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Gaeumannomyces tritici , an ascomycete soilborne fungus, causes a devastating root disease in wheat. Carabrone, a botanical bicyclic sesquiterpenic lactone, is a promising fungicidal agent that can effectively control  G. tritici . However, the mechanism of action of carabrone against  G. tritici  remains largely unclear. Here, we used immunogold for subcellular localization of carabrone and the results showed that carabrone is subcellularly localized in the mitochondria of  G. tritici . We then explored the functional analysis of genes  GtCytc  1 ,  GtCytb , and  GtIsp  of the mitochondrial respiratory chain cytochrome  bc  1  complex in  G. tritici  by RNA silencing as a possible target of carabrone. The results showed that the silenced mutant ∆GtIsp is less sensitive to carabrone compared to ∆GtCytc 1  and ∆GtCytb. Compared with the control, the activities of complex III in all the strains, except ∆GtIsp and carabrone‐resistant isolate 24‐HN‐1, were significantly decreased following treatment with carabrone at EC 20  and EC 80  in vitro (40%–50% and 70%–80%, respectively). The activities of mitochondrial respiratory chain complex III and the mitochondrial respiration oxygen consumption rates in all the strains, except ∆GtIsp and 24‐HN‐1, were higher with respect to the control when treated with carabrone at EC 20  in vivo. The rates of mitochondrial respiration of all strains, except ∆GtIsp, were significantly inhibited following treatment with carabrone at EC 80  (ranging from 57% to 81%). This study reveals that the targeting of the iron–sulphur protein encoded by  GtIsp  is highly sensitive to carabrone and provides a direction for the research of carabrone's target.

molecular plant pathology

A functional analysis of mitochondrial respiratory chain cytochrome  bc  1  complex in  Gaeumannomyces tritici  by RNA silencing as a possible target of carabrone