BACKGROUND: Rice sheath blight caused by Rhizoctonia solani is a devastating disease of rice in China. However, indiscriminate use of chemical fungicides applied to control the disease raise major environmental and food safety issues. Ecofriendly biocontrol alternatives are urgently needed. Eugenol, one of the main ingredients in Syzygium aromaticum, has attracted much attention owing to its antifungal properties. However, its mode of action is still not clear. Herein, the antifungal activity and mode of action of eugenol against R. solani were investigated.RESULTS: Results confirmed that the mycelia of R. solani treated with eugenol shrank and became dehydrated, the cytoplasmic wall separated, and the vacuoles and mitochondria decreased or dissolved. Moreover, we found that eugenol downregulated expression of C-4 methyl sterol oxidase, inhibited synthesis of ergosterol, increased membrane permeability and impaired the transportation of amino acids and glucose across the cell membrane. In addition, eugenol decreased the mitochondrial membrane potential and initiated an oxidative stress reaction by increasing reactive oxygen species and malondialdehyde, which together with membrane damage contribute to the antifungal activity of eugenol. Meanwhile, eugenol might inhibit R. solani by affecting oxidative phosphorylation and the tricarboxylic acid cycle (TCA cycle). CONCLUSION: In view of its multitarget properties against R. solani, eugenol provides an alternative approach to chemical control strategies against rice sheath blight.
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-[(3chlorophenyl)amino]-phenyl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, was equivalent to the commercial fungicide thifluzamide and its EC 50 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.
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