Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2008
Publication Date: 7/26/2008
Citation: Ma, J., Haudenshield, J.S., Hill, C., Hartman, G.L. 2008. A quantitative PCR assay for Macrophomina phaseolina [abstract]. Phytopathology. 98:S95.
Technical Abstract: Charcoal rot of soybean is caused by Macrophomina phaseolina and crop damage may be considerable under conditions of high temperature or drought. This soilborne fungus has a wide host range, with numerous isolates infecting about 500 plant species in more than 100 families throughout the world. In order to develop a rapid and accurate DNA-based method for the specific detection of M. phaseolina, we selected the rRNA internal transcribed spacer (ITS) region as a target site, amplified this region from isolate MP3-3-1-1 using primers M1 and M4, and sequenced the resulting amplicon. That sequence was then aligned with the corresponding data available for other pathogenic fungi of soybean, and the primers Macr1 and Macr2 were chosen and synthesized, along with a dual-labeled linear hydrolysis probe MacrP1, to provide a 5’-fluorogenic exonuclease assay specific to M. phaseolina, isolate MP3-3-1-1. The DNA from nine other plant pathogenic fungi was tested and none produced a positive result, supporting the specificity of this assay. An endpoint dilution series of purified M. phaseolina total DNA exhibited accurate quantification using this technique to a detection threshold of approximately 100 fg DNA. These results demonstrate that the assay we have developed can be a useful tool in rapid detection and quantification of M. phaseolina.