Submitted to: Alfalfa Improvement Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 5/1/2000
Publication Date: 7/1/2000
Citation: LARSEN, R.C., VANDEMARK, G.J., GRITSENKO, M.A., HOLLINGSWORTH, C.R., GRAY, F.A. USE OF PCR-BASED MARKERS FOR THE IDENTIFICATION OF PHOMA SCLEROTIODES CAUSING BROWN ROOT ROT DISEASE OF ALFALFA. PROCEEDINGS OF THE 37TH NORTH AMERICAN ALFALFA IMPROVEMENT CONFERENCE, JULY 2000, P. 309. 2000.
Technical Abstract: Phoma sclerotiodes, the causal organism of Brown Root Rot, has been shown to be responsible for severe winter kill in alfalfa fields located in high elevation growing areas of Wyoming. Because isolation, and identification of P. sclerotiodes is considerably time consuming, the objective of this work was to develop a fast and efficient method using DNA sequence characterized amplified regions (SCARs) for the detection and identification of the pathogen. Eighteen isolates of P. sclerotiodes collected from 6 regions in Wyoming and an ATCC isolate from Canada were used in the evaluations. DNA was extracted from cultures grown on potato dextrose agar. Five RAPD primers amplified products in PCR reactions specific to all isolates of P. sclerotiodes and did not react with 5 other common soil-inhabiting pathogens. SCAR primers were designed from amplification products obtained from 2 selected RAPD reactions. After optimization of thermocycling conditions, SCAR primers OpC15-F30 and OpC15 R29 amplified a single DNA product approximately 2000 bp from each of the 19 P. sclerotiodes isolates and including 2 isolates of P. medicagensis. The second primer pair OpB12-F27 and OpB12-R26 amplified a 499 bp product specific to P. sclerotiodes but not P. medicagensis. In addition, neither SCAR amplified products in any of the control soilborne microorganisms. We anticipate that the SCARs developed here will be useful in rapid identification of P. sclerotiodes in soils collected during field surveys from potentially infested areas. Pre-planting diagnosis should help prevent growers from sustaining unacceptable losses resulting from planting alfalfa in fields infested with P. sclerotiodes.