Effect of Natural Bio-treatments on Physiological and Metabolomic Response of Citrus Under Semi-Arid Conditions
Abstract
Citrus cultivation in semi-arid regions is increasingly constrained by drought, salinity, and high temperatures, reducing plant performance and productivity. The aim of this study was to evaluate the effects of three natural bio-treatments based on stone pine (Pinus pinea L.), rosemary (Rosmarinus officinalis L.), and shrimp chitin on the physiological, metabolomic, and soil responses of citrus plants grown under semi-arid conditions. Photosynthetic pigments, proline, soluble sugars, secondary metabolites (LC-MS/MS), and soil physicochemical properties were assessed to characterize plant responses and treatment-induced changes in nutrient availability. The results demonstrated that bio-treatments significantly affected citrus adaptation through distinct mechanisms. Seasonal evaluation revealed that TRPP consistently maintained the highest chlorophyll content (4.14 mg g⁻¹ FM in June), whereas TRSC promoted proline accumulation during the summer periods (25.97 mg g⁻¹ FM). Soluble sugars increased in treated plants during the second half of the experimental period, indicating improved osmotic adjustment. TRPP increased potassium and phosphorus availability (K=38.81 mg L⁻1, P2O5= 217.55 ppm), reduced sodium accumulation (Na= 15.12 mg L⁻1), and promoted the accumulation of flavonoids and phenolic compounds implicated in antioxidant protection. TRSC enhanced the abundance of defence-related metabolites, including caffeic, ferulic, Cis-p coumaric, and salicylic acids. TRRO was mainly associated with lower soil electrical conductivity (EC= 0.51 dS m⁻1) improved iron availability (Fe= 12.7 mg L⁻1) and higher levels of rutin, myricetin, kojic acid, and folic acid. Multivariate analyses identified TRPP and TRSC as the most effective modulators of secondary metabolism. Overall, the findings demonstrate that natural bio-treatments improve citrus resilience in semi-arid conditions through coordinated regulation of photosynthetic activity, osmotic adjustment, secondary metabolism, soil nutrition availability and nutrient homeostasis.