Determination of Salmonella enterica Leaf Internalization Varies Substantially According to the Method and Conditions Used to Assess Bacterial Localization

Abstract

In a previous study, comparing the internalization of S. enterica serovar Typhimurium in various leaves by confocal microscopy, we have demonstrated that the pathogen failed to internalize tomato leaves. Numerous reasons may account for these findings, yet one such factor might be the methodology employed to quantify leaf internalization. To this end, we have systematically studied leaf localization of a Green-fluorescent protein-labeled Salmonella strain in tomato, lettuce, and Arabidopsis leaves by surface sterilization and enumeration of the surviving bacteria, side by side, with confocal microscopy observations. Leaf sterilization was performed using either sodium hypochlorite, silver nitrate, or ethanol for 1 to 7min. The level of internalization varied according to the type of disinfectant used for surface sterilization and the treatment time. Treatment of tomato leaves with 70% ethanol for up to 7min suggested possible internalization of Salmonella, while confocal microscopy showed no internalization. In the case of in lettuce and Arabidopsis leaves, both the plate-count technique and confocal microscopy demonstrated considerable Salmonella internalization thought different sterilization conditions resulted in variations in the internalization levels. Our findings highlighted the dependency of the internalization results on the specific disinfection protocol used to determine bacterial localization. The results underscore the importance of confocal microscopy in validating a particular surface sterilization protocol whenever a new pair of bacterial strain and plant cultivar is studied.

Xanthomonas infection transforms the apoplast into an accessible and habitable niche for Salmonella enterica

“…The apoplast can be protective and provide bacteria with increased nutrient availability, however, this environment is exclusive to microbes that can successfully migrate there and tolerate the host immune response. Tomato leaves are relatively restrictive to S. enterica ingression (26,27), yet we found that S. enterica can access an apoplast that is watersoaked either by Xanthomonas infection or abiotically. Additionally, comparison of S. enterica populations between experiments sampled at 2.5 hours post-arrival and 3 days post-arrival indicates that S. enterica populations are stable within a healthy apoplast and increase within an infected one.…”

“…The flagella- dependent localization of S. enterica to internal tissue in response to light has been demonstrated via confocal microscopy of lettuce leaf pieces submerged in S. enterica inoculum (25). However, tomato leaves have been shown to be impermeable to S. enterica internalization when compared with lettuce leaves under comparable conditions (26, 27). Whether or not phytopathogen infection could enhance the permeability of leaves to enteric pathogens was not addressed by these studies.…”

Pre‐heated blades for harvesting baby‐leaves reduce the risk of Escherichia coli internalization in leaves

“…17,26 The plant tissue can therefore protect cells against decontamination procedures. In addition, because the cells can enter into leaves from wounds and open stomata, 20,21 the harvest cutting site may also be responsible for the bacterial internalization in leaves.…”

“…The cutting site may also be responsible for bacterial internalization in leaves, in addition to wounds and open stomata. 20,21 In industry, salads and baby-leaves can be cut using a mechanized harvester (Fig. 1A).…”

RpoS contributes in a host-dependent manner to Salmonella colonization of the leaf apoplast during plant disease

“…These studies have also shown that S. enterica colonization of plants is not limited to external plant surfaces. S. enterica can become internalized through roots and is able to actively bypass stomata to colonize the leaf apoplastic space ( Gu et al, 2011 ; Karmakar et al, 2018 ; Rodrigues Oblessuc et al, 2019 ; Chahar et al, 2021 ).…”