In planta immunolabelling of three types of peripheral vesicles in cells of Phytophthora cinnamomi infecting chestnut roots

Abstract

Micropropagated plantlets of an improved selection of chestnut (Castanea sativa) that was susceptible to Phytophthora root rot, were inoculated in vitro at the root tip with zoospores of Phytophthora cinnamomi. Plantlets were then incubated for up to 4 d, during which time infection was examined by clearing and staining whole roots or immunolabelling cryosections with monoclonal antibodies specific for fungal vesicular components. Hyphae had colonized entire root systems by 3 d after inoculation and plantlets were dead by 4 d after inoculation. Three types of fungal vesicles, large peripheral, dorsal and ventral vesicles, were immunofluorescently labelled in hyphae infecting roots from I d after inoculation with zoospores. Their appearance correlated spatially and temporally with the production of sporangia and chlamydospores. Vesicles were not observed in fungal cells at the advancing hyphal front, but appeared ∼5 mm behind the growing front. Large peripheral vesicles were also immunolabelled in oospores formed after 10 wk in roots of plantlets inoculated with A1 and A2 mating type isolates of P. cinnamomi, either together or alone.