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An Assay to Study Botrytis cinerea-infected Grapevine Leaves Primed with Pseudomonas fluorescens

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Molecular Plant Microbe Interactions
Oct 2015



Grapevine (Vitis vinifera L.) is susceptible to an array of diseases among them the grey mold caused by the necrotrophic fungus Botrytis cinerea that decreases grape productivity and quality. To ensure a satisfactory yield and harvest quality numerous chemical fungicides are required, but they have serious drawbacks. One alternative is the use of beneficial bacteria to improve plant health. Pseudomonas fluorescens has been shown to trigger a plant-mediated resistance response in aboveground plant tissues against fungal, oomycete, bacterial, and viral pathogens. Triggered plant resistance exploits mechanisms of the plant immune system through a priming state that provides plants with enhanced capacity for rapid and strong activation of defense responses after pathogen infection, resulting in a lower fitness-cost. The primed responses by beneficial bacteria include induced expression of defense-related genes, cell wall reinforcement, and the production of secondary metabolites after pathogen infection. In this protocol, we describe the experimental design to evaluate the priming state of grapevine plants by the beneficial bacterium Pseudomonas fluorescens PTA-CT2 and their resistance level to Botrytis cinerea according to Verhagen et al. (2011) and Gruau et al. (2015).

Keywords: Biocontrol (生物防治), Pseudomonas (铜绿假单胞菌), Grapevine (小道消息), Priming (启动), Botrytis (灰)

Materials and Reagents

  1. 25 mm culture tubes (VWR, catalog number: 212-0304 )
  2. Conical tubes (50 ml) (Corning, Falcon®, catalog number: 352070 )
  3. Sterile spatula (VWR, catalog number: 612-1561 )
  4. Falcon® cell strainer 100 µm nylon (Corning, Falcon®, catalog number: 352360 )
  5. 1.5 ml microtubes (Treff, catalog number: 96.07246.9.01 )
  6. 2 ml microtubes (Treff, catalog number: 96.09329.9.01 )
  7. Whatman paper ashless discs 90 mm diameter (Sigma-Aldrich, catalog number: WHA1441090 )
  8. Petri dishes (90 x 14 mm) (VWR, catalog number: 391-0439 )
  9. Grapevine (Vitis vinifera cv. Chardonnay 7535) plants obtained by micropropagation from nodal explants on agar Murashige & Skoog (MS) medium (Aziz et al., 2006) in 25 mm culture tubes
  10. Pseudomonas fluorescens PTA-CT2 (Trotel-Aziz et al., 2008)
  11. Botrytis cinerea conidia (Bc 630) isolated from infected grape at ripening by INRA Versailles, France
  12. Distilled water from Aquadem blue range (Véolia Water Aquadem)
  13. Distilled water sterilized with autoclave Avor Getinge
  14. Ethanol (from Alcool absolu) (Charbonneaux Brabant, catalog number: 3077319001001 )
  15. Murashige & Skoog medium (MS) (Duchefa Biochimie, catalog number: M0231.0001 )
  16. Glycerol (VWR, catalog number: 24387-292 )
  17. Luria Bertani liquid medium (LB) (Duchefa Biochimie, catalog number: L1703.0500 )
  18. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M2773 )
  19. BactoTM agar (BD, catalog number: 214010 )
  20. Agar tomato medium (see Recipes)


  1. Plant growth chamber with photoperiod (SANYO, model: MLR-351 H )
  2. Horizontal laminar flow cabinet (ASTEC Microflow, model: Horizontal laminar flow workstation )
  3. Long sterile metal clamp (VWR, catalog number: 232-0084 )
  4. Magenta boxes (Sigma-Aldrich, catalog number: V8505 )
  5. Microbiological safety cabinet (Angelantoni Life Science, model: VBH Compact )
  6. Sterile pipettes (VWR, catalog number: VHPA23004 )
  7. 28 °C incubator shaker (New Brunswick Scientific, model: C24 )
  8. Variable speed refrigerated centrifuge with rotor having number of rotor cavities x nominal largest load (capacity: 8 x 50 ml) (Eppendorf, model: 5804R )
  9. Spectrophotometer (Bio-Rad Laboratories, model: SmartSpec 3000 )
  10. Vortex mixer (Vortex Genie 2 untimed mixer) (VWR, catalog number: 58815-232 )
  11. Malassez counting chamber (Marienfeld, catalog number: 0640610 )
  12. Glass atomizer (VWR, catalog number: 552-0031 )
  13. Camera (Nikon, model: D7000 )


  1. APS Assess 2.0 software (APS, Item No: 43696M5, ISBN 978-0-89054-369-6)


  1. Preparation of plant material
    Grapevine vitro-plantlets (Vitis vinifera L. cv. Chardonnay clone 7535) were micropropagated from nodal explants grown on 15 ml of agar-modified Murashige-Skoog (MS) medium (with 0.7% of Bacto-agar) (Aziz et al., 2003) in 25 mm culture tubes (Figure 1).
    Plants were grown at 25 °C with a 16- and 8-h photoperiod. Under sterile conditions (horizontal laminar flow cabinet) 8-week-old vitro-plantlets are gently removed from the MS agar and transferred with a long sterile metal clamp to sterile Magenta boxes containing sterile liquid MS medium, then closed for 24 h before treatment for acclimation. Each Magenta box contains four sterile lids filled with 9 ml of sterile liquid MS medium and the roots of each plant were immersed individually in each medium (Figure 1).

    Figure 1. Procedure of inoculation with Pseudomonas fluorescens. A. Grapevine plants (Vitis vinifera L. cv. Chardonnay) were sensitive to Botrytis cinerea. B. Grapevine plants transferred to sterile Magenta boxes and inoculated with the bacterium Pseudomonas fluorescens PTA-CT2 at root level.

  2. Bacterial inoculum preparation and inoculation
    1. Withdraw Pseudomonas fluorescens PTA-CT2 strain (Trotel-Aziz et al., 2008) from -80 °C glycerol stock (30%) and in the microbiological safety cabinet use a sterilized pipette tip to inoculate the bacterium in sterile Luria Bertani (LB) liquid medium (100 μl of glycerol stock for 10 ml of culture medium) and grow for 18 h in a 28 °C incubator shaker at 110 rpm.
    2. Use this preculture for 10 new LB cultures by inoculating 1 ml aliquots in 10 x 50 ml sterile tubes containing each 40 ml of LB under the same conditions as in paragraph B1.
    3. Centrifuge the bacterial suspension at 5,000 x g for 10 min, and in the microbiological safety cabinet wash the pellet with sterile 10 mM MgSO4 solution and resuspend it in 10 ml sterile 10 mM MgSO4 solution or MS medium.
    4. Vortex the cell suspension well and check its optical density at 450 and 600 nm (OD450 and OD600) by using a spectrophotometer. To prepare the inoculum, adjust the density of the bacterial suspension to 2 x 108 cfu/ml in 10 mM MgSO4 or in liquid MS medium.
    5. With a sterile pipet add 1 ml of bacterial suspension to the roots of each plantlet placed in lids in sterile conditions, to reach a final concentration of 2 x 107 cfu/ml (Figure 2A). For control plantlets, add 1 ml of 10 mM MgSO4 or of liquid MS medium.
    6. Place bacterized and control plantlets in a growth chamber at 22 °C with a 16- and 8-h photoperiod.
    7. For statistical analysis, prepare a minimum of twelve biological replicates.

  3. Fungal inoculum preparation and inoculation
    1. Withdraw conidial suspension of Botrytis cinerea 630 from -80 °C glycerol stock (30%) and in the microbiological safety cabinet use a sterilized pipette tip to prick out conidia (10 µl) on a Petri dish containing tomato agar medium and incubate it for 21 days at 22 °C under continuous light for sporulation.
    2. Put 10 ml sterile water on sporulated B. cinerea and scrape with a sterile spatula to dislodge the conidia and mycelium of B. cinerea.
    3. Transfer the mixture of mycelium and conidia into the 50 ml conical tube, then shake it by vortexing several times to obtain conidia in water.
    4. Filter the obtained suspension using a filter tube (50 ml Falcon® tube containing 100 µM nylon Falcon® cell strainer) to remove mycelium.
    5. Measure the concentration of conidia in the suspension using a Malassez counting chamber and dilute with sterile water to a concentration of 1 x 106 conidia per ml water.
    6. For priming experiments and B. cinerea development assays, spray leaves of root-treated plants with P. fluorescens PTA-CT2 or control plants with the inoculation suspension of B. cinerea at 1 x 106 conidia per ml using an atomizer (Figure 2B), until homogenous coverage of the leaves is reached.
    7. Keep plants in a growth chamber at 22 °C with a 16- and 8-h photoperiod.
    8. Collect samples at 24 h post inoculation and grind the leaves with a mortar-pestle in liquid nitrogen to obtain a thin powder kept frozen.
    9. Weigh 50 mg of the frozen fresh leaf powder in a precooled 2 ml microtube before storage at -80 °C for further mRNA extraction and quantification of Actin gene expression of B. cinerea (EDN28275.1, GenBank) by qRT-PCR (Figure 2D) as described in Gruau et al. (2015).
    10. For the evaluation of long lasting resistance to B. cinerea, detach leaves from 5 day bacterized or control plants (Figure 2C).
    11. Put detached leaves with the upper face in contact with humidified Whatman paper discs in Petri dishes.
    12. Apply one needle-prick wound to the middle part of each leaf (Figure 2C), and cover the fresh wounds with 5-μl drops of a suspension of 1 x 106 conidia/ml.
    13. Keep Petri dishes containing leaves in a growth chamber at 22 °C with a 16- and 8-h photoperiod for 72 h post infection.
    14. Take pictures of the upper side of inoculated leaves with a Nikon camera D7000 and scan them, then measure the necrotic lesion areas formed during B. cinerea infection by Image Analysis Software, APS Assess 2.0 software (American Phytopathological Society Press, St. Paul, MN, USA) for necrosis surface quantification (Figure 2E).

      Figure 2. Procedure of priming plants with Pseudomonas fluorescens after challenge with B. cinerea. Five days post inoculation of grapevine plants with P. fluorescens PTA-CT2 in Magenta boxes at root level (A), leaves from control and treated plants were either sprayed with B. cinerea conidia (B), or detached and drop-inoculated with B. cinerea conidia (C). Evaluate the pathogen development during the first 24 hpi with the spray of B. cinerea conidia by quantifying the Actin transcript level of B. cinerea (as in D), or the mean necrosis area formed at 72 hpi in drop-infected leaves (as in E). 

Data analysis

Constitutive Actin gene of B. cinerea in leaves of intact plants was analyzed by quantitative real-time RT-PCR using specific primers (see Grau et al., 2015). The reference genes EF1α (BQ799343, GenBank) and 60RSP (XM_002270599, GenBank) were used as internal controls and leaves of infected plantlets at zero time correspond to the reference sample (1x expression level). The results presented are means from three independent experiments.
Necrosis surface provoked by B. cinerea on detached leaves was acquired with the APS Assess 2.0 software. About 30 leaves were used for each condition in one experiment and the results are means of six independent experiments. ANOVA test was performed using Duncan’s multiple range test; P < 0.05.


  1. Agar tomato medium
    25% (v/v) commercial 100% tomato juice
    2.4% (v/v) agar


This work was partially supported by the Vineal 2 program, and grants to C. Gruau and B. Verhagen from the Champagne-Ardenne Region and the City of Reims. This protocol was adapted from Gruau et al. (2015) and Verhagen et al. (2011).


  1. Aziz, A., Poinssot, B., Daire, X., Adrian, M., Bezier, A., Lambert, B., Joubert, J. M. and Pugin, A. (2003). Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola. Mol Plant Microbe Interact 16(12): 1118-1128.
  2. Aziz, A., Trotel-Aziz, P., Dhuicq, L., Jeandet, P., Couderchet, M., and Vernet, G. (2006). Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew. Phytopathology 96 (11):1188-1194.
  3. Gruau, C., Trotel-Aziz, P., Villaume, S., Rabenoelina, F., Clement, C., Baillieul, F. and Aziz, A. (2015). Pseudomonas fluorescens PTA-CT2 triggers local and systemic immune response against Botrytis cinerea in grapevine. Mol Plant Microbe Interac 28(10): 1117-1129.
  4. Trotel-Aziz, P., Couderchet, M., Biagianti, S. and Aziz, A. (2008). Characterization of new bacterial biocontrol agents Acinetobacter, Bacillus, Pantoea and Pseudomonas spp. mediating grapevine resistance against Botrytis cinerea. Environ and Exp Bot 64(1): 21-32.
  5. Verhagen, B., Trotel-Aziz, P., Jeandet, P., Baillieul, F. and Aziz, A. (2011). Improved resistance against Botrytis cinerea by grapevine-associated bacteria that induce a prime oxidative burst and phytoalexin production. Phytopathology 101(7): 768-777.


葡萄(葡萄(Vitis vinifera)L.)易受一系列疾病的影响,其中包括由坏死性真菌葡萄孢菌引起的灰霉病,其降低葡萄生产力和质量。为了确保令人满意的产量和收获质量,需要许多化学杀真菌剂,但它们具有严重的缺点。一个替代方案是使用有益细菌来改善植物健康。已经显示荧光假单胞菌在地上植物组织中触发植物介导的对真菌,卵菌,细菌和病毒病原体的抗性反应。触发的植物抗性通过启动状态利用植物免疫系统的机制,其提供植物在病原体感染后快速和强烈地激活防御反应的增强能力,导致较低的适合度成本。有益细菌的引发应答包括防御相关基因的诱导表达,细胞壁增强和病原体感染后次生代谢产物的产生。在该方案中,我们描述了根据Verhagen评价有益细菌荧光假单胞菌PTA-CT2对葡萄植​​物的引发状态及其对灰葡萄孢的抗性水平的实验设计等。 (2011)和Gruau等人。 (2015)。

关键字:生物防治, 铜绿假单胞菌, 小道消息, 启动, 灰


  1. 25mm培养管(VWR,目录号:212-0304)
  2. 锥形管(50ml)(Corning,Falcon ,目录号:352070)
  3. 无菌刮刀(VWR,目录号:612-1561)
  4. Falcon 细胞过滤器100μm尼龙(Corning,Falcon ,目录号:352360)
  5. 1.5ml微管(Treff,目录号:96.07246.9.01)
  6. 2ml微管(Treff,目录号:96.09329.9.01)
  7. Whatman纸无灰盘90mm dimeter(Sigma-Aldrich,目录号:WHA1441090)
  8. 培养皿(90×14mm)(VWR,目录号:391-0439)
  9. 葡萄(葡萄(Vitis vinifera)cv.Chardonnay 7535)植物,通过从琼脂Murashige& 在25mm培养管中的Skoog(MS)培养基(Aziz等人,2006)
  10. Pseudomonas fluorescens PTA-CT2(Trotel-Aziz等人,2008)
  11. 分生孢子(Bc 630)分离自感染的葡萄,由INRA法国凡尔赛成熟
  12. Aquadem蓝色系列蒸馏水(VéoliaWater Aquadem)
  13. 蒸馏水用高压灭菌器Avor Getinge灭菌
  14. 乙醇(来自Alcool absolu)(Charbonneaux Brabant,目录号:3077319001001)
  15. Murashige& Skoog培养基(MS)(Duchefa Biochimie,目录号:M0231.0001)
  16. 甘油(VWR,目录号:24387-292)
  17. Luria Bertani液体培养基(LB)(Duchefa Biochimie,目录号:L1703.0500)
  18. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:M2773)
  19. (BD,目录号:214010)
  20. 琼脂番茄培养基(见配方)


  1. 具有光周期的植物生长室(SANYO,型号:MLR-351H)
  2. 水平层流柜(ASTEC Microflow,型号:水平层流工作站)
  3. 长无菌金属夹(VWR,目录号:232-0084)
  4. 品红盒(Sigma-Aldrich,目录号:V8505)
  5. 微生物安全柜(Angelantoni Life Science,型号:VBH Compact)
  6. 无菌移液管(VWR,目录号:VHPA23004)
  7. 28℃培养摇床(New Brunswick Scientific,型号:C24)
  8. 具有转子数目的转子的可变速度冷冻离心机×标称最大负载(容量:8×50ml)(Eppendorf,型号:5804R)
  9. 分光光度计(Bio-Rad Laboratories,型号:SmartSpec 3000)
  10. 涡旋混合器(Vortex Genie 2 untimed mixer)(VWR,目录号:58815-232)
  11. Malassez计数室(Marienfeld,目录号:0640610)
  12. 玻璃雾化器(VWR,目录号:552-0031)
  13. 相机(尼康,型号:D7000)


  1. APS评估2.0软件( APS,产品编号:43696M5, ISBN 978-0-89054-369-6


  1. 植物材料的制备
    在15ml琼脂修饰的Murashige-Skoog(MS)培养基(含有0.7%Bacto琼脂)上的结节外植体中微量繁殖葡萄体外植物小植株(葡萄种子枸杞Chardonnay克隆7535) )(Aziz等人,2003)在25mm培养管中(图1)。

    图1.用荧光假单胞菌接种的程序A.葡萄植物(葡萄(Vitis vinifera)L.cv.Chardonnay)对灰葡萄孢(Botrytis cinerea)敏感。 B.葡萄植物转移到无菌Magenta盒中,并用根细菌接种荧光假单胞菌 PTA-CT2。

  2. 细菌接种物的制备和接种
    1. 从-80℃甘油储液(30%)中取出荧光假单胞菌PTA-CT2菌株(Trotel-Aziz等人,2008),在微生物安全柜中使用灭菌的移液管尖端以在无菌Luria Bertani(LB)液体培养基(100μl的甘油储备液,用于10ml培养基)中接种细菌,并在28℃培养摇床中以110rpm生长18小时。
    2. 通过在与B1段相同的条件下在含有每40ml LB的10×50ml无菌管中接种1ml等分试样,将该预培养物用于10个新的LB培养物。
    3. 在5,000xg离心细菌悬浮液10分钟,并在微生物安全柜中用无菌的10mM MgSO 4溶液洗涤沉淀物并将其重悬于10ml无菌的10mM MgSO 4溶液或MS培养基。
    4. 涡旋细胞悬浮液,并通过使用分光光度计检查其在450和600nm(OD <450和OD <600)的光密度。为了制备接种物,在10mM MgSO 4或液体MS培养基中将细菌悬浮液的密度调节至2×10 8 cfu/ml。
    5. 用无菌移液管将无菌条件下置于盖子中的每个小植物的根部加入1ml细菌悬浮液,以达到2×10 7 cfu/ml的最终浓度(图2A)。 对于对照植物,加入1ml 10mM MgSO 4或液体MS培养基
    6. 将细菌和对照植物放在22℃,16和8小时光周期的生长室中。
    7. 对于统计分析,至少准备12个生物重复
  3. 真菌接种物制备和接种
    1. 从-80℃甘油储备液(30%)中取出分生孢子灰霉病菌的分生孢子悬浮液(30%),并在微生物安全柜中使用灭菌的移液管尖端在含有番茄的培养皿上刺破分生孢子 琼脂培养基中,并在22℃下在连续光下孵育21天以形成孢子
    2. 将10 ml无菌水置于孢子形成的B上。灰霉病菌,并用无菌刮刀刮去B的分生孢子和菌丝体。 cinerea 。
    3. 将菌丝体和分生孢子的混合物转移到50ml锥形管中,然后通过涡旋振荡数次以获得水中的分生孢子。
    4. 使用过滤管(50ml含有100μM尼龙Falcon 细胞过滤器的管)过滤所获得的悬浮液以除去菌丝体。
    5. 使用Malassez计数室测量悬浮液中分生孢子的浓度,并用无菌水稀释至每ml水1×10 6分生孢子的浓度。
    6. 用于引发实验和B。灰霉病发展测定,用根瘤处理的植物的叶子喷雾。荧光素PTA-CT2或具有B的接种悬浮液的对照植物。使用雾化器(图2B)在1×10 6分生孢子/ml的情况下进行灰化,直至达到叶的均匀覆盖。
    7. 保持植物在22℃的生长室与16和8小时光周期
    8. 在接种后24小时收集样品,并用研钵在液氮中研磨叶子,得到保持冷冻的稀粉末。
    9. 在预冷的2ml微量管中称取50mg冷冻的新鲜叶粉末,然后在-80℃下储存以进一步进行mRNA提取和定量B的肌动蛋白基因表达。 (Gruau等人)中描述的通过qRT-PCR(图2D)来检测细胞灰质(EDN28275.1,GenBank)。 (2015)。
    10. 用于评价对B的持久耐久性。灰霉病,从5天的细菌或对照植物分离叶(图2C)
    11. 将分离的叶子与上表面接触加湿的Whatman纸碟在培养皿中
    12. 应用一个针刺伤口到每个叶的中间部分(图2C),并用5μl滴1×10 6分生孢子/ml的悬浮液覆盖新鲜的伤口。 >
    13. 保持培养皿中含叶子的培养皿在22℃的生长室,16和8小时的光周期感染后72小时。
    14. 用尼康照相机D7000拍摄接种叶的上侧的照片,并扫描它们,然后测量B期间形成的坏死损伤区域。通过图像分析软件,APS评估2.0软件(美国植物病理学会出版社,圣保罗,MN,美国)进行坏死表面定量(图2E)的灰质感染。

      图2.用B攻击后用荧光假单胞菌(Pseudomonas fluorescens)引发植物的程序。灰霉病。用葡萄植物接种后5天。荧光素PTA-CT2在根部水平的洋红盒中(A),来自对照的叶子和处理的植物用B喷雾。灰霉病菌(cinerea)分生孢子(B),或用B分离和滴注。灰霉病菌(C)。在第一次24 hpi期间用喷雾剂B评估病原体的发展。通过定量 B的肌动蛋白转录物水平来确定灰质分生孢子。灰霉病(如在D中),或在感染的叶中(如E中)在72hpi形成的平均坏死区。 


B的组成型肌动蛋白基因。通过使用特异性引物的定量实时RT-PCR分析完整植物叶片中的灰霉病细胞(参见Grau等人,2015)。使用参照基因EF1α(BQ799343,GenBank)和60RSP(XM_002270599,GenBank)作为内部对照,并且零时感染的植株的叶对应于参考样品(1x表达水平)。所给出的结果是来自三个独立实验的平均值 坏死面由B引发。 灰皮埃在分离叶上获得与APS评估2.0软件。 在一个实验中对每种条件使用约30片叶子,结果是6次独立实验的平均值。 ANOVA检验使用Duncan's多范围检验; 0.05。


  1. 琼脂番茄培养基
    25%(v/v)商业100%番茄汁 2.4%(v/v)琼脂


这项工作部分得到Vineal 2计划的支持,并授予来自香槟 - 阿登地区和兰斯市的C. Gruau和B. Verhagen。 该方案改编自Gruau等人。 (2015)和Verhagen 等。 (2011)。


  1. Aziz,A.,Poinssot,B.,Daire,X.,Adrian,M.,Bezier,A.,Lambert,B.,Joubert,JM and Pugin,A。(2003)。  Laminarin在葡萄中引发防御反应,并诱导对灰葡萄孢(Botrytis cinerea)和 Plasmopara viticola 。 Mol Plant Microbe Interact 16(12):1118-1128。
  2. Aziz,A.,Trotel-Aziz,P.,Dhuicq,L.,Jeandet,P.,Couderchet,M.,and Vernet,G.(2006)。  壳聚糖低聚物和硫酸铜诱导葡萄树防御反应和对灰霉病和霜霉病的抗性。植物病理学 96(11):1188-1194。
  3. Gruau,C。,Trotel-Aziz,P.,Villaume,S.,Rabenoelina,F.,Clement,C.,Baillieul,F. and Aziz,A。(2015)。  Pseudomonas fluorescens PTA-CT2引发局部和全身免疫反应, Mol Plant Microbe Interac 28(10):1117-1129。
  4. Trotel-Aziz,P.,Couderchet,M.,Biagianti,S. and Aziz,A.(2008)。  新的细菌生物防治剂不动杆菌,芽孢杆菌 ,泛菌和假单胞菌 介导对灰葡萄孢的葡萄抗性。 环境和遗传学64(1):21-32。
  5. Verhagen,B.,Trotel-Aziz,P.,Jeandet,P.,Baillieul,F.and Aziz,A。(2011)。  通过葡萄相关细菌改善对灰葡萄孢菌的抗性,诱导初次氧化爆发和植物抗毒素生产。 em> Phytopathology 101(7):768-777。
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引用:Gruau, C., Trotel-Aziz, P., Verhagen, B., Villaume, S., Rabenoelina, F., Courteaux, B., Clément, C., Baillieul, F. and Aziz, A. (2016). An Assay to Study Botrytis cinerea-infected Grapevine Leaves Primed with Pseudomonas fluorescens . Bio-protocol 6(19): e1943. DOI: 10.21769/BioProtoc.1943.