Revisiting the Source of Wilt Symptoms: X-Ray Microcomputed Tomography Provides Direct Evidence ThatRalstoniaBiomass Clogs Xylem Vessels

Abstract

Ralstonia cause wilt diseases by colonizing xylem vessels and disrupting water transport. The dogma is that bacterial biomass clogs vessels and reduces the flow of xylem sap due to Ralstonia abundance. However, the physiological mechanism of xylem disruption during bacterial wilt is untested. Using a tomato and Ralstonia pseudosolanacearum GMI1000 model, we visualized and quantified spatiotemporal dynamics of xylem disruption during bacterial wilt. First, we measured stomatal conductance of leaflets on mock-inoculated and wilt-symptomatic plants. Wilted leaflets had reduced stomatal conductance, as did turgid leaflets on the same petiole as wilted leaflets. Next, we used X-ray microcomputed tomography (X-ray microCT) and light microscopy to differentiate between mechanisms of xylem disruption: blockage by bacterial biomass, blockage by vascular tyloses, or sap displacement by gas embolisms. We imaged intact plant stems to quantify embolized vessels. Embolized vessels were rare, but infected plants with low bacterial populations had a non-significant trend of more vessel embolisms. To test that vessels are clogged during bacterial wilt, we imaged excised stems after brief dehydration. Most vessels in mock-infected plants emptied their contents after excision, but non-conductive clogged vessels were abundant in infected plants by 2 days post infection. At wilt onset when bacterial populations exceeded 5x108 cfu/g stem tissue, approximately half of the vessels were clogged with electron-dense bacterial biomass. We found no evidence of tyloses in X-ray microCT reconstructions or from light microscopy of preserved stems. Therefore, bacterial blockage of vessels appears to be the principal cause of xylem disruption during Ralstonia wilt.