Detection and quantification of Fusarium solani f. sp glycines in soybean roots with real-time quantitative polymerase chain reaction

Fusarium solani f. sp. glycines is the causal organism of soybean sudden death syndrome (SDS). This organism is difficult to detect and quantify because it is a slow-growing fungus with variable phenotypic characteristics. Reliable and fast procedures are important for detection of this soybean pathogen. Protocols were optimized for extraction of DNA from pure fungal cultures and fresh or dry roots. A new procedure to test polymerase chain reaction (PCR) inhibitors in DNA extracts was developed. Novel real-time quantitative PCR (QPCR) assays were developed for both absolute and relative quantification of F solani f. sp. glycines. The fungus was quantified based on detection of the mitochondrial small-subunit rRNA gene, and the host plant based on detection of the cyclophilin gene of the host plant. DNA of F solani f. sp. glycines was detected in soybean plants both with and without SDS foliar symptoms to contents as low as 9.0 x 10(-5) ng in the absolute QPCR assays. This is the first report of relative QPCR using the comparative threshold cycle (Ct) method to quantify the DNA of a plant pathogen relative to its host DNA. The relative QPCR assay is reliable if care is taken to avoid reaction inhibition and it may be used to further elucidate the fungus-host interaction in the development of SDS or screen for resistance to the fungus.