The combined application of ethylenediurea and arbuscular mycorrhizal fungi alleviates ozone damage to Medicago sativa L

• EDU reduces O 3 sensitivity of alfalfa by mediating antioxidant enzyme activities. • AM symbiosis increases stomatal conductance and plant O 3 sensitivity. • AM fungi increase stomatal conductance by increasing plant stomatal density. • AM inoculation combined with EDU can mitigate negative effects of O 3 on plants. Ozone (O 3 ) is a phytotoxic air pollutant, both ethylenediurea (EDU) and arbuscular mycorrhizal (AM) fungi can affect plant O 3 sensitivity. However, the underlying mechanisms of EDU and AM fungi on plant O 3 sensitivity are unclear, and whether the combined application of the two can alleviate O 3 damage has not been verified. In this study, an open-top chamber experiment was conducted to examine the effects of EDU and AM inoculation on growth and physiological parameters of alfalfa ( Medicago sativa L.) plants under O 3 enrichment. The results showed that EDU significantly decreased O 3 visible injury (28.67%–68.47%), while AM inoculation significantly increased O 3 visible injury. Mechanistically, the reduction of plant O 3 sensitivity by EDU was mediated by antioxidant enzyme activities rather than stomatal conductance. Although AM inoculation increased antioxidant enzyme activities (4.99%–211.23%), it significantly increased stomatal conductance (42.69%) and decreased specific leaf weight (12.98%), the negative impact was overwhelming. Therefore, AM inoculation increased alfalfa’s O 3 sensitivity. Furthermore, we found AM inoculation increased stomatal conductance by increasing stomatal density. The research indicated EDU was sufficient to counteract the negative effects of AM inoculation on O 3 sensitivity. The combined application of EDU and AM fungi could largely alleviate the adverse effects of O 3 on plant performance.