Extracellular matrices associated with conidia and germ tubes of Botrytis fabae (Sard.) sporelings grown on Vicia faba L. leaves were clearly 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 against B. cinerea and B. fabae, and are known to recognize carbohydrate epitopes on a glycoprotein. Both BC-KH4 and BC-FD7-G9 also labelled matrix material located at the surface of penetration and infection hyphae inside the leaf tissue by epi-fluorescence microscopy. Such matrix material was not visible by DIC microscopy.

Immunoelectron microscopy of B. fabae-infected leaf tissue, prepared by progressive low-temperature dehydration and embedding in acrylic resin, allowed further investigation of the spatial distribution of the antibody-binding sites. An abundance of BC-KH4 and BC-FD7-G9 antigenic sites were observed throughout the fibrillar-like matrix material surrounding the germ tubes on the leaf surface and the infection hyphae inside the host cells. However, close examination of the V. faba–B. fabae interface inside the host tissue showed that this fibrillar material extended some distance from the surface of the infection hyphae and through the swollen epidermal and mesophyll cell walls. Such fibrillar matrix material is thought to be of fungal origin. The possible role(s) of this matrix material in the infection process are discussed.

Double-immunolabelling studies using the BC-KH4 MAb and a polyclonal antiserum directed against oligosaccharides containing β-(1→3)-glucose were carried out in order to localize and distinguish between the fungal extracellular matrix material and translucent cell wall respectively. This technique allowed a closer examination of the interactions of the fungal matrix components with the host walls and degenerate host cytoplasm. Finally, inward curling of the leaf cuticle suggested that mechanical pressure is involved in the penetration process.

Immunoelectron microscopy using the anti-pectin monoclonal antibody JIM 7 confirmed earlier observations that pectin degradation is a primary event in the process of host cell wall breakdown during the development of chocolate spot disease (causal agent: Botrytis fabae (Sard.)) of broad bean. Close examination of infected and non-infected Vicia faba L. leaves indicated a loss of JIM 7-labelling, and therefore, methyl-esterified pectin, from swollen walls of infected and contiguous epidermal cells. Modified mesophyll walls also possessed less methyl-esterified pectin than healthy walls. Enzymes which attack methyl-esterified pectin appeared to be most active in regions of host tissue close to sites of fungal infection.

Ultrastructural studies using the enzyme, cellobiohydrolase conjugated to gold (CBH1-Au) revealed that the cellulose microfibrils of outer epidermal walls of non-infected V. faba leaf tissue were heavily masked by other components of the plant cell wall. Such material was most probably pectin because the cellulose microfibrils of swollen epidermal and modified mesophyll walls of infected host tissue were heavily labelled with CBH1-Au. These results were confirmed by double-labelling studies using JIM 7 and CBH1-Au. At early stages of the infection process, limited cellulose degradation was observed in infected leaf tissue.

Double-labelling experiments using the monoclonal antibody BC-KH4 directed against Botrytis matrices and a marker for the plant cell wall (JIM 7 or CBH1-Au) confirmed previous observations that the fungal matrices extended through modified host walls and degenerate cytoplasm. It is suggested that the wall-modifying action of the pectin-degrading enzymes produced during the infection process might facilitate pervasion of matrix material associated with the infection hyphae through host cell walls. Possible role(s) of such matrix material during the post-penetration processes of the V. faba–B. fabae relationship are discussed.