Quantification of Thrips Damage Using Ilastik and ImageJ Fiji

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.