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Title: DEVELOPMENT OF PCR-BASED SCAR MARKERS FOR THE DETECTION AND IDENTIFICATION OF PHOMA SCLEROTIODES, THE CAUSE OF BROWN ROOT ROT OF ALFALFA

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

Submitted to: Proceedings of the 2002 Mycological Society of America Annual Meeting
Publication Type: Research Notes
Publication Acceptance Date: 5/1/2002
Publication Date: 7/1/2002
Citation: LARSEN, R.C., VANDEMARK, G.J., HOLLINGSWORTH, C.R., GRITSENKO, M., GRAY, F.A. DEVELOPMENT OF PCR-BASED SCAR MARKERS FOR THE DETECTION AND IDENTIFICATION OF PHOMA SCLEROTIODES, THE CAUSE OF BROWN ROOT ROT OF ALFALFA. PROCEEDINGS OF THE 2002 MYCOLOGICAL SOCIETY OF AMERICA ANNUAL MEETING, p. 56. 2002.

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 sclerotioides and has been detected in Wyoming and other high altitude areas near the Rocky Mountain range. BRR is responsible for severe decline due to winter kill of alfalfa stands. Symptoms on alfalfa roots are 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 agar 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. sclerotioides 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, up to 100 days are required for growth of the fungus before definitive structures are produced that allow 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 may otherwise result in large yield and financial losses.

Technical Abstract: Brown root rot of forage legumes is caused by the soilborne fungal organism Phoma sclerotioides. The disease has caused severe mortality rates in alfalfa crops grown in southwest Wyoming. In order to circumvent the lengthy process required for proper identification of the pathogen, a fast and efficient method using DNA sequence characterized amplified regions (SCARs) was developed. Initially, five RAPD primers amplified products in PCR reactions specific to 19 isolates of P. sclerotioides. SCAR primer pair PSB12(499) designed from a resulting RAPD sequence amplified a 499 bp product specific to P. sclerotioides but no product was produced from Phoma medicaginis or Phoma betae. A 499 bp amplification product was also produced from root tissue known to be infected with the fungus as verified by microscopic examination. A similar PCR product was obtained from soil samples collected from fields with an established infection of P. sclerotioides on alfalfa. A second set of SCAR primers PSC15(2600) amplified a single 2600 bp DNA product from each of the 19 P. sclerotioides isolates tested including two isolates of P. medicaginis and four isolates of Phoma betae. Neither SCAR primer pair amplified products in other soilborne root rotting microorganisms included as controls in the evaluation. This PCR-based assay enables detection of P. sclerotioides from alfalfa root tissue and in 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.