NOTE TO THE EDITOR Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp

The form genus Rhizoctonia D.C. is regarded as a heterogeneous assemblage of filamentous fungal taxa that do not produce asexual spores and share a number of common features in their anamorphic states (Garcia et al. 2006). It includes several of the most devastating crop pathogens such as Rhizoctonia solani Kühn [teleomorph = Thanatephorus cucumeris (A.B. Frank) Donk]. In the absence of a perfect state, the number of nuclei in the vegetative hyphal cells is one of the anamorphic features that separate the former from other Rhizoctonia-like fungi. Anamorphs of R. solani are typically multinucleate, whilst Rhizoctonia spp., with a Ceratobasidium perfect state, are binucleate or more rarely uninucleate (Garcia et al. 2006). However, even within the same isolate, the nuclei number can vary from one cell to another in the same hypha (Sanford and Skoropad 1955; Flentje et al. 1970). Nowadays, the determination of the nuclear condition in Rhizoctonia is a straightforward procedure: applying a nuclear stain that fluoresces upon excitation by UV light, such as DAPI (4¢,6¢-diamidino-2-phenylindole) and Hoechst Dye 33258, both specific to AT-rich regions, which is visualized with epifluorescence microscopy (Hua’an et al. 1984; Kangatharalingam and Ferguson 1991). Accurate determination of hyphal walls and of the septa from a flat thin layer of disentangled mycelium is also necessary, but can be tedious when dealing with a large number of isolates. A solution is to add a fluorophore that binds the hyphal walls. The two most commonly used are Calcofluor White M2R or Solophenyl Flavine 7GFE 500 (Hoch et al. 2005). Calcofluor, in