Quantitative polymerase chain reaction (Q-PCR) and fluorescent in situ hybridization (FISH) detection of soilborne pathogen Sclerotium rolfsii

Abstract

Early detection of soilborne disease is essential for prediction of disease development and successful disease management. Traditional methods of monitoring soilborne diseases based on plant growth and symptoms did not take into account the presence and inoculum potential of pathogens in the soil, which generally delay the prevention of diseases. To detect and quantify soil populations of Sclerotium rolfsii, the causal agent of southern blight of tomatoes, quantitative polymerase chain reaction (Q-PCR) was used to measure the amount of soil general fungal and specific S. rolfsii DNA. Significantly higher amounts of total fungal and S. rolfsii DNA were detected in inoculated than in uninoculated soil in a greenhouse inoculation experiment. Fluorescent in situ hybridization (FISH) was successfully developed to detect the abundance of S. rolfsii through whole cell hybridization and was further applied for visual detection of the abundance of S. rolfsii in the soil samples of greenhouse inoculation experiment. Natural abundance of S. rolfsii in soil with a DNA amount of 0.06 pg µl−1 DNA extraction, which was equivalent to 8 pg g−1 soil, was successfully detected. The hybridization signal detected in soil for S. rolfsii had a great correlation with the amount of infested DNA levels of S. rolfsii. Molecular detection and quantification of soilborne pathogens in plant roots and in the soil could provide valuable information to predict disease development and apply management practices for soilborne diseases.