Ca2+ Efflux Is Involved in Cinnamaldehyde-Induced Growth Inhibition of Phytophthora capsici

As a destructive fungus-like plant pathogen, the oomycetePhytophthora capsiciis unable to synthesize its own ergosterol as the potential target of fungicide cinnamaldehyde (CA). In this study, CA exerted efficient inhibitory effects on both mycelial growth (EC50=0.75 mM) and zoospore germination (MIC=0.4 mM) ofP. capsici . CA-induced immediate Ca 2+ efflux from zoospores could be confirmed by the rapid decrease in intracellular Ca 2+ content determined by using Fluo-3 AM and the increase in extracellular Ca 2+ concentration determined by using ICP-AES (inductively coupled plasma atomic emission spectrometry). Blocking Ca 2+ influx with ruthenium red and verapamil led to a higher level of CA-induced Ca 2+ efflux, suggesting the simultaneous occurrence of Ca 2+ influx along with the Ca 2+ efflux under CA exposure. Further results showed that EGTA-induced decrease in intracellular Ca 2+ gave rise to the impaired vitality ofP. capsiciwhile the addition of exogenous Ca 2+ could suppress the growth inhibitory effect of CA. These results suggested that Ca 2+ efflux played an important role in CA-induced growth inhibition ofP. capsici . The application of 3-phenyl-1-propanal, a CA analog without α,β- unsaturated bond, resulted in a marked Ca 2+ influx in zoospores but did not show any growth inhibitory effects. In addition, exogenous cysteine, an antagonist against the Michael addition (the nucleophilic addition of a carbanion or another nucleophile) between CA and its targets, could attenuate CA-induced growth inhibition ofP. capsiciby suppressing Ca 2+ efflux. Our results suggest that CA inhibits the growth ofP. capsiciby stimulating a transient Ca 2+ efflux via Michael addition, which provides important new insights into the antimicrobial action of CA.