A synergistic pomegranate-biopolymer bio-stimulant enhances barley growth, heat stress resilience, and antimicrobial activity through physiological and molecular modulation.

内容摘要

Global agricultural challenges necessitate sustainable strategies to enhance crop productivity and resilience under environmental stress. This study investigated the synergistic effects of a novel bio-stimulant formulation consisting of pomegranate (Punica granatum L.) leaf extract, chitosan, and sodium alginate on the growth, physiological performance, molecular variation, and heat stress tolerance of barley (Hordeum vulgare L.). Compared with the individual treatments, the combined formulation produced the greatest improvements in plant performance, increasing seed germination from 60% in the untreated control to 80% under normal conditions and maintaining up to 90% germination under moderate heat stress (35-40 °C). The combined treatment also significantly enhanced vegetative growth and improved key physiological attributes, including chlorophyll index (12.83 to 28.17 SPAD units), total protein (0.536 to 0.725 mg g⁻1), proline (0.329 to 1.167 µg g⁻1 FW), nitrogen content (3.93% to 8.67%), soluble sugars (1.354 to 1.812 mg g⁻1), total phenolics (0.259 to 0.392 mg GAE g⁻1), and antioxidant capacity, with the DPPH IC₅₀ decreasing from 0.307 to 0.106 mg mL⁻1. Start codon targeted (SCoT) marker analysis generated 26 amplification products, of which 29.17% were polymorphic, indicating detectable molecular variation while preserving overall genomic stability. GC-MS profiling identified 19 phytochemical constituents, dominated by linoleic acid and its derivatives, which were supported by molecular docking analyses showing strong binding affinities toward microbial targets (SecY2, OmpF, CYP51) and the plant auxin receptor TIR1, providing mechanistic insights into both antimicrobial and growth-promoting activities. The ethanolic extract exhibited potent antimicrobial activity, producing inhibition zones of up to 22.5 mm against Candida albicans, 20.0 mm against Escherichia coli, and 18.5 mm against Staphylococcus aureus, with additional support from the chitosan-alginate matrix. Collectively, these findings demonstrate that integrating biodegradable biopolymers with pomegranate leaf phytochemicals provides a multifunctional, eco-friendly bio-stimulant capable of enhancing barley growth, physiological performance, antimicrobial activity, and tolerance to heat stress, highlighting its potential for sustainable climate-resilient agriculture.