[Objective] To optimize the transformation system of a phosphate-solubilizing fungus Penicillium brocae and modify the strain with molecular tag for root colonization analysis; to evaluate the growth promotion effects of P. brocae. [Methods] We optimized ATMT (Agrobacterium tumefaciens-mediated transformation) parameters to get P. brocae transformants, we confirmed the transformants and its root colonization with molecular tags; we used TAIL-PCR (thermal asymmetric interlaced-PCR) to characterize T-DNA insertion site; we evaluated the growth promotion effects of P. brocae by Petunia hybrida pot experiments with treatments of T1 (10 mL water), T2 (10 g/L phosphate rock, 10 mL), T3 (10⁶ spores/mL, 10 mL) or T4 (10 g/L phosphate rock and 10⁶ spores/mL, 10 mL).[Results] We got 44 transformants per 10⁶ spores by parameters optimization and confirmed the transformants with gfp and hyg gene cloning, GFP fluorescence detecting and GUS staining. We observed the colonization of P. brocae on P. hybrida root surface by GUS staining. The insertion site flanking sequence of a transformant characterized with phosphate solubilization capacity significantly decreasing was obtained by TAIL-PCR. P. hybrida fresh weight of T4 were 35.4%, 35.6% and 21.4% higher than that of T1, T2 and T3, the dry weight of T4 were 25.1%, 37.8% and 26.0% higher than that of T1, T2 and T3, respectively. There was no significant difference among the fresh or dry weight of T1, T2 and T3 (P ≤ 0.05). [Conclusion] We optimized the ATMT system and observed the root colonization of P. brocae. The application of P. brocae spores and phosphate rock together but not alone, could promote the growth of P. hybrida and this gave a hint that P. brocae may enhance the fertilizer efficiency of phosphate rock in soil.