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The understanding of the effects of nitrogen sources on lignin synthesis in sweet potato during storage root formation is limited. In this study, we investigated the effects of different nitrogen source on sweet potato storage root formation and development, as well as lignin synthesis in potential storage roots. The sweet potato cultivars Shangshu 19 and Jixu 23 were used in field experiments in 2019 and 2020. Three treatments were tested: (a) no nitrogen fertilizer application (control); (b) 60 kg hm⁻² ammonium nitrogen; and (c) 60 kg hm⁻² amide nitrogen. The results indicate that during sweet potato storage root formation, ammonium nitrogen significantly enhanced root activity compared to that of the control. The ammonium nitrogen treatment promoted IbEXP1 and inhibited Ibkn1 and Ibkn2 expression during the early stages of storage root formation, then increased gibberellic acid and decreased zeatin riboside content, enhanced phenylalanine ammonia lyase and peroxidase activities, and promoted lignin synthesis in potential storage roots. The opposite effects of ammonium nitrogen treatment on gene expression, hormone contents, and enzyme activity were observed in the late stages of storage root formation. Relative to the control, the ammonium nitrogen treatment significantly increased the number of storage roots during canopy closure. The ammonium nitrogen treatment produced the highest storage root yield and number of storage roots per plant. These results indicated that the ammonium nitrogen can inhibit root lignin synthesis, then promote storage root formation and increase the yield of sweet potato. Full article