Salmonella Establishment in Agricultural Soil and Colonization of Crop Plants Depend on Soil Type and Plant Species

Abstract

Human pathogenic bacteria, such as Salmonella enterica, are able to colonize crop plants. So far, not much is known about biotic and abiotic factors influencing this colonization in field soil. This understanding, however, is imperative for the provision of safe fresh produce to the consumer. In this study, we investigated the effects of soil type, organic fertilization, plant species and the way of Salmonella entry into the plant production system, on the survival of S. enterica in soil as well as the colonization of plants. The selected S. enterica serovar Typhimurium strain 14028s, S. Typhimurium strain LT2 and S. Senftenberg were able to persist in soil for several weeks. Salmonella's persistence in soil was prolonged in loamy, if compared to sandy soil, and when applied together with organic fertilizer. The leaves of lettuce and corn salad were colonized by S. enterica providing evidence for internalization from the soil via the root. Colonization rates were affected by soil type, plant species and S. enterica strain. Overall, S. enterica was detected in leaves of 0.5-0.9% of the plants, while lettuce was more frequently colonized than corn salad. Plants grown in sandy soil were more often colonized than plants grown in loamy soil. After spray inoculation, S. enterica could be detected on and in leaves for several weeks by cultivation-depending methods, confirmed by confocal microscopy using GFP-labeled S. Typhimurium 14028s. On the one hand, transcriptome data from S. Typhimurium 14028s assessed in response to lettuce medium or lettuce root exudates showed an upregulation of genes associated with biofilm formation and virulence. On the other hand, lettuce inoculated with S. Typhimurium 14028s showed a strong upregulation of genes associated with plant immune response and genes related to stress response. In summary, these results showed that organic fertilizers can increase the persistence of Salmonella in soil and that soil type and plant species play a crucial role in the interactions between human pathogens and crop plants. This understanding is therefore a starting point for new strategies to provide safe food for the consumer.

Keywords: Salmonella; crop plants; internalization; persistence; plant defense; soil.

FIGURE 1

CFU counts of S. Typhimurium 14028s in DS and AL soil. Exemplarily shown is the decrease of S. Typhimurium 14028s colony forming unit (CFU) counts in diluvial sand (DS) (A) and alluvial loam (AL) (B) soil treated with or without pig manure. The Salmonella strain was introduced together with manure application (stored) or on day 0 directly before planting of lettuce (fresh). Shown are the mean values for treatments and respective standard deviations (n = 8). Samples with lettuce and corn salad as crop plants were averaged. Solid and dashed lines indicate linear regressions for soil treated with manure or without manure, respectively.

FIGURE 2

Colonization rates of lettuce and corn salad plants by Salmonella. Two plants were pooled into one single 50 mL screw cap centrifugation tube. Only leaves that were not in contact with the soil were sampled. The range of colonized plants (Salmonella detected in leaf samples after enrichment) is indicated by colored bars showing the range between the minimal and maximal possible colonization rate. The data are grouped, representing the assessed factors: soil type [diluvial sand (DS)/alluvial loam (AL)], plant (lettuce/corn salad), Salmonella strain (14028s/LT2/Senftenberg), time of Salmonella application [mixing day (stored)/day zero (fresh)] and fertilizer application (pig manure/chicken litter/without). In total, 3,024 plants were sampled excluding the control plants without Salmonella inoculation.

FIGURE 3

Time-dependent colonization of plants by Salmonella. The maximum proportion (maximum range) of lettuce and corn salad plants colonized by S. enterica over the experimental period is shown. The plant and soil types as well as the Salmonella strains are averaged. The solid line shows the linear regression, which in tendency is decreasing but not significantly (linear model analysis, p > 0.05, R² = 0.53).

FIGURE 4

Persistence of S. Typhimurium 14028s on lettuce and corn salad leaves. Bars display Salmonella CFU counts per g leaf fresh weight (fw) and corresponding standard deviations (n = 4) during the days after spray inoculation of the leaves. Asterisks indicate a detection of Salmonella only after enrichment. Different cell counts of Salmonella (CFU per mL) sprayed on day 0 are indicated by different shades of gray.

FIGURE 5

Colonization pattern of Salmonella on roots. S. Typhimurium 14028s GFP on root of lettuce (A,B) and corn salad (C,D) grown under sterile conditions. Plants were inoculated with GFP-expressing Salmonella for 3 days. Maximum projection images (A,C) and orthogonal scalings (B,D) indicate nucleus in blue, cell walls in red and GFP-labeled Salmonella cells in green. Orthogonal scalings show S. Typhimurium 14028s GFP cells in the cavity between primary and secondary root (B,D). The scale bars indicate 30 μm (A,B) and 40 μm (C,D).

FIGURE 6

Colonization pattern of Salmonella on lettuce leaves. Hydathode regions of lettuce leaves inoculated with S. Typhimurium 14028s GFP. Leaves were inoculated with S. Typhimurium 14028s GFP for 24 h. Maximum projection images (A,C) and orthogonal scalings (B,D) show hydathodes, nucleus and cuticula in blue, autofluorescence of chloroplasts in red and GFP-labeled Salmonella cells in green. Orthogonal scalings show S. Typhimurium 14028s GFP cells inside the hydathode openings (B,D). The scale bars indicate 20 μm.

FIGURE 7

Colonization pattern of S. Typhimurium 14028s GFP on corn salad leaves. Representative images were taken 6 days after spray inoculation with S. Typhimurium 14028s GFP. Maximum projection image (A) and orthogonal scaling (B) show stomatal openings and cuticula in blue, autofluorescence of chloroplasts in red and GFP-labeled Salmonella cells in green. Orthogonal scaling shows S. Typhimurium 14028s GFP cells attached to the cuticula but also inside of spongy parenchyma and stomatal cavities (B). The scale bars indicate 20 μm.

FIGURE 8

Differentially expressed lettuce genes in response to S. Typhimurium 14028s. Sterile lettuce plants were exposed to S. Typhimurium 14028s or 10 mM MgCl₂ for 24 h. The 30 genes with the highest increase in fold change and the 10 with the highest decrease in fold change are shown. Standard errors are indicated by error bars (p < 0.05, n = 3).

FIGURE 9

Lettuce response to S. Typhimurium 14028s. Changes in signaling pathways related to plant defense response after 24 h exposure to S. Typhimurium 14028s, compared to exposure to 10 mM MgCl₂. Genes were translated to amino acid sequences and those were assigned to Arabidopsis protein orthologs. Up- and down regulated genes in the particular pathways are indicated in green and red, respectively. White background indicates genes not found in the analysis. The figure was based on KEGG and Pathview.

FIGURE 10

Euler diagram of differentially expressed Salmonella genes in lettuce medium and lettuce exudates. The up- and downregulated genes from S. Typhimurium 14028s exposed to lettuce-based medium (LM) and lettuce exudates (LE) compared to the minimal medium (MM) as control are shown in green and red, respectively. Indicated are genes with significantly higher or lower expression (p < 0.05) as well as genes for which the expression was not detected in the treatment or the control. The bar plot indicates significantly enriched GO-terms of Salmonella from genes that were differentially regulated in expression after exposure to LE compared to MM as control.