Authors: Isabelle A Kagan
Publish Date: 2016/06/02
Volume: 147, Issue: 1, Pages: 1-6
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 pathogenspecific 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 01 ng DNA per reaction was needed to produce a detectable band With the primers to the 368bp 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 mockinoculated 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 alfalfaI thank the R Creamer laboratory for providing the sequences of GenBank accessions KM376908 KM376909 and KM376910 prior to release R Dinkins for designing primers and providing helpful suggestions and D Samac for providing DNA from Colletotrichum trifolii and Medicago sativa as well as helpful suggestions This project was funded by the United States Department of Agriculture
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