The United States of America as Represented by the Secretary of

United States of America, as Represented by the Secretary of Agriculture

Morris R Bonde

Norman W Schaad

Paul W Tooley

Gary L Peterson

Efstathios Hatziloukas

Reid D Frederick

Raymond J Beck

Oney P Smith

David A Knorr

Marisa A Ferreira

Karnal bunt of wheat is caused by,Mitra. Recently, teliospores morphologically resembling,were isolated from bunted ryegrass seeds and wheat seed washes. Previously developed PCR assays failed to differentiate,from the newly discovered ryegrass pathogen,,. The nucleotide sequence of a 2.3-kb region of mitochondrial DNA, previously amplified by PCR only from,, was determined for three isolates of,and for three isolates of,. There was greater than 99% identity within either the,group or the,group of isolates, whereas there was approximately 3% divergence between isolates of these two Tilletia species. Five sets of PCR primers were made specific to,, and three sets were designed specifically for,based upon nucleotide differences within the mtDNA region. In addition, a 212 bp amplicon was developed as a target sequence in a fluorogenic 5&prime; nuclease PCR assay using the TaqMan system for the detection and discrimination of,and

Method for differentiating between the casual agents of karnal bunt wheat fungus and ryegrass smut using PCR

Mitochondrial DNA of five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3 kb- EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, T. barclayana. The clone was partially sequenced, primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3 kb fragment from total DNA of 17 T. indica isolates from India, Pakistan and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, T. foetida, T. caries, T. fusca and T. controversa) did not produce any bands as detected by ethidium bromide-stained agarose gels and Southern hybridizations. Sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds.

Species-specific mitochondrial sequences for identification of Tilletia

The polymerase chain reaction (PCR) was used to identify Tilletia indica, the causal agent of Karnal bunt of wheat. The method uses two sets of oligonucleotide primers developed by sequence analysis of cloned Dra I 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 species. In addition, the primer pair TI57M1 (5'-TTTTCCCTCTCTC-CTTTTTTCA-3') and TI57M2 (5'-AGCAAAGACAAAGTAGGCTTCC-3'), derived from clone pTI-MD57, produced a product of 118 bp which was unique to T. indica.

Development of a PCR-based method for identification of Tilletia indica,

Growing community of inventors

Middletown, MD, United States of America

Morris R Bonde