3.3.28
DIFFERENTIATION BETWEEN TILLETIA INDICA, THE CAUSAL ORGANISM OF KARNAL BUNT OF WHEAT, AND A RELATED RYEGRASS SMUT USING PCR

RD FREDERICK, PW TOOLEY, Y BERTHIER-SCHAAD, GL PETERSON, MR BONDE and NW SCHAAD

USDA-ARS-NAA, Foreign Disease-Weed Science Research Unit, 1301 Ditto Ave., Fort Detrick, Frederick, MD 21702, USA

Background and objectives
Karnal bunt of wheat, caused by Tilletia indica Mitra, is an important disease of international trade. Grain and grain products from countries with the disease are subject to quarantine by countries free of the disease. A rapid and sensitive assay based upon PCR has been developed in our laboratory for detection of the pathogen [1,2]. In 1996, these primers were used in PCR tests to confirm the presence of T. indica in the southwestern United States. Strict quarantine measures were placed into effect by the Animal and Plant Health Inspection Service of the United States Department of Agriculture to limit the spread of the pathogen from the infested areas of Arizona and California bordering Mexico. In 1997, teliospores that morphologically resembled T. indica and tested positive for Karnal bunt by PCR were isolated from bunted ryegrass seed from Oregon and from wheat samples containing ryegrass seed from the southeastern United States. Upon further inspection, bunted ryegrass seed also was discovered in the wheat samples from the southeastern United States. The goal of this study was to develop a diagnostic test using improved PCR primers that would differentiate between T. indica and the smut isolated from ryegrass seed.

Results and conclusions
The nucleotide sequence of a 2.3 kb region of mitochondrial DNA (mtDNA), previously amplified by PCR only from T. indica[1], was determined for three isolates each of T. indica and the ryegrass smut pathogen. Among the T. indica isolates, or the ryegrass isolates obtained from bunted ryegrass seeds, there was greater than 99% identity within the mtDNA region. However, a comparison of the T. indica isolates with the ryegrass isolates revealed approximately 3% divergence. Oligonucleotide primers were designed for PCR that would distinguish between T. indica and the ryegrass smut pathogen based upon nucleotide differences within the 2.3 kb mtDNA region. Five sets of PCR primers were made for T. indica, and three sets were designed for the smut from ryegrass. PCR tests were conducted on total DNA extracted from T. indica isolates from India, Pakistan, and Mexico, and from ryegrass isolates from Oregon and the southeastern United States. PCR products of the expected sizes were amplified from all T. indica isolates examined with each of the five T. indica-specific primer combinations, whereas no PCR products were detected with the ryegrass smut isolates. Similarly, PCR products were found from all of the ryegrass isolates and none of the T. indica isolates using the ryegrass smut-specific PCR primer sets. We conclude that T. indica and the ryegrass smut recently isolated in the United States are two genetically distinct populations of organisms that can be distinguished using PCR.

References
1. Ferreira MASV, Tooley PW, Hatziloukas E, Castro C, and Schaad NW. 1996. Applied and Environmental Microbiology 62, 87-93.
2. Smith OP, Peterson, GL, Beck, RJ, Schaad, NW, and Bonde MR. 1996. Phytopathology 86, 115-22.