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

As a destructive fungus-like plant pathogen, the oomycete Phytophthora capsici is 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) of P. capsici. CA-induced immediate Ca2+ efflux from zoospores could be confirmed by the rapid decrease in intracellular Ca2+ content determined by using Fluo-3 AM and the increase in extracellular Ca2+ concentration determined by using ICP-AES (inductively coupled plasma atomic emission spectrometry). Blocking Ca2+ influx with ruthenium red and verapamil led to a higher level of CA-induced Ca2+ efflux, suggesting the simultaneous occurrence of Ca2+ influx along with the Ca2+ efflux under CA exposure. Further results showed that EGTA-induced decrease in intracellular Ca2+ gave rise to the impaired vitality of P. capsici while the addition of exogenous Ca2+ could suppress the growth inhibitory effect of CA. These results suggested that Ca2+ efflux played an important role in CA-induced growth inhibition of P. capsici. The application of 3-phenyl-1-propanal, a CA analog without alpha,beta-unsaturated bond, resulted in a marked Ca2+ 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 of P. capsici by suppressing Ca2+ efflux. Our results suggest that CA inhibits the growth of P. capsici by stimulating a transient Ca2+ efflux via Michael addition, which provides important new insights into the antimicrobial action of CA.