Technical Abstract: In the absence of perfect state, the number of nuclei in their vegetative hyphae is one of the anamorphic features that separate Rhizoctonia solani from other Rhizoctonia-like fungi. Anamorphs of Rhizoctonia solani are typically multinucleate while the other Rhizoctonia species are binucleate. However, the nuclear number can vary among isolates or even within the same isolate, requiring an accurate estimation of the number of nuclei per hypha cells. During the course of a project to better characterize some Rhizoctonia strains, a new fluorescence-based method was developed to simplify the determination of the nuclear condition of these strains. This currently needs to be performed either using two 'uorescent dyes, which is more costly and time-consuming, or using only one 'uorescent dye, which is less accurate. Nuclei in all Rhizoctonia isolates are brightly stained by DAPI under UV excitation. The hyphal walls and septa, when exposed to green excitation, fluoresce intensely red, with no photobleaching. This property was exploited for the first time to determine the number of hyphal cells in the mycelium slide culture. By overlapping images obtained with DAPI fluorescence and with red fluorescence, the number of nuclei per hypha cells in the Rhizoctonia strains was determined more accurately than the conventional DAPI 'uorescence used alone. This bi-'uorescence imaging strategy provides a sensitive tool for determining the nuclear condition of Rhizoctonia strains. Its safety and simplicity is a key advantage when there are numerous cultures to be examined.