The United States of America as Represented by the Secretary of

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

Paul W Tooley

Norman W Schaad

Efstathios Hatziloukas

Morris R Bonde

Reid D Frederick

Gary L Peterson

Marie Carras

David A Knorr

Britt Bunyard

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

Phythophthora species which infect potatoes may result in the devastating disease potato late blight or in pink rot. Primers specific for Phythophthora infestans (late blight), and for Phytophthora erythroseptica and Phytophthora nicotianae (pink rot) have been designed which are useful for detecting the presence of the microorganisms by polymerase chain reaction methods. The primers were derived from the internal transcribed spacer region of Phytophthora ribosomal DNA and may be used to confirm the presence of the microorganisms or to distinguish among them.

Species specific method for the PCR detection of phythophthora

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

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Braddock Heights, MD, United States of America

Paul W Tooley