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

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The Plant Cell
Aug 2017



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)


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

  1. 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 )
  2. Pipette tips
  3. Micropore tape (3M, MicroporeTM, catalog number: 1530S-1 )
  4. Nutritional soil (Miracle-Gro® Garden Soil for Vegetables with total N 0.68%, P2O5 0.27% and K2O 0.36%)
  5. Tomato (Solanum lycopersicum) cv M82
  6. B. cinerea B05.10
  7. V8 juice (Campbell, 100%, original vegetable juice)
  8. Calcium carbonate (CaCO3) (Sigma-Aldrich, catalog number: V900138 )
  9. Bacto-agar (BD, BactoTM, catalog number: 214010 )
  10. Mycological peptone (Sigma-Aldrich, catalog number: 77199 )
  11. Sodium phosphate (Sigma-Aldrich, catalog number: 342483 )
  12. Maltose (Sigma-Aldrich, catalog number: M5885 )
  13. 2x V8 agar medium (see Recipes)
  14. SMB medium (see Recipes)
  15. 0.8% agar medium (see Recipes)


  1. Pipettes (Gilson, Pipetman® G)
  2. Water bath (YIHENG, model: DK-80 )
  3. Autoclave (Panasonic Healthcare, model: MLS-3781L )
  4. 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
  5. Centrifuge (Eppendorf, models: 5810 R and 5424 )
  6. Microscope (ZEISS, model: Axio Imager Z2 ; 20x objective plan-APOCHROMAT; 10x objective EC plan-NEOFLUAR) or other light microscopes
  7. Counting chamber (QIUJING® KB-K-25, 0.1 mm, 1/400 mm2)


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


  1. Preparation of seedling plants for infection
    1. Seeds treatment and germination
      1. Put tomato seeds into a cheesecloth or nylon bag and then incubate in a 50 °C water bath for 25 min. Briefly submerge the seeds in tap water to cool down to room temperature.
        After that, transfer the seeds to a 10% trisodium phosphate (TSP) solution for 15 min and rinse (at least 5 times) in autoclaved distilled water for 5 min at room temperature to remove residual disinfectant.
      2. Germinate tomato seeds for 48 h on a wet filter paper at room temperature in the dark (Figure 1B). Sow the germinated seeds in soil and cultivate in growth chambers under cycles of 16 h light at 25 °C and 8 h darkness at 18 °C. (The light intensity for light cycle was 200 μM photons m-2 sec-1 in our growth chamber.) For disease assay, the tomato seedlings are grown for 4 weeks (Figure 1C).
        Note: In our lab, we use Miracle-Gro® Garden Soil (for Vegetables) for plant incubation, which is pre-sterile and less labor cost. Because plants grow faster in this soil, thus we use 4-week plants for the disease assay.

        Figure 1. Preparation of tomato seedlings for infection. A. Customized transparent plastic box (edge highlighted with black lines) for plant inoculation, with 50 x 50 x 50 centimeter (cm). B. Tomato seeds on wet filter paper for germination after treatments. Scale bar = 1 cm. C. A 4-week-old tomato plant for inoculation assay.

  2. Fungal strains and growth conditions
    1. B. cinerea B05.10 used in this study is routinely maintained on 2x V8 agar for 21 days at 20 °C under a 12-h photoperiod prior to spore collection (Figure 2A).
    2. Harvest spores by scrubbing the plates with pipette tips until the tips have about 2 ml amount of tissues, then suspend in 5 ml 1% SMB. Filter the suspensions through nylon mesh on a funnel to remove mycelia (Figure 2B, Video 1). 

      Video 1. How to collect spores

    3. Measure the concentration of spores with a counting chamber. Figure 2B shows the small squares of counting chamber we used under 20x objective. The counting chamber is 0.1 mm sample depth. The small square is 1/400 mm2 as shown in Figure 2B. It makes the volume of each small square 2.5 x 10-7 ml/square. Each spore in a small square is equal to 4 x 106 spores/ml. Adjust the concentration to 1 x 106 spores/ml for inoculation.
    4. Set a solution of 1% SMB without spores as the control treatment.

  3. Plant infection assay
    The 4-week-old plants are selected for infection assay (Figure 1C).
    For the pathogenicity test on detached leaves:
    1. Individually harvest the leaflet of the third true leaves of 4-week-old plants and gently place in Petri dishes containing 25 ml 0.8% agar, with the petiole embedded in the medium (Video 1).
    2. Inoculate each leaflet with a single 5 μl spore suspensions droplet on the right or left of main midrib (Video 2). Cover plates with lids, followed by sealing with Micropore tape.

      Video 2. How to place a detached leaflet on medium

    3. Place the plates under the same conditions as for plant growth (Figure 2C). Monitor the disease development by scanning the plates 3 days after infection. The size of the infected area was measured with ImageJ.

    For the pathogenicity test on living plants
    1. Place 4-week-old plants in a transparent box (Figure 1A), which keeps the humidity at 100%. Inoculate the third true leaves with B. cinerea. Each leaflet is infected with a single 5 μl spore suspensions droplet on right or left of the main midrib (Video 3). Cover the box with lid and keep it under the same conditions used for plant growth (Figure 2D, Video 3).

      Video 3. Infecting a leaflet with a droplet of spore suspension

    2. Harvest the infected leaflets at different time points (0, 3, 6, 9, 12, 18, 24, 30, 36 h) after infection (HAI) for profiling resistance gene expression or at 3 days after infection (DAI) for monitoring disease development by imaging the leaflets.
    3. Then measure the size of the infected area on those leaflets with ImageJ (see Data analysis), following the procedure of the imageJ User Guide (https://imagej.nih.gov/ij/docs/index.html).

      Figure 2. B. cinerea inoculation assay. A. 3-week-old B. cinerea strain on 2x V8 medium, scale bar for 1 cm. B. The spores of B. cinerea on counting chamber, scale bar for 50 µm. C. Pathogenicity test on detached leaves, the right leaflet was inoculated with a single 5 μl spore suspensions (106 spores/ml) droplet on the right or left of main midrib, while the left leaflet was set as control. The scale bar stands for 1 cm. D. Pathogenicity test on living plants, each leaflet was inoculated with a single 5 μl spore suspensions (106 spores/ml) droplet on the right or left of the main midrib. The areas infected by B. cinerea that developed for 3 days were highlighted with red circles.

Data analysis

Images of the diseased leaflets were imported to software ImageJ. “Polygon selections” was used to select diseased area on the image. Then chose “measure” function from “Analyze” menu in software imageJ to calculate the lesion area. Data analysis was conducted in software Microsoft Excel.
Three independent experiments were conducted, each experiment included at least six replicates. All data from independent experiments were analyzed. The statistical test included average, STDEV and Student’s t-test calculation in excel. The t-test value of 0.05 is considered as significant difference and 0.01 for very significant difference. In the experiment, a droplet of spore suspension on leaf surface should develop to a diseased lesion with round shape. In some cases, the droplet spreads along with leaf vein and developed to an irregular shaped lesion, which should be avoided for analysis. Otherwise, all samples were collected for data analysis.


  1. Freshly produced spore should be used for the infection. The spore suspensions should be gently placed on leaf surface to prevent liquid spreading along with the tiny leaf vein.
  2. For detached leaf infection, the leaflets should cling to agar medium to maintain the moisture. After infection, the medium plates with infected leaves should be sealed with breathable tape to prevent the anaerobic respiration of leaves and keep moisture.


  1. 2x V8 agar medium
    360 ml V8 juice
    2.00 g CaCO3
    20 g Bacto-agar
    Add ddH2O to 1,000 ml
    Autoclave at 121 °C for 15 min
  2. SMB medium
    10.00 g Mycological peptone
    40.00 g Maltose
    Add ddH2O to 1,000 ml
    Final pH (at 25 °C): 5.6 ± 0.2
    Autoclave at 121 °C for 15 min
  3. 0.8% agar medium
    8.00 g Agar
    Add ddH2O to 1,000 ml
    Autoclave at 121 °C for 15 min


This work was supported by the National Key Research and Development Program of China (2016YFD0100603-10), by Tai-Shan Scholar Program from the Shandong Province. This protocol was adapted appropriately from previous work (Du et al., 2017). The authors declare that they have no competing interests.


  1. Elad, Y., Vivier, M. and Fillinger, S. (2016). Botrytis, the good, the bad and the ugly. In: Fillinger, S. and Elad, Y. (Eds). Botrytis: The fungus, the pathogen and its management in agricultural systems. Springer 1-15.
  2. Dahmen, H., Staub, Th. and Schwinn, F. J. (1982). Technique for long-term preservation of phytopathogenic fungi in liquid nitrogen. Phytopathol 73: 6.
  3. Dean, R., Van Kan, J. A., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P. D., Rudd, J. J., Dickman, M., Kahmann, R., Ellis, J. and Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13(4): 414-430.
  4. Du, M., Zhao, J., Tzeng, D. T. W., Liu, Y., Deng, L., Yang, T., Zhai, Q., Wu, F., Huang, Z., Zhou, M., Wang, Q., Chen, Q., Zhong, S., Li, C. B. and Li, C. (2017). MYC2 orchestrates a hierarchical transcriptional cascade that regulates jasmonate-mediated plant immunity in tomato. Plant Cell 29(8): 1883-1906.
  5. Rosli, H. G. and Martin, G. B. (2015). Functional genomics of tomato for the study of plant immunity. Brief Funct Genomics 14(4): 291-301.
  6. Ryan, C. A. (2000). The systemin signaling pathway: differential activation of plant defensive genes. Biochim Biophys Acta 1477(1-2): 112-121.
  7. Sun, J. Q., Jiang, H. L. and Li, C. Y. (2011). Systemin/Jasmonate-mediated systemic defense signaling in tomato. Mol Plant 4(4): 607-615.
  8. van Kan, J. A., Stassen, J. H., Mosbach, A., Van Der Lee, T. A., Faino, L., Farmer, A. D., Papasotiriou, D. G., Zhou, S., Seidl, M. F., Cottam, E., Edel, D., Hahn, M., Schwartz, D. C., Dietrich, R. A., Widdison, S. and Scalliet, G. (2017). A gapless genome sequence of the fungus Botrytis cinerea. Mol Plant Pathol 18(1): 75-89.


灰葡萄孢(Botrytis cinerea)( B. cinerea )攻击许多经济重要作物,代表了研究最广泛的坏死营养病原体之一。 接种 B。 cinerea 和植物抗性的表型分析是研究植物免疫机制的关键程序。 这里我们描述一个用于 B的协议。 基于我们使用番茄B的研究,在培养基和植物上接种灰霉病菌。 cinerea 系统。

【背景】 B中。在全世界超过200种作物中,包括许多重要的蔬菜和小型水果作物,“灰霉病”导致严重损失。广谱病原体可以感染植物茎,叶,花和果实以产生孢子(Dean等人,2012; van Kan等人,2017),其中喜欢在高湿度下发生(Elad等人,2007年)。产生的孢子对多种宿主构成持久的威胁(Elad et al。,2007)。基于其科学和经济的重要性, B。被评为第二大植物致病真菌(Dean等,2012)。在 B中。 cinerea宿主植物,番茄(Solanum lycopersicum)是一种经济上有价值的物种,也可作为研究植物免疫力的经典模型(Ryan,2000; Sun等人, em>,2011; Rosli和Martin,2015)。为了研究植物对 B的免疫分子基础。我们采用常规程序来生产B组。灰霉素孢子在人工培养基上。此外,我们提供详细的方法,使用收集的孢子感染番茄植物或脱落的叶子,并控制其强度,并量化疾病的发展。该协议已成功用于揭示茉莉酸介导的植物免疫中主调节物MYC2的转录调控(Du等人,2017)。

关键字灰葡萄孢菌, 番茄, 接种


  1. 一般实验室材料,包括:
    1.5 ml微量离心管(USA Scientific,目录号:4036-3204)

    15 ml离心管(Corning,目录号:430790)
    50 ml离心管(上海柯尔根公司,产品编号:KG2821)
  2. 移液器吸头
  3. 微孔带(3M,Micropore TM TM,目录号:1530S-1)
  4. 营养土(用于蔬菜的Miracle-Gro 花园土,总N为0.68%,P 2 O 5:0.27%和K 2
    O 0.36%)
  5. 番茄( Solanum lycopersicum )cv M82
  6. B中。灰霉病 B05.10
  7. V8果汁(坎贝尔,100%原汁原味的蔬菜汁)
  8. 碳酸钙(CaCO 3)(Sigma-Aldrich,目录号:V900138)
  9. 细菌琼脂(BD,Bacto TM,目录号:214010)
  10. 真菌蛋白胨(Sigma-Aldrich,目录号:77199)
  11. 磷酸钠(Sigma-Aldrich,目录号:342483)
  12. 麦芽糖(Sigma-Aldrich,目录号:M5885)
  13. 2x V8琼脂培养基(见食谱)
  14. SMB介质(请参阅食谱)
  15. 0.8%琼脂培养基(见食谱)


  1. 移液器(Gilson,Pipetman®) G)
  2. 水浴(益恒,型号:DK-80)

  3. 高压灭菌器(松下医疗,型号:MLS-3781L)
  4. 定制的透明塑料盒(材料:聚甲基丙烯酸甲酯,L / W / H:50/50/50厘米,顶部开口,图1A)或任何可替代使用的培养箱
  5. 离心机(Eppendorf,型号:5810 R和5424)
  6. 显微镜(ZEISS,型号:Axio Imager Z2; 20x物镜规划-APOCHROMAT; 10倍物镜EC计划-NEOFLUAR)或其他光学显微镜
  7. 计数室(QIUJING ® KB-K-25,0.1 mm,1/400 mm 2 )


  1. ImageJ( https://imagej.nih.gov/ij/download.html )< br />
  2. Microsoft Excel


  1. 制备感染的秧苗
    1. 种子处理和发芽
      1. 将番茄种子放入干酪布或尼龙袋中,然后在50°C水浴中孵育25分钟。将种子简短地浸入自来水中冷却至室温。
      2. 在室温下在黑暗中在湿滤纸上发芽番茄种子48小时(图1B)。将萌发的种子播种在土壤中,并在25℃16小时光照和18℃黑暗8小时的循环下在生长室中培养。 (在我们的生长室中光循环的光强度为200μM光子m-2秒-1)。对于疾病测定,番茄幼苗生长4周(图1C)。
        注意:在我们的实验室中,我们使用Miracle-Gro ® 用于植物培育的Garden Soil(用于蔬菜)无菌且劳动力成本较低。由于植物在这片土壤中生长得更快,因此我们使用4周植物进行疾病分析。

    图1.用于感染的番茄幼苗的制备A.用于植物接种的定制透明塑料盒(用黑线突出的边缘),具有50×50×50厘米(cm)。 B.在湿滤纸上的番茄种子在处理后发芽。比例尺= 1厘米。 C.用于接种测定的4周龄番茄植物。

  2. 真菌菌株和生长条件
    1. B中。在孢子收集之前,在本研究中使用的B05.10在20℃下在12-h光周期下常规维持在2x V8琼脂上21天(图2A)。
    2. 通过用移液管端部擦洗板来收获孢子,直到尖端具有约2ml量的组织,然后悬浮在5ml 1%SMB中。通过漏斗上的尼龙网过滤悬浮液以去除菌丝体(图2B,视频1)。


    3. 用计数室测量孢子浓度。图2B显示了我们在20倍物镜下使用的计数室的小方格。计数室的样本深度为0.1毫米。如图2B所示,小方形是1/400mm 2。它使每个小方块的体积为2.5×10 -7毫升/平方。小方块中的每个孢子等于4×10 6孢子/ ml。
      调整浓度至1×10 6孢子/毫升接种。
    4. 设置1%SMB无孢子溶液作为对照处理。

  3. 植物感染分析
    1. 单独收获4周龄植物的第三片真叶的小叶,并轻轻地置于含有25ml 0.8%琼脂的培养皿中,并将叶柄嵌入培养基中(视频1)。
    2. 在主中脉的右侧或左侧接种单个5μl孢子悬浮液小滴(视频2)。盖上盖子,然后用Micropore胶带密封。

    3. 将板放置在与植物生长相同的条件下(图2C)。感染后3天通过扫描板来监测疾病发展。使用ImageJ测量感染区域的大小。

    1. 将4周龄的植物置于透明的盒子中(图1A),使湿度保持在100%。用B接种第三片真叶。孢。每个小叶感染主中脉右侧或左侧的单个5μl孢子悬浮液滴(视频3)。将盖子盖上盖子,并保持在与植物生长相同的条件下(图2D,视频3)。


    2. 在感染后(HAI)的不同时间点(0,3,6,9,12,18,24,30,36 h)收集感染的小叶以用于分析抗性基因表达或在感染后3天(DAI)监测疾病通过对传单成像进行发展。
    3. 然后按照imageJ用户指南( https://imagej.nih.gov/ij/docs/index.html )。

    图2. B。灰霉病接种试验。 :一种。 3周龄的 B。在2×V8培养基上的灰霉病菌株,比例尺为1cm。 B.乙的孢子。 Cinerea 放在计数室中,比例尺为50μm。 C.对离体叶片进行致病性试验,右小叶接种单个5μl孢子悬浮液(10 6孢子/ ml)滴在主中脉的右侧或左侧,而左侧小叶被设定作为控制。比例尺代表1厘米。 D.对活植物的致病性测试,每个小叶在主中脉的右侧或左侧接种单个5μl孢子悬浮液(10 6孢子/ ml)液滴。由 B感染的区域。开发3天的cinerea 用红色圆圈突出显示。


患病传单的图像被导入ImageJ软件。使用“多边形选择”来选择图像上的病变区域。然后从软件imageJ的“分析”菜单中选择“测量”功能来计算病变面积。数据分析在Microsoft Excel软件中进行。
进行了三次独立实验,每个实验至少包括六次重复。分析来自独立实验的所有数据。统计学测试包括平均值,STDEV和excel中的Student's t 测试计算。 0.05的测试值被认为是显着性差异,而0.01是非常显着的差异。在实验中,叶表面上的一滴孢子悬浮液应该发展成圆形的病变病灶。在某些情况下,液滴会随着叶脉扩散并发展为不规则形状的病变,应避免进行分析。否则,收集所有样品进行数据分析。


  1. 新感染的孢子应该用于感染。
  2. 对于分离的叶子感染,小叶应贴在琼脂培养基上以保持湿度。感染后,带感染叶片的培养基板应用透气胶带密封,以防止叶片无氧呼吸并保持水分。


  1. 2x V8琼脂培养基
    将ddH <2> O加入1,000 ml

  2. SMB媒体
    将ddH <2> O加入1,000 ml

  3. 0.8%琼脂培养基

    8.00克琼脂 将ddH <2> O加入1,000 ml





  1. Elad,Y.,Vivier,M.和Fillinger,S。(2016)。 Botrytis ,好,坏和丑陋的。在:Fillinger,S.和Elad,Y.(Eds)中。 Botrytis:农业系统中的真菌,病原体及其管理。 Springer 1-15。
  2. Dahmen,H.,Staub,Th。和Schwinn,F.J。(1982)。 在液氮中长期保存植物病原真菌的技术。 a> Phytopathol 73:6。
  3. Dean,R.,Van Kan,JA,Pretorius,ZA,Hammond-Kosack,KE,Di Pietro,A.,Spanu,PD,Rudd,JJ,Dickman,M.,Kahmann,R.,Ellis,J。和Foster ,GD(2012)。 分子植物病理学中十大真菌病原体分子植物病理学 13(4):414-430。
  4. Du,M.,Zhao,J.,Tzeng,DTW,Liu,Y.,Deng,L.,Yang,T.,Zhai,Q.,Wu,F.,Huang,Z.,Zhou,M.,Wang ,Q.,Chen,Q.,Zhong,S.,Li,CB和Li,C。(2017)。 MYC2协调调控番茄中茉莉酸介导的植物免疫的等级转录级联。 Plant Cell 29(8):1883-1906。
  5. Rosli,H.G和Martin,G.B。(2015)。 番茄功能基因组学研究植物免疫力 简介功能基因组学 14(4):291-301。
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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.