Use of REMI andAgrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus,Coniothyrium minitans

Abstract

Restriction enzyme mediated integration (REMI) and Agrobacterium-mediated transformation (ATMT) were used to transform protoplasts or germinated conidia of the mycoparasite Coniothyrium minitans to hygromycin resistance. Using REMI, up to 32 transformants μg DNA⁻¹ were obtained, while 37.8 transformants 5 × 10⁵ germlings⁻¹ were obtained using ATMT. Single-copy integrations occurred in 8% and 40% of REMI and ATMT transformants, respectively. A novel microtitre plate-based test was developed to expedite screening of 4000 REMI and ATMT C. minitans transformants. Nine pathogenicity mutants that displayed reduced or no pathogenicity on sclerotia of Sclerotinia sclerotiorum were identified.