To begin, gently rinse the maize leaf sheaths inoculated with fungal spores in deionized water. Place the sheath on a clean glass microscope slide with the adaxial surface facing down and the abaxial surface uppermost. Using a single-edge razor blade, trim approximately one centimeter off the sheath ends and remove most lamina tissue on either side of the midrib.
Hold the razor blade at a 90-degree angle to shave tissue from the abaxial midrib, exposing the epidermal layer on the adaxial surface above the inoculated spot. Carefully lift the sheath section from one edge and transfer it onto a new slide with the adaxial surface facing the uppermost closest to the objective lens. Apply 60 microliters of deionized water to the section and place a glass cover slip without introducing air bubbles.
Stenocarpella maydis rapidly invaded epidermal cells directly via undifferentiated hyphal swellings. While Colletotrichum graminicola produced melanized appressoria. Trypan blue staining revealed the nature and extent of fungal hyphae colonization, with C.graminicola initially invading via thick primary hyphae and later switching to a necrotrophic stage.
C.graminicola invades living maize cells using thick primary hyphae separated from the host cytoplasm by a membrane. In contrast, S.maydis produces a phytotoxin that kills the epidermal cells before invading them. In C.graminicola, the production of dark brown precipitate in the maize leaf sheaths indicated an oxidative burst related to a host-resistance response.
