Use of fusarium oxysporum f.sp. dianthi transformed with marker genes to follow colonization of carnation roots

A highly pathogenic strain of F. oxysporum f.sp. dianthi (Fod) has been transformed with genes coding for Green Fluorescent Protein (GFP) and Red Fluorescent Protein (DsRedFP). Expression of GFP and DsRedFP resulted in bright green or red cytoplasmic fluorescence of mycelium when excited by fluorescent light. Stable transformants, selected on the basis of brightness and stability of fluorescence, were inoculated on roots of a partially resistant cultivar of carnation. Root and vessel colonization was evaluated, at regular times, by fluorescent microscopic observation of plant tissue sections. Autofluorescence of the carnation root was very high at the emission wavelength of the DsRed-Express, so that it was not possible to distinguish infecting hyphae within plant tissues. GFP-transformed Fod was easily visualized on and within root tissues. The fungus invaded the root tip and its growth seemed to proceed randomly in the sub-apical zone. The hyphae were confined within the vascular cylinder by the endodermal cells beginning from the zone of differentiation of vascular tissues, and were able to grow inside vessels.