Use of DAPI for Anastomosis Group Typing of Strains of the Fungus Rhizoctonia solani

Strains of Rhizoctonia solani, a common soil-borne, pathogenic fungus of plants, are assigned to one of 11 anastomosis groups (AGs) based on the occurrence of imperfect fusions (anastomoses) between hyphae of a non-typed strain and a tester strain of one of the 11 AG's. Imperfect fusion is characterized by the death of one or more cells in each of the hyphae involved in the fusion. Although hyphae from branches of the same strain of JR. solani may fuse with each other (self-fusion), cell death does not occur. Cell death is accompanied by nuclear degradation and granulation, or plasmolysis of the cytoplasm, which often is not visible using bright-field microscopy. When the DNA-binding fluorochrome DAPI (4', 6-diamidino-2-phenylindole) is used and the hyphal fusions viewed under fluorescence microscopy, no nuclei are observed in fused hyphal cells from two strains of the same AG of R. solani Because DAPI reacts only with living nuclei, lack of staining is presumptive evidence that the fused cells are dead as a result of imperfect fusion. The use of DAPI reduces the time required for making AG determinations compared to standard methods because it eliminates the need to assess cell wall dissolution and cytoplasmic fusion. Also, it is not necessary to trace the hyphae involved in the fusion to their respective origins to ensure that self-fusion has not occurred.