Detecting Callose Deposition in Soybean Lateral Roots During Fungal Infections

Callose is a β-1,3-glucan polysaccharide deposited at the plant cell wall interface. It is involved in numerous plant physiological processes and responses to both biotic and abiotic stresses. Callose deposition under biotic stress conditions is typically associated with pattern-triggered immunity, which is activated upon recognition of pathogen-associated molecular patterns or damage-associated molecular patterns at the cell wall interface. These depositions reinforce compromised and damaged cell walls caused by pathogen invasion. The standard method for visualizing callose deposition in various plant tissues involves aniline blue staining. Aniline blue fluorochrome preferentially binds to β-1,3-glucans, which enables this staining technique to specifically locate callose deposition. Although multiple protocols for callose detection using aniline blue are available in various model plants, such as Arabidopsis, there is no optimized method for lignified lateral root tissues after fungal infection. Lignification of roots can hinder the clear visualization of callose depositions; therefore, it is essential to remove them for improved callose detection. Here, we have optimized a robust and reliable method for detecting callose deposition in soybean lateral roots during Macrophomina phaseolina infections. M. phaseolina is a filamentous, soil-borne, necrotrophic fungus that causes charcoal rot disease in soybean and other crop plants. Here, we also provide a detailed methodology for soybean root infection with M. phaseolina using the root-dip method of inoculation, followed by aniline blue staining. Furthermore, we provide a detailed workflow for employing open-source Fiji software together with the Trainable Weka Segmentation (TWS) plugin to detect and count callose structures in fungal-infected root tissues. This protocol may be applicable for detecting callose deposition in other crop plants during fungal infections. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Infection assay with M. phaseolina using root dip method of inoculation Basic Protocol 2: Staining of M. phaseolina-infected soybean roots with aniline blue and imaging of stained soybean roots using fluorescence microscopy Support Protocol: Callose quantification using ImageJ software combined with the TWS plugin.