The class of Qo-inhibiting fungicides (QoIs) act as respiration inhibitors by binding to the Qo center of cytochrome b. The longevity of these fungicides has been challenged by the selection of fungal sub-populations resisting high doses of QoI fungicides, with a G143A amino acid exchange in the cytochrome b target site identified as the most common cause of resistance. In contrast, the mechanism of alternative respiration, as another mechanism of fungal QoI resistance, has thus far not been affiliated with practical resistance. In the present study, azoxystrobin-resistant mutants of Magnaporthe grisea were generated and characterized. Emergence of these spontaneous mutants was facilitated when resting melanized mycelia were allowed to escape full inhibition by azoxystrobin. This escape was related to the intactness of alternative respiration, indicating that residual expression of this rescue mechanism was involved in the spontaneous emergence of target-site mutants. The two mutants characterized resisted high doses of the QoI, azoxystrobin, with resistance factors exceeding 1,000. Two different mutations of the cytochrome b gene were identified as exchanges of guanine, leading to a G143A or a G143S amino acid exchange. Resistance of both target-site mutants remained stable during four consecutive disease cycles in the absence of azoxystrobin. Several parameters tested to measure fitness penalties inherent to the mutational changes revealed that the G143A mutant was not compromised. In contrast, the conidia production of the G143S mutant was significantly lower under both saprophytic and pathogenic conditions of reproduction.

BACKGROUNDFenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated.RESULTSUK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides.CONCLUSIONFenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Plants and numerous fungi including Magnaporthe grisea protect mitochondria from interference by respiration inhibitors by expressing alternative oxidase, the enzymatic core of alternative respiration. The alternative oxidase gene AOXMg of M. grisea was disrupted. Several lines of evidence suggested that the disruption of AOXMg was sufficient to completely curb the expression of alternative respiration. In the infection of barley leaves, several AOXMg-minus and, thus, alternative respiration-deficient mutants of M. grisea retained their pathogenicity without significant impairment of virulence. However, differences between the wild-type strain and an AOXMg-minus mutant were apparent under oxidative stress conditions generated by the treatment of infected barley leaves with the commercial respiration inhibitor azoxystrobin. Symptom development was effectively suppressed on leaves infected with the alternative respiration-deficient mutant, while lesions on leaves infected with the wild-type strain continued to develop at much higher inhibitor doses. However, respective lesions rarely developed to the stage of full maturity. The results did not conform to a previous model implying that expression of alternative respiration is silenced during pathogenesis by the presence of constitutive plant antioxidants. Rather, alternative respiration provided protection from azoxystrobin during both saprophytic and infectious stages of the pathogen. The nature of similar oxidative stress conditions in the ecology of M. grisea remains an open question.