Abstract: Large-scale cultivation of microalgae is a core issue of microalgal biodiesel production, with contamination by biological pollutants difficult to avoid in outdoor cultivation. Graesiella sp. WBG-1 is an oleaginous microalga suitable for large-scale cultivation in outdoor open ponds. However, infection by the pathogenic fungus Amoeboaphelidium sp. can seriously affect growth and lipid accumulation, even leading to the entire collapse of the Graesiella sp. WBG-1 culture. In this study, MTT staining was used to stain sporangia of the pathogenic fungus, with the infected algal cells then detected and counted under microscopy, and the infection rate (ratio of infected cells to total cells) calculated to evaluate the infection ability of the fungus under different culture conditions of temperature, light intensity, pH and air flux (turbulence of algal liquid). The different culture conditions were set to investigate the effects of environmental factors on the infectivity of the pathogenic fungus. The results demonstrated that MTT staining was easy and suitable for the detection of pathogenic fungal infection in the mass culture of Graesiella sp. WBG-1. Temperature, light intensity, pH and air flux all had significant effects on the infectivity of the pathogenic fungus. High temperature, high light intensity, slight acid environments, and algal suspension without stirring had adverse effects on the infection of Graesiella sp. WBG-1. Successive transfer culture of the fungus infecting Graesiella sp. WBG-1 was successfully established at a temperature of 30℃, light intensity of 140 μmol·m^-2·s^-1, air flux of 1.0 L·min^-1 and pH of 9.0±0.5. After 3-4 d of transfer culture, the infection rate developed from 0 to above 90%, with almost all algal cells infected. The successive transfer culture of algae coupled with artificial infection by the pathogenic fungus simulated the infection procedure that occurs in the mass culture of Graesiella sp.WBG-1 in the field. Therefore, these results provide a good platform for studies on the infection behaviors of pathogenic fungi and the mechanism of infection.