Mechanism of Action of Novel Pyrazole Carboxamide Containing a Diarylamine Scaffold against <i>Rhizoctonia solani</i>

In the last few decades, <i>Rhizoctonia solani</i> causing rice sheath blight has resulted in a lot of economic losses in the world. Therefore, many novel pyrazole carboxamide fungicides have been intensively researched and employed to fight against it. In this regard, in recent years, our group reported a novel pyrazole carboxamide containing a diarylamine scaffold with good antifungal activity against rice sheath blight in the pot test and field trial. Following this project, the antifungal mechanism of action of the pyrazole carboxamide has been elucidated in this work. The antifungal result showed that compound SCU2028, <i>N</i>-[2-[(3-chlorophenyl)amino]-phenyl]-3-(difluoromethyl)-1-methyl-1<i>H</i>-pyrazole-4-carboxamide, was equivalent to the commercial fungicide thifluzamide and its EC₅₀ value was 0.022 mg/L against <i>R. solani</i>. Also, the observation results by scanning electron microscopy and transmission electron microscopy showed that it could destroy the fungus' cell walls or membranes and result in the leakage of contents and increase of the number of mitochondria and abnormal morphology. Meanwhile, the result on the mitochondrial membrane potential (MMP) showed that it could decrease <i>R. solani</i>'s MMP. Furthermore, the results by label-free quantitative proteomic analysis showed that 1153 proteins were found after <i>R. solani</i> was treated with compound SCU2028, including 212 proteins in the control group and 257 proteins in the treatment group. A total of 142 differential proteins were obtained, of which 92 proteins were upregulated and 50 proteins were downregulated. The differentially expressed proteins affected a series of physiological and biochemical pathways in the mitochondria, endoplasmic reticulum, ribosome, and other related GO and KEGG pathways. In particular, the inhibition of the respiratory chain caused by the TCA cycle and oxidative phosphorylation KEGG pathway indicated that complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase) might be compound SCU2028's main action targets. In addition, multiple experiments of qRT-PCR, enzyme activity detection, and molecular docking confirmed complex II and complex IV as targets. It could be seen that these findings provided a theoretical support for further research and development of the pyrazole carboxamide fungicides.