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.

Root cell types as an interface for biotic interactions

“…(B) Tyloses block the lumen of the xylem vessel, forming a potential barrier for vascular wilt pathogens, limiting their spread along the root longitudinal axis in the shootward direction and further progression to the shoot [49,54]. The effectiveness of tyloses for plant resistance is controversial [55,56].…”

“…(B) Vascular wilt pathogens induce coating of the xylem vessel with lignin, callose, and phenolic compounds which block these pathogens from entry into the vasculature and/or spread from vessel to vessel [50,51]. Tyloses have been suggested to form additional barriers in xylem [49,54], but their effectiveness remains controversial [55,56]. (C,D) Differentiation programs of root cells act as innate barriers for pathogens.…”

Trophic preferences of the pathogen Ralstonia solanacearum and consequences on its growth in xylem sap

“…It then progressively invades the xylem vessels and disperses rapidly to the aerial part of the plant (stems and leaves) through the vascular system (Peeters et al, 2013 ; Plener et al, 2012 ). Wilting symptoms probably result from the extensive bacterial colonization of the xylem and the production of a large amount of exopolysaccharides (EPS) leading to the obstruction of the vessels and plant water transport (Ingel et al, 2021 ).…”

Whole genome sequencing suggests that “non-pathogenicity on banana (NPB)” is the ancestral state of the Ralstonia solanacearum IIB-4 lineage

“…The Ralstonia solanacearum species complex is a group of related tropical and temperate pathogens that cause plant wilt diseases after they invade and colonize the xylem (Prior et al 2016; Lowe-Power et al 2018; Ingel et al 2022). Collectively, the species complex has a remarkably broad host range, capable of infecting hundreds of host species in dozens of taxonomic families (Hayward 1991; Lowe-Power et al 2020).…”