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

Wenzhao  Wang; Ningxiao  Li; Xingzhong  Liu; Seogchan   Kang

doi:10.21769/BioProtoc.3166

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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

WW Wenzhao Wang ^*

Ningxiao Li ^*

XL Xingzhong Liu email

SK Seogchan Kang email

(*contributed equally to this work) 发布: 2019年02月20日第9卷第4期 DOI: 10.21769/BioProtoc.3166 浏览次数: 6951

评审: Zhaohui LiuDemosthenis ChronisTimo A Lehti

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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

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

Equipment

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

Software

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

Procedure

English

中文翻译

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如何引用

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.