Activation of the phenylpropanoid pathway and enhanced powdery mildew resistance in honeysuckle induced by inactivated Erysiphe lonicerae spores

Honeysuckle (Lonicera japonica) is a widely utilized traditional medicinal plant that is highly susceptible to powdery mildew. This study explores whether artificially inactivated spores of Erysiphe lonicerae, the causal agent of honeysuckle powdery mildew, can function as a biostimulant to elicit the plant's immune response and promote the accumulation of secondary metabolites essential for its medicinal quality. To evaluate the effects and underlying mechanisms, we conducted secondary metabolite profiling, gene expression analysis (qPCR), powdery mildew resistance assays, and high-throughput proteomic and metabolomic analyses. Following 60 days of treatment with inactivated E. lonicerae spores applied at 3-day intervals, proteomic analysis revealed significant upregulation of nine key enzymes involved in the phenylpropanoid pathway, along with the upstream regulatory protein calmodulin. These proteomic changes were corroborated at the transcript level by qPCR. Metabolomic analysis further demonstrated a pronounced accumulation of phenylpropanoid pathway metabolites, increased activity of phenylalanine ammonia-lyase (PAL), and elevated levels of downstream flavonoids, collectively indicating robust activation of this pathway. Additionally, treated plants exhibited significant alterations in oxidative stress markers, specifically hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), along with sustained increases in the activities of four major antioxidant enzymes, resulting in a marked enhancement of total antioxidant capacity. Notably, pretreatment with inactivated spores at least 20 days prior to pathogen inoculation significantly improved resistance to powdery mildew, with earlier pretreatment providing stronger protection. These findings demonstrate that artificially inactivated E. lonicerae spores can enhance honeysuckle's resistance to powdery mildew while activating the phenylpropanoid biosynthetic pathway, thereby promoting the accumulation of medicinally valuable secondary metabolites.