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Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/23/2016 Publication Date: 6/2/2016 Citation: Kagan, I. 2016. Use of the polymerase chain reaction to help determine the presence of blackpatch (Rhizoctonia leguminicola) in inoculated red clover leaves. European Journal of Plant Pathology. 147:1-6. Interpretive Summary: Rhizoctonia leguminicola is a fungus that infects red clover and other legumes. It causes blackpatch, a disease whose symptoms include brown to black spots on leaves and the presence of patches of diseased plants in the field. Livestock grazing the diseased clover may start to slobber profusely, due to a compound called slaframine that is produced by the fungus. Because the slobbering can result in dehydration, many horse farmers try to avoid having red clover in their pastures, despite the nutritional benefits of red clover protein. A first step to estimating the risk of finding blackpatch in Kentucky pastures would be to study the distribution of R. leguminicola in different locations and under different environmental conditions. To do such a study, a simple diagnostic test for the presence of R. leguminicola is needed. Standard methods of identification include microscopy, and extraction of slaframine from infected leaves followed by separation from other components of the chemical extract. Identification by microscopy requires considerable skill, and the equipment for chemical extraction and separation may not be available to confirm the microscopic diagnosis. This study describes a method to identify R. leguminicola in infected leaves by extracting DNA from 2 to 3 infected leaves with the aid of a kit, then using the polymerase chain reaction (PCR) to amplify a DNA sequence that is unique to this fungus. The DNA sequence was successfully amplified in DNA from the fungus, and from a red clover leaf infected with the fungus. The fact that the fungal DNA sequence was amplified in a mixture of plant and fungal DNA suggests that this technique could be used to confirm a visual diagnosis of blackpatch. Technical Abstract: Rhizoctonia leguminicola, the causal agent of blackpatch of red clover, produces alkaloids that cause livestock to salivate excessively. Its presence is generally confirmed by microscopy, disappearance of symptoms after removal of the suspect forage, and chromatographic analysis of slaframine in extracts. A method is presented for identifying R. leguminicola by polymerase chain reaction (PCR) of DNA extracts, using primers for a 368 base pair fragment of the internal transcribed spacer (ITS) gene. This method could complement the microscopic and chemical analyses generally used to identify the pathogen in legume forages. |
