Generation of tomato seeds artificially infected with a GFP - tagged strain of Clavibacter michiganensis subsp. michiganensis

Abstract

Clavibacter michiganensis subsp. michiganensis (Cmm) is an economically-important seed-borne pathogen that causes bacterial canker and bacterial wilt of tomato. In this study, a procedure was developed to generate tomato seed (internally-) infected with Cmm. These seeds can be used to evaluate methods for detection of Cmm in seed extracts, to study the effect of seed treatments and to study transmission from seed to seedlings. Seeds were subsequently wounded by scarification, softened by incubation on wetted blotting paper and inoculated with a Cmm suspension by vacuum-infiltration. To enhance growth of the Cmm in the seeds, they were incubated on wetted blotting paper or on a Cmm-selective medium. Directly after inoculation, densities were 104 cfu seed-1 before or 103 cfu seed-1 if seeds were disinfected. Incubation of seeds resulted in a strong increase of Cmm populations to densities up to 1010 cfu seed-1 after three days. A GFP-tagged Cmm strain was used to visualise Cmm in plant material. The GFP signal could be observed by epifluorescence stereomicroscopy in germinated seeds on the seed coat, cotyledons, stem and roots. Colonisation of xylem vessels in stems and roots was observed with confocal laser scanning microscopy, indicating systemic infections of germinated seeds.