A Staining Technique for Nuclei ofRhizoctonia Solaniand Related Fungi

The species concept of Rhizoctonia solani Kuhn, as reviewed by Parmeter and Whitney (1), stipulates a multinucleate condition in young vegetative hyphae. Fungi resembling R. solani but possessing predominantly binucleate cells may be characterized readily as distinct species if the nuclear condition is ascertained (2). Nuclear-staining techniques previously available for R. solani and related fungi involve complex procedures and preparation times ranging from 30 min to more than 1 h (4, 6). Such methods may be prohibitive if a number of isolates are to be stained. Tu and Kimbrough (5) devised a rapid-staining technique using an acidified solution of aniline blue in 50% glycerine. We have found that this technique is satisfactory for some isolates but results in poor resolution with others. Therefore, the following rapid-nuclear-staining technique was developed. Fifty-four isolates of R. solani and 98 isolates of binucleate R. solanilike fungi, including Ceratobasidium cornigerum Rogers, C. anceps (Bres. & H. Syd.) Jacks., and C. ramicola Tu & Kimbr., were grown on 15 ml of Difco potato-dextrose agar (PDA) in 9-cm plastic Petri dishes for 4 d at 21 C. Seven-mm mycelial plugs were cut from the edges of the fungal colonies and transferred to the center of sterile Petri dishes containing 15 ml of 1.5% water agar (ICN Pharmaceuticals, Cleveland, Ohio). The fungi were incubated at 21 C until colonies were ca 1 cm from the edge of the dishes. A small drop of 0.5% aniline blue or trypan blue in lactophenol, prepared according to the methods of Phillips and Hayman (3), was placed directly on the mycelium midway between the center and the periphery of the fungal colony (FIG. 1). The stained area was affixed with a 18 x 18 mm cover slip. Hyphal cells were examined microscopically at 400x either by placing Petri dishes directly on the micro1281