Abstract
Quantification of insect damage is an essential measurement for identifying resistance in plants. In screening for host plant resistance against thrips, the total damaged leaf area is used as a criterion to determine resistance levels. Here we present an objective novel method for analyzing thrips damage on leaf disc using the freely available software programs Ilastik and ImageJ. The protocol was developed in order to screen over 40 Capsicum lines for resistance against Frankliniella occidentalis (Western Flower Thrips) and Thrips tabaci (Onion thrips).
Keywords: Insect resistance, Insect damage, Image analyses
Background
Quantification of insect damage is an essential measurement for identifying resistance in plants. In screening programs for host-plant resistance against thrips, the total damaged leaf area is used as a criterion to determine resistance levels. Thrips damage is characterized by silvery spots that show high contrast with the intact leaf area, but the feeding spots also include darker areas ranging from dark green to brown. These gradual discolorations of the leaf are too subtle to precisely quantify with programs such as Winfolia (http://www.regentinstruments.com/assets/winfolia_software.html) or ImageJ (Rasband, 2011) alone. As a result, thrips damage is commonly scored by individuals that rate the samples. Samples are classified into categories signifying the amount of damage (Mirnezhad et al., 2010; Maharijaya et al., 2011 and 2012), or damage is estimated to the nearest 1 mm2 (Leiss et al., 2009; Mirnezhad et al., 2010; Maharijaya et al., 2011 and 2012). These subjective measurements make comparison between studies/screening programs difficult. Moreover, they are time consuming and thus costly for breeding companies. Here we present an objective high-throughput standardized screening method to measure leaf surface damage caused by thrips using the freely available software programs ImageJ Fiji (Schindelin et al., 2012) and Ilastik (Sommer et al., 2011). Ilastik has a wide range of applications ranging from cell biology (Fabrowski et al., 2013), where it is used to compute the amount of surface flattening of epithelial cells, to biomechanics (Bongiorno et al., 2014), where it is used to identify boundaries of human mesenchymal stem cells. It is an easy-to-use, self-learning image processing program that allows segmentation and classification of two-dimensional surfaces based on labels provided by the user (Sommer et al., 2011). ImageJ is often used to quantify the amount of removed leaf area by chewing herbivores and the total leaf surface of intact leaves (Meyer and Hull-Sanders, 2008; Morrison and Lindell, 2012). However, it is rarely used to quantify feeding damage caused by thrips. Thrips feeding causes rather subtle discolorations on the leaves. ImageJ is limited in quantifying such color differences, for which Ilastik provides a more suitable alternative.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Before reporting the thrips damage of your leaf discs, you should correct for the average error that Ilastik makes. This average error is determined by calculating the average thrips damage on the leaf discs with no thrips (control discs). (Table 1) Corrected data Corrected thrips damage per leaf disc: Image_1_T = 20 – 2 = 18 Image_1_T = 15 – 2 = 13 Image_1_T = 17 – 2 = 15 The corrected thrips damage can be used for further statistical analysis. Table 1. Numerical example of obtained data
Recipes
Acknowledgments
This work was supported by the Stichting voor de Technische Wetenschappen (STW) which is part of the Green defense Against Pest (GAP) program, project 13552. Nicole M. van Dam gratefully acknowledges the support of the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig funded by the German Research Foundation (FZT 118). This protocol has been used in Entomologia Experimentalis et Applicata (Visschers et al., 2018). The authors have declared no conflicts of interest.
References
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