High-Throughput Fluorescence Microscopy Using Aniline Blue Staining to Study the Maize–Exserohilum turcicum Pathosystem

Abstract

Maize is a globally important grain crop that is important for food and fuel. Northern corn leaf blight, caused by Exserohilum turcicum, is an important fungal foliar disease of maize that is highly prevalent and causes yield losses globally. Microscopy can be used to visualize plant–fungal interactions on a cellular level, which enables pathology and genetics studies. Host resistance and isolate aggressiveness can be characterized at different stages of disease development, which enables a more detailed understanding of the pathogenesis process and host–pathogen interactions. Our protocol outlines an efficient, cost-effective method for staining E. turcicum tissue on inoculated maize leaves and visualizing samples using a compound fluorescence microscope. This protocol uses KOH treatment followed by aniline blue staining, which stains glucans present in plant and fungal cell walls, and samples are visualized using fluorescence microscopy. Quantitative data about fungal structures including the conidia, hyphal structures, and appressoria, the structures formed to push through the plant leaf surface after conidia have germinated, can be obtained from the images generated using this technique. Visualization of these structures can help pathologists understand plant–pathogen interactions for maize and E. turcicum. This method has advantages over other methods because the stain is less toxic than other available stains, samples can be processed in a more high-throughput manner than other protocols, and the required supplies are relatively inexpensive.