Bioluminescent Xanthomonas hortorum pv. gardneri as a Tool to Quantify Bacteria in Planta, Screen Germplasm, and Identify Infection Routes on Leaf Surfaces

Abstract

Imaging technology can provide insight into biological processes governing plant-pathogen interactions. We created and used a bioluminescent strain of Xanthomonas hortorum pv. gardneri (Xgb) to quantify infection processes in plants using tomato as a model. An X. hortorum pv. gardneri is one of the four Xanthomonas species that causes bacterial spots in tomatoes. We used Xgb to quantify bacterial growth in planta, to assess disease severity in resistant and susceptible tomato lines, and to observe infection routes in leaves. A positive and significant linear correlation r (67) = 0.57, p ≤ 0.0001 was observed between bioluminescence signals emitted by Xgb in planta and bacterial populations determined through dilution plating. Based on bioluminescence imaging, resistant and susceptible tomato lines had significantly different average radiances. In addition, there was a positive and significant correlation r = 0.45, p = 0.024 between X. hortorum pv. gardneri-inoculated tomato lines evaluated by bioluminescence imaging and tomatoes rated in the field using the Horsfall-Barrat Scale. Heritability was calculated to compare the genetic variance for disease severity using bioluminescence imaging and classical field ratings. The genetic variances were 25 and 63% for bioluminescence imaging and field ratings, respectively. The disadvantage of lower heritability attained by bioluminescence imaging may be offset by the ability to complete germplasm evaluation experiments within 30 days rather than 90–120 days in field trials. We further explored X. hortorum pv. gardneri infection routes on leaves using spray and dip inoculation techniques. Patterns of bioluminescence demonstrated that the inoculation technique affected the distribution of bacteria, an observation verified using scanning electron microscopy (SEM). We found significant non-random distributions of X. hortorum pv. gardneri on leaf surfaces with the method of inoculation affecting bacterial distribution on leaf surfaces at 4 h postinoculation (hpi). At 18 hpi, regardless of inoculation method, X. hortorum pv. gardneri localized on leaf edges near hydathodes based on bioluminescence imaging and confirmed by electron microscopy. These findings demonstrated the utility of bioluminescent X. hortorum pv. gardneri to estimate bacterial populations in planta, to select for resistant germplasm, and to detect likely points of infection.

Arabidopsis hydathodes are sites of intense auxin metabolism and nutrient scavenging

“…Healthy hydathodes have been reported to host a rich microbial community (Perrin et al, 1972). Several vascular bacterial pathogens of the Xanthomonas and Clavibacter genera have evolved to colonize this niche and can infect hydathodes (Lewis and Goodman, 1966; Carlton et al, 1998; Hugouvieux et al, 1998; Cerutti et al, 2017; Bernal et al, 2021; Mullens and Jamann, 2021). In contrast to stomata, hydathode pores seem unable to fully close and may thus facilitate pathogen entry and infection (Cerutti et al, 2017).…”

Phylogenetic Distribution and Evolution of Type VI Secretion System in the Genus Xanthomonas

“…While X. hortorum pv. gardneri is not known to colonize vascular tissues, Bernal et al (2021) recently showed colonization of hydathodes upon inoculation of tomato plants with X. hortorum pv. gardneri.…”

ScAnalyzer: an image processing tool to monitor plant disease symptoms and pathogen spread in Arabidopsis thaliana leaves

“…Attempts to quantify the bacterial luminescence signal have relied on using luminometers to measure photon emission by bacteria that colonize a single leaf or plant (22). In some cases, the image processing software ImageJ is used to obtain numerical data from images obtained with ultra-sensitive CCD cameras (21,28).…”