Immunocytochemical studies of fungal cell surface molecules and the vicia faba-Botrytis interaction
Immunofluorescence and immunoelectron microscope studies of fungal cell surface molecules and the Vicia fabci-Botrytis-interaction were conducted using the Bottytis-MAbs BC-KH4, BC-AF7 and BC-FD7-G9. The Botrytis-MAbs were raised against surface washings of Botrytis cinerea and directed against B. cinerea and B. fabae (Bossi & Dewey, 1992; Plant Pathology 41, 472-482). All three MAbs bound to fibrillar matrices associated with conidia, germ-tubes and hyphae of B. cinerea and B. fabae grown on agar, glass slides (conidia inoculated in aqueous glucose) and V. faba leaves. These matrices provided firm attachment of sporelings to glass and leaf surfaces. Antibody-binding was observed at fungal walls, including septa; however, no labelling was detected inside the fungal cell or at hyphal tips. These surface antigens may be synthesised and/or modified in muro. Cryo-fixation and low- temperature freeze-drying of fungal material enhanced the preservation of fibrillar matrices and retention of surface antigens for immunocytochemistry. Light and electron microscopy of the infection processes of wet and dry conidia inoculated onto glass and V. faba leaves showed that in all cases conidia germinated to produce one or more germ tubes. However, the subsequent growth of the developing germ tubes varied according to mode of inoculation applied. In the presence of aqueous glucose, conidia developed long germ tubes. In contrast, dry-inoculated conidia produced short germ tubes only, which penetrated the leaf surface directly. Transmission electron microscopy revealed an amorphous pad of matrix material around short germ tubes on the leaf surface. This pad appeared to consist of fibrillar material in a condensed form. Observation of both wet- and dry-infections revealed that fibrillar material was associated with both penetration and infection hyphae. This fibrillar material was also immunolabelled with the Botrytis-MAbs. Fibrils often extended some distance from the penetration and infection hyphae and pervaded the degraded host cell walls. Double-labelling methods using BC- KH4 and specific markers for plant wall components allowed the host-pathogen interface to be investigated further. Interactions between fibrillar matrices and host walls and/or cytoplasm were visualised clearly. These studies provided further support for the wallmodifying action of pectic enzymes. The possible role(s) for Botrytis extracellular matrices are discussed.
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