ARS | Publication request: Insertional Mutagenesis of Sclerotinia Sclerotiorum Through Agrobacterium Tumefaciens-Mediated Transformation.

Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: March 31, 2005
Publication Date: N/A

Technical Abstract: Stem rot caused by Sclerotinia sclerotiorum is one of the most serious diseases of many economically important crops under conducive conditions. Despite extensive investigations on the disease, the genetic factors that control the pathogenesis of S. sclerotiorum are incompletely understood, except for the recent studies on the pac1 gene and the pg1 gene. S. sclerotiorum was transformed using protoplast-based protocols by Rollins (2003). A more convenient transformation system without protoplast preparation is sought in order to study other genetic factors involved in pathogenesis. An insertional mutagenesis protocol using Agrobacterium tumefacience-mediated transformation is developed to transform S. sclerotiorum. Both mycelial fragments and ascospores were successfully used in transformation with hygromycin phosphotransferase (hph) gene as a selection marker. Two promoters were compared for their efficiency in expressing the hph gene in S. sclerotiorum, and the Aspergillus nidulans trpC promoter was better than the Cauliflower mosaic virus 35S promoter CaMV35s. Transformants were tested for number of insertions by Southern hybridization and for stability of hygromycin resistance, and are being screened for altered pathogenicity on lentil plants and for production of oxalic acid.