Identification and differentiation of Tilletia indica and T. walkeri usingthe polymerase chain reaction

Karnal bunt of wheat, caused by Tilletia indica, was found in regions of th e southwestern United States in 1996. Yield losses due to Karnal bunt are s light, 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 morpholo gically resembling T. indica were isolated from bunted ryegrass seeds and w heat seed washes. Previously developed PCR assays faired differentiate T. i ndica from the recently discovered ryegrass pathogen, T. walkeri. The nucle otide sequence of a 2.3 kb region of mitochondrial DNA, previously amplifie d by PCR only from T. indica, was determined for three isolates of T. indic a and three isolates of T. walkeri. There was greater than 99% identity wit hin either the T. indica group or the T. walkeri group of isolates, whereas there was approximate to 3% divergence between isolates of these two Tille tia 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 ampl icon was developed as a target sequence in a fluorogenic 5' nuclease PCR as say using the TaqMan system for the detection and discrimination of T. indi ca and T. walkeri.