9(3): 166-175.
  13. Li, N., Wang, W., Bitas, V., Subbarao, K., Liu, X. and Kang, S. (2018b). Volatile compounds emitted by diverse Verticillium species enhance plant growth by manipulating auxin signaling. Mol Plant Microbe Interact 31(10): 1021-1031.
  14. Ryu, C. M., Farag, M. A., Hu, C. H., Reddy, M. S., Wei, H. X., Pare, P. W. and Kloepper, J. W. (2003). Bacterial volatiles promote growth in Arabidopsis. Proc Natl Acad Sci U S A 100(8): 4927-4932.
  15. Stoppacher, N., Kluger, B., Zeilinger, S., Krska, R. and Schuhmacher, R. (2010). Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride by HS-SPME-GC-MS. J Microbiol Methods 81(2): 187-193.
  16. Vaishnav, A., Kumari, S., Jain, S., Varma, A. and Choudhary, D. K. (2015). Putative bacterial volatile-mediated growth in soybean (Glycine max L. Merrill) and expression of induced proteins under salt stress. J Appl Microbiol 119(2): 539-551.
  17. Xie, X., Zhang, H. and Pare, P. W. (2009). Sustained growth promotion in Arabidopsis with long-term exposure to the beneficial soil bacterium Bacillus subtilis (GB03). Plant Signal Behav 4(10): 948-953.
  18. Zhang, H., Kim, M. S., Krishnamachari, V., Payton, P., Sun, Y., Grimson, M., Farag, M. A., Ryu, C. M., Allen, R., Melo, I. S. and Pare, P. W. (2007). Rhizobacterial volatile emissions regulate auxin homeostasis and cell expansion in Arabidopsis. Planta 226(4): 839-851.
  19. Zhang, H., Xie, X., Kim, M. S., Kornyeyev, D. A., Holaday, S. and Pare, P. W. (2008). Soil bacteria augment Arabidopsis photosynthesis by decreasing glucose sensing and abscisic acid levels in planta. Plant J 56(2): 264-273.

简介

生物挥发性化合物(VC)介导植物,动物和微生物中各种类型的关键的种内和种间相互作用,因为它们能够穿过空气,液体和多孔土壤。 为了研究由土霉菌(Verticillium dahliae)(一种土壤真菌病原体)产生的VC如何影响植物生长和发育,我们稍微修改了一种先前用于研究细菌VC对植物生长的影响的方法。 该方法涉及在I板中培养微生物细胞和植物以仅允许VC介导的相互作用。 修改后的方案易于设置并产生可重复的结果,便于研究这种研究较少的植物 - 真菌相互作用形式。 我们还使用固相微萃取(SPME)与气相色谱 - 质谱联用(GC-MS)优化了提取和鉴定真菌VC的条件。
【背景】已经显示或建议挥发性化合物(VC)在介导王国内部和跨王国的生物相互作用中发挥各种和关键的作用。植物依靠VC来吸引传粉媒介,种子传播者和寄生蜂(Baldwin,2010; Herrmann,2010)。动物已经进化出复杂的嗅觉系统,通过挥发性线索检测和应对食物,威胁和配偶(Buck,2004)。同样,微生物VC似乎具有多种功能,如抑制竞争对手,调节其种群密度和控制形态转变(Bailly和Weisskopf,2012; Bennett et al。,2012; Bitas et al 。,2013)。微生物VC在植物生长,发育和应激反应中的作用已经使用几种实验设置进行了研究,这些实验设置将微生物细胞与植物物理分离,因此只能发生VC介导的相互作用(Ryu et al。,2003 ; Kai和Piechulla,2009; Xie et al。,2009; Hung et al。,2013; Vaishnav et al。,2015)。其中,最常使用的是采用二分Petri板(也称为I板)的方法。我们采用I plate来研究土壤真菌病原体产生的VC对植物生长,发育以及对生物和非生物胁迫的反应的影响(Bitas et al。,2015; Li and Kang,2018; Li 等人,2018b)。此外,我们优化了VC提取和分析方案,以帮助鉴定负责调节植物生长和发育的真菌VC(Li et al。,2018b)。

在这里,我们提供了一个详细的协议,用于建立一个I平板测定,用于评估黄萎病菌产生的VC的影响,黄萎病菌是一种破坏性的土壤真菌病原体,感染数百种植物,拟南芥拟南芥拟南芥。该协议能够快速,直接地确定真菌VC是否以及如何影响植物。我们还描述了通过固相微萃取(SPME)捕获VC并通过气相色谱 - 质谱(GC-MS)分析提取的VC的方案。结合起来,这些方案将有助于探索由不同真菌产生的VC如何影响植物,并且还可以用于研究VC介导的微生物之间的相互作用。

关键字:拟南芥, GC-MS, I板, 植物真菌互作, SPME, 大丽轮枝菌, 挥发性化合物

材料和试剂

  1. 手术刀片#10和#11
  2. Parafilm(Bemis,目录号:PM-99)
  3. 纸巾
  4. 10μl和1,000μl微量移液器吸头
  5. 100 x 15 mm I板(VWR,目录号:25384-310)
  6. 100 x 100 mm方板(VWR,目录号:10799-140)
  7. 100 x 15 mm(VWR,目录号:25384-302)和60 x 15 mm(VWR,目录号:25384-092)培养皿
  8. 1.7毫升微量离心管(VWR,目录号:87003-294)
  9. 25毫升血清移液管(VWR,目录号:89130-900) 
  10. 带0.2μM纤维素膜的过滤装置(Nalgene,目录号:121-0020)
  11. 1.5ml样品瓶(Shimadzu,目录号:221-34274-91),带隔膜的白色盖子
  12. 诉dahliae 菌株PD322和PD413(在20%甘油中分生孢子悬浮液并储存在-80°C)
  13. 甲。拟南芥生态型Col-0种子(Lehle Seed Co.) 
  14. 无菌MilliQ水
  15. Murashige和Skoog(MS)基础培养基(Sigma-Aldrich,目录号:M0404-10L)
  16. 蔗糖(Alfa Aesar,目录号:A15583)
  17. 颗粒状琼脂(Difco,目录号:214530)
  18. 马铃薯葡萄糖琼脂(PDA)(Difco,目录号:213400)
  19. 200标准乙醇(KOPTEC,目录号:64-17-5)
  20. 6%次氯酸钠(CLOROX) 
  21. 正己烷(EMSURE,目录号:1043744000)
  22. C 7 -C 30 饱和烷烃(Sigma-Aldrich,目录号:49451-U) 
  23. 99.99%纯氦气 
  24. 0.5x PDA介质(参见食谱)
  25. MS琼脂培养基(见食谱)

设备

  1. 耐热玻璃瓶
  2. 10μl和1,000μl微量移液管
  3. 解剖刀
  4. 钳子
  5. 软木钻孔机(直径5毫米)
  6. SPME纤维支架(Supelco,目录号:57330-U)
  7. 15毫升透明玻璃小瓶(Supelco,目录号:27159),带PTFE /硅胶隔垫的螺帽 
  8. SPME纤维组件,带50 /30μmDVB/ CAR / PDMS纤维涂层(Supelco,目录号:57328-U)
  9. 电动移液器控制器(Drummond Scientific Co.,目录号:4-000-110-TC)
  10. Vortex(VWR,型号:Genie 2)
  11. 台式离心机(Eppendorf,型号:5417C) 
  12. 台式振动台(VWR,目录号:57018-754)
  13. 分析天平(Mettler Toledo,型号:AE-100)
  14. 解剖显微镜(Zeiss,型号:Stemi 2000-C)
  15. 孵化器(Sheldon Manufacturing,型号:1510E)
  16. 植物生长室(Conviron,型号:CMP5090)
  17. 柔性臂电极座(Mettler Toledo,目录号:30266628)
  18. GC-MS系统(Shimadzu,型号:GCMS-QP2010 ultra)配备AOC-20i自动注射器(Shimadzu,目录号:221-72315-48) 
  19. Rtx-Wax毛细管(60 m,0.25 mm ID和0.25μmdf)色谱柱(Restek,目录号:12426)
  20. 4°C冰箱
  21. 高压灭菌器

软件

  1. ImageJ(版本1.52a) 
  2. GC-MS解决方案(Shimadzu,版本2.72),一个支持GC-MS实时和运行后分析的软件包
  3. 美国国家标准与技术研究院(NIST)质谱库(Shimadzu,版本11)

程序

  1. 我用平板测定法(图1)
    1. 使用无菌的10μl移液管尖端划线 V. dahliae 在0.5x PDA(配方1)板上以Z字形图案储存,并在22°C下孵育10天。 
    2. 表面消毒 A.拟南芥种子如下:
      1. 将1ml 95%乙醇加入含有种子的1.7ml微量离心管中,涡旋并孵育1分钟。
      2. 除去乙醇后,用无菌MilliQ水洗涤一次并弃去水。
      3. 加入1ml 6%次氯酸钠溶液,涡旋,并以100rpm振荡孵育15分钟。
      4. 在除去次氯酸钠溶液后,用无菌MilliQ水洗涤两次。
      5. 将种子在1 ml无菌MilliQ水中于4°C在黑暗中孵育3天。 
    3. 用MS琼脂(配方2)准备方形板,并使用带刀片#10的无菌手术刀将介质切成10 x 10 mm的碎片。
    4. 使用抽吸将一粒种子保持在10μl微量移液管的尖端,然后将种子释放到每个琼脂片上。用两层Parafilm密封平板并置于设置在22℃,12小时光照的植物生长室中(4,500勒克斯,60μmol光子m -2 s -1 )和60%相对湿度7天。
    5. 通过将8ml MS琼脂加入一个隔室并将8ml 0.5x PDA加入另一个隔室来制备I盘。 
    6. 转移五个 A.将thaliana 幼苗(大小和生长阶段相似)连同附着的琼脂片一起使用带有刀片#10的无菌解剖刀到I板的MS侧。
    7. 使用加热灭菌的软木蛀虫沿 V的活跃生长边缘生成培养塞。 dahliae 培养并使用带刀片#11的无菌手术刀将一个塞子(倒置)放到I板的PDA侧的远端。
    8. 用两层Parafilm密封接种的I板,并在每个实验中将其放置在植物生长室中指定的时间量(具体实施例参见Li 等人,2018b)。


    图1.基于I板的分析的工作流程

  2. 提取由 V产生的VC。 dahliae (图2)
    1. 接种 V的插头。在8ml PDA上的大丽花培养物在15ml透明玻璃小瓶中倾斜。用Parafilm密封小瓶。 
    2. 在22°C孵育直至培养物完全覆盖PDA倾斜表面,通常需要8天。
    3. 用含有PTFE /硅胶隔垫的螺帽更换Parafilm并孵育一天。
    4. 通过将SPME针放入设定在230°C的GC进样口1小时,在VC萃取前调节SPME纤维。
    5. 将经过调节的SPME光纤插入进样口并启动表1中所示的GC温度程序。在进样口中将光纤解吸5分钟。 5分钟后收回光纤并取出针头,并在程序完成后检查生成的色谱图。背景峰的强度(=空白样本)应该非常低。否则,重复解吸纤维。 
    6. 将SPME纤维留在取样瓶的顶部空间中,提取VC 1小时。使用柔性臂电极夹固定SPME光纤夹持器的位置,使所有提取的光纤插入深度均匀。 


    图2.挥发性化合物提取的工作流程

    表1.气相色谱 - 质谱分析的条件


  3. 提取的VC的GC-MS分析(图3)
    1. 按照表1中所述的条件,使用GC-MS系统(手动进样模式,图3A)分析提取的VC。在将SPME针从每个取样瓶中收回后立即将其插入GC进样口。 Desorb VC与SPME纤维结合5分钟。使用Rtx-Wax毛细管色谱柱,因为它表现出更好的保留和分离极性VC。 
    2. 将光纤放入GC进样口20分钟进行解吸,然后再提取另一个VC样品。
    3. 使用自动样品注射模式分析C 7 -C 30 饱和烷烃标准品(图3B)。将AOC-20i自动注射器连接到GC进样口。将1.5ml含有0.5ml标准溶液(用正己烷1:50稀释)的样品瓶放在自动注射器的支架上。将1μl溶液注入GC-MS系统,并使用相同的色谱柱和条件进行分析(表1)。


    图3.气相色谱 - 质谱分析 A.从 V中提取的挥发性化合物(VC)。 dahliae 通过手动注射模式进行分析。 B.使用自动进样模式分析Alkane标准品。

数据分析

  1. 个体VC处理后根系生长和发育的评估
    1. 共培养后,用镊子轻轻拉动 A. thaliana 幼苗来自培养基而不损伤其根。
    2. 用纸巾去除根部多余的水分。
    3. 使用镊子从枝条上分离根部,使用分析天平立即称量根部。 
    4. 将根部安装在平坦的表面上(例如,100 x 15 mm培养皿)并在其上加3 ml水。 
    5. 使用镊子轻轻地展开根部,使主根和侧根不重叠。
    6. 使用60 x 15 mm培养皿的底部使根部变平(图4A),以便进行后续测量: 
      1. 在解剖显微镜下计算侧根的数量,包括所有分支。 
      2. 拍摄根部照片并将它们导入ImageJ。按照ImageJ中的说明测量主根长度(图4B)。
    7. 通过将侧根数除以主根长度来计算每个样本的侧根密度。


      图4. A的分析。用 V产生的挥发性化合物处理后的拟南芥根。 dahliae 。 A.扁平 A。通过用60×15mm培养皿压制来制备拟南芥。 B.用于在VC处理后测量主根长度(由黄线表示)的ImageJ数据的快照。使用红色箭头指示主根的开始和结束。

  2. 鉴定个别化合物
    1. 使用GC-MS Solution包中的运行后分析软件进行峰发现,峰积分和保留时间校正。
    2. 使用以下方法测定每种化合物(峰)的推定特性。
      1. 将得到的质谱图与NIST质谱库(版本11)中存档的参考数据进行比较。匹配因子≥90%的最高命中率被列入暂定标识化合物的“肯定列表”。
      2. 使用以下等式计算每种化合物的保留指数(RI)(用于程序升温色谱法)(Kováts,1958)。


        哪里,
        x =样品中未知化合物
        n =未知化合物前的烷烃中的碳数
        N =未知化合物后烷烃中的碳数
        Tx =未知化合物的保留时间
        Tn =前一烷烃的保留时间
        TN =以下烷烃的保留时间

      将实验获得的每个峰的RI与NIST Chemistry WebBook中的RI进行比较( href="https://webbook.nist.gov/" target="_blank"> https://webbook.nist.gov ,使用极柱)。为了确认同一性,使用了与公布值的±2%的最大相对偏差(Stoppacher et al。,2010)。

笔记

  1. I平板测定可能不适合研究由快速生长的真菌产生的VC的影响,因为真菌菌丝体可能在I板的中央分隔物上生长并污染MS培养基。当我们研究生长速度比 V的真菌时,我们从I板分隔器的两侧去除琼脂条以防止这种污染。 dahliae (Bitas et al。,2015)。然而,对于像 Trichoderma 这样的真菌,这种方法还不够(Li et al。,2018a)。
  2. 由于I板的空间有限,在其早期生长阶段只有小型植物(例如, A.thaliana 和 Nicotiana benthamiana )是合适的用于此测定。 
  3. 在使用的生长条件下,对 A的生长促进作用。早在与 V共培养7天后,拟南芥就变得显着了。 dahliae (Li et al。,2018b)。 
  4. 因为 A.在共培养14天后,拟南芥开始引发花序发育,我们建议共培养的持续时间不应超过14天。 
  5. 在这里,我们仅描述了如何分析VC暴露后的根系生长和发育。还可以分析VC暴露植物的其他性状,如枝条重量,叶绿素含量,生理和分子变化( eg ,Zhang et al。,2007和2008 ; Li et al。,2018b)。 
  6. 可能需要评估几种SPME纤维和GC色谱柱以优化VC提取和分析方案。在我们的研究中,DVB / CAR / PDMS 50 /30μm纤维涂层提取了最多的VC,Rtx-Wax毛细管柱比DB-5柱更好地分离了提取的VC。
  7. 从未接种的PDA倾斜中提取的VC的分析对于排除源自培养基和环境的VC是必要的。

食谱

  1. 0.5x PDA中等(1升)
    19.5克PDA
    将高压灭菌的培养基冷却至50°C后倒入 
  2. MS琼脂培养基(1升)
    4.5克MS和7.5克琼脂
    将高压灭菌的培养基冷却至50°C后,加入5 ml 50%(w / v)蔗糖(过滤灭菌)
    浇注前要好好混合

致谢

我们对真菌风险投资的研究得到了美国农业部 - 特种农作物多州计划(AM170200XXXXG006)的资助,以及来自Storkan-Hanes-McCaslin基金会的NL和NL的哈克学位论文研究奖。 I plate试验和VC提取方法分别采用Ryu 等人(2003)和Stoppacher 等人(2010)。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

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引用:Wang, W., Li, N., Liu, X. and Kang, S. (2019). I Plate-based Assay for Studying How Fungal Volatile Compounds (VCs) Affect Plant Growth and Development and the Identification of VCs via SPME-GC-MS. Bio-protocol 9(4): e3166. DOI: 10.21769/BioProtoc.3166.
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