ABSTRACT Peronospora tabacina is an obligate plant pathogen that causes downy mildew disease on several species of Nicotiana, including N. tabacum (tobacco). The primary objective of this study was to use gnotobiotic associations to describe interactions between the pathogen and roots of either N. tabacum (cv. KY14) or N. repanda. We found that the pathogen was capable of moving systemically from shoots to roots of both host species and emerged from the root tissues as hyphae. We also demonstrated that root-associated hyphae were infectious on roots of nearby plants and resulted in new systemic infections. Following overnight darkness, sporulation of the pathogen was observed on infected roots exposed to air on both host species. We also found that within 2 months in culture, structures resembling resting stages of Peronospora tabacina were produced on hyphae emerging from roots of N. repanda but not N. tabacum. These findings appear relevant to both the epidemiology of the disease and to future studies of this and other downy mildew pathosystems.