| Summary: | <i>Streptomyces alfalfae</i> XN-04 has been reported for the production of antifungal metabolites effectively to control Fusarium wilt of cotton, caused by <i>Fusarium oxysporum</i> f. sp. <i>vasinfectum</i> (<i>Fov</i>). In this study, we used integrated statistical experimental design methods to investigate the optimized liquid fermentation medium components of XN-04, which can significantly increase the antifungal activity and biomass of XN-04. Seven variables, including soluble starch, KNO<sub>3</sub>, soybean cake powder, K<sub>2</sub>HPO<sub>4</sub>, MgSO<sub>4</sub>·7H<sub>2</sub>O, CaCO<sub>3</sub> and FeSO<sub>4</sub>·7H<sub>2</sub>O, were identified as the best ingredients based on one-factor-at-a-time (OFAT) method. The results of Plackett–Burman Design (PBD) showed that soluble starch, soybean cake powder and K<sub>2</sub>HPO<sub>4</sub> were the most significant variables among the seven variables. The steepest climbing experiment and response surface methodology (RSM) were performed to determine the interactions among these three variables and fine-tune the concentrations. The optimal compositions of medium were as follows: soluble starch (26.26 g/L), KNO<sub>3</sub> (1.00 g/L), soybean cake powder (23.54 g/L), K<sub>2</sub>HPO<sub>4</sub> (0.27 g/L), MgSO<sub>4</sub>·7H<sub>2</sub>O (0.50 g/L), CaCO<sub>3</sub> (1.00 g/L) and FeSO<sub>4</sub>·7H<sub>2</sub>O (0.10 g/L). A verification experiment was then carried out under the optimized conditions, and the results revealed the mycelial dry weight of <i>S. alfalfae</i> XN-04 reaching 6.61 g/L. Compared with the initial medium, a 7.47-fold increase in the biomass was achieved using the optimized medium. Moreover, the active ingredient was purified from the methanol extract of <i>S. alfalfae</i> XN-04 mycelium and then identified as roflamycoin (a polyene macrolide antibiotic). The results may provide new insights into the development of <i>S. alfalfae</i> XN-04 fermentation process and the control of the Fusarium wilt of cotton and other plant diseases.
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