Coordinated mechanisms of the Acremonium strictum SS71-induced ROS accumulation in strawberry elucidated via optimized dual-treatment detached leaf assays.

The oxidative burst is likely a pivotal early event in immune activation in strawberry, a non-model plant, yet the underlying mechanisms remain largely unknown. Here we optimized a detached-leaf system for independent application of ROS inducers and pharmacological inhibitors to investigate the roles of NADPH oxidases, SOD, and calcium in ROS accumulation induced by the avirulent fungus Acremonium strictum SS71. Our results show that the dynamics of SS71-induced ROS accumulation in the detached-leaf system was consistent with previous whole-plant strawberry studies, reaching maximum levels at 6 hpi. The efficient scavenging of paraquat-induced ROS by vascular application of ascorbic acid further supports the validity of this model compared with the other delivery methods assayed. The pharmacological inhibitors used were DDC (a Cu,Zn-SOD inhibitor), DPI (a NADPH oxidase inhibitor), and EGTA (a calcium chelator). Concentrations lacking phytotoxic effects were selected based on cellular damage assessments following petiolar uptake, and the functional efficacy of these treatments was validated by SOD activity assays (DDC) and stomatal closure assays (DPI and EGTA). Finally, the effects of pharmacological inhibitors on SS71-induced ROS accumulation were evaluated. Our findings suggest that NADPH oxidases contribute to SS71-induced superoxide accumulation, although additional sources may also involve. SS71-induced hydrogen peroxide accumulation appears to be mainly dependent on Cu,Zn-SOD activity. Extracellular calcium influx does not appear to be required for SS71-induced ROS accumulation under the experimental conditions tested. These findings provide new insights into strawberry defense signaling and establish a useful detached-leaf system for studying cellular mechanisms in non-model species.