Clonostachys rosea is a promising biocontrol agent that parasitizes various fungal plant pathogens. In this paper, transaldolase gene Tal67 was found to be greatly upregulated in C. rosea isolate 67-1 during mycoparasitism of Sclerotinia sclerotiorum sclerotia. Quantitative real-time PCR revealed a significant increase in expression at 0-48 h after induction by sclerotia, and the level peaked at 13.9-fold higher than the control at 24 h. Gene disruption led to a decrease in the growth rate of the Tal67-deficient strain Delta Tal67 to 5.3 mm/day, which was much lower than the wild type and the complemented strain Delta Tal67+ (P < 0.05). The antagonistic activity of Delta Tal67 against Botrytis cinerea was 15.8% lower than the wild type, and the parasitic rate to S. sclerotiorum decreased by 24.6%. However, reinsertion of the transaldolase gene recovered the fungicidal activity of C rosea. The efficacy of the mutants against soybean Sclerotinia stem rot was evaluated in the greenhouse, and the control efficiency of isolate 67-1 reached 65.3%, while the efficiency of the Delta Tal67 strain decreased sharply to 17.8%, and the complemented strain Delta Tal67+ recovered to 64.8%. These results suggest that Tal67 plays an important role in the growth and biocontrol activity of C. rosea. (C) 2016 Elsevier Inc. All rights reserved.