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A Rapid Method Using PCR-Based SCAR Markers for the Detection and Identification of Phoma sclerotioides: The Cause of Brown Root Rot Disease of Alfalfa

A rapid technique for identification and detection of Phoma sclerotioides, the causal agent of brown root rot of alfalfa, has been developed using polymerase chain reaction (PCR). Amplification products obtained from random amplified polymorphic DNA (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. A single 499-bp DNA amplification product was consistently obtained from primers PSB12₄₉₉ that was specific for 19 isolates of P.sclerotioides but was not produced from Phoma medicaginis or Phoma betae, or from other soilborne pathogens including Aphanomyces euteiches, Rhizoctonia solani, Fusarium oxysporum, Pythium ultimum, or Phytophthora infestans. 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. 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 with standard methods that require up to 100 days for identification using agar media.