Abstract: 【Objective】 The effects of two kinds of alfalfa leaf spot pathogens Phoma medicaginis and Stemphylium vesicarium on the mycelial growth biological characteristics of the two strains. 【Method】 Leaf spot fungus P. medicaginis (representing strain PM-1) on different medium, temperature, carbon source, and nitrogen source conditions, were cultured 7 days and 5 dayscolony and mycelium growth during culture. The diameter of colonies was measured by the cross method. Finally, the statistical analysis software SPSS 20.0 was used to analyze the differences between the treatments. 【Result】 PM-1 grow on 11 medium, 8 carbon sources and 12 nitrogen sources. The optimal medium GPA and PSA, the optimal carbon source maltose, and the optimal nitrogen source L-gluten Amine amide. There significant differences between different culture media, carbon sources, sources on the growth of colonies. The temperature that P. medicaginis grow 5℃ to 30℃, while the optimal temperature 25℃; the lethal temperature of mycelium 55℃. The complete illumination was conductive to mycelium growth. SV-1 grow on 11 media, 8 carbon sources, and 12 nitrogen sources. The optimal medium GPA, the optimal carbon source inulin, and the optimal nitrogen source L-gluten Amine amide. There significant differences between different culture media, carbon sources, nitrogen sources on the growth of colonies; The temperature that S. vesicarium grow 5℃ to 35℃, while the optimal temperature 25℃. The lethal temperature of mycelium 60℃; while significant of mycelium growth between different illumination treatments. 【Conclusion】 P. medicaginis (representing strain PM-1) optimal culture conditions were temperature 25℃, GPA or PSA medium, illumination, carbon and nitrogen sources were maltose and L-gluten Amine amidum. S. vesicarium (representing strain SV-1) optimal culture conditions were temperature 25℃, GPA medium, carbon and nitrogen were inulin and L-glutamine.