Cmm-tomato interactions: Visualization during infection, biofilm formation and epiphytic fitness

The Gram-positive actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of tomato wilt disease, an economically important disease worldwide. Pathogenicity of Cmm is associated with two plasmids, pCM1 and pCM2 and a chp/tomA pathogenicity island. Cmm colonization of tomato plant by microscopic methods, as well as epiphytic fitness on tomato leaves and biofilm formation were examined. Confocal laser scanning microscopy revealed that GFP-labeled Cmm extensively colonized the xylem vessels with preferential attachment to spiral rings and tracheary elements of wilted leaves. Scanning electron imaging showed aggregates of Cmm cells between the spiral elements at 7days post-inoculation, while after 15 days Cmm densely colonized the whole xylem vessels. Moreover, Cmm cells were observed on remnants of cell wall which appeared perforated and might result from cell wall degrading enzymes. Crystal violet staining assay showed that biofilm formation of Cmm grown in xylem sap was significantly higher as compared to Luria Broth and minimal medium by 2.5 and 1.5 fold, respectively. In addition of being an efficient endophyte, Cmm showed high epiphytic fitness on tomato as compared to bean leaves. These results were supported by the observation that GFP-labeled Cmm cells densely colonized tomato leaf surfaces. This is the first study in which GFP-labeled Cmm was employed to demonstrate endophytic and epiphytic characteristics of this phytopathogen.