AimsThe aims of this study were to investigate the biocontrol effects of Penicillium griseofulvum strain CF3 and its mechanisms against soil-borne root pathogens (Fusarium oxysporum and Sclerotium rolfsii) of the medical plant Aconitum carmichaelii Debx. Methods and ResultsThe effects of P. griseofulvum strain CF3 were evaluated with regard to the hyphal growth of S. rolfsii and F. oxysporum, the sclerotial formation and germination of S. rolfsii and its expression of sclerotia-formation-related genes. A field experiment was conducted to explore how strain CF3 controls the severity of soil-borne diseases, promotes the growth of A. carmichaelii plants and mediates shifts in the culturable rhizosphere microbial populations. The results showed that treatment with a cell-free culture filtrate of strain CF3 considerably inhibited the hyphal growth of both S. rolfsii and F. oxysporum, in addition to limiting the sclerotial formation and germination of S. rolfsii. Three genes related to sclerotial formation (ArsclR, ArnsdD1 and ArnsdD2) were predicted in S. rolfsii and their expression was found suppressed by the CF3 treatment. Field application of the CF3 biocontrol agent in a powder form (1.9 x 10(10) conidia per gram of substrate) reduced soil-borne disease severity by 15.0%. The shoot and root growth of A. carmichaelii plants was promoted by 61.6 and 83.1% respectively, as the biocontrol strain massively colonized the rhizosphere soil. The CF3 treatment also markedly reduced the density of some known species harmful to plants while increasing the density of some beneficial species in the rhizosphere soil. Significance and Impact of the StudyGenes related to sclerotia formation of S. rolfsii are predicted for the first time and their expression patterns in the presence of P. griseofulvum strain CF3 are evaluated. This comprehensive study provides a candidate fungal biocontrol strain and reveals its potential mechanisms against S. rolfsii and F. oxysporum in A. carmichaelii plants.