Microbial exopolysaccharide EPS66A inducing walnut (Juglans regia) resistance to bacterial blight

•A comprehensive analysis of walnuts leaves flavonoid biosynthesis and metabolism, which 11 differentially expressed metabolites and 14 differentially expressed genes were identified and annotated to the flavonoid biosynthesis pathway.•Streptomyces-derived polysaccharide EPS66A improves walnut disease resistance to bacterial blight by regulating the biosynthesis of walnut flavonoids and phenylpropane.•This study provides insight into the flavonoid biosynthesis mechanism of EPS66A-induced resistance in walnuts and contributes to our understanding of the plant immune response in the EPS66A-mediated defense response. Bacterial blight caused by Xanthomonas arboricola pv. juglandis is a major obstacle to walnut production. EPS66A, derived from Streptomyces sp. strain HL-66, has various beneficial properties, including broad-spectrum microbe inhibition and plant disease resistance induction. To understand the effects of Xaj and EPS66A on walnut, a comprehensive analysis of the metabolome and transcriptome was conducted. While EPS66A did not directly inhibit Xaj on agar media, applying it at 200 μg/mL, 24 h after Xaj inoculation on walnut leaves, significantly reduced bacterial blight in a greenhouse. Additionally, EPS66A increased phenolic and flavonoid concentrations and enhanced enzymatic activities associated with resistance, such as catalase, superoxide dismutase, peroxidase, and phenylalanine ammonia lyase. Differential expression of eleven metabolites and fourteen genes related to flavonoid biosynthesis pathway was observed. Consequently, EPS66A application induced systemic resistance in walnuts, effectively preventing Xaj infection. This study provides insights into the flavonoid biosynthesis mechanism underlying EPS66A-induced resistance in walnuts.