DEVELOPMENT OF A SCAR MARKER FOR DETECTION VERTICILLIUM ALBO-ATRUM IN ALFALFA CULTIVARS AND SUBSEQUENT QUANTIFICATION OF THE PATHOGEN DNA USING REAL-TIME PCR.

Authors: Larsen, Richard; Vandemark, George; Hughes, Teresa

Submitted to: North American Alfalfa Improvement Conference
Publication Type: Proceedings
Publication Acceptance Date: 6/29/2004
Publication Date: 8/5/2004
Citation: Larsen, R.C., Vandemark, G.J., Hughes, T.J. 2004. Development of a SCAR marker for detection verticillium albo-atrum in alfalfa cultivars and subsequent quantification of the pathogen DNA using real-time PCR. Abstracts of the 39th North American Alfalfa Improvement Conference. A48.

Interpretive Summary: Verticillium albo-atrum (VA) is a serious fungal pathogen of alfalfa that causes severe wilt and eventual death of the plant in established stands. In fields where susceptible varieties are planted, losses of 100% are not uncommon. To develop a rapid method for disease detection in plants that can also be used to quantify levels of resistance in alfalfa varieties and germplasm, a molecular marker system was developed. DNA from five isolates of VA was extracted and used to screen a set of random primers using polymerase chain reaction (PCR). Using DNA from 5 other related fungi and DNA from the alfalfa standard check varieties, a molecular marker referred to as a SCAR was developed that unambiguously detected VA from all other DNA samples tested. The marker should be useful in phytosanitary issues involving movement of Verticillium-free seed. In addition, we were also able to show using real-time PCR that significantly more pathogen DNA was detected in the susceptible check Saranac than in the other two check cultivars, and significantly more pathogen DNA was detected in Vertus (moderately resistant) than in Oneida VR (highly resistant). This technology can be used to quantitatively evaluate new alfalfa germplasm and available cultivars for resistance to the pathogen.

Technical Abstract: Verticillium albo-atrum is a serious pathogen of alfalfa (Medicago sativa L.) and other forages and can significantly reduce forage yields due to stand decline and eventual death of the plant. Although that pathogen can be identified by isolation and growth on agar media in 7-10 days, use of molecular markers can facilitate rapid detection, especially when multiple samples are to be examined. Five isolates of Verticillium albo-atrum isolated from alfalfa were used to screen a set of RAPD primers. Total DNA also extracted from fungal pathogens Aphanomyces euteiches, Fusarium oxysporum, Mycospheriella spp., Rhizoctonia solanum, Pythium ultimum and P. aphanadermatum and DNA from alfalfa standard check cultivars Oneida VR (HR), Vertus (R), and Saranac (S) were used as controls. A fragment approximately 1500 bp was identified and subsequently converted to a SCAR marker. The SCAR unambiguously identified the five isolates of V. albo-atrum but did not react with any DNA from the other fungi or alfalfa used as controls. The marker successfully identified the pathogen from 12 dried alfalfa stem and leaf samples supplied from Wisconsin. It is presumed that the marker can be used for phytosanitary issues involving movement of Verticillium-free seed. To further exploit the utility of the SCAR, V. albo-atrum DNA extracted from a set of the standard alfalfa check cultivars was quantified using a real-time PCR assay. Significantly more pathogen DNA was detected in the susceptible check Saranac than in the other two check cultivars, and significantly more pathogen DNA was detected in Vertus than in Oneida VR. The correlation between pathogen DNA content in stems and disease severity index (DSI) ratings was positive (0.43) and highly significant (P < 0.0001).