A Modified Slide-Embedded Scanning Electron Microscopy Preparation Method to Visualize Antagonistic Interactions Between Trichoderma viride and Fusarium sp.

Background

Mycoparasitism is one of the major mechanisms underlying fungus–fungus antagonism, in which one living fungus derives nutrients from another living fungal host. Species of Trichoderma are among the most extensively studied mycoparasitic fungi because of their broad host range and strong antagonistic activity against several phytopathogenic fungi [1]. Fungicolous fungi are commonly categorized as symbionts, saprophytes, neutral associates, or mycoparasites depending on the nature of their interaction with other fungi [2,3]. Mycoparasitism is considered an ancestral trait of Trichoderma spp., contributing significantly to their effectiveness as biological control agents against plant pathogens.

In addition to mycoparasitism, Trichoderma spp. exhibit multiple biocontrol mechanisms, including antibiosis, induced systemic resistance (ISR), and competitive exclusion. These fungi behave predominantly as necrotrophs toward their hosts through the secretion of cell wall–degrading enzymes and secondary metabolites that suppress pathogenic fungal growth. Important enzymes produced by Trichoderma spp. include β-glucanases, proteases, cellulases, hemicellulases, and small secreted proteins, whereas secondary metabolites such as gliotoxin contribute to antagonistic activity [4,5].

Visualization of filamentous fungal interactions using scanning electron microscopy (SEM) plays an important role in understanding the structural and mechanistic aspects of mycoparasitism, including hyphal coiling, penetration, attachment, and distortion of host hyphae. Conventionally, SEM studies of fungal interactions have employed filter paper–based systems in which fungi grow over removable substrates prior to fixation and imaging [6]. However, these methods often present several limitations, including fragility of the substrate, difficulty during handling, and distortion of delicate hyphal structures during fixation and dehydration procedures.

To overcome these challenges, alternative techniques such as slide culture methods, cellophane membrane overlays, and coverslip-based systems have been developed to improve structural preservation and facilitate clearer visualization of fungal interaction zones [7,8]. Among these, the slide culture technique is considered one of the most reliable and feasible approaches for studying localized fungal interactions because of its simple setup and improved preservation of fungal morphology [8].

The present protocol describes a modified slide-embedded technique adapted from the novel slide culture approach reported by Bhat 2017 for studying mycoparasitic interactions between filamentous fungi [9]. In this method, pre-cut sterile glass slide fragments are embedded directly into PDA medium, allowing fungal growth and interaction to occur on a stable glass surface suitable for SEM analysis. Compared with conventional methods, this approach offers several advantages: (i) simplified experimental setup without the need for elaborate slide culture assemblies; (ii) improved sample handling and reduced structural deformation during SEM preparation; (iii) enhanced preservation of hyphal interactions; (iv) better visualization of early antagonistic events such as hyphal coiling and penetration; and (v) consistent localization of interaction zones using orientation-marked slide fragments. Consequently, this modified slide-embedded method provides a practical, reproducible, and user-friendly approach for SEM-based visualization of fungal mycoparasitic interactions. In this study, we investigate and visualize the mycoparasitic interaction between Fusarium sp. and the biocontrol fungus Trichoderma viride using SEM.