Phoma macdonaldii, the causal agent of black stem disease of sunflower (Helianthus annuus), also attacks roots and collars of the plants, resulting in early death. Totally resistant lines do not exist for infection of the aerial parts, but tolerant lines have been characterised. This paper presents a study on colonisation of a partially resistant and a susceptible sunflower line by P. macdonaldii. The fungus was transformed with a constitutively expressed reporter gene encoding the jellyfish green fluorescent protein via Agrobacterium tumefaciens, and colonisation of sunflower roots by this transformed strain was studied by various microscopy techniques including confocal and scanning electron microscopy. The results show that penetration of the fungus into the root occurred through natural fissures or through the epidermis and was similar in both lines. In contrast, the colonisation rate of the stele was reduced in the partially resistant line, and the morphology of the fungal hyphae was also affected. The effect on hyphal morphology was strongest in the stele, indicating a localised production of defence compounds in this line.