DEVELOPMENT OF A PCR-BASED METHOD FOR IDENTIFICATION OF TILLETIA-INDICA, CAUSAL AGENT OF KARNAL BUNT OF WHEAT

The polymerase chain reaction (PCR) was used to identify Tilletia indi ca, the causal agent of Karnal bunt of wheat. The method used two sets of oligonucleotide primers developed by sequence analysis of cloned D raI fragments of mitochondrial DNA of I: indica. The primer pair TI17M 1 (5'-TCCCCTTGGATCAGAACGTA-3') and TI17M2 (5'-AGAAGTCTAACTCCCCCCTCT-3' ), derived from clone pTI-MD17, amplified a single 825-bp product from all isolates of T. indica and no products for other Tilletia spp. In addition, the primer pair TI57M1 (5'-TTTTCCCTCTCTCCTTTTTTCA-3') and TI 57M2 (5'-AGCAAAGACAAAGTAGGCTTCC-3'), derived from clone pTI-MD57, prod uced a product of 118 bp which was unique to I indica. Specificity of the primers was evaluated using 78 isolates of T.: indica and 79 isola tes of five other Tilletia spp., including 69 isolates of T. barclayan a, from geographically diverse locations. The specificity of amplifica tion products for I: indica was confirmed by Southern-blot hybridizati on using pTI-MD17 or pTI-MD57 as P-32-labeled probes. The method also employed a control PCR assay that used primers to conserved binding si tes that amplified an internal transcribed spacer (ITS) region of ribo somal DNA reported in the literature for several groups of fungi. All Tilletia spp. produced a 420-bp product using the primers ITS3 and ITS 4 in the control assay. These results demonstrated that the negative P CR results obtained with T. barclayana and other Tilletia spp. using I : indica-specific primers were not associated with mycelial DNA degrad ation or the presence of PCR inhibitors. Using teliospores germinated from a seed wash extraction method of infested grain, we demonstrated that ?: indica can be reliably detected at an infestation level of fiv e teliospores per 50-g grain sample.