Use of the polymerase chain reaction to help determine the presence of blackpatch (Rhizoctonia leguminicola) in inoculated red clover leaves

Abstract

Rhizoctonia leguminicola, the traditional name for the causal agent of blackpatch of red clover (Trifolium pratense) and other legumes, produces alkaloids, one of which causes livestock to salivate excessively. Fungal presence is generally confirmed by microscopy, disappearance of symptoms in livestock after removal of suspect forage, and chromatography of the alkaloid slaframine, in legume tissue. Use of the polymerase chain reaction (PCR) to amplify a pathogen-specific DNA fragment would complement the other methods of pathogen identification. Primers were designed to the R. leguminicola ITS region, sequence provided by another laboratory. Two separate primer pairs each amplified a different fragment–one ~250 bp long (expected length 249 bp), and the other 300 to 400 bp long (expected length 368 bp)–in DNA extracted from cultures of R. leguminicola. Under the experimental conditions, the primers to the larger fragment amplified a stronger band, and a minimum of 0.1 ng DNA per reaction was needed to produce a detectable band. With the primers to the 368-bp fragment, a band 300 to 400 bp long was also amplified in DNA extracted from red clover (cultivar Kenland) inoculated with R. leguminicola and harvested 70 h post inoculation. No amplification with this primer set occurred in DNA extracted from mock-inoculated red clover plants, supporting the likelihood that the primers amplified R. leguminicola DNA extracted from inoculated red clover. This primer set did not amplify DNA extracted from a red clover isolate of the legume pathogen Stemphylium sarcinaeforme, or DNA extracted from two isolates of the legume pathogen Colletotrichum trifolii, indicating specificity for R. leguminicola DNA. Lack of amplification of alfalfa DNA indicated that the R. leguminicola primers will be useful for testing for the presence of blackpatch in alfalfa.