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

Abstract

A PCR (polymerase chain reaction) assay was developed to distinguish isolates of the sclerotial mycoparasite Coniothyrium minitans. In particular, the assay uniquely identified isolate A69 which was of interest due to its potential as a biocontrol agent for the plant pathogen Sclerotinia sclerotiorum. Random amplification of polymorphic DNA (RAPD) was used to screen isolates of C. minitans for polymorphic products which distinguished A69 from other 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 isolates indicating that it was a dispersed repetitive element. Novel PCR primers were designed from the sequences flanking the tandem repeats. These primers 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 study the ecology of C. minitans in soil.

Key Words:

• biocontrol
• mycoparasite
• RAPD