A nonpathogenic mutant of Colletotrichum acutatum exhibited extended germ tube growth prior to appressoria formation on solid surface and strawberry leaves in the absence of an external nitrogen source, but grew necrotrophically when inoculated directly onto wounded sites. The deduced amino acid sequence of the impaired gene product was characterized as a transcriptional regulator of nitrogen metabolism. Inoculation of leaves with wildtype or the mutant in the presence of a preferred nitrogen source resulted in massive epiphytic hyphal production, appressoria formation and rapid symptom development. Proteins patterns of different developmental stages and growth conditions revealed variation in abundance of proteins belonging to the ROS (Reactive oxygen species) scavenger machinery. CuZn superoxide dismutase and glutathione peroxidase were upregulated at the appressoria formation stage and nitrogen limiting conditions, compared to growth in complete nutrient supply, whereas abundance of bifunctional catalase was up-regulated predominantly at the appressoria formation stage. Fungal ROS was detected within germinating conidia during strawberry host prepenetration, penetration and colonization stages. Application of an exogenous antioxidant quenched ROS production and reduced appressoria formation frequency. This study indicates that developmental processes occurring under nutritional deprivation in C. acutatum infecting strawberry are facilitated by metabolic shifts which may be mediated by fungal ROS production.