Enhancing copper and arsenic phytoextraction from calcareous soil through the synergistic interaction of ethylenediaminetetraacetic acid application timing and metal-resistant plant growth-promoting rhizobacteria inoculation.

Remediating calcareous soils co-contaminated with copper and arsenic remains a major challenge due to low metal bioavailability and the risk of chelator-induced phytotoxicity, which can severely limit phytoremediation success. This study investigated a synergistic strategy combining heavy metal-resistant plant growth-promoting rhizobacteria (PGPR) with precisely timed ethylenediaminetetraacetic acid (EDTA) application to improve phytoextraction efficiency in corn (Zea mays). Eight PGPR isolates were obtained from contaminated soil, and three strains (Stenotrophomonas sp. A22, Pseudomonas sp. A2 and A5) were selected based on their high resistance to Cu (up to 400 mg L-1) and As (up to 250 mg L-1), as well as multiple plant growth-promoting traits. In a controlled pot experiment, we evaluated bacterial inoculation and EDTA application at 20, 35, or 45 days after planting on plant growth, physiological performance, and metal uptake. Early EDTA addition (day 20) caused severe phytotoxicity, markedly reducing root and shoot biomass and depressing photosynthetic efficiency (Fv/Fm). In contrast, delaying EDTA application to days 35 or 45 substantially alleviated these adverse effects. PGPR inoculation, particularly with strain A5, further mitigated EDTA-induced stress and improved biomass production and physiological status. The combined PGPR-EDTA treatments significantly increased soil metal bioavailability and enhanced plant uptake, with maximum shoot Cu (214 mg kg-1) and As (99 mg kg-1) concentrations observed with strains A5 or A2 and EDTA application at day 20. Sequential extraction confirmed that these PGPR-EDTA treatments shifted metals from residual and oxide-bound pools into more soluble and exchangeable/carbonate-bound fractions. The findings support a mechanistically informed, optimized phytoremediation strategy for calcareous soils, based on the optimal timing of EDTA application and inoculation with metal-resistant PGPR.