Charcoal rot of soybean, caused by the fungus Macrophomina phaseolina, is a growing issue worldwide and also causes significant yield loss in soybean production regions of the U.S. In a soil survey conducted in 143 soybean fields throughout 51 counties in Ohio, M. phaseolina was detected in more than 95% of the samples. The prevalence and distribution of this pathogen is alarming. The overall aims of this project are to reveal the strategies that M. phaseolina uses to infect soybean, and to show how these strategies are distributed across populations sampled from Ohio soybean fields. While other major soybean pathogens have received significant research attention to understand the genetic and molecular mechanisms of pathogenicity and virulence, these types of studies are lacking in M. phaseolina. This knowledge will reveal potential avenues for intervention to improve genetic resistance to M. phaseolina and applying advanced genetic editing strategies in soybean.