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Maize significantly contributes to food and fuel production. Yields can be reduced due to foliar diseases, which reduce photosynthetic leaf area. The bacterial foliar disease Goss's wilt (caused byClavibacter nebraskensis) can cause significant yield losses in susceptible maize varieties.C. nebraskensiscan infect leaves through wounds and colonize the vascular tissue of the leaf. We present a protocol that replicates this process with the use of a “clapper” with pins on one end to create wounds and a sponge soaked in inoculum on the other end, which allows for efficient field inoculations of maize leaves. Disease severity is then rated on a percentage scale multiple times over the season to generate an area under disease progress curve (AUDPC). Genetic host resistance is one of the most effective forms of foliar disease control in maize, as there are few effective forms of chemical control for bacterial diseases that affect maize. Screening for resistance in diverse germplasm, or for fine mapping a specific resistance gene, requires inoculating large populations in the field for obtaining phenotypic data. Our high-throughput protocol allows for large-scale disease evaluations and is useful for finding forms of genetic resistance or to understand plant–pathogen interactions of bacterial foliar pathogens.
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This content will become publicly available on June 3, 2026
High-Throughput Fluorescence Microscopy Using Aniline Blue Staining to Study the Maize –Exserohilum turcicum Pathosystem
Maize is a globally important grain crop that is important for food and fuel. Northern corn leaf blight, caused byExserohilum 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 stainingE. turcicumtissue 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 andE. 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.
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- Award ID(s):
- 2154872
- PAR ID:
- 10608739
- Publisher / Repository:
- Cold Spring Harbor Laboratory Press
- Date Published:
- Journal Name:
- Cold Spring Harbor Protocols
- ISSN:
- 1940-3402
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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