Development and Application of E6, a Bacillus subtilis Metabolite–Based Biostimulant, for Priming Cold Tolerance in Apple Through MAPK Signalling and Phenylpropanoid Metabolism

Late-spring cold stress disrupts vegetative physiology in apple (Malus domestica), a major horticultural crop in continental regions such as Xinjiang, China. Current cold-protection strategies, including physical heating and chemical sprays, are costly, labor-intensive, and environmentally unfriendly, while microbial metabolite-based elicitors have emerged as sustainable alternatives but remain underexplored in apple cold tolerance. This study aimed to develop and characterize a novel Bacillus subtilis BS-Z15 metabolite extract (B-MES)-based elicitor (E6) for priming cold tolerance in apple leaves, and to elucidate its molecular mechanisms through integrated transcriptome and metabolome analysis. E6 was optimized using an L18(37) orthogonal array design (18 experimental runs, 7 factors at 3 levels each) and validated across three biological scales (detached branches, potted seedlings, adult-tree-derived branches) in two apple cultivars. E6 pretreatment significantly increased SOD and POD activities and proline content while reducing MDA content and relative electrolyte leakage and preserving chlorophyll under 0 °C stress. Integrated transcriptome and metabolome analysis revealed concurrent activation of PTI–like signaling, phenylpropanoid metabolism, glutathione metabolism, and MAPK signaling pathways. A rosmarinic acid-laccase module showed sustained positive correlation during priming and recovery, which is indicative of a sustained priming response. Cross-cultivar validation in ‘Wanglin No. 1’ confirmed the generality of the protective mechanism. This study provides a novel candidate biostimulant and mechanistic insights into elicitor-induced cold priming in apple leaves; further validation at the reproductive tissue and orchard scale is warranted.