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Title: Real-time PCR detection of sorghum ergot pathogens Claviceps africana, C. sorghi, and C. sorghicola

item Tooley, Paul
item Carras, Marie
item Sechler, Aaron
item RAJASAB, A - Gulgarga University

Submitted to: Journal of Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/4/2010
Publication Date: 4/5/2010
Citation: Tooley, P.W., Carras, M.M., Sechler, A.J., Rajasab, A.H. 2010. Real-time PCR detection of sorghum ergot pathogens Claviceps africana, C. sorghi, and C. sorghicola. Journal of Phytopathology. doi: 10.1111/j.1439-0434.2010.01683.x

Interpretive Summary: This paper describes the development of a rapid and sensitive detection assay for species of fungi that cause a disease of sorghum called ergot. Ergot disease has caused large losses to the sorghum industry and when infection occurs in seed production areas shipment of the infected material is subject to quarantine. Sorghum ergot is caused by three different fungal species which are difficult to tell apart under the microscope. We used methods based on the DNA within the fungal species to identify and distinguish between the three different species causing sorghum ergot. The DNA-based method we employed for detection is called real-time polymerase chain reaction, and is a method now being applied successfully to a variety of fungal plant pathogens. Using this method, the three species can be differentiated with greater speed and accuracy than when using other methods. Application of this detection assay to future outbreaks of sorghum ergot will streamline the detection process and allow government officials to make quarantine and regulatory decisions in a more timely fashion.

Technical Abstract: Sorghum ergot is a serious disease that has caused major losses in sorghum growing regions worldwide. Claviceps africana is now the most widely distributed species causing ergot in many countries including the U.S., whereas both C. africana and C. sorghi exist in India. A third species (C. sorghicola) has been described causing sorghum ergot in Japan. As the three species are morphologically very similar, a DNA-based assay is desirable for rapid identification in cases where ergot-infected sorghum is found entering the U.S. and other countries. We designed primers and probes specific for the above three Claviceps species and tested them using purified DNA and ergot samples from the greenhouse and field. Real-time PCR was performed using an Applied Biosystems Prism 7700 Sequence Detection System in a total volume of 25 microliters. Species-specific primers and probes were developed from the intron 3 region of the beta tubulin gene (C. africana, C. sorghi) or the elongation factor 1 alpha gene intron 4 (C. sorghicola). No amplification was obtained in any of the three ergot species-specific assays when DNA from six other Claviceps species was used as template. The lower limit of detection was 1 pg genomic template DNA in all three assays. The assays we describe will provide useful tools for detecting sorghum ergot in seed and grain shipments and for determining which species are present in the samples, thereby aiding in the regulatory decision-making process.