Invasion of Clavibacter michiganensis into the Xylem and Surrounding Tissues Drives Histological Alterations and Disease Development in Tomato.

Abstract

Clavibacter michiganensis is a xylem-colonizing and seed-borne phytopathogen that causes bacterial canker disease in tomato plants. In this study, we aimed to determine the colonization patterns of C. michiganensis, as well as the histological changes in tomato plants during infection. To achieve these, GFP-tagged C. michiganensis was inoculated into tomato plants using three methods: stem injection, soil drenching with or without prior root cutting, and cotton-pad inoculation. After inoculation, bacterial colonization was observed using GFP signals, and histological changes were examined using propidium iodide staining. Following the stem injection, C. michiganensis colonized the xylem and spread to adjacent pith tissues, causing degradation and canker symptoms at the inoculation sites, while largely limited to the xylem in the upper stem regions. After soil drenching with prior root cutting, C. michiganensis initially colonized the xylem, spread systemically, and later caused tissue collapse and wilting, subsequently being detected in adjacent pith tissues. Using the cottonpad method, C. michiganensis entered through epidermal structures, initially colonized the cortex tissues, and subsequently reached the xylem. Although wilting was occurred, histological changes were primarily confined to the cortex tissues. Regardless of the inoculation methods, C. michiganensis colonized the xylem for systemic spread and caused wilting and sometimes canker symptoms, probably resulting from collapse of cortex, vascular, and pith tissues. These findings suggest that C. michiganensis enters through natural openings or wounds, then colonizes the cortex, invades the xylem, and subsequently colonizes the pith, resulting in tissue alterations and symptom development in tomato plants.