A species-specific polymerase chain reaction assay for rapid and sensitive detection of Sclerotium rolfsii

The failure to adequately identify plant pathogens from culture-based morphological techniques has led to the development of culture-independent molecular approaches. The timely and accurate detection of pathogens is critical in the study of epidemiology and management of plant diseases. A polymerase chain reaction (PCR)-based method was developed for the identification and detection of Sclerotium rolfsii in Amorphophallus paeoniifolius plants. A PCR primer pair specific for S. rolfsii was designed based on the sequence of the internal transcribed spacer region. The designed primer pair SCR-F/SCR-R amplified a 540-bp product from S. rolfsii DNA and did not amplify DNA from A. paeoniifolius or several other fungi pathogenic to A. paeoniifolius. In conventional PCR, the limit of detection of pure fungal gDNA was 6 pg ml−1, which was reduced 2-fold within a plant DNA background. S. rolfsii DNA was detected in inoculated A. paeoniifolius and 12 h after inoculation in symptomless tuber samples. The protocol was assessed for the detection of S. rolfsii in infected soils.