IMMUNOCYTOCHEMICAL STUDIES OF THE INFECTION MECHANISMS OF BOTRYTIS-FABAE - I - THE FUNGAL EXTRACELLULAR-MATRIX IN PENETRATION AND POST-PENETRATION PROCESSES

Extracellular matrices associated with conidia and germ tubes of Botry tis fabae (Sard.) sporelings grown on Vicia faba L. leaves were clearl y visualized by epi-fluorescence microscopy following immunolabelling with the monoclonal antibodies, BC-KH4 and BC-FD7-G9. These antibodies were raised against surface washings of B. cinerea, are directed agai nst B. cinerea and B. fabae, and are known to recognize carbohydrate e pitopes on a glycoprotein. Both BC-KH4 and BC-FD7-G9 also labelled mat rix material located at the surface of penetration and infection hypha e inside the leaf tissue by epi-fluorescence microscopy. Such matrix m aterial was not visible by DIC microscopy. Immunoelectron microscopy o f B. fabae-infected leaf tissue, prepared by progressive low-temperatu re dehydration and embedding in acrylic resin, allowed further investi gation of the spatial distribution of the antibody-binding sites. An a bundance of BC-KH4 and BC-FD7-G9 antigenic sites were observed through out the fibrillar-like matrix material surrounding the germ tubes on t he leaf surface and the infection hyphae inside the host cells. Howeve r, close examination of the V. faba-B. fabae interface inside the host tissue showed that this fibrillar material extended some distance fro m the surface of the infection hyphae and through the swollen epiderma l and mesophyll cell walls. Such fibrillar matrix material is thought to be of fungal origin. The possible role(s) of this matrix material i n the infection process are discussed. Double-immunolabelling studies using the BC-KH4 MAb and a polyclonal antiserum directed against oligo saccharides containing beta-(1 --> 3)-glucose were carried out in orde r to localize and distinguish between the fungal extracellular matrix material and translucent cell wall respectively. This technique allowe d a closer examination of the interactions of the fungal matrix compon ents with the host walls and degenerate host cytoplasm. Finally, inwar d curling of the leaf cuticle suggested that mechanical pressure is in volved in the penetration process.