Abstract: Aspergillus terreus is effective in controlling plant-parasitic nematodes and fungi, making it a filamentous fungus with good biocontrol potentiality. In this study, A. terreus strain AT9 was used as the experimental material to investigate in vitro inhibitory activity against Fusarium oxysporum. The effects of types of osmotic pressure stabilizer, enzymatic hydrolysis system, mycelial age, enzymatic digestion temperature, time and rotational speed on preparation of strain AT9 protoplasts were systematically examined. Based on these results, an efficient genetic transformation system for strain AT9 would be established and verified. The results indicated that strain AT9 culture and its crude extract from fermentation exhibited significant inhibitory effects against F. oxysporum. Strain AT9 cultured in YES medium at 28 ℃ for 17 hours yielded a maximum of 2.49×10⁷ cfu/mL protoplasts, using 0.7 mol/L KCl + 0.27 mol/L CaCl₂ as the osmotic pressure stabilizer, with enzymatic hydrolysis by20 mg/mL Lysing Enzymes, 20 mg/mL snail enzyme, and 20 mg/mL cellulolytic enzyme at 26 ℃ and 160 r/min for 9 h. Using PEG-mediated transformation, the plasmid pch-sGFP was introduced into the AT9 strain. Fluorescence observation and PCR identification confirmed that the exogenous gene had been successfully integrated into the genome of the AT9 strain, and an efficient transformation system for A. terreus AT9 was successfully established, resulting in the acquisition of a green fluorescent protein（GFP）-labeled strain GFP-AT9. This study successfully established a genetic transformation system for A. terreus and obtained the GFP-AT9 strain, providing crucial technical support for analyzing the biocontrol function and conducting genetic improvement.

Key words: Aspergillus terreus, biological control, protoplast preparation, genetic transformation, GFP