Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus

Abstract

Fusarium rot caused by Fusarium oxyspo- rum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in bulbs in storage. In order to study the mechanisms of path- ogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B phosphotransferase (hph) gene and fluorescence reporter genes EGFP (green), EYFP (yel- low) or ECFP (cyan) using the AGL-1 strain of A. tumefaciens. Hygromycin B (100 μg/ml) resistant col- onies were observed only when acetosyringone was added to the co-cultivation medium. Transformed col- onies are more clearly visible when co-cultivated on cellophane membrane than on Hybond -N + membrane. Transformed lines were stably maintained through four serial passages on medium containing hygromycin B, andtheyexpressedgreen,yelloworcyano fluorescence. PCR with hph-specific primers and Southern blotting with an hph-specific probe were positive for Hyg R lines but not for the untransformed isolate. The cyano fluo- rescenceoftheECFP-transformed isolate was clearly distinguishable from the green autofluorescence of Gladiolus roots, signifying the potential of these lines for further histopathological investigations. Transformed lines will be useful for identifying patho- genicity related genes, screening transgenic resistance, and in studies of host-pathogen interactions.