A Modified Slide-Embedded Scanning Electron Microscopy Preparation Method to Visualize Antagonistic Interactions...

Mycoparasitism is an important mechanism of fungal antagonism in which one fungus parasitizes another. This type of interaction plays a major role in the biocontrol activity of Trichoderma spp. against phytopathogenic fungi. Detailed visualization of these interactions is essential for understanding the structural mechanisms involved in fungal antagonism, including hyphal attachment, coiling, penetration, and cellular distortion. Scanning electron microscopy (SEM) is widely used for structural examination of fungal interactions; however, conventional preparation methods such as filter paper systems, membrane overlays, and agar block techniques often result in structural distortion, fragile sample handling, and difficulty in locating defined interaction zones. Here, we describe a modified slide-embedded technique for SEM visualization of mycoparasitic interactions between filamentous fungi. The protocol is adapted from previously reported slide culture approaches and involves embedding pre-cut sterile glass slide fragments directly into potato dextrose agar (PDA), followed by sequential inoculation of Fusarium sp. and Trichoderma viride. Fungal interactions occurring directly on the glass surface are subsequently subjected to fixation with 2.5% glutaraldehyde, graded ethanol dehydration, sputter coating, and SEM observation. Compared with conventional methods, the present approach provides improved handling stability, better preservation of native hyphal architecture, reduced deformation during processing, and easier localization of interaction zones during microscopy. The protocol also enables clear visualization of early antagonistic events such as hyphal coiling, penetration, and surface colonization. Due to its simplicity, reproducibility, and minimal technical complexity, this method serves as a practical and efficient approach for SEM-based investigation of fungal-fungal interactions and can be readily adapted for studying diverse mycoparasitic systems. Key features • A simple, cost-effective method to visualize filamentous fungus-fungus interactions, particularly mycoparasitism, using minimal reagents and basic laboratory equipment. • A standardized inoculation strategy with pathogen inoculation 48 h before the mycoparasite ensures reproducible fungal interaction and mycoparasitic development. • Blunt-cut square glass slide pieces embedded in PDA enable easy sample handling, storage, transport, and direct preparation for scanning electron microscopy. • SEM-compatible fixation and ethanol dehydration preserve fungal ultrastructure, enabling high-resolution visualization of hyphal coiling, distortion, and other mycoparasitic interactions.