Co-cultivation of broccoli endophytic fungi with broccoli hairy roots enhances accumulation of the anticancer compound sulforaphane and its precursor glucoraphanin

Glucoraphanin (GRA), the primary aliphatic glucosinolate in broccoli, serves as the precursor to sulforaphane (SF), a compound widely recognized for its potent anticancer properties. Endophytic fungi, increasingly acknowledged as a valuable source of bioactive secondary metabolites, can modulate the biosynthesis and accumulation of plant-derived compounds. However, studies investigating the dynamic changes in host metabolite profiles during such fungal interactions remain scarce. Here, endophytic fungi isolated from broccoli were initially assessed for their GRA and SF content. These fungi were subsequently co-cultured with broccoli hairy roots to evaluate their effects on GRA and SF biosynthesis and accumulation. The dominant strain, EL-07 ( Penicillium citrinum ), significantly enhanced GRA and SF biosynthesis during the early co-culture phase (215.99% and 361.05%, respectively). Following successful colonization of hairy roots by P. citrinum , a CERK1-LYK5-mediated transition in the plant's immune/symbiotic status was associated with a marked decline in GRA and SF content. Furthermore, P. citrinum elicited oxidative stress and defense responses and upregulated the expression of genes involved in pathogen defense mechanisms, GRA and SF biosynthesis, and related transporters. Overall, co-culturing hairy roots with P. citrinum engages an “immunity-symbiosis” trade-off mechanism that effectively enhances both the intracellular accumulation and extracellular secretion of GRA and SF. • Endophytic fungi isolated from broccoli are capable of producing glucoraphanin and sulforaphane. • Co-culturing these fungi with broccoli hairy roots significantly enhances the accumulation of the target metabolites. • Stable fungal colonization is established after the peak production of secondary metabolites has occurred. • The fungal elicitor upregulates the expression of plant defense genes and ABC transporters. • This live fungal co-culture system offers an efficient and low-cost strategy for producing anti-cancer compounds.