|Atallah, Z - UW-MADISON|
|Bae, J - UW-MADISON|
|Rouse, D - UW-MADISON|
|Stevenson, W - UW-MADISON|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 5, 2007
Publication Date: July 1, 2007
Citation: Atallah, Z.K., Bae, J.J., Jansky, S.H., Rouse, D.I., Stevenson, W.R. 2007. Multiplex real-time quantitative PCR methodology to assist in the breeding of potato lines with resistance to Verticillium wilt. Phytopathology. 97(7):865-872. Interpretive Summary: Verticillium wilt is a serious disease of potato caused by a soil-borne fungus. It causes early death of potato plants and can result in dramatic yield reductions. The fungus is found in all major potato growing regions and can survive in the soil for a decade or more. Consequently, Verticillium wilt is a problem for most potato growers every year. The only effective method of control is soil fumigation, which is expensive and can be harmful to the environment. The development of Verticillium wilt resistant varieties offers a cost-effective and environmentally-friendly alternative control method. However, major varieties in the United States are not resistant to the fungus. Breeding for resistance requires a reliable high-throughput method for the identification of clones that are able to suppress pathogen growth in their stems. Currently, clones are screened by plating sap or dried stems on a selective medium, incubating the cultures, and counting the number of Verticillium colonies that grow on the plate. This is a time-consuming procedure and it does not always distinguish between pathogenic and non-pathogenic fungi. In this study, we have developed a PCR-based assay that reliably quantifies fungal biomass in potato tissues. It provides a more sensitive and time-effective method for identifying clones with resistance to Verticillium wilt.
Technical Abstract: Potato early dying (PED), caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide and PED-resistant cultivars currently represent only a small percentage of potato production. In this study we developed a real-time quantitative PCR (Q-PCR) approach to detect and quantify V. dahliae. The efficiency of the designed primer pair was greater than 95%, in monoplex Q-PCR and duplex (using Plexor technology) procedures with primers for the potato host. As few as 148 fg of V. dahliae DNA were detected and quantified, which is equivalent to five nuclei. Q-PCR detected V. dahliae in naturally infected air-dried potato stems and fresh inoculated stems. Spearman correlations indicated a high correlation (upwards of 80%) between V. dahliae quantifications using Q-PCR and the currently used NPX plating assay. Moreover, QPCR substantially reduced the variability observed in the NPX plating assay, and allowed for the detection of V. dahliae in 10% of stem samples found pathogen-free on the culture medium. The described Q-PCR approach should provide breeders with a more sensitive and less variable alternative to the time-consuming plating assays, hence enhancing efforts to develop new cultivars with resistance to PED.