| Summary: | Leaf spot disease caused by <i>Alternaria</i> pathogens seriously threatens peony production. The physiological mechanism of peony resistance to the pathogen is little reported. This study aimed to reveal the defensive mechanism of peonies in response to the pathogen <i>Alternaria tenuissima</i>. The disease-resistant (R) variety ‘Zi Fengyu’ and susceptible (S) variety ‘Heihai Botao’ were employed, and some parameters in the leaves were analyzed after inoculation with <i>A. tenuissima</i>, mainly including the hypersensitive response (HR), activity of defensive enzymes, and expression of disease-resistance genes. The results showed that compared with the responses in the S genotype, HR occurred more rapidly in the R genotype. Meanwhile, the activity of antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) and other two defense enzymes (polyphenol oxidase and phenylalanine ammonia-lyase) increased more significantly, and the expression of <i>pathogenesis-related</i> (<i>PR</i>) genes (<i>PlPR1</i>, <i>PlPR2</i>, <i>PlPR4B</i>, <i>PlPR5</i>, and <i>PlPR10</i>) and two <i>WRKY</i> genes (<i>PlWRKY13</i> and <i>PlWRKY65</i>) was more strongly induced. These responses collectively contributed to the disease resistance of the R genotype. These findings provided a theoretical basis for understanding the intrinsic mechanism of peony resistance to <i>Alternaria</i> leaf spot disease and breeding the disease-resistant peony varieties using a molecular approach.
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