IMMUNOMAGNETIC ISOLATION OF VIABLE INTRACELLULAR HYPHAE OF COLLETOTRICHUM-LINDEMUTHIANUM (SACC AND MAGN) BRIOSI AND CAV FROM INFECTED BEAN-LEAVES USING A MONOCLONAL-ANTIBODY

A method is described for isolating intracellular hyphae (IH, i.e. inf ection vesicles and primary hyphae) of Colletotrichum lindemuthianum ( Sacc. and Magn.) Briosi & Cav. from infected leaves of bean (Phaseolus vulgaris L.). IH were recovered from homogenates of infected leaves a fter filtration through a 45 mu m nylon mesh and isopycnic centrifugat ion on Percoll. IH were then affinity-purified by immunomagnetic separ ation using Dynabeads coated with monoclonal antibody UB25, specific f or IH surface glycoproteins. The method yielded 7 x 10(4) IH g(-1) f. wt leaf tissue, with 27% purity and 62% viability, as judged by staini ng with fluorescein diacetate. The viability of isolated IH was confir med by their ability to grow in nutrient medium and by the normal ultr astructure of their cytoplasm. The host plasma membrane and matrix lay er 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, alpha-linked mannose residues and beta- linked polysaccharides, including chitin and beta-1,3-glucans. Potenti al uses of the isolated IH are discussed.