Immunomagnetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected bean leaves using a monoclonal antibody.

A method is described for isolating intracellular hyphae (IH, i.e. infection vesicles and primary hyphae) of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected leaves-of bean (Phaseolus vulgaris L.). 1H were recovered from homogenates of infected leaves after filtration through a 45 μm nylon mesh and isopyenic centrifugation on Percoll. 1H were then affinity-purified by immunomagnetic separation using Dynabeads coated with monoclonal antibody UB25, specific for IH surface glycoproteins. The method yielded 7 × 10 IH g f. wt leaf tissue, with 27% purity and 62% viability, as judged by staining with fluorescein diacetate. The Viability of isolated IH was confirmed by their ability to grow in nutrient medium and by the normal ultrastructure of their cytoplasm. The host plasma membrane and matrix layer which surround IH in planta were absent from isolated IH. Staining with lectins. Calcofluor and aniline blue showed that the walls of IH contain N-acetylgalactosamine. α-linked mannose residues and β-linked polysaccharides, including-chitin and β-1,3-glucans. Potential uses of the isolated IH are discussed.