Root mediated uptake of Salmonella is different from phyto-pathogen and associated with the colonization of edible organs

Abstract

Background: Pre-harvest contamination of fruits and vegetables by Salmonella in fields is one of the causes of food-borne outbreaks. Natural openings like stomata, hydathodes and fruit cracks are known to serve as entry points. While there are reports indicating that Salmonella colonize and enter root through lateral root emerging area, further investigations regarding how the accessibility of Salmonella to lateral root is different from phyto-pathogenic bacteria, the efficacy of lateral root to facilitate entry have remained unexplored. In this study we attempted to investigate the lateral root mediated entry of Salmonella, and to bridge this gap in knowledge.

Results: Unlike phytopathogens, Salmonella cannot utilize cellulose as the sole carbon source. This negates the fact of active entry by degrading plant cellulose and pectin. Endophytic Salmonella colonization showed a high correlation with number of lateral roots. When given equal opportunity to colonize the plants with high or low lateral roots, Salmonella internalization was found higher in the plants with more lateral roots. However, the epiphytic colonization in both these plants remained unaltered. To understand the ecological significance, we induced lateral root production by increasing soil salinity which made the plants susceptible to Salmonella invasion and the plants showed higher Salmonella burden in the aerial organs.

Conclusion: Salmonella, being unable to degrade plant cell wall material relies heavily on natural openings. Therefore, its invasion is highly dependent on the number of lateral roots which provides an entry point because of the epidermis remodeling. Thus, when number of lateral root was enhanced by increasing the soil salinity, plants became susceptible to Salmonella invasion in roots and its transmission to aerial organs.

Keywords: Epidermis remodeling; Lateral roots; Salinity; Salmonella; Tomato.

Fig. 1

In-vitro colonization of Salmonella is dependent on lateral roots. (a) Representative image depicting Salmonella (mcherry tagged) colonization near the lateral root emerging area. Root is stained with DAPI. (b) Number of lateral roots upon inoculation of various organisms on Day 1, 5 and 10. ANOVA was used to analyze the data. The comparison shown is between uninfected and infected plants at 10th day. *** = P < 0.001; ns = not significant. (c) Salmonella colonization upon inhibition and induction of lateral roots by NAA for Col-0 and axr 1–3 respectively. ANOVA was used to evaluate the significance. *** = P < 0.001. Each experiment was repeated 3 times with 5 replicate each. (d) Correlation of Salmonella colonization with lateral root (at 95% CI). Ralstonia solanacearum ΔhrpB was used as positive control and E.coli was used as non phytopathogenic bacteria control respectively. N = 50 for each organism for calculating correlation

Fig. 2

In-situ colonization of Salmonella is dependent on lateral roots. (a) Colonization of Salmonella in rhizoplane (left) and root tissue (right) of Col-0 and axr1–3 grown in soil. ANOVA was used to analyze the data. *** = P < 0.001; ns = not significant. Best fit line was plotted (at 95% CI). (b) Invasion index (Log CFU root tissue/Log CFU rhizoplane) for Col-0 and axr 1–3 was plotted over different days. One way ANOVA was used to evaluate the significance. *** = P < 0.001. Each experiment was repeated 3 times with 3 replicate each. (c) One-to-one correlation for root tissue colonization upon co-transplantation of Col-0 and axr 1–3 in same pot containing Salmonella (10⁸cells/g soil). The experiment was repeated 3 times with 5 replicate each. Wilcoxan rank test was used to analyse the data. *** = P < 0.001. (d) Representative image showing remodeling of epidermis and lateral root emergence with Salmonella colonization. The red arrow head shows the space created when lateral root (LR) emerges from primary root (PR). (e) Representative image of transverse section of Arabidopsis root showing Salmonella in between the epidermis (Ep) and cortical cells (Co). (f) Transverse section of emerged lateral root in tomato with 3 representative Z-stacks: i. Surface view of lateral root (LR) and primary root (PR); ii. Remodeling of epidermis (White arrow head) and Salmonella entry through the space between PR and LR; iii. Salmonella colonization in between epidermis and cortical cell layer

Fig. 3

Salinity renders the plant susceptible to Salmonella. (a) Number of lateral roots upon treatment with varing amount of NaCl. (b) Salmonella colonization on arabidopsis roots treated with 50 mM and 100 mM NaCl. One way ANOVA followed by Bonferroni’s post test was used to analyse the data. Different letters shows significance at P < 0.05. (c) Correlation between salt stress and Salmonella colonization on arabidopsis root (at 95% CI). The values in parenthesis represent number of lateral root (Mean ± SD). (d) Number of lateral root of tomato plotted against varying salt stress. One way ANOVA followed by Bonferroni’s post test was used to analyse the data. P < 0.001 = ***; ns = not significant. (e) Tomato fruits infected with Salmonella upon mild salinity stress (25 mg NaCl/g soil; EC = 2.25dS/m). Each experiment was repeated 3 times with 3 replicate each. Mann-Whitney test was used to analyse the data. P < 0.05 = *