Regulation of multi-metal stress responses in spinach by malic acid and Moringa leaf extract: implications for plant performance and phytoextraction

Multi-metal contamination is a major limiting factor for phytoextraction with high biomass plants like spinach ( Spinacia oleracea L.) from wastewater used in the surgery industry. This study investigated the integrated application of malic acid (10 mM) from rhizosphere and Moringa oleifera leaf extract (MLE) on the growth, antioxidant defense and multi-metal accumulation in spinach under 50% and 100% authentic surgical wastewater containing higher concentrations of Ni, Cu and Pb. Untreated 100% wastewater reduced plant height by 65.8%, it caused oxidative damage and suppressed biomass production. The dual treatment of 10 mM malic acid + MLE completely reversed these effects such as plant height which recovered by 42.8%; root SOD activity enhancement from 34.25 to 53.06 U mg -1 protein under 100% wastewater; ROS reduction and maintenance of photosynthetic pigments. At the same time, malic acid increased the bioavailability of the metals leading to significant increases in absolute accumulation by roots (under 100% wastewater, root Cu was 7046.67 µg and root Ni was 10510.69 µg). The combined rhizosphere–foliar application of malic acid and MLE against the surgical wastewater (SWW) containing several metals, which has been used in real conditions, has not yet been studied in spinach. This is the first report of the simultaneous rhizosphere application of malic acid and foliar application of Moringa oleifera leaf extract under authentic multi-metal surgical wastewater. The dual-route strategy not only enhanced absolute accumulation of Cu, Pb and Ni but also significantly alleviated oxidative stress and restored growth and photosynthetic performance, suggesting a low-cost approach for phytoextraction with a high-biomass crop.