Pyraclostrobin resistance in tobacco anthracnose pathogens: Risk assessment and molecular mechanism

Tobacco anthracnose, caused by Colletotrichum spp., is an aggressive disease that reduces tobacco leaf quality and yield, primarily controlled by chemical fungicides. Pyraclostrobin, a typical quinone outside inhibitor (QoI), acts by inhibiting cytochrome bc1 complex to block energy transfer, but its efficacy against Colletotrichum spp. remains understudied. This study determined antifungal activity of pyraclostrobin against 102 Colletotrichum spp. isolates, with EC₅₀ values ranging from 0.0302 to 0.1286 μg/mL (average 0.0511 ± 0.0178 μg/mL), confirming strong antifungal activity. Nine high-level pyraclostrobin-resistant mutants with Cytb-G143A mutation were obtained from three sensitive parental isolates via chemical taming. Molecular docking revealed that Cytb-G143A mutation alters the binding mode of Cytb and pyraclostrobin, reducing binding affinity. Resistant mutants exhibited generally reduced fitness in mycelial growth (at 25 °C and 30 °C), virulence, and sporulation, although the effects varied among mutants. Positive cross-resistance was observed between pyraclostrobin and azoxystrobin or trifloxystrobin, while no cross-resistance was detected between pyraclostrobin and tebuconazole, pydiflumetofen, or fludioxonil. Overall, Colletotrichum spp. have a moderate resistance risk to pyraclostrobin. These findings deepen our understanding of the molecular mechanisms of Colletotrichum spp. resistance to pyraclostrobin, and provide an important reference for field chemical control of tobacco anthracnose.