Revisiting the source of wilt symptoms: X-ray microcomputed tomography provides direct evidence that Ralstonia biomass clogs xylem vessels

Authors: INGEL, BRIAN - University Of California, Davis; CALDWELL, DENISE - Purdue University; DUONG, FIONA - University Of California, Davis; PARKINSON, DILWORTH - Lawrence Berkeley National Laboratory; MCCULLOH, KATHERINE - University Of Wisconsin; IYER-PASCUZZI, ANJALI - Purdue University; McElrone, Andrew; LOWE-POWER, TIFFANY - University Of California, Davis

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/23/2021
Publication Date: 1/12/2022
Citation: Ingel, B., Caldwell, D., Duong, F., Parkinson, D.Y., McCulloh, K.A., Iyer-Pascuzzi, A.S., McElrone, A.J., Lowe-Power, T.M. 2022. Revisiting the source of wilt symptoms: X-ray microcomputed tomography provides direct evidence that Ralstonia biomass clogs xylem vessels. Phytofrontiers. 2(1):41-51. https://doi.org/10.1094/PHYTOFR-06-21-0041-R.
DOI: https://doi.org/10.1094/PHYTOFR-06-21-0041-R

Interpretive Summary:

Technical Abstract: Plant pathogenic Ralstonia cause wilt diseases by colonizing xylem vessels and disrupting their function. Due to the abundance of Ralstonia cells in xylem vessels, the dogma is that bacterial biomass clogs vessels and reduces the flow of xylem sap. However, the physiological mechanism of xylem dysfunction during bacterial wilt disease is untested. Although Ralstonia is abundant within xylem vessels, Ralstonia biofilm is highly fluidal, so it remains unclear whether Ralstonia cells and biofilm directly restrict sap flow. Using a tomato and Ralstonia pseudosolanacearum GMI1000 model, we visualized and quantified the spatiotemporal dynamics of xylem dysfunction during bacterial wilt disease. Gas exchange measurements showed that wilted leaflets had little-to-no stomatal conductance. Visually turgid leaflets on symptomatic, infected plants had lower stomatal conductance than turgid leaflets on healthy plants. Moreover, proximity to wilted leaflets influenced the stomatal conductance of visually turgid leaflets. Turgid leaflets co-located on a petiole with wilted leaflets had significantly lower stomatal conductance than leaflets elsewhere on the plant. Parallel results were obtained in tomato plants inoculated with three diverse R. solanacearum strains. We used X-ray microcomputed tomography (X-ray microCT) and microscopy to differentiate between mechanisms of xylem dysfunction: blockage by bacteria, blockage by vascular tyloses, or sap displacement by gas embolisms. We analyzed stems from healthy and Ralstonia-infected tomato plants at 1 and 2 days post inoculation. We found no evidence of tyloses in the X-ray microCT reconstructions or light microscopy on the preserved stems. At wilt onset when bacterial populations exceeded 5x108 cfu per g stem tissue, approximately half of the xylem vessels were clogged with electron-dense bacterial biofilm. Although there was a slight trend of increased xylem vessel embolisms in pre-symptomatic infected plants, bacterial blockage of the vessels appears to be the predominant cause of vascular decline.