Evolution of multiple resistance under long-term herbicide exposure reshapes Mediterranean perennial agroecosystems

Decades of intensive herbicide use in perennial cropping systems have imposed substantial environmental pressures and accelerated the evolution of herbicide-resistant weed populations. In Andalusia (southern Spain), citrus and olive orchards rely heavily on glyphosate and other herbicide modes of action, resulting in chronic ecosystem exposure, declining herbicide efficacy, and increasing chemical inputs. We surveyed ten dominant weed species across eleven orchards to characterize resistance patterns and investigate underlying physiological mechanisms relevant to environmental persistence and contaminant cycling. Rapid screening assays using six widely applied herbicides (atrazine, 2,4-D, glyphosate, oxyfluorfen, quizalofop-ethyl, and tribenuron-methyl) revealed widespread multiple and cross-resistance in both dicots and monocots. Amaranthus albus, A. viridis, Conyza bonariensis, and Solanum nigrum displayed resistance to atrazine, glyphosate, and tribenuron-methyl, while Lolium rigidum and Sorghum halepense showed broad-spectrum resistance involving target-site alterations and enhanced metabolic detoxification, partially reversed by the cytochrome P450 inhibitor malathion. Complementary biochemical assays targeting PSII, ALS, ACCase, and EPSPS, together with diagnostic tests for PPO- and auxin-related herbicides, indicated diverse non-target-site resistance pathways, suggesting increased herbicide transformation and altered degradation dynamics in soil-plant systems. The emergence of complex resistance mechanisms reflects prolonged selection pressure from intensive herbicide regimes and implies elevated risks of environmental accumulation, off-target effects, and reduced sustainability of chemical-based weed control. These findings highlight the need to reduce herbicide dependence and adopt integrated weed management strategies that minimize pollutant loads in Mediterranean agroecosystems.