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Title: A RAPID METHOD USING PCR BASED SCAR MARKERS FOR THE DETECTION AND IDENTIFICATION OF PHOMA SCLEROTIODES-THE CAUSE OF BROWN ROOT ROT DISEASE OF ALFALFA

Author
item Larsen, Richard
item HOLLINGSWORTH, C - UNIV WYOMING, LARAMIE, WY
item Vandemark, George
item GRITSENKO, M - WSU-IAREC, PROSSER, WA
item GRAY, F - UNIV WYOMING, LARAMIE, WY

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2002
Publication Date: 11/1/2001
Citation: LARSEN, R.C., HOLLINGSWORTH, C.R., VANDEMARK, G.J., GRITSENKO, M.A., GRAY, F.A. A RAPID METHOD USING PCR BASED SCAR MARKERS FOR THE DETECTION AND IDENTIFICATION OF PHOMA SCLEROTIODES-THE CAUSE OF BROWN ROOT ROT DISEASE OF ALFALFA. PLANT DISEASE, 86:928-932. 2001.

Interpretive Summary: A new soilborne disease was reported in alfalfa in the continental United States during 1997. The disease, called brown root rot (BRR) of alfalfa, is caused by the fungus Phoma sclerotiodes and has been detected in Wyoming and other high altitude areas near the Rocky Mountain range. BRR is responsible for severe decline of alfalfa stands. Symptoms on alfalfa roots sare easily confused with those caused by Phytophthora root rot. Detection and diagnosis of the disease is difficult and time consuming due to the slow growth of the organism on artificial media. We have developed a rapid method using molecular tools called SCARs (sequence-amplified characterized regions) that allow us to rapidly evaluate and accurately identify the organism. The SCAR DNA primers are used in PCR (polymerase chain reaction) assays that unambiguously discriminate between P. sclerotiodes and at least six other soilborne pathogens tested by producing a single fluorescent DNA band visualized on agarose gels. No bands are produced in the other non- target organisms when these SCAR primers are used. We have been successful in detecting the pathogen from infected alfalfa root tissue and from as little as one half gram of field soil samples. Typically, 30-60 days are required for growth of the fungus before definitive structures are produced that allows investigators to accurately identify the pathogen. Utilizing this technology, the organism can be detected in a single day. In addition, field soil samples can be evaluated for the BRR organism prior to establishment of alfalfa fields that would otherwise result in large potential yield and financial losses.

Technical Abstract: A rapid technique for identification and detection of Phoma sclerotiodes, the causal agent of brown root rot of alfalfa, has been developed using polymerase chain reaction (PCR). Amplification products obtained from RAPD reactions were cloned and sequenced and two extended primer sets were designed from the resulting data that were used to detect sequence- characterized DNA markers. Primers PSC152600 amplified a 2600 bp product from 19 isolates of P. sclerotiodes genomic DNA as well as DNA from Phoma medicagensis, and Phoma betae. A single 499 bp DNA amplification product was consistently obtained from primers PSB12499 that was specific for the 1 isolates of P. sclerotiodes but was not produced from P. medicagensis or P, betae. No PCR products using PSB12499 or PSC152600 were detected from genom genome DNA from soilborne pathogens Aphanomyces euteiches, Rhizoctonia solani, Fusarium oxysporum, or Pythium ultimum. Primers PSB12499 were also used to detect P.sclerotiodes from alfalfa root tissue and soil samples collected from fields in Farson, WY. A 499 bp amplification produce was produced from all root tissue known to be infected with the fungus and was verified by microscropic examination. A similar product was obtained from soil samples collected from fields with an established infection of P. sclerotiodes on alfalfa, but not from sterile soil. The detection system can detect P. sclerotiodes from root tissue and soil samples in a single day including extraction of DNA compared to standard methods that require up to 60 days for identification using artificial media.