Trehalose tetraester-producing Rhodococcus erythropolis KB1 enhances alfalfa-assisted rhizosphere remediation of phenanthrene-contaminated soil through plant metabolic reprogramming and microbiome assembly.

Biosurfactant-assisted rhizosphere remediation is a promising strategy for polycyclic aromatic hydrocarbon (PAH)-contaminated soils; however, the mechanisms by which biosurfactants regulate host plant responses to PAH stress and drive rhizosphere microbiome assembly remain unclear. Here, we constructed an alfalfa-assisted rhizosphere remediation system using novel trehalose tetraesters (TTEs) and TTE-producing Rhodococcus erythropolis KB1 to remediate phenanthrene-contaminated soil and decoded mechanisms via multi-omics. The colonization of KB1 and TTE-mediated rhizosphere regulation enhanced plant-microbe cooperation under phenanthrene stress. The system achieved a 98.1% phenanthrene removal rate, buffered soil pH, upregulated key enzymes (>1.3-fold), and suppressed stress signaling (e.g., salicylic acid), restoring alfalfa photosynthetic capacity to 80%. Mechanistically, TTEs and KB1 induced root metabolic reprogramming involving glycerophospholipid metabolism and flavonoid biosynthesis. This promoted the release of root exudates, including sesamin and cafestol. The exudate shift was associated with the selective assembly of a functional rhizosphere microbiome enriched in potential phenanthrene-degrading taxa such as Sporacetigenium and Rhodococcus. These results support a cascade regulatory pathway of "root metabolic reprogramming → targeted root exudation → functional microbiome assembly", revealing that TTE-producing KB1 enhances alfalfa-assisted rhizosphere remediation by helping plants actively reshape rhizosphere homeostasis. This study provides a mechanistic basis for precision bioremediation of PAH-contaminated soils.