番茄离体及活体灰葡萄孢菌接种试验

Jiajie Lian; Hongyu Han; Jiuhai Zhao; Chuanyou  Li

doi:10.21769/BioProtoc.2810

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Peer-reviewed

In-vitro and in-planta Botrytis cinerea Inoculation Assays for Tomato

番茄离体及活体灰葡萄孢菌接种试验

JL Jiajie Lian ^*

HH Hongyu Han ^*

JZ Jiuhai Zhao email

Chuanyou Li email

(*contributed equally to this work) 发布: 2018年04月20日第8卷第8期 DOI: 10.21769/BioProtoc.2810 浏览次数: 13216

评审: Tuan Minh TranAnonymous reviewer(s)

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Abstract

Botrytis cinerea (B. cinerea) attacks many crops of economic importance, represents one of the most extensively studied necrotrophic pathogens. Inoculation of B. cinerea and phenotypic analysis of plant resistance are key procedures to investigate the mechanism of plant immunity. Here we describe a protocol for B. cinerea inoculation on medium and planta based on our study using the tomato-B. cinerea system.

Keywords: Botrytis cinerea (灰葡萄孢菌)

Tomato (番茄)

Inoculation (接种)

Background

B. cinerea causes serious loss in more than 200 crops worldwide, including many important vegetables and small fruit crops. The broad-spectrum pathogen can infect plant stem, leaf, flower and fruit to produce spores (Dean et al., 2012; van Kan et al., 2017), which prefer to occur under high humidity (Elad et al., 2007). The produced spores pose long lasting threat to diverse hosts (Elad et al., 2007). Based on its scientific and economic importance, B. cinerea was ranked as the second most important plant-pathogenic fungus (Dean et al., 2012). Among B. cinerea host plants, tomato (Solanum lycopersicum), an economically valuable species, also serves as a classic model to study plant immunity (Ryan, 2000; Sun et al., 2011; Rosli and Martin, 2015). To investigate the molecular basis of plant immunity to B. cinerea, we employ a routine procedure to produce B. cinerea spores on artificial media. In addition, we provide detailed methods to infect tomato plants or detached leaves with a controlled strength using the collected spores and quantify disease development. This protocol has been successfully used to reveal the transcriptional regulation of master regulator MYC2 in Jasmonate-mediated plant immunity (Du et al., 2017).

Materials and Reagents

General lab materials, including:
Nylon mesh (Solarbio, catalog number: YA0964 )
Petri dish (9 cm)
Square Petri dish (10 x 10 cm)
Funnel (Conventional type, upper diameter: 8 cm)
1.5 ml microcentrifuge tube (USA Scientific, catalog number: 4036-3204)
Manufacturer: Eppendorf, catalog number: 022363204 .
15 ml centrifuge tube (Corning, catalog number: 430790 )
50 ml centrifuge tube (Shanghai Kirgen, catalog number: KG2821 )
Pipette tips
Micropore tape (3M, Micropore^TM, catalog number: 1530S-1 )
Nutritional soil (Miracle-Gro^® Garden Soil for Vegetables with total N 0.68%, P₂O₅ 0.27% and K₂O 0.36%)
Tomato (Solanum lycopersicum) cv M82
B. cinerea B05.10
V8 juice (Campbell, 100%, original vegetable juice)
Calcium carbonate (CaCO₃) (Sigma-Aldrich, catalog number: V900138 )
Bacto-agar (BD, Bacto^TM, catalog number: 214010 )
Mycological peptone (Sigma-Aldrich, catalog number: 77199 )
Sodium phosphate (Sigma-Aldrich, catalog number: 342483 )
Maltose (Sigma-Aldrich, catalog number: M5885 )
2x V8 agar medium (see Recipes)
SMB medium (see Recipes)
0.8% agar medium (see Recipes)

Equipment

Pipettes (Gilson, Pipetman^® G)
Water bath (YIHENG, model: DK-80 )
Autoclave (Panasonic Healthcare, model: MLS-3781L )
Customized transparent plastic box (materials: Polymeric Methyl Methacrylate, L/W/H: 50/50/50 centimeter, open at the top, Figure 1A) or any incubators that can be used alternatively
Centrifuge (Eppendorf, models: 5810 R and 5424 )
Microscope (ZEISS, model: Axio Imager Z2 ; 20x objective plan-APOCHROMAT; 10x objective EC plan-NEOFLUAR) or other light microscopes
Counting chamber (QIUJING^® KB-K-25, 0.1 mm, 1/400 mm²)

Software

ImageJ (https://imagej.nih.gov/ij/download.html)
Microsoft Excel

Procedure

English

中文翻译

文章信息

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如何引用

Lian, J., Han, H., Zhao, J. and Li, C. (2018). In-vitro and in-planta Botrytis cinerea Inoculation Assays for Tomato. Bio-protocol 8(8): e2810. DOI: 10.21769/BioProtoc.2810.