Dual beneficial effects of Clonostachys rosea GU-9, a natural riboflavin overproducer, on Glycyrrhiza uralensis

Amidst increasing emphasis on environmental sustainability in agriculture, the eco-friendly biocontrol fungus Clonostachys rosea is gaining prominence. In this study, we report for the first time that the endophytic fungus C. rosea strain GU-9, isolated from Glycyrrhiza uralensis , can naturally overproduce riboflavin. We assessed the riboflavin production capacity of GU-9, elucidated its biosynthetic pathway, and investigated its effects on the host plant. Both GU-9 and another isolate, GU-D4, were morphologically and molecularly identified as C. rosea . Notably, GU-9 secreted copious amounts of a yellow pigment during cultivation, distinguishing it from GU-D4 and other documented C. rosea strains. UPLC-MS/MS analysis revealed that this pigment was riboflavin. After optimizing fermentation conditions, GU-9 achieved a riboflavin yield of 249.97 mg/L. Whole genome sequencing identified the putative complete riboflavin biosynthesis pathway in GU-9 and ten putative key enzyme-encoding genes. The expression of key genes ( RIB3 , RIB4 , and RIB5 ) was significantly upregulated during the late cultivation stage, coinciding with a substantial increase in riboflavin yield. In plant-microbe interaction assays, GU-9 exhibited superior performance to GU-D4, significantly promoting the growth of suspension cells and seedlings and significantly increasing the accumulation of bioactive compounds in the host plant. Following Ashbya gossypii , GU-9 represents a newly discovered naturally occurring riboflavin overproducer among filamentous fungi. Unlike traditional biocontrol agents, GU-9 confers potent growth-promoting effects and enhances the accumulation of valuable bioactive compounds in plants, demonstrating its potential as a novel multifunctional biocontrol agent. • Clonostachys rosea GU-9 is confirmed as a natural riboflavin overproducer. • The riboflavin yield of GU-9 is 249.97 mg/L. • The complete riboflavin biosynthetic pathway was identified in GU-9’s genome. • Expression levels of RIB3 , RIB4 and RIB5 correlated highly with riboflavin yield. • GU-9 better promoted plant growth and bioactive component accumulation than GU-D4.