Real-time PCR Detection of Sorghum Ergot Pathogens Claviceps africana, Claviceps sorghi and Claviceps sorghicola

Abstract

Sorghum ergot is a serious disease that has caused major losses in sorghum growing regions worldwide. Claviceps africana, originally reported from Zimbabwe, is now the most widely distributed species causing ergot in many countries including the United States of America, whereas both C. africana and Claviceps sorghi exist in India. A third species (Claviceps sorghicola) has been described causing sorghum ergot in Japan. As the three species show morphological similarities, a DNAbased assay is desirable for rapid identification in cases where ergot-infected sorghum is found by regulatory authorities. We designed PCR primers and probes from the intron 3 region of the b-tubulin gene (for C. africana and C. sorghi) and the intron 4 region of EF-1a (for C. sorghicola) and tested them by real-time PCR with purified DNA and ergot samples from the field and greenhouse. The primer and probe sets specifically amplified DNA from the respective species with a detection limit of c. 1 pg DNA. Genomic DNA from six other Claviceps species did not amplify in any of the three ergot species-specific assays. The assays we describe will provide useful tools for detecting sorghum ergot pathogens in seed and grain shipments and for determining which species are present in the samples, thereby aiding in the regulatory decisionmaking process.