Fusarium oxysporum f. sp. dianthi transformed with marker genes as a tool for studying resistance in Dianthus

Fusarium oxysporum Schlechtend:Fr. f. sp. dianthi (Prill. & Delacr.) W. C. Snyd. & H. N. Hans. (Fod), the causal agent of a vascular disease known as Fusarium wilt, represents the most important pathogen of carnation (Dianthus caryophyllus L.), causing severe yield losses in susceptible cultivars. The pathogen is present in the soil profile in which carnation roots are distributed. Various biological races of Fod are currently known on the basis of their selective virulence against different carnation cultivars. In Italy, as in the rest of Europe, race 2 of the pathogen is the most prevalent1. The most important factor influencing wilt development is the cultivar resistance; different carnation cultivars are known to show from low to high levels of resistance against the different races of Fod. Reporter proteins as Green Fluorescent Protein (GFP) and the Red Fluorescent Protein (DsRedFP) have been previously used to follow interactions of filamentous fungi with other organisms2 3. In this work, a highly pathogenic F. oxysporum f. sp. dianthi (Fod) strain has been transformed with genes coding for GFP and DsRedFP, as a tool to evaluate the colonization process and localize the pathogen in the root structure of carnation plants. Expression of both proteins resulted in bright green or red cytoplasmic fluorescence of mycelium when observed under 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 it was not possible to distinguish infecting hyphae within plant tissues. GFP transformed Fod was easily visualized on and within root tissues. The fungus invades the root apex without any preferential entry point and its growth seems to proceed randomly in the sub-apical zone. The hyphae are confined within the vascular cylinder by the endodermis cells beginning from the zone of differentiation of vascular tissues and are able to grow inside vessels. The availability of a marked F. oxysporum f. sp. dianthi strain allows the monitoring of carnation root colonisation and may be a powerful tool for studying resistance processes easily and accurately. Furthermore the possibility to follow plant-microbe interactions can help in defining biological control measures by beneficial antagonistic microorganisms