Objective This study aims to elucidate the regulatory mechanism of root exudates from island cotton lines with differing resistance levels on the gene expression of Fusarium oxysporum f. sp. vasinfectum, clarify the molecular basis of the root exudate–pathogen interaction, and provide a theoretical foundation for breeding new resistant island cotton varieties and rhizosphere ecological regulation.

Methods Three island cotton lines with significantly different disease resistance (resistant line 10702, tolerant line 10765, and susceptible line 10744) preserved at Xinjiang Agricultural University were used as research subjects. A pathogen model was established by culturing Fusarium oxysporum f. sp. vasinfectum (st89) with root exudates, and transcriptome sequencing technology was employed to analyze the regulatory effects of root exudates from different island cotton lines on the gene expression of the pathogen.

Results The root exudates of the resistant line significantly downregulated the expression of key genes involved in ribosomal metabolism, membrane transport, and redox pathways, resulting in marked inhibition of mycelial growth and spore reproduction. The exudates of the tolerant line exhibited a time-dependent inhibitory-to-promoting effect. In contrast, the exudates of the susceptible line consistently promoted pathogen growth. Functional enrichment analysis identified key molecular targets and metabolic pathways.

Conclusion Root exudates from island cotton lines with different resistance levels differentially regulate the metabolism of Fusarium oxysporum f. sp. vasinfectum, thereby determining the pathogenic mechanism: the resistant line continuously suppresses basal metabolism and pathogenic signals of the pathogen, the susceptible line fully activates infection-related pathways, and the tolerant line, due to insufficient early inhibition, leads to a delayed burst of pathogenic genes in the later stage. The study screened 124 core DEGs in the resistant line (R), 149 in the susceptible line (S), and 168 in the tolerant line (T), providing important candidate targets for molecular breeding of resistant island cotton and for the control of Fusarium wilt.