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Spore germination and appressorium formation are important steps in the process of fungal development and pathogenesis. These prepenetration events, which begin with spore attachment and culminate with appressorium maturation, a common scheme for many pathogenic fungi, are prerequisites for penetration of host external barriers and subsequent colonization. Conditions for in vitro spore germination and appressorium development in Colletotrichum trifolii are described. In addition, effects of Ca(sup2+) and calmodulin on these processes have been examined. Results indicate that, as for other pathogenic fungi, appressorium development is induced on a hard surface. The data suggest that disturbance of calcium homeostasis, by ethylene-bis(oxy-ethylenenitrolo)tetraacetic acid (EGTA) or calcium channel blockers, impairs appressorium development. Moreover, calmodulin inhibitors affect both germination and differentiation, implying that the Ca(sup2+)/calmodulin signal transduction pathway is important in the early development of C. trifolii on the plant host surface.