|Ferreira, M - UNIV.BRASILIA, BRAZIL|
|Castro, Carlos - CENARGEN/EMBRAPA, BRAZIL|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 24, 1995
Publication Date: N/A
Interpretive Summary: Karnal bunt of wheat is a plant fungal disease of major quarantine significance, which could have very damaging effects on foreign trade if the disease were found in U.S. wheat. To detect the fungus which causes the disease and verify its identity, a sensitive and specific method is needed which can differentiate the pathogen from similar fungi which may be efound in wheat shipments, railroad boxcars, and grain elevators. We have developed methodology based on DNA sequence analysis and the polymerase chain reaction (PCR) which allows specific detection of the Karnal bunt fungus starting with very low quantities of DNA. DNA can be extracted from germinated fungal spores found in wheat shipments and the PCR test performed to determine whether Karnal bunt is present. Thus, development of this method provides a tool for positive identification of the Karnal bunt fungus.
Technical Abstract: 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 Tilletia 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.