A MATLAB-Based Image Processing Protocol for Quantitative Differentiation of Diseased and Healthy Plant Tissue From...

Accurate quantification of plant disease severity is essential for evaluating host-pathogen interactions and assessing the effectiveness of disease management strategies. Traditional visual scoring methods and manual estimation of infected tissue are widely used but are often subjective and prone to observer bias. Digital image analysis offers an objective alternative by enabling automated identification and quantification of symptomatic plant tissues based on color and spatial characteristics. Here, we present a MATLAB-based image processing protocol for differentiating diseased and healthy plant tissue from digital leaf images. The workflow involves acquisition of standardized leaf images, conversion of RGB images into hue-saturation-value (HSV) color space, segmentation of diseased tissue using defined HSV thresholds, refinement of the segmented mask through morphological operations, and extraction of the whole leaf area. The protocol then calculates the diseased area and total leaf area in pixels and computes the percentage of infected tissue. The method uses MATLAB together with the Image Processing Toolbox and can be implemented using simple scripts. This protocol enables rapid and reproducible quantification of disease severity in plant leaves exhibiting visually distinct symptoms such as necrotic lesions or blight patches. By minimizing observer bias and providing quantitative measurements of infected area, the protocol offers a practical and reproducible approach for plant disease phenotyping and evaluation of disease management strategies across diverse plant-pathogen systems where diseased tissues can be clearly distinguished from healthy tissues under reasonably controlled imaging conditions. Key features • A reproducible MATLAB-based workflow for separating diseased and healthy plant tissue using color-space segmentation. • Applicable to plant diseases where symptomatic tissue contrasts clearly with healthy tissue (necrosis, blight lesions, rot patches). • Requires digital leaf images, MATLAB, and the MATLAB Image Processing Toolbox for image processing and disease quantification. • Enables rapid calculation of diseased leaf area and disease severity using automated pixel-based quantification.