Pumpkin rootstock confers multilayered defense against root-knot nematode in grafted cucumber

Greenhouse cucumber production faces significant constraints due to infestations of Meloidogyne incognita . Grafting onto nematode-resistant rootstocks represents an effective strategy for nematode control. To evaluate the nematode resistance potential of pumpkin rootstocks, we developed pumpkin-grafted cucumber plants (PGC) by grafting onto a pumpkin rootstock. Comprehensive evaluation through in vivo nematode assays, reactive oxygen species (ROS) quantification, chemotaxis tests, qPCR analysis, and field trials revealed that PGC reduced root galls by 83.8% ( P  < 0.001) and completely suppressed egg mass formation. Nematode penetration decreased by 73.2% at 3 days post-inoculation, with inhibited female development (body width decreased 39.3%, P  < 0.001). ​qPCR analysis demonstrated significant upregulation of defense genes in PGC roots, with pathogenesis-related protein 1( Pr1 ) expression increasing 5.3-fold, callose synthase ( Cals ) 4.8-fold, and lipoxygenase ( Lox1 ) 3.7-fold, while ethylene signaling modulator constitutive triple response 1( Ctr1 ) was downregulated.​​ Chemotaxis assays showed 45.2% higher nematode repellence ( P  < 0.01). Field trials demonstrated 56.9% disease control efficacy and 31.8% yield increase. These results indicate multi-layered resistance through callose deposition, ROS burst, coordinated SA/JA/ET signaling, and chemoreception disruption, providing the first evidence of nematode resistance in pumpkin rootstocks.