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Poplar canker, mainly caused by Botryosphaeria species, is a serious disease that has resulted in the reduced productivity and death of poplar worldwide. Different Populus species have varied resistance levels to poplar canker; however, whether phenolic compounds in poplar are involved in this resistance remains uncertain. Here, we determined the concentrations of phenolic compounds and their antifungal activities in canker-resistant P. tomentosa and canker-susceptible P. beijingensis. We identified 29 phenolic compounds with significantly different concentrations between the two species. Salicylic acid (SA), tremuloidin, salicin, and poplin were dominant in P. tomentosa, while benzoic acid (BA) and catechol were dominant in P. beijingensis. These six phenolic compounds were further tested for antifungal activities. SA, BA, and catechol showed significant antifungal activities against Botryosphaeria dothidea …