Building upon previous structure-activity relationships about the fungicidal amide and hydrazide lead structures, 24 novel amide-hydrazide compounds were designed and synthesized with L-isoleucine as the initial skeleton to explore the impact of substituents in the hydrazide bridge on the fungicidal activity. Among these compounds, A5 exhibited excellent and broad spectrum inhibitory activity, along with satisfactory in vivo protective efficiency against R. solani at concentrations of 200 and 50 mu g<middle dot>mL-1. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed that compound A5 induced significant morphological changes in the R. solani mycelium coupled with vacuole rupture and cytoplasmic inhomogeneity in cellular structures. Transcriptomic and metabolomic analyses indicated that, following A5 treatment, the differentially expressed genes and metabolites were significantly enriched in carbohydrate metabolism-related pathways as well as in lipid metabolism-associated pathways, including glycerophospholipid metabolism, steroid biosynthesis, arachidonic acid metabolism, and sphingolipid metabolism. Additionally, compound A5 demonstrated low toxicity to zebrafish, with survival rates of 100% and 60% at concentrations of 1 and 10 mu g<middle dot>mL-1, respectively, over a period of 7 days. The above results provide theoretical guidance for the development of novel green hydrazide fungicidal candidates.
