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item Larsen, Richard
item Vandemark, George
item HUGHES, T
item GRAV, C

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/21/2007
Publication Date: 11/1/2007
Citation: Larsen, R.C., Vandemark, G.J., Hughes, T.J., Grav, C.R. 2007. Development of a real-time pcr assay for quantifying verticillium albo-atrum dna in resistant and susceptible alfalfa. Phytopathology. 97:1519-1525.

Interpretive Summary: Verticillium wilt disease in alfalfa is caused by the fungal pathogen Verticillium albo-atrum, and is the cause of severe yield losses and stand decline in the U.S. The fungus is seed transmitted which results in phytosanitary concerns on movement of potentially infected seed lots. At the USDA-ARS, Prosser lab, we have developed a molecular marker that can be used to rapidly detect the pathogen without having to carry out a time-consuming biological assay. In addition, DNA sequence obtained from the marker has enabled us to design molecular probes for use in real-time PCR assays. The assay is very precise and a very significant positive correlation has been shown to occur between the amount of pathogen DNA detected and the severity of disease in infected alfalfa plants. Resistant plants had significantly less pathogen DNA than susceptible plants. However, not all results correlated with rankings previously assigned to two cultivars tested, suggesting that real-time PCR would be useful for more accurate determination of commercial variety disease ratings. In addition, this assay will assist breeders to more accurately select the most resistant plants for use in breeding programs, and will accelerate the development of extremely resistant alfalfa cultivars.

Technical Abstract: A RAPD PCR amplification product specific to Verticillium albo-atrum was identified and subsequently converted to a SCAR marker. The resulting SCAR primer set Svert1513 amplified a 1513 DNA product in each of five V. albo-atrum isolates but not from DNA extracted from Aphanomyces euteiches, Fusarium oxysporum, Mycospheriella spp., Rhizoctonia solanum, Pythium ultimum, P. aphanadermatum, or from healthy alfalfa controls. A primer and probe set was subsequently derived from the SCAR sequence for use in real-time PCR assays to quantify V. albo-atrum DNA in six alfalfa cutivars. The assay discriminated between the three standard check cultivars Saranac (susceptible), Vertus (moderately resistant) and Oneida VR (highly resistant), and three additional commercial cultivars Vernema, Wrangler, and Samauri, based on analysis of DNA extracted from stems of individual plants inoculated with three different isolates of the pathogen. Spearman rank correlations between pathogen DNA content and disease severity index was positive and highly significant (P<0.0001) between the amount of V. albo-atrum DNA detected in the three DSI classes of the standard check cultivars. Significantly less pathogen DNA was detected in two separate experiments in the moderately resistant plants than in susceptible plants, and significantly less DNA was detected in highly resistant plants than in moderately resistant plants (P<0.0001). Not all DSI results correlated with rankings previously assigned to two cultivars, suggesting that real-time PCR would be useful for more accurate determination of commercial disease ratings.