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

Alexandre  Perochon; Fiona M. Doohan

doi:10.21769/BioProtoc.2065

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Peer-reviewed

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

Alexandre Perochon email

Fiona M. Doohan

发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2065 浏览次数: 11784

评审: Arsalan DaudiMalou FraitureBaohua Li

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Dec 2015

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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

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

Equipment

Incubator (20 °C)
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)
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)
Digital camera (Nikon, model: COOLPIX P500 with a NIKKOR 36X wide optical ZOOM ED VR, 4.0 - 144 mm, 1:3.4 - 5.7)
Copy stand (RPS Studio RS-CS920 copy stand)
Lights (cold-light fluorescent lighting system)

Software

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

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

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.
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.