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Development of a PCR-Based Method for Identification of Tilletia indica, Causal Agent of Karnal Bunt of Wheat. Oney P. Smith, Department of Biology, Hood College, 401 Rosemont Avenue, Frederick, MD 21701; Gary L. Peterson(2), Raymond J. Beck(3), Norman W. Schaad(4), and Morris R. Bonde(5). (2)(3)(4)(5)USDA-ARS, Foreign Disease-Weed Science Research, Fort Detrick, Frederick, MD 21702. Phytopathology 86:115-122. Accepted for publication 9 October 1995. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1996. DOI: 10.1094/Phyto-86-115.

The polymerase chain reaction (PCR) was used to identify Tilletia indica, the causal agent of Karnal bunt of wheat. The method used two sets of oligonucleotide primers developed by sequence analysis of cloned DraI fragments of mitochondrial DNA of T. indica. The primer pair TI17M1 (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 TI57M2 (5'-AGCAAAGACAAAGTAGGCTTCC-3'), derived from clone pTI-MD57, produced a product of 118 bp which was unique to T. indica. Specificity of the primers was evaluated using 78 isolates of T. indica and 79 isolates of five other Tilletia spp., including 69 isolates of T. barclayana, from geographically diverse locations. The specificity of amplification products for T. indica was confirmed by Southern-blot hybridization using pTI-MD17 or pTI-MD57 as 32P-labeled probes. The method also employed a control PCR assay that used primers to conserved binding sites that amplified an internal transcribed spacer (ITS) region of ribosomal DNA reported in the literature for several groups of fungi. All Tilletia spp. produced a 420-bp product using the primers ITS3 and ITS4 in the control assay. These results demonstrated that the negative PCR results obtained with T. barclayana and other Tilletia spp. using T. indica-specific primers were not associated with mycelial DNA degradation or the presence of PCR inhibitors. Using teliospores germinated from a seed wash extraction method of infested grain, we demonstrated that T. indica can be reliably detected at an infestation level of five teliospores per 50-g grain sample.