Ethyl acetate extract of Artemisia argyi improves the resistance of cotton to Verticillium dahliae by activating the immune response

Verticillium wilt, a significant pathogen affecting cotton, has historically been challenging to control, posing a substantial threat to the sustainable development of the cotton industry. This study demonstrates that resistance to Verticillium dahliae in cotton can be enhanced by treating the roots with an ethyl acetate extract (EAAA) extracted from Artemisia argyi. The mechanisms by which EAAA activates immunity in cotton were elucidated by examining the expression levels of resistance genes post-treatment, evaluating salicylic acid (SA) and jasmonic acid (JA) levels, analyzing transcriptome data, and employing virus-induced gene silencing (VIGS) technology. Additionally, pot experiments were conducted to validate the efficacy of EAAA in controlling Verticillium wilt. The flavonoid content in EAAA was qualitatively analyzed using Ultra-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UPLC-MS/MS), identifying three specific flavonoids that were further screened to verify their roles in activating cotton immunity. Cotton plants treated with EAAA exhibited reduced leaf chlorosis and browning in the vascular bundles. Genes involved in SA and JA synthesis and signaling in the root system were highly expressed, resulting in increased levels of SA and JA. Transcriptome analysis revealed that most upregulated differentially expressed genes were primarily enriched in the Mitogen-Activated Protein Kinase (MAPK) signaling pathway. Two specific genes, RLK and MAPKKK18, were identified through VIGS technology as key regulators of the immune pathway in cotton. The flavonoid monomer activation experiment demonstrated that eupatilin, hispidulin, jaceosidin, and a mixture of these three could induce the expression of cotton-related resistance genes. Collectively, these findings provide a research basis for the development of EAAA as a natural plant immune-inducing agent against cotton Verticillium wilt.