Enhanced acid fuchsin staining for visualization of foliar nematodes using brightfield and fluorescence microscopy

Authors: Reis Vieira, Paulo; Waldo, Benjamin; WOLF, EMILY - Oak Ridge Institute For Science And Education (ORISE)

Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/23/2026
Publication Date: 4/23/2026
Citation: Reis Vieira, P.C., Waldo, B.D., Wolf, E. 2026. Enhanced acid fuchsin staining for visualization of foliar nematodes using brightfield and fluorescence microscopy. Journal of Nematology. 58(1):44-54. https://doi.org/10.2478/jofnem-2026-0008.
DOI: https://doi.org/10.2478/jofnem-2026-0008

Interpretive Summary: Foliar nematodes are recognized as significant plant pathogens capable of causing substantial economic losses and yield reductions in many plants. Staining of nematodes within plant tissues is a standard procedure for the study of plant-parasitic nematodes. This process allows the visualization of nematodes, facilitating both research and nematode screening efforts. Hereby, we assessed two microscopic approaches for the detection of foliar nematodes by focusing on the interaction between the beech leaf disease nematode and beech leaf tissues. We illustrate how these simple approaches can be adapted to image foliar nematodes with high-resolution across distinct leaf symptoms. In doing so, we not only enhance the visual characterization of nematode distribution but also uncover novel and biologically significant aspects of their life cycle and interaction with host tissues. These approaches will be used by extension personnel, plant pathologists, and others engaged in the detection and monitoring of plant diseases induced by foliar nematodes.

Technical Abstract: Foliar nematodes are economically important plant pathogens affecting a wide range of hosts, including food crops, ornamentals, and forest trees. Among these, Litylenchus crenatae (family Anguinidae), the causal agent of beech leaf disease (BLD), poses a growing threat to American beech forests. Traditional nematode staining methods, such as acid fuchsin staining, have been widely used for root plant-parasitic nematodes, but their application to foliar tissues remains limited due to differences in tissue structure and stain permeability. In this study, we optimized two acid fuchsin-based protocols for brightfield and fluorescence microscopy to improve the visualization of L. crenatae in symptomatic beech leaves as a model system for foliar nematodes. The improved staining methods provided high-resolution visualization of L. crenatae at the surface and within the leaf tissues using both dissected and whole-mount samples. These enhanced visualization methods offer a valuable tool for investigating the distribution and dynamics of foliar nematodes with distinct layers of the leaf.