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Rice (Oryza sativa) is a highly significant cereal crop on a global scale. Crop plants usually respond to the biotic challenges with altered metabolic composition and physiological perturbations. We have deciphered altered metabolite composition, modulated metabolic pathways and identified metabolite biomarkers in M. graminicola-challenged susceptible rice variety HUR-917 using NMR (Nuclear magnetic resonance) mass spectrophotometry-based metabolomics. In this study, we conducted a comprehensive analysis of the metabolome of susceptible rice plants challenged with the pathogen M. graminicola to unravel complex metabolic changes, identify key biosynthetic pathways, and pinpoint metabolite biomarkers. Through statistical analysis, we identified 100 significant metabolites, with 48 upregulated and 52 downregulated metabolites at a fold change threshold of ≥ 2.0. Multivariate analyses, including Partial Least Squares Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), revealed clear discrimination between control and treated samples, with high predictive ability for annotated discriminant metabolites. Pathway enrichment analysis unveiled prominent involvement of metabolic pathways such as nicotine and nicotinamide metabolism and valine, leucine, and isoleucine degradation. Furthermore, putatively annotated biomarkers identified through multivariate ROC curve analysis included metabolites like Thymol, Glycylproline, N-acetylglutamate, and Betaine, among others. These biomarkers, along with pathway enrichment results, underscored the intricate defense mechanisms employed by rice plants in response to M. graminicola infection. Notably, upregulated metabolites such as betaines, histamine, and 5-hydroxytryptophan were implicated in plant defense responses, while downregulated metabolites like thymol and N-acetylglutamate may contribute to increased susceptibility to nematode infection. Nicotinic acid downregulation is crucial in enhanced susceptibility of rice against M. graminicola. Pathway mapping highlighted the enrichment of crucial metabolic pathways involved in primary and secondary metabolism, emphasizing the shift from growth-related processes to defense-related responses like nicotine and nicotinamide metabolism under stress conditions. Overall, our findings provide valuable insights into the metabolic dynamics of rice plants during pathogen invasion, identifying potential biomarkers and elucidating key metabolic pathways involved in plant defense mechanisms. This research contributes to a deeper understanding of plant-nematode interactions and holds implications for the development of effective strategies for root knot management in rice cultivation.