Identification of Coniothyrium minitans isolates using PCR amplification of a dispersed repetitive element

A PCR (polymerase chain reaction) assay was developed to distinguish isolat es of the sclerotial mycoparasite Coniothyrium minitans. In particular; the assay uniquely identified isolate A69 which was of interest due to its pot ential as a biocontrol agent for the plant pathogen Sclerotinia sclerotioru m. Random amplification of polymorphic DNA (RAPD) was used to screen isolat es of C, minitans for polymorphic products which distinguished A69 from oth er C. minitans isolates. A 1.4 kb A69-specific RAPD fragment was cloned and sequenced. The sequence was found to contain two tandemly repeated 114 bp units separated by a single base. Southern blot analysis of C. minitans DNA showed that the cloned fragment hybridized to multiple bands in all isolat es indicating that it was a dispersed repetitive element. Novel PCR primers were designed from the sequences flanking the tandem repeats. These primer s were used to develop a PCR assay which distinguished some isolates of C. minitans and uniquely identified isolate A69. This assay will be used to st udy the ecology of C. minitans in soil.