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

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 plant-parasitic nematodes found in roots, 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. 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 practical tool for investigating the spatial distribution and dynamics of foliar nematodes within distinct layers of the leaf.