Calcium cytotoxicity, protoplast viability and the role of calcium in soft-rot of Brassica napus due to Botrytis cinerea Pers

Abstract

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 m m external Ca 2+, whereas high viability and O 2-consumption were maintained without added external Ca 2+ or when higher concentrations (5–200 m m) of Ca 2+ were used. It was concluded that Ca 2+ 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 45Ca 2+ into swede protoplasts, but binding to the plasmalemma only occurred at 10 and 200 mM 45Ca 2+ in the external medium. Scatchard analysis showed swede protoplasts had a high affinity and binding capacity for Ca 2− at the plasma membrane. Swede protoplasts showed significantly reduced Ca 2+ uptake when treated with the Ca 2+ channel antagonists bepridil and verapamil, which implied voltage-gated Ca 2+ 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 n m remaining unchanged when the external Ca 2+ concentration was altered. The uptake of calcium ions by B. cinerea varied with the external concentration, and at high concentrations (100–200 m m), 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 Ca 2+ during plant pathogenesis.