The Biochemical Mode of Action of the Novel Selective Fungicide Cyazofamid: Specific Inhibition of Mitochondrial Complex III in Pythium spinosum

To elucidate the background of the highly selective fungicidal activity of cyazofamid (ISO proposed common name), 4-chloro-2-cyano-N,N-dimethyl-5-p-tolylimidazole-1-sulfonam ide, the biochemical mode of action of this fungicide in Pythium spinosum was investigated. Cyazofamid inhibited mycelial growth of P. spinosum on the order of 1 mu M on agar medium containing gelatin, on water agar, and on potato dextrose agar. The mycelial growth inhibition was markedly potentiated in the presence of salicylhydroxamic acid (SHAM), an inhibitor of mitochondrial alternative oxidase. Oxygen consumption of P. spinosum mycelia treated with 4 mu M cyazofamid was reduced by about 60%. At 60 min after the treatment, the oxygen consumption was recovered, but the respiration was resistant to potassium cyanide and sensitive to SHAM. From the effect of cyazofamid on electron transport activity of P. spinosum mitochondria, it was revealed that this fungicide specifically interferes with cytochrome bc sub(1) complex (complex III) activity (I sub(50): 0.04 mu M). Cyazofamid, however, exhibited no inhibition of complex III activities in mitochondria isolated from other biological sources such as Botrytis cinerea, Saccharomyces cerevisiae, rat liver, and potato tuber. Thus, the highly selective activity of cyazofamid appeared to be due to a difference in the inhibitor susceptibility at the target enzyme. To identify the binding site of cyazofamid in complex III, reduction kinetics of cytochrome b hemes was investigated with P. spinosum mitochondria. The addition of cyazofamid immediately reduced cytochrome b hemes and the extent of reduction was higher than that without cyazofamid. Reduction of b hemes was markedly inhibited by the combined use of cyazofamid and azoxystrobin (Q sub(o) center inhibitor). These results suggest that cyazofamid binds to the Q sub(i) center of complex III. Copyright 2001 Academic Press.