Methods to Observe Plant Tissue Colonization by Fusarium oxysporum

Abstract Fusarium oxysporum , an important soil‐borne pathogen, causes vascular wilts in more than 100 plant species, leading to billions of dollars in annual yield losses. Controlling Fusarium wilt diseases is challenging due to the persistence of pathogen spores in infested fields and the growing resistance to available fungicides. Understanding the molecular interactions between F. oxysporum and its host plants is crucial for developing novel control strategies, but studying these interactions is difficult because F. oxysporum invades plant roots long before wilt symptoms can be detected in above‐ground tissues. To illuminate the hidden interactions between F. oxysporum and its plant hosts, we present three confocal microscopy protocols for visualizing fungal colonization in plant tissues and the associated plant responses. The first protocol employs wheat germ agglutinin–Alexa Fluor 488 and propidium iodide to stain fungal cells and plant host tissues, respectively. The second uses sirofluor to detect deposition of callose, a (1,3)‐β‐glucan polymer found in plant cell walls that plays a significant role in plant defense. The third utilizes fluorescent protein–tagged fungal isolates and a stable transgenic Arabidopsis thaliana line, providing a clean and easily accessible system for visualizing early infection stages. The protocols described here will shed light on underground plant‐pathogen interactions, aiding researchers in unraveling the complex dynamics between diverse F. oxysporum pathotypes and their plant hosts.© 2025 Wiley Periodicals LLC. Basic Protocol 1 : Observation of F. oxysporum cells in the tomato stem vasculature Basic Protocol 2 : Observation of callose deposition in F. oxysporum –colonized tomato plant roots Basic Protocol 3 : Observation of fungal colonization in an F. oxysporum – A. thaliana model system