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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Publications at this Location » Publication #60065
Title: DEVELOPMENT OF A PCR-BASED METHOD FOR IDENTIFICATION OF TILLETIA INDICA, CAUSAL AGENT OF KARNAL BUNT OF WHEAT

Author
item Smith, Oney
item Peterson, Gary
item BECK, R - FORMER ARS EMPLOYEE
item Schaad, Norman
item Bonde, Morris

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/9/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Karnal bunt of wheat, a fungal disease of quarantine significance, is not present in the United States. However, the disease is present in Northwestern Mexico and poses a significant threat to U.S. agriculture. Accurate identification of the pathogen, Tilletia indica, requires extensive, time-consuming pathogenicity tests. We describe a simple molecular method utilizing the polymerase chain reaction (PCR) for accurate identification of the pathogen. Suspected spores of the pathogen are germinated and the resulting growth extracted to obtain DNA for testing. The entire procedure, including germination of the spores and molecular test, requires 6-14 days, considerably less time than the 2-3 months to conduct pathogenicity tests. We presently are investigating the use of the method to detect pathogen spores in seed samples

Technical Abstract: Polymerase chain reaction (PCR) technology was used to identify Tilletia indica, the causal agent of Karnal bunt of wheat. The technique uses total DNA extracted from fungal mycelia and two sets of oligonucleotide primers that were developed by sequence analysis of cloned Dra I fragments of mitochondrial DNA of T. indica. Primer specificity was evaluated using 78 isolates of T. indica and 79 isolates of five other Tilletia species, including 69 isolates of T. barclayana, from geograph- ically diverse locations. The primer pair TI17M1 (5'-TCCCCTTGGATCAGAACGTA-3') and TI17M2 (5'-AGAAGTCTAACTCCCCCCTCT-3'), derived from sequence analysis of clone pTI-MD17, amplified a single 825-bp product from all isolates of T. indica and no products for other Tilletia species. In addition, the primer pair TI57M1 (5'-TTTTCCCTCTCTCCTTTTTTCA-3') and TI57M2 (5'-AGCAAAGACAAAGTAG- GCTTCC-3'), derived clone pTI-MD57, produced a 118-bp product unique to T. indica. The specificity of products was confirmed by Southern-blot hybridization using pTI-MD17 or pTI-MD57 as probes. The method also employs a control PCR assay using primers that amplify an internal transcribed spacer (ITS) region (420 bp) of ribosomal DNA common to all Tilletia species tested. This control assay demonstrates that the negative PCR results obtained using T. indica-derived primers are not associated with mycelial DNA degradation or the presence of PCR inhibitors