In the last few decades, Rhizoctonia solani 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, N-[2-[(3-chlorophenyl)amino]-phenyl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, was equivalent to the commercial fungicide thifluzamide and its EC₅₀ value was 0.022 mg/L against R. solani. 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 R. solani’s MMP. Furthermore, the results by label-free quantitative proteomic analysis showed that 1153 proteins were found after R. solani 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.

中文翻译：

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新型的含二芳基胺支架的吡唑羧酰胺对茄枯萎菌的作用机理。

在过去的几十年中，引起水稻鞘枯萎病的茄枯萎病已在世界范围内造成许多经济损失。因此，已经对许多新颖的吡唑羧酰胺类杀真菌剂进行了深入的研究并用于对抗它。在这方面，近年来，我们的小组在盆栽试验和田间试验中报道了一种新型的吡唑甲酰胺，该吡唑甲酰胺含有二芳基胺支架，对稻米鞘枯病具有良好的抗真菌活性。在该项目之后，吡唑羧酰胺的抗真菌作用机理已经阐明。抗真菌结果表明，化合物SCU2028，N- [2-[（3-氯苯基）氨基]-苯基] -3-（二氟甲基）-1-甲基-1 H吡唑-4-甲酰胺，相当于商业杀真菌剂的thifluzamide和其EC ₅₀值是针对0.022 mg / L的水稻纹枯病菌。此外，通过扫描电子显微镜和透射电子显微镜的观察结果表明，它可以破坏真菌的细胞壁或膜，并导致内容物的泄漏和线粒体数目的增加以及形态异常。同时，线粒体膜电位（MMP）的结果表明，它可以降低茄形假单胞菌的MMP。此外，无标记定量蛋白质组学分析的结果表明，在R. solani后发现了1153种蛋白质。用化合物SCU2028处理了Aβ1，SCU2028包括对照组中的212种蛋白质和治疗组中的257种蛋白质。总共获得142个差异蛋白，其中92个蛋白被上调，而50个蛋白被下调。差异表达的蛋白质影响线粒体，内质网，核糖体以及其他相关的GO和KEGG途径中的一系列生理和生化途径。特别是，由TCA循环和氧化磷酸化KEGG途径引起的呼吸链抑制表明复合物II（琥珀酸脱氢酶）和复合物IV（细胞色素氧化酶）可能是化合物SCU2028的主要作用靶标。此外，qRT-PCR，酶活性检测和分子对接的多次实验确认复合物II和复合物IV为靶标。