Many plant pathogenic fungi produce an extracellular matrix (ECM). The ECM is known to play a role in pathogenesis through adhesion of the conidiospore and/or germling to its host. The ECM may contribute to pathogenesis in other ways once the fungus has entered the host tissues. We suspect this is true for the maize pathogen Cochliobolus heterostrophus because a mutant that lacks the full ECM is unable to form normal lesions after it enters the leaf. Otherwise, it behaves normally in culture and on the leaf surface. The ECM of the wild type consists of two layers: an inner layer that binds to a wide variety of stains; and an outer layer that appears in negative relief with India ink. The mutant lacks the outer layer and this provides a good control for testing the hypothesis that the outer layer plays a role in pathogenesis within host tissues. Study of the outer ECM layer within host tissues has been unsuccessful due to the difficulty of visualizing it. Our research has focused on further characterizing the entire ECM and rendering the outer layer visible using a variety of staining techniques on germlings incubated on cellophane membranes, and on hyphae within maize leaf lesions produced after conidiospore inoculation. Both were studied in the fresh state, as well as after conventional fixation and cryofixation followed by freeze substitution. Visualization with light microscopy included nigrosin and India ink, silver-enhanced, gold-labeled antibody and colloidal gold staining, and differential interference contrast. Localization techniques with the electron microscope included enbloc staining using tannic acid and/or uranyl acetate, enbloc staining with ruthenium red or alcian blue followed by ruthenium red and osmium tetroxide; colloidal gold staining, Thiery technique, and antibody labeling. Our results are presented pictorially.

Key words: Cochliobolus, ECM, LM, pathogenic fungus, ruthenium red, TEM