Institute of Food Safety and Monitoring Technology, Jiangsu Academy of Agricultural Sciences, Nanjing, China ; School of Food Science, Henan Institute of Science and Technology, Xinxiang, China.
PLoS One. 2013 Oct 1;8(10):e76264. doi: 10.1371/journal.pone.0076264. eCollection 2013.
As a destructive fungus-like plant pathogen, the oomycete Phytophthoracapsici 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 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 of P. capsici while 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 of P. 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 of P. capsici by suppressing Ca(2+) efflux. Our results suggest that CA inhibits the growth of P. capsici by stimulating a transient Ca(2+) efflux via Michael addition, which provides important new insights into the antimicrobial action of CA.
作为一种具有破坏性的真菌样植物病原体，卵菌 Phytophthora capsici 无法合成自身的麦角固醇，而麦角固醇是杀真菌剂肉桂醛 (CA) 的潜在靶标。在这项研究中，CA 对 P. capsici 的菌丝生长 (EC50=0.75 mM) 和游动孢子萌发 (MIC=0.4 mM) 均具有高效的抑制作用。通过使用 Fluo-3 AM 测定细胞内 Ca2+ 含量的快速下降和 ICP-AES (电感耦合等离子体原子发射光谱法) 测定细胞外 Ca2+ 浓度的增加，可以证实 CA 诱导的游动孢子中 Ca2+ 的即刻外排。用钌红和维拉帕米阻断 Ca2+ 内流会导致 CA 诱导的 Ca2+ 外排水平更高，这表明在 CA 暴露下同时发生 Ca2+ 内流和 Ca2+ 外排。进一步的结果表明，EGTA 诱导的细胞内 Ca2+ 减少导致 P. capsici 的活力受损，而添加外源性 Ca2+ 可以抑制 CA 的生长抑制作用。这些结果表明，Ca2+ 外排在 CA 诱导的 P. capsici 生长抑制中起重要作用。3-苯基-1-丙醛，一种没有α,β-不饱和键的 CA 类似物，会导致游动孢子中 Ca2+ 的大量内流，但没有表现出任何生长抑制作用。此外，作为 CA 与其靶标之间迈克尔加成 (碳负离子或另一种亲核试剂的亲核加成) 的拮抗剂的半胱氨酸，可以通过抑制 Ca2+ 外排来减轻 CA 对 P. capsici 生长抑制的作用。我们的研究结果表明，CA 通过迈克尔加成刺激短暂的 Ca2+ 外排来抑制 P. capsici 的生长，这为 CA 的抗菌作用提供了重要的新见解。
