Identification and Differentiation of Tilletia indica and T. walkeri Using the Polymerase Chain Reaction

ABSTRACT Karnal bunt of wheat, caused by Tilletia indica, was found in regions of the southwestern United States in 1996. Yield losses due to Karnal bunt are slight, and the greatest threat of Karnal bunt to the U.S. wheat industry is the loss of its export market. Many countries either prohibit or restrict wheat imports from countries with Karnal bunt. In 1997, teliospores morphologically resembling T. indica were isolated from bunted ryegrass seeds and wheat seed washes. Previously developed PCR assays failed to differentiate T. indica from the recently discovered ryegrass pathogen, T. walkeri. The nucleotide sequence of a 2.3 kb region of mitochondrial DNA, previously amplified by PCR only from T. indica, was determined for three isolates of T. indica and three isolates of T. walkeri. There was greater than 99% identity within either the T. indica group or the T. walkeri group of isolates, whereas there was =3% divergence between isolates of these two Tilletia species. Five sets of PCR primers were made specific to T. indica, and three sets were designed specifically for T. walkeri based upon nucleotide differences within the mitochondrial DNA region. In addition, a 212 bp amplicon was developed as a target sequence in a fluorogenic 5' nuclease PCR assay using the TaqMan system for the detection and discrimination of T. indica and T. walkeri.