ISOLATION OF A SPECIES-SPECIFIC MITOCHONDRIAL-DNA SEQUENCE FOR IDENTIFICATION OF TILLETIA-INDICA, THE KARNAL BUNT OF WHEAT FUNGUS

Mitochondrial DNA (mtDNA) from five isolates of Tilletia indica was is olated and digested with several restriction enzymes. A 2.3-kb EcoRI f ragment was chosen, cloned, and shown to hybridize with total DNA rest ricted with EcoRI from T, indica and not from a morphologically simila r smut fungus, Tilletia barclayana. The clone was partially sequenced, and primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2,3-kb fragment fr om total DNA of 17 T. indica isolates from India, Pakistan, and Mexico , DNA from 25 isolates of other smut fungi (T. barclayana, Tilletia fo etida, Tilletia caries, Tilletia fusca, and Tilletia controversa) did not produce any bands, as detected by ethidium bromide-stained agarose gels and Southern hybridizations. The sensitivity of the assay was de termined and increased by using a single nested primer in a second rou nd of amplification, so that 1 pg of total mycelial DNA could be detec ted, The results indicated that the primers which originated from a cl oned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contam inating wheat seeds.