Metabolomics-Based Analysis Reveals Flavonoid-Mediated Defence in Alfalfa (Medicago sativa ‘Gannong No. 5’) Against Pea Aphid (Acyrthosiphon pisum) Infestation

The pea aphid (Acyrthosiphon pisum) is a destructive phloem-feeding pest of alfalfa. Its overreliance on chemical insecticides necessitates the development of sustainable, plant-based resistance strategies. Plant flavonoids are important in induced defense, but their dynamic responses to aphid feeding and subsequent insecticidal effects remain unclear. Using the highly resistant alfalfa variety ‘Gannong No. 5’ (GN5), this study integrated behavioral assays, performance bioassays, untargeted and targeted metabolomics, and exogenous flavonoid feeding assays to investigate how pea aphid infestation alters flavonoid metabolism and thereby affects aphid performance. Bioassays confirmed strong antibiosis of GN5 against A. pisum. Behavioral choice assays showed that, at 2 h post-release, aphids significantly avoided plants pre-infested for 12 h and 48 h, but not those pre-infested for 24 h; by 8 h post-release, they significantly avoided all pre-infested plants regardless of infestation duration. Prolonged pre-infestation (48 h) also reduced average fecundity per female. Non-targeted metabolomics revealed substantial metabolic reprogramming after 48 h of aphid feeding, with most flavonoids and isoflavonoids significantly upregulated. Targeted metabolomics identified 28 flavonoids, among which only sakuranetin and chrysin were significantly upregulated after 48 h, indicating their specific induction. Finally, performance bioassays confirmed insecticidal effects in a concentration-dependent manner: sakuranetin at 0.1 μg/μL reduced reproduction, and at 10.0 μg/μL reduced survival; chrysin at 0.1 μg/μL reduced both survival and reproduction. Collectively, these results demonstrate that pea aphid feeding triggers the induction of specific defensive flavonoids in GN5, which may contribute to antibiosis. This study provides a theoretical basis for exploring flavonoid-based approaches in sustainable aphid management.