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Internal Transcribed Spacer Sequence-Based Phylogeny and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Differentiation of Tilletia walkeri and T. indica

First and fourth authors: USDA-APHIS PPQ National Plant Germplasm Quarantine Center, Beltsville, MD 20705; second author: USDA-ARS Systematic Botany and Mycology Laboratory, Beltsville, MD 20705; and third and fifth authors: Department of Plant Pathology, Washington State University, Pullman 99164

A polymerase chain reaction-restriction fragment length polymorphism assay to distinguish Tilleita walkeri, a rye grass bunt fungus that occurs in the southeastern United States and Oregon, from T. indica, the Karnal bunt fungus, is described. The internal transcribed spacer (ITS) region of the ribosomal DNA repeat unit was amplified and sequenced for isolates of T. indica, T. walkeri, T. horrida, and a number of other taxa in the genus Tilletia. A unique restriction digest site in the ITS1 region of T. walkeri was identified that distinguishes it from the other taxa in the genus. Phylogenetic analysis of the taxa based on ITS sequence data revealed a close relationship between T. indica and T. walkeri, but more distant relationships between these two species and other morphologically similar taxa.