CALCIUM CYTOTOXICITY, PROTOPLAST VIABILITY AND THE ROLE OF CALCIUM IN SOFT-ROT OF BRASSICA-NAPUS DUE TO BOTRYTIS-CINEREA PERS

The role of calcium during necrotrophic pathogenesis of Brassica napus (swede) storage organs infected with Botrytis cinerea was examined using a model system. The viability and oxygen consumption of swede protoplasts was significantly reduced when incubated in the presence of 1 mm external Ca2+, whereas high viability and O2-consumption were maintained without added external Ca2+ or when higher concentrations (5-200 mm) of Ca2+ were used. It was concluded that Ca2+ may be a contributory factor in the death of swede tissues, since viability was reduced by calcium concentration within the range estimated in the apparent free space of the host during disease. There was significant transport of 1 mM 45Ca2+ into swede protoplasts, but binding to the plasmalemma only occurred at 10 and 200 mM 45Ca2+ in the external medium. Scatchard analysis showed swede protoplasts had a high affinity and binding capacity for Ca2- at the plasma membrane. Swede protoplasts showed significantly reduced Ca2+ uptake when treated with the Ca2+ channel antagonists bepridil and verapamil, which implied voltage-gated Ca2+ diffusion channels in the plasmalemma. Estimations were made of cytosolic free calcium concentration of swede protoplasts using indo-1 with the mean value of 14 nm remaining unchanged when the external Ca2+ concentration was altered. The uptake of calcium ions by B. cinerea varied with the external concentration, and at high concentrations (100-200 mm), beyond those arising in infected swede, reduced the viability of germling mycelium. The results are discussed with particular reference to plasmalemma integrity and cell responses towards potentially cytotoxic levels of Ca2+ during plant pathogenesis. © 1992.