Peony (Paeonia suffruticosa Andr.) is a traditional ornamental flower in China. Peony potted culture has become mainstream. The development of agricultural and forestry waste instead of a nonrenewable peat matrix as the substrate is an important direction for the future industry of potted peonies. However, the mechanism of the effects of different substrates on the growth and development of peonies remains unclear. In this experiment, “Luhehong”, a peony variety from Heze, Shandong Province, was selected as the main experimental material. The response mechanism of peony roots to different substrate proportions was analyzed using untargeted metabolomics of potted peonies. Results showed that the potential for pot growth of planted peonies using the mixed-straw mixing matrix (Matrix K) is better than that of the rice husk matrix (Matrix G) and the control matrix. Moreover, the comparative analysis of the metabolic groups indicates that the number of peony root secretions in the three matrices has a remarkable difference, possibly related to the nutrition supply levels of the peony roots, in the different matrices of adaptive response and adjustment. Furthermore, (2r)-3-hydroxyisovaleroylcarnitine, trigonelline, 2-acetylresorcinol, and dehydroascorbic acid (oxidized vitamin C)—four metabolites with the most substantial intergroup differences—are considered key metabolites. The metabolic pathway analysis of propanoate, and ascorbate and aldarate metabolism, and the two pathways related to an environmental response, namely, ABC transporters and phosphatidylinositol signaling system, indicated that these pathways were remarkably enriched. Both may be the key way to affect the growth and development of potted peonies. This study can provide an important reference for the rational allocation of the substrate of agricultural and forestry waste in potted peony cultivation and application.
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