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Assessment of Wheat Resistance to Fusarium graminearum by Automated Image Analysis of Detached Leaves Assay
通过离体叶片的自动图像分析评估小麦对禾谷镰刀菌的抗性   

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Abstract

Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the image analysis. The protocol provides the different steps of a rapid, automatic and quantitative image analysis to evaluate leaf area infected by Fusarium graminearum.

Keywords: Detached leaf assay(离体叶片测定), Disease assessment(疾病评估), Fusarium graminearum(禾谷镰刀菌), Image analysis(图像分析), Fiji(Fiji软件), Pathogenicity(致病性), Wheat(小麦)

Background

Evaluation of wheat FHB resistance at the whole plant level is estimated by visual scoring at flowering stage which is laborious, time consuming and requires space. Therefore in vitro methods that expedites disease assessment for FHB resistance at early plant stage have been developed, such as seed germination assay (Browne, 2009), coleoptiles assay (Shin et al., 2014), detached leaf assay (Browne and Cooke, 2004) and seedling assay (Li et al., 2010). Detached leaves assay is commonly used to assess host responses to Fusarium and was successful in identifying components of FHB resistance (Browne and Cooke, 2004). In such assays pathogen establishment is visually assessed which is time-consuming and limits accurate measurement. The method described recently by Perochon et al. (2015) and detailed here resolved these two limitations by using an automatic method that quantifies leaf area infected by image analysis based on particle size.

Materials and Reagents

  1. Petri dish, triple vent 94 x 15mm (Greiner Bio One, catalog number: 633 185 )
  2. Filter paper (Whatman, catalog number: 1001-090 )
  3. Parafilm (Parafilm, catalog number: PM992 )
  4. Plant container pots, 3 L (National Agrochemical Distributors, catalog number: POTS34 )
  5. John Innes compost No. 2 (Westland Horticulture)
  6. Square Petri dish 100 x 100 x 20 mm (SARSTEDT, catalog number: 82.9923.422 )
  7. Glass Pasteur pipette (VWR, catalog number: 14673-010 )
  8. Fusarium graminearum strain GZ3639 (Proctor et al., 1995)
  9. Plant agar (Duchefa, catalog number: P1001.1000 )
  10. Benzimidazole (Stock solution at 500 mM in ethanol stored in aliquots at -20 °C) (Sigma-Aldrich, catalog number: 194123 )
  11. Ethanol (Sigma-Aldrich, catalog number: E7023 )
  12. Tween-20 (Sigma-Aldrich, catalog number: P2287 )

Equipment

  1. Incubator (20 °C)
  2. Glasshouse (Cambridge HOK production glasshouse) (20-22 °C with a 16 h light/8 h dark photoperiod at 300 μmol m-2 s-1 and 70% relative humidity)
  3. Plant growth room (20 °C with a 16 h light/8 h dark photoperiod at 200 μmol m-2 s-1 and 70% relative humidity)
  4. Digital camera (Nikon, model: COOLPIX P500 with a NIKKOR 36X wide optical ZOOM ED VR, 4.0 - 144 mm, 1:3.4 - 5.7)
  5. Copy stand (RPS Studio RS-CS920 copy stand)
  6. Lights (cold-light fluorescent lighting system)

Software

  1. Fiji (http://fiji.sc/)
    Note: Fiji is an open source software using the same functionality as ImageJ with many bundled plugins. For that reason the image analysis presented here could be executed with ImageJ.

Procedure

  1. Place wheat seeds in a 94 mm diameter Petri dish plate containing 2 filter papers and 6 ml of sterile water.
  2. Seal the plates with a piece of Parafilm and germinate in the dark for 3 days at 20 °C in an incubator.
  3. Transplant seedlings to 3 L pots containing moistened John Innes compost No 2 with maximum 6 seedlings/pot.
  4. Grow the plants in the glasshouse under controlled environment conditions at 20-22 °C with a 16 h light/8 h dark photoperiod at 300 μmol m-2 s-1 and 70% relative humidity.
  5. After approximately 20 days at the 3-leaf-stage (growth stage 13; [Zadoks et al., 1974]), cut an 8 cm section from the second leaf at a fixed distance from the leaf base (Figure 1A).
    Note: 8 leaf sections fit in the square Petri dish used.


    Figure 1. Setup of the detached leaf assay. A. Section of a wheat second leaf at the 3-leaf-stage to be used for the assay. B. Different leaf sections are placed in a square Petri dish with their cut ends held between an upper and lower agar section.

  6. Immediately after cutting, place leaf sections with the adaxial side facing upwards on the surface of a square Petri dish. Put the cut ends between a sandwich of 1 % plant agar pH 5.7 containing 0.5 mM benzimidazole (Figure 1B, Video 1).
    Note: Removing the agar from the center of the plate prevent excessive fungal growth at the point of leaf inoculation, additionally removed agar section that could be used to form the sandwich. Benzimidazole is used to delay the leaf senescence.

    Video 1. Placement of the leaf cut ends between agar sections

  7. Puncture the center of each leaf section with the tip of a glass Pasteur pipette and treat with a 4 μl droplet of 0.02 % (v/v) Tween-20 solution with 106 conidia/ml of F. graminearum strain GZ3639 prepared as previously described (Ali et al., 2015).
  8. Add 2 ml of water into the plate under the leaf sections to keep high humidity and seal the plates with a piece of Parafilm before to incubate at 20 °C under a 16 h light/8 h dark cycle in a growth room.
    Note: Try to place your plates always at the same level and position in your growth room or growth cabinet. The level of condensation inside your plates may differ and affect reproducible disease development.
  9. Analyse the leaf sections at 4 days post-inoculation (Figure 2).
    Note: You can follow as well the progress of infection during a time course. In that case photograph your leaf section every day and process analysis as described below (Data analysis).
  10. Repeat the experiment at least three times, each time including 6 plates per treatment, and each plate including two leaf sections per wheat genotype.

Data analysis

  1. Place the digital camera on the copy stand to calibrate the height and the light to illuminate completely the square petri dish from the side at a 45° angle.
    Note: It’s important to make sure that the lighting is even on all the leaf sections particularly on the infected area. Uneven lighting will affect an accurate delineation of the infected area and thus, its measurement. For more details about how to use a copy strand go to: http://www.jiscdigitalmedia.ac.uk/toolkit/digitisation-equipment/the-copy-stand
  2. Remove the cover plates and photograph the leaf sections with a ruler placed inside the field of view. Camera settings were as follows: ISO 160, F-stop 3.4, shutter speed 1/30.
    Note: The quality of the image is an important determinant of accurate measurement. Adjust the camera settings according to your light conditions. It’s important to make sure the infected area is in focus to allow your image to be sharp and thus your measurement to be accurate.
  3. Collect all the pictures of your experiment save in TIFF format and measure the disease leaf area using Fiji software (Schindelin et al., 2012).
  4. Open all your individual pictures in Fiji and create a stack of all your images (‘Image’ > ‘Stacks’ > ‘Image to stacks’).
  5. Convert your RGB images to 8-bit binary images (‘Image’ > ‘Type’ > ‘8-bit’).
  6. Set the scale using a known distance of your photographed ruler. Select the button Straight and draw a line along a known distance of your ruler. Set the scale (‘Analyse’ > ‘Set scale’). Add the known distance and select the unit of your choice (e.g., cm).
    Note: To make sure your scale is correct, reuse the tool Straight and measure a known distance of your ruler and compare your result (‘Analyse’ > ‘Measure’).
  7. Crop your images to focus your analysis only on the leaf sections. Select the button ‘Rectangular’ and define your area of interest. Make sure that all your leaf sections from your different images are included in your selection before cropping the image (‘Image’ > ‘Crop’).
  8. To perform an automatic particles analysis, the first step is to set a threshold for the image. This tells the particle analysis what are the objects of interest (infected area) as distinct from the background (non-infected area). Manually calibrate the threshold until the black area fits within your infected area (‘Image’ > ‘Adjust’ > ‘Threshold’) (Figure 2).
  9. To automatically measure the infected area, use the tool ‘Analyse particles’ (‘Analyse’ > ‘Analyse particles’). Set the minimum and maximum area in the box ‘Size’ (Figure 2). Select inside the box ‘Show’ the setting ‘Overlay outlines’ and tick the boxes ‘Display results’, ‘Summarize’ and ‘In situ show’ (Figure 2).
    Note: To know what minimum size to choose, you can manually measure the smallest particle in your pictures with the freehand selection tool.
  10. The results will appear in a table and the corresponding particles measured will be identified on your images. The results from the table can be copy-and-pasted into an Excel file.


    Figure 2. Fiji image analysis pipeline. At four days post-inoculation leaf sections were analysed by image analysis. Corresponding RGB images were converted to binary images. In order to measure automatically the infected area, a threshold was manually set prior to analysis.

Notes

  1. If you want to compare different genotypes or treatments, avoid the block effect by randomising the leaves within the Petri dishes.

Acknowledgments

This work was supported by the Science Foundation Ireland (project Nos. 10/IN.1/B3028 and 14/1A/2508). Parts of this protocol were adapted from previously described detached leaf disease experiments (Browne and Cooke, 2004; Perochon et al., 2015). The authors thank Pierre Grandjean for technical assistance and Emmanuel G. Reynaud (UCD) for assistance with image analysis.

References

  1. Ali, S. S., Gunupuru, L. R. and Doohan, F. M. (2015). Visual assessment of the severity of Fusarium seedling blight (FSB) and fusarium head blight (FHB) disease in barley. Bio-protocol 5(12): e1507.
  2. Browne, R. A. (2009). Investigation into components of partial disease resistance, determined in vitro, and the concept of types of resistance to Fusarium head blight (FHB) in wheat. Eur J Plant Pathol 123(2): 229-234.
  3. Browne, R. A. and Cooke, B. M. (2004). Development and evaluation of an in vitro detached leaf assay forc pre-screening resistance to Fusarium head blight in wheat. Eur J Plant Pathol 110: 91-102.
  4. Li, X., Zhang, J. B., Song, B., Li, H. P., Xu, H. Q., Qu, B., Dang, F. J. and Liao, Y. C. (2010). Resistance to Fusarium head blight and seedling blight in wheat is associated with activation of a cytochrome p450 gene. Phytopathology 100(2): 183-191.
  5. Perochon, A., Jianguang, J., Kahla, A., Arunachalam, C., Scofield, S. R., Bowden, S., Wallington, E. and Doohan, F. M. (2015). TaFROG encodes a Pooideae orphan protein that interacts with SnRK1 and enhances resistance to the mycotoxigenic fungus Fusarium graminearum. Plant Physiol 169(4): 2895-2906.
  6. Proctor, R. H., Hohn, T. M. and McCormick, S. P. (1995). Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol Plant Microbe Interact 8(4): 593-601.
  7. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  8. Shin, S., Kim, K. H., Kang, C. S., Cho, K. M., Park, C. S., Okagaki, R. and Park, J. C. (2014). A simple method for the assessment of Fusarium head blight resistance in Korean wheat seedlings inoculated with Fusarium graminearum. Plant Pathol J 30(1): 25-32.
  9. Zadoks, J. C., Chang, T. T. and Konzak, C. F. (1974). A decimal code for the growth stages of cereals. Weed Research 14: 415-421.

简介

镰孢霉病引起的枯萎病(FHB)病原体是全球重要的谷物疾病。为了研究镰刀菌致病性和宿主抗病性,需要用于疾病评估和定量的强大方法。这里我们描述强调图像分析的分离叶分析的过程。该方案提供了快速,自动和定量的图像分析的不同步骤,以评估禾谷镰孢感染的叶面积。

背景 通过在开花期的视觉评分估计整个植物水平的小麦FHB抗性,这是费力,耗时且需要空间的。因此,已经开发了加速早期植物阶段对FHB抗性的疾病评估的方法,例如种子发芽测定(Browne,2009),胚芽鞘试验(Shin et al。 ,2014),脱叶测定(Browne和Cooke,2004)和幼苗测定(Li等人,2010)。分离叶分析通常用于评估宿主对镰刀菌的反应,并且成功鉴定了FHB抗性的组分(Browne和Cooke,2004)。在这样的测定中,目视评估病原体的建立是耗时且限制精确测量的。最近由Perochon等人描述的方法。 (2015),并在此详细解析了这两个限制,通过使用自动方法,量化基于粒径的图像分析感染的叶面积。

关键字:离体叶片测定, 疾病评估, 禾谷镰刀菌, 图像分析, Fiji软件, 致病性, 小麦

材料和试剂

  1. 培养皿,三通孔94 x 15mm(Greiner Bio One,目录号:633 185)
  2. 滤纸(Whatman,目录号:1001-090)
  3. 石蜡膜(Parafilm,目录号:PM992)
  4. 植物容器盆,3升(国家农化部经销商,目录号:POTS34)
  5. 约翰·内斯堆肥2号(Westland Horticulture)
  6. 方形培养皿100 x 100 x 20毫米(SARSTEDT,目录号:82.9923.422)
  7. 玻璃巴斯德移液器(VWR,目录号:14673-010)
  8. 镰刀菌菌株GZ3639(Proctor等人,1995)
  9. 植物琼脂(Duchefa,目录号:P1001.1000)
  10. 苯并咪唑(在-20℃下以等分试样储存的乙醇中的500mM储备溶液)(Sigma-Aldrich,目录号:194123)
  11. 乙醇(Sigma-Aldrich,目录号:E7023)
  12. 吐温-20(Sigma-Aldrich,目录号:P2287)

设备

  1. 孵化器(20°C)
  2. 玻璃屋(剑桥HOK生产玻璃温室)(20-22℃,16小时光/8小时深光光周期,在300μmol/平方米以上)和70%相对湿度)
  3. 植物生长室(20℃,具有16小时光/8小时200微米/秒的深光周期和70%相对湿度)
  4. 数码相机(尼康,型号:COOLPIX P500,NIKKOR 36X宽光学ZOOM ED VR,4.0 - 144 mm,1:3.4 - 5.7)
  5. 复印台(RPS Studio RS-CS920复印架)
  6. 灯(冷光荧光照明系统)

软件

  1. 斐济( http://fiji.sc/
    注意:斐济是一款使用与许多捆绑插件相同功能的开源软件。因此,此处提供的图像分析可以使用ImageJ执行。

程序

  1. 将小麦种子放在含有2张滤纸和6 ml无菌水的94 mm直径的培养皿中
  2. 用一片石蜡膜密封板,并在20℃下在培养箱中在黑暗中发芽3天。
  3. 将移栽幼苗移植到含有最多6棵幼苗/盆的含有约翰·内斯堆肥2号的3L盆中。
  4. 在温度为20-22℃的温度控制环境条件下,在300μmol/平方米的高光照条件下,在16小时光/8小时暗光照条件下,在温室内种植植物, 70%相对湿度。
  5. 在3叶期(生长阶段13; [Zadoks等人,1974])约20天后,从距离叶基部固定距离的第二叶切割8cm的部分(图1A) 注意:8个叶片适合所使用的方格培养皿。


    图1.分离叶测定的设置 A.用于测定的3叶期的小麦第二叶的部分。 B.将不同的叶片部分放置在方形培养皿中,其切割端保持在上部和下部琼脂部分之间。

  6. 切割后立即将正面侧面朝上的叶片放置在正方形陪替氏培养皿的表面。将切割的末端放在含有0.5mM苯并咪唑的1%植物琼脂pH 5.7的夹心之间(图1B,视频1)。
    注意:从板材中心除去琼脂可防止在叶片接种时过多的真菌生长,另外除去可用于形成三明治的琼脂部分。苯并咪唑用于延缓叶片衰老。

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  7. 用玻璃巴斯德移液管的尖端刺穿每个叶片部分的中心,并用含有10分钟/分钟分子/ml的0.02%(v/v)吐温-20溶液的4微升滴定液> F。禾谷镰刀菌菌株GZ3639如前所述(Ali等人,2015)制备。
  8. 在叶片下面的板中加入2ml水以保持高湿度,并用一块石蜡膜密封板,然后在生长室中在20℃,16小时光/8小时黑暗循环下孵育。 > 注意:尝试将您的铭牌始终保持在同一水平,并在您的成长室或成长柜内。您的平板内的冷凝水平可能会有所不同,并影响可重现的疾病发展。
  9. 在接种后4天分析叶片部分(图2) 注意:您也可以跟踪感染过程中的进度。在这种情况下,每天照片你的叶节和下面的过程分析(数据分析)。
  10. 重复实验至少三次,每次每次处理包括6个平板,每个平板包括每个小麦基因型的两个叶片段。

数据分析

  1. 将数码相机放置在复印机架上,校准高度和光线,以45°角从侧面完全照亮方形陪替氏培养皿。
    注意:重要的是确保照明在所有的叶片部分,特别是感染的区域。不均匀的照明将影响感染区域的准确描述,从而影响其测量。有关如何使用副本的更多详细信息,请访问: http://www.jiscdigitalmedia.ac.uk/toolkit/digitisation-equipment/the-copy-stand
  2. 拆下盖板,并用放置在视野内的标尺拍摄叶片部分。相机设置如下:ISO 160,F-stop 3.4,快门速度1/30。
    注意:图像的质量是准确测量的重要决定因素。根据您的光线条件调整相机设置。重要的是要确保受感染的区域是焦点,以使您的图像清晰,从而使您的测量准确。
  3. 以TIFF格式收集实验的所有照片,并使用斐济软件测量疾病叶面积(Schindelin等人,2012年)。
  4. 打开斐济的所有照片,并创建一堆所有图像('Image'>'Stacks'>'Image to stacks)。
  5. 将RGB图像转换为8位二进制图像('Image'>'Type'>'8-bit')。
  6. 使用您拍摄的标尺的已知距离设置刻度。选择按钮直线,沿着标尺的已知距离画一条线。设置比例('分析'>'设置比例')。添加已知距离并选择您选择的单位(例如,cm)。
    注意:为了确保您的比例是正确的,请重复使用"直线"工具,并测量标尺的已知距离并比较结果("分析">"度量")。

  7. 裁剪您的图像,将您的分析仅集中在叶片部分。选择"矩形"按钮,并定义您感兴趣的区域。裁剪图像('Image'>'Crop')之前,确保您的不同图像中的所有叶片都包含在您的选择中。
  8. 要执行自动粒子分析,第一步是设置图像的阈值。这告诉粒子分析感兴趣的对象(感染区域)与背景(非感染区域)不同。手动校准阈值,直到黑色区域适合感染区域("图像">"调整">"阈值")(图2)。
  9. 要自动测量感染区域,请使用"分析粒子"("分析">"分析粒子")工具。在"大小"框中设置最小和最大面积(图2)。在"显示"设置"覆盖轮廓"框内选中,然后勾选"显示结果","总结"和"原位展示"框(图2)。
    注意:要知道要选择的最小尺寸,您可以用手写选择工具手动测量图片中最小的粒子。
  10. 结果将显示在表格中,并且您的图像上将标识相应的测量颗粒。表格中的结果可以复制并粘贴到Excel文件中。


    图2.斐济图像分析管道。在接种后四天,通过图像分析分析叶片切片。将相应的RGB图像转换为二进制图像。为了自动测量感染区域,在分析之前手动设置阈值。

笔记

  1. 如果您想比较不同的基因型或治疗方法,通过随机分配培养皿中的叶子来避免阻滞作用。

致谢

这项工作得到了爱尔兰科学基金会(项目编号10/IN.1/B3028和14/1A/2508)的支持。该协议的一部分是从先前描述的分离的叶病实验(Browne和Cooke,2004; Perochon等人,2015)中改编的。作者感谢Pierre Grandjean的技术援助和Emmanuel G. Reynaud(UCD)协助形象分析。

参考文献

  1. Ali,SS,Gunupuru,LR and Doohan,FM(2015)。  Visual评估镰刀菌幼苗(FSB)和禾谷镰刀菌病(FHB)病在大麦中的严重程度。 5(12):e1507。
  2. Browne,RA(2009)。  调查成为部分抗病性的成分,在体外确定,以及小麦中对镰孢霉病(FHB)的耐药性的概念。 Eur J Plant Pathol 123(2):229-234。
  3. Browne,RA和Cooke,BM(2004)。< a class ="ke-insertfile"href ="http://link.springer.com/article/10.1023%2FB%3AEJPP.0000010143.20226.21"target =" _blank">开发和评估体外分离的叶片测定法,在小麦中预先筛选抗镰孢霉病疫苗。 Eur J Plant Pathol 110:91-102。
  4. Li,X.,Zhang,JB,Song,B.,Li,HP,Xu,HQ,Qu,B.,Dang,FJ and Liao,YC(2010)。  抗镰刀菌小麦中的枯萎病和幼苗枯萎与细胞色素的激活有关 p450 基因。 植物病理学 100(2):183-191。
  5. Perochon,A.,Jianguang,J.,Kahla,A.,Arunachalam,C.,Scofield,SR,Bowden,S.,Wallington,E.and Doohan,FM(2015)。< a class =插入文件"href ="http://www.ncbi.nlm.nih.gov/pubmed/26508775"target ="_ blank"> TaFROG 编码了一种Pooideae 孤儿蛋白,与SnRK1相互作用,并增强对真菌致病性真菌镰刀菌禾本科植物的抵抗力。禾本科植物。植物生理学169(4):2895-2906。 br />
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Perochon, A. and Doohan, F. M. (2016). Assessment of Wheat Resistance to Fusarium graminearum by Automated Image Analysis of Detached Leaves Assay. Bio-protocol 6(24): e2065. DOI: 10.21769/BioProtoc.2065.
  2. Perochon, A., Jianguang, J., Kahla, A., Arunachalam, C., Scofield, S. R., Bowden, S., Wallington, E. and Doohan, F. M. (2015). TaFROG encodes a Pooideae orphan protein that interacts with SnRK1 and enhances resistance to the mycotoxigenic fungus Fusarium graminearum. Plant Physiol 169(4): 2895-2906.
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