A Simple and Reproducible ImageJ Workflow for Measuring Areas of Irregularly Shaped Necrotic Lesions on Plant Leaves.

Accurately quantifying the areas of necrotic lesions on plant leaves is essential for evaluating plant-pathogen interactions and disease resistance. Although digital image analysis methods using ImageJ are widely employed, they often require case-specific optimization and may not be readily applicable across different experimental conditions. Furthermore, many studies have used ImageJ for lesion measurement without providing methodological details, which limits reproducibility. Here, we present a simple, step-by-step ImageJ workflow for measuring irregular necrotic lesions using a standard personal computer and mouse. The procedure relies on manual lesion selection using the freehand selection tool, followed by Gaussian smoothing, binarization, and automated particle analysis to extract lesion area measurements. By balancing manual isolation with computational thresholding, this protocol eliminates the need for extensive parameter tuning. This approach provides an accessible, reliable alternative to time-consuming color thresholding methods, thereby improving transparency and reproducibility in lesion quantification. The workflow's reproducibility has been confirmed through both intra-user and inter-user analyses. Key features • Relies on simple manual freehand selection combined with minimal image processing, requiring only a standard computer and mouse without specialized software or advanced training. • Enables accurate quantification of irregular necrotic lesions in conditions where automated thresholding methods require time-consuming optimization. • Provides a fully detailed, reproducible ImageJ workflow addressing common gaps in published methods, facilitating direct implementation. • Demonstrates high reproducibility, validated by intra-user and inter-user statistical analyses, ensuring reliable lesion quantification regardless of the operator.