ABSTRACT

We investigated whether the co-occurrence of phytopathogens (Clavibacter michiganensis subsp. michiganensis [Cmm] and Xanthomonas gardneri [Xg]) frequently encountered in tomato production and Salmonella enterica subsp. enterica serotype Typhimurium (strain JSG626) affects the persistence of these pathogens in tomato plant tissues during the early stages of plant growth. Cmm increased the recovery of Salmonella Typhimurium (up to 1.8 log CFU per plant at 21 days postinoculation [DPI]) from coinoculated tomato plants compared with plants inoculated with Salmonella Typhimurium alone (P < 0.05). Xg had no effect on Salmonella Typhimurium persistence in the plants. Increased persistence of Salmonella Typhimurium was also observed when it was inoculated 7 days after Cmm inoculation of the same plant (P < 0.05). In contrast, Salmonella Typhimurium reduced the population of both Cmm and Xg (up to 1.5 log CFU per plant at 21 DPI; P < 0.05) in coinoculated plants compared with plants inoculated with Cmm or Xg alone. The Xg population increased (1.16 log CFU per plant at 21 DPI; P < 0.05) when Salmonella Typhimurium was inoculated 7 days after Xg inoculation compared with plants inoculated with Xg alone. Our findings indicate that the type of phytopathogen present in the phyllosphere and inoculation time influence the persistence of Salmonella Typhimurium JSG626 and its interactions with phytopathogens cocolonized in tomato plants. Salmonella reduced the phytopathogen load in plant tissues, and Cmm enhanced the recovery of Salmonella from the coinoculated plant tissues. However, further investigations are needed to understand the mechanisms behind these interactions.

HIGHLIGHTS
  • Salmonella Typhimurium inhibited two phytopathogens coinoculated in tomato plant tissues.

  • Inhibition of the phytopathogens might be associated with an antimicrobial effect.

  • C. michiganensis subsp. michiganensis enhanced Salmonella Typhimurium persistence.

Nontyphoid Salmonella strains are a leading cause of foodborne gastroenteritis worldwide and in the United States account for 1.2 million illness cases and 450 deaths and costs of $360 million annually (8, 9). Current social trends highlight the substantial health benefits of fresh produce in our daily diet (12, 69). However, fresh produce (fruits, seeds, and sprouts) also have been associated with outbreaks of salmonellosis worldwide (ca. 15% of all salmonellosis cases) over the last several decades (9, 10, 13, 15, 16, 30, 32, 41, 68, 71). Nevertheless, salmonellosis outbreaks associated with fresh produce remain infrequent, suggesting that specific biotic conditions (e.g., phytobiome, genotype, inoculum, and surrounding vectors) (4, 5, 14, 24, 60, 70) and abiotic conditions (e.g., environmental conditions, agricultural inputs, physicochemical properties of Salmonella surroundings, and nutrient availability) (20, 23, 36, 39, 4244, 46) are required for the contamination of crops by Salmonella enterica. Disinfecting agents (e.g., chlorine) have limited impact on the persistence of Salmonella internalized in host tissues because of the fragile nature of fresh produce (21, 45). Therefore, it is important to understand the conditions enhancing the persistence of Salmonella in plant tissues to minimize risk and assure safe consumption of fresh and minimally processed produce.

To persist in plant tissues, Salmonella first must compete for space and nutrients with the phytobiome and overcome basal plant defenses (5, 22, 25, 35). Plant tissues with constant epidermal remodeling (e.g., root tips) are preferential survival and internalization sites for Salmonella because of weaker cell walls, the presence of wounds, the availability of nutrients, and reduced microbial competition due to low pH (11, 20, 37, 38). Specific histological features (e.g., trichome quality and density, hydathodes, and stomata) enhance the attachment, persistence, and internalization of Salmonella in the foliage and provide shelter from adverse environmental conditions (5, 17, 31).

Salmonella also must compete with the epiphytic microorganisms in the phytosphere where water and nutrients can be limited (60). Enterobacteria produce toxic peptides (bacteriocins) that inhibit the growth of closely related microorganisms by affecting the integrity of the cell envelope. Bacteriocins facilitate the colonization of a specific environment and the scavenging of nutrients (62). Therefore, the production of bacteriocins provides a competitive advantage for the installation of exogenous organisms in a hostile environment colonized by a native microbial community. The presence of an undisturbed native phytobiome and high microbial diversity reduced the persistence of Salmonella in plant tissues (14, 23, 40, 50, 52, 55, 61, 66). Various plant pathogens (Xanthomonas perforans, Xanthomonas campestris, Xanthomonas vescicatoria, Pseudomonas syringae, Pectobacterium carotovorum, lettuce mosaic virus, Ralstonia solanacearum, and Erwinia herbicola) can significantly enhance Salmonella persistence in plant tissues (1, 7, 26, 59, 60, 72). Plant pathogenic bacteria suppress plant defenses through the type III secretion system (TTSS) and weaken basal plant defenses, which release plant cell contents and increase free-water content in the infected area (65). These alterations may create a more conducive environment for the persistence of Salmonella in plant tissues, increase its competitiveness against native microbial populations of the phyllosphere, and facilitate its internalization in plant tissues (23, 60). Therefore, plants infected with a phytopathogen might be at higher risk for foodborne pathogen infection, which can affect food safety. However, little is known concerning the nature of the interactions between Salmonella, phytopathogens, and the plant host.

This study was conducted to analyze the impact of phytopathogenic bacteria that cause local infections (Xanthomonas gardneri strain SM775.12 [Xg]) or systemic infections (Clavibacter michiganensis subsp. michiganensis strain C290 [Cmm]) on the recovery of S. enterica subsp. enterica serotype Typhimurium (strain JSG626) from tomato plants and vice versa for 3 weeks postinoculation.

Bacterial strains

S. enterica Typhimurium LT2 (strain JSG626; responsible for gastroenteritis in humans), Xg (strain Xg SM775.12; local pathogen that causes bacterial spot of tomato) (51), and Cmm (strain C290; systemic pathogen that causes bacterial canker of tomato) (73) were used as models to understand the interactions between Salmonella and phytopathogens (Xg and Cmm) in plant tissues. Salmonella Typhimurium JSG626 (Dr. John Gunn, The Ohio State University, Columbus) is a well characterized and commonly used strain of Salmonella Typhimurium (1921, 56). This strain was selected for this study because among the seven Salmonella Typhimurium strains tested in our preliminary studies (unpublished data), it was the most persistent over time when tomato seedlings were either spray or clip inoculated. Use of this strain also allows further characterization of bacterial genes implicated in the persistence of Salmonella in plant tissues and the interactions between Salmonella and phytopathogens. Salmonella Typhimurium JSG626 was grown in Luria-Bertani broth for 24 h at 28°C, and the phytopathogens were grown in nutrient broth with yeast extract (NBY) for 48 h at 28°C to prepare the inocula.

Tomato varieties

‘Tiny Tim' (Johnny's Selected Seeds, Winslow, ME) tomato plants were used as a model to investigate interactions between Salmonella and phytopathogens. Tiny Tim is a dwarf tomato plant variety that produces a high number of ripe cherry tomatoes within a relatively short period (ca. 7 to 8 weeks), making this a useful model plant to study host-pathogen interactions, particularly with in vivo imaging. Fresh market tomatoes are consumed raw with minimal processing steps and have been recognized as a source of salmonellosis (32). Tiny Tim seeds were sown and grown in a greenhouse (22 to 28°C, 20 to 80% relative humidity, 12-h photoperiod) as previously described (20, 21, 47).

Interaction between Salmonella and Xg or Cmm in tomato plants

The impact of systemic (Cmm) and local (Xg) phytopathogens on the persistence of Salmonella Typhimurium JSG626 in inoculated tomato plant tissues was studied using coinoculation and sequential inoculation strategies to mimic conditions where both the foodborne pathogen and the phytopathogens are introduced at the same time or at different time points using contaminated agricultural inputs (e.g., water or cutting tools).

Interaction between Salmonella and Xg or Cmm in tomato plants: coinoculation

Three-week-old Tiny Tim tomato plants (five- to six-leaf stage) were inoculated with Salmonella Typhimurium JSG626 and Xg or Cmm at the same time using the same inoculation method (ca. 1 mL per plant; 100:1) (Fig. 1). Plants were inoculated either by spraying leaves with Xg-contaminated water (ca. 1 × 106 CFU/mL) (48) or by cotyledon clipping with scissors dipped in Cmm-contaminated water (ca. 1 × 106 CFU/mL) (73). Plants inoculated with either sterile water, Salmonella, Xg, or Cmm alone at the same level were used as controls. Two trials were conducted (six treatment groups, 16 plants per group, 96 plants per trial), and samples were collected from four plants at 0, 7, 14, and 21 days postinoculation (DPI) at each time in each trial. The experiments were conducted for 21 days because in our earlier study we found that Salmonella Typhimurium JSG626 can persist in inoculated plant tissues under the same growing conditions for up to 21 DPI (20).

FIGURE 1

Experiment design. Tomato plants were inoculated by (A) the foliage spray method (ca. 1 mL per plant) with Xanthomonas gardneri SM775.12 (Xg; 106 CFU/mL) and (B) the cotyledon clip method with Clavibacter michiganensis subsp. michiganensis C290 (Cmm; 106 CFU/mL). Salmonella enterica subsp. enterica serotype Typhimurium JSG626 (108 CFU/mL) was inoculated into tomato plants with the same methods used for the phytopathogens. (C) Time points and procedures for coinoculation of Salmonella and a phytopathogen (Xg or Cmm). DPI, days postinoculation. (D) Time points and procedures for sequential inoculation of Salmonella and a phytopathogen (Xg or Cmm) when the phytopathogen was inoculated 1 week before Salmonella inoculation.

FIGURE 1

Experiment design. Tomato plants were inoculated by (A) the foliage spray method (ca. 1 mL per plant) with Xanthomonas gardneri SM775.12 (Xg; 106 CFU/mL) and (B) the cotyledon clip method with Clavibacter michiganensis subsp. michiganensis C290 (Cmm; 106 CFU/mL). Salmonella enterica subsp. enterica serotype Typhimurium JSG626 (108 CFU/mL) was inoculated into tomato plants with the same methods used for the phytopathogens. (C) Time points and procedures for coinoculation of Salmonella and a phytopathogen (Xg or Cmm). DPI, days postinoculation. (D) Time points and procedures for sequential inoculation of Salmonella and a phytopathogen (Xg or Cmm) when the phytopathogen was inoculated 1 week before Salmonella inoculation.

Close modal

Interaction between Salmonella and Xg or Cmm in tomato plants: sequential inoculation

In a similar experiment, the plants were inoculated with Cmm or Xg 7 days before inoculation with Salmonella Typhimurium JSG626 instead of at the same time (Fig. 1). The same inoculation methods and growth conditions were used. Two trials (six treatment groups, 20 plants per group, 120 plants per trial) were conducted, and samples were collected from four plants per group at −7, 0, 7, 14, and 21 DPI at each time in each trial.

Processing of plant tissues

Samples were processed as previously described (20, 21). Soil attached to the roots was manually removed by shaking the root system and by rinsing the roots in tap water (pH 7, 50 to 100 mL depending on the size of the root system). For plants inoculated by cotyledon clip with Cmm, cotyledons, leaves, stem, and roots were collected into separate Whirl-Pak sample bags (Nasco, Fort Atkinson, WI) containing sterile water and processed individually. However, for plants spray inoculated with Xg, the whole plant was processed as one sample. The size of the bag and the volume of sterile water added were based on the amount of plant tissue (207- to 709-mL bag, 1 to 10 mL of sterile water). Tissues were macerated and serially diluted 10-fold. For Salmonella, samples were plated on xylose lysine Tergitol 4 agar plates and incubated for 36 h at 37°C. For Cmm and Xg, samples were plated on yeast dextrose calcium carbonate (YDC) agar plates and incubated for 72 h at 28°C.

Antimicrobial activity of Salmonella whole culture and Salmonella supernatant on Xg and Cmm in vitro as determined with agar well diffusion assays

The antimicrobial activity of Salmonella Typhimurium JSG626 against Cmm and Xg was tested with an agar well diffusion assay (3). Salmonella whole culture was obtained by growing Salmonella Typhimurium JSG626 in YDC for 72 h at 28°C. YDC was used to obtain the Salmonella whole culture because the phytopathogens were evaluated on this medium in plant studies. A suspension of Cmm or Xg (100 μL) normalized at ca. 1 × 106 CFU/mL in fresh NBY medium was spread homogeneously on YDC agar plates. Wells (10 mm in diameter) were created in the YDC agar plate with a 20-μL pipette tip (Mettler-Toledo Rainin, Oakland, CA), and 100 μL of Salmonella Typhimurium JSG626 whole culture was transferred into each well (ca. 108 CFU per well). Plates were incubated up to 5 days at 28°C. Zones of growth inhibition (radius in millimeters from the edge of the well) were measured daily. Wells filled with medium, 1× phosphate-buffered saline, and a whole culture of Cmm or Xg (108 CFU per well under growing conditions used for Salmonella whole culture) were used as controls. The experiment was conducted three times.

Because Salmonella whole culture had antibacterial effects on Cmm and Xg in the agar gel diffusion assay, the antimicrobial activity of Salmonella Typhimurium JSG626 supernatant against Cmm and Xg was also evaluated using the agar well diffusion assay (3). To obtain Salmonella cell-free supernatant, bacteria were grown in YDC broth as above, and culture supernatant was centrifuged at 10,000 × g for 7 min and filtered through a 0.2-μm-pore-size filter membrane. The experiment was conducted three times.

Statistical analyses

For each experiment, plants were randomized between treatment groups after inoculation. Bacterial data were log transformed. Statistical analyses were performed with JMP PRO 12/14 software (SAS Institute, Cary, NC). A one-way analysis of variance combined with a Tukey test was used to assess the differences in bacterial populations between the treatment groups for the plant tissues and time points studied. All plant experiments were conducted twice.

Xg had no effect on the recovery of Salmonella Typhimurium from tomato plants

The interaction between Xg and Salmonella Typhimurium JSG626 was studied in Tiny Tim tomato plants at 3 to 6 weeks after seeds were sown (Figs. 1 and 2). When plants were spray inoculated with Salmonella Typhimurium JSG626 and Xg (100:1) together on the leaves, the presence of Xg did not affect the recovery of Salmonella Typhimurium JSG626 from tomato plants over time compared with plants inoculated with Salmonella Typhimurium JSG626 alone (Fig. 2A). In the presence or absence of Xg, the Salmonella Typhimurium JSG626 population was significantly decreased at 7 DPI compared with 0 DPI (Fig. 2A) (P < 0.05) and remained stable at 14 and 21 DPI compared with the previous sampling time.

FIGURE 2

Interactions between Salmonella enterica subsp. enterica serotype Typhimurium JSG626 and Xanthomonas gardneri SM775.12 (Xg) inoculated into tomato plants. The two pathogens were coinoculated at the same time (A, C) or sequentially, i.e., Xg was inoculated 7 days before Salmonella inoculation (B, D). Histograms show populations of Salmonella (A, B) and Xg (C, D) in tomato plants. Salmonella was inoculated using the same spray method as used for Xg (100:1, Salmonella:Xg). Bars indicate standard deviations (four plants per group per time point). Asterisks indicate significant differences in Xg populations between coinoculated plants and plants inoculated with Xg alone (P < 0.05) at each time point. Differences between time points are not indicated.

FIGURE 2

Interactions between Salmonella enterica subsp. enterica serotype Typhimurium JSG626 and Xanthomonas gardneri SM775.12 (Xg) inoculated into tomato plants. The two pathogens were coinoculated at the same time (A, C) or sequentially, i.e., Xg was inoculated 7 days before Salmonella inoculation (B, D). Histograms show populations of Salmonella (A, B) and Xg (C, D) in tomato plants. Salmonella was inoculated using the same spray method as used for Xg (100:1, Salmonella:Xg). Bars indicate standard deviations (four plants per group per time point). Asterisks indicate significant differences in Xg populations between coinoculated plants and plants inoculated with Xg alone (P < 0.05) at each time point. Differences between time points are not indicated.

Close modal

A similar trend was observed when plant leaves were spray inoculated with Xg 7 days before Salmonella Typhimurium JSG626 inoculation (sequential inoculation) (Figs. 1 and 2B). Salmonella Typhimurium JSG626 population in plant tissues did not change in the presence of Xg. However, the Salmonella population was significantly decreased at 7 DPI compared with 0 DPI and at 21 DPI compared with 14 DPI in the presence or absence of Xg (Fig. 2B) (P < 0.05).

Salmonella Typhimurium altered Xg populations in tomato plants

The population of Xg was also monitored in plants coinoculated with Salmonella Typhimurium JSG626 (Fig. 2C). When Xg was spray inoculated alone on leaves, the Xg population significantly increased in the plant tissues (foliage and root system processed together) at 7 DPI compared with 0 DPI and remained the same at 14 and 21 DPI compared with the previous sampling time (P < 0.05). In contrast, the presence of Salmonella Typhimurium JSG626 significantly reduced Xg levels in coinoculated plants (by ca. 0.5 and 1.5 log CFU per plant at 7 and 21 DPI, respectively) compared with plants inoculated with Xg alone (Fig. 2C) (P < 0.05). The Xg population in coinoculated plants was similar between 0 and 21 DPI.

Different trends were observed when plant leaves were spray inoculated with Xg 7 days before Salmonella Typhimurium JSG626 inoculation (sequential inoculation) (Fig. 2D). The Xg population significantly increased at 21 DPI (by ca. 1.16 log CFU per plant) when Xg and Salmonella Typhimurium JSG626 were sequentially inoculated compared with plants inoculated with only Xg (P < 0.05). In the presence or absence of Salmonella Typhimurium JSG626, Xg populations were similar between −7 and 7 DPI and were significantly increased at 14 DPI compared with 7 DPI (P < 0.05).

Cmm enhanced the recovery of Salmonella Typhimurium from tomato plants

Similar experiments were performed with Cmm and Salmonella Typhimurium JSG626 in which plants were inoculated by clipping a cotyledon with scissors dipped in inoculum (Figs. 1 and 3). When Salmonella Typhimurium JSG626 was clip inoculated alone, Salmonella populations were significantly decreased at 7 DPI compared with 0 DPI (Fig. 3A) (P < 0.05) and remained stable at 14 and 21 DPI compared with the previous sampling time. In contrast, the presence of Cmm significantly increased Salmonella Typhimurium JSG626 populations in coinoculated plants at 7, 14, and 21 DPI (by ca. 1.0, 2.5, and 1.8 log CFU per pair of cotyledons [PC]) compared with plants inoculated with Salmonella Typhimurium JSG626 alone (Fig. 3A) (P < 0.05).

FIGURE 3

Interactions between Salmonella enterica subsp. enterica serotype Typhimurium JSG626 and Clavibacter michiganensis subsp. michiganensis C290 (Cmm) inoculated into tomato plants. The two pathogens were coinoculated at the same time (A, C) or sequentially, i.e., Cmm was inoculated 7 days before Salmonella inoculation (B, D). Histograms show populations of Salmonella (A, B) and Cmm (C, D) in tomato plants. Salmonella was inoculated using the same cotyledon clip method as used for Cmm (100:1, Salmonella:Cmm). PC, pair of cotyledons. Bars indicate standard deviations (four plants per group per time point). Asterisks indicate significant differences in Salmonella and Cmm populations between coinoculated plants and plants inoculated with either pathogen alone (P < 0.05) at each time point. Differences between time points are not labeled. Arrows indicate estimated time point of wilting and detachment of inoculated cotyledons from the plant.

FIGURE 3

Interactions between Salmonella enterica subsp. enterica serotype Typhimurium JSG626 and Clavibacter michiganensis subsp. michiganensis C290 (Cmm) inoculated into tomato plants. The two pathogens were coinoculated at the same time (A, C) or sequentially, i.e., Cmm was inoculated 7 days before Salmonella inoculation (B, D). Histograms show populations of Salmonella (A, B) and Cmm (C, D) in tomato plants. Salmonella was inoculated using the same cotyledon clip method as used for Cmm (100:1, Salmonella:Cmm). PC, pair of cotyledons. Bars indicate standard deviations (four plants per group per time point). Asterisks indicate significant differences in Salmonella and Cmm populations between coinoculated plants and plants inoculated with either pathogen alone (P < 0.05) at each time point. Differences between time points are not labeled. Arrows indicate estimated time point of wilting and detachment of inoculated cotyledons from the plant.

Close modal

Similar increases in Salmonella Typhimurium JSG626 populations were observed at 7 DPI compared with 0 DPI and at 14 DPI compared with 7 DPI (by ca. 1.07 and 0.82 log CFU per PC, respectively) when plants were clip inoculated with Cmm 7 days before Salmonella Typhimurium JSG626 inoculation compared with plants clip inoculated with only Salmonella Typhimurium JSG626 (Fig. 3B) (P < 0.05). However, Salmonella Typhimurium JSG626 populations still significantly decreased over time for both conditions (Salmonella Typhimurium JSG626 in the presence or absence of Cmm). Nevertheless, Salmonella Typhimurium JSG626 was still detected at 14 DPI in inoculated cotyledons in the presence of Cmm (ca. 0.8 log CFU per PC) but was not detected when Salmonella Typhimurium JSG626 was introduced alone (Fig. 3B). Salmonella Typhimurium JSG626 was not detected in inoculated cotyledons 21 DPI because of the senescence and detachment of the cotyledons from the plants between 14 and 21 DPI (Fig. 3B, arrow), as observed with mock-inoculated plants (data not shown).

Salmonella Typhimurium reduced Cmm populations in tomato plants when coinoculated

The populations of Cmm were also monitored in plants coinoculated with Salmonella Typhimurium JSG626. The presence of Salmonella Typhimurium JSG626 significantly decreased Cmm populations in coinoculated cotyledons at 7 and 14 DPI (by ca. 1.38 and 0.7 log CFU per PC, respectively) compared with plants inoculated with Cmm alone (Fig. 3C) (P < 0.05). When Cmm was clip inoculated alone, its population was significantly increased 7 DPI compared with 0 DPI and 14 DPI compared with 7 DPI (Fig. 3C) (P < 0.05). When Cmm was clip inoculated with Salmonella Typhimurium JSG626, the Cmm population was still significantly increased at 7 DPI compared with at 0 and 14 DPI (Fig. 3C) (P < 0.05). The Cmm population was the same in the presence or absence of Salmonella Typhimurium JSG626 in the noninoculated tissues (stem, roots, and foliage) (data not shown).

No reductions in Cmm populations were detected in cotyledons in the presence or absence of Salmonella Typhimurium JSG626 when plants were clip inoculated with Cmm 7 days before Salmonella Typhimurium JSG626 inoculation (Fig. 3D). Cmm populations were significantly higher at 0 DPI compared with −7 DPI and remained the same at 7 and 14 DPI compared with the previous sampling time in presence or absence of Salmonella Typhimurium JSG626 (Fig. 3D) (P < 0.05). Cmm was not detected in the inoculated cotyledons at 21 DPI because of the senescence and detachment of the cotyledons from the plants between 14 and 21 DPI, as observed with the mock-inoculated plants. The Cmm population was the same in the presence or absence of Salmonella Typhimurium JSG626 in the noninoculated tissues (stem, roots, and foliage).

Salmonella Typhimurium inhibited the growth of Xg and Cmm in vitro

Xg and Cmm were challenged in vitro (agar well diffusion assay) with Salmonella Typhimurium JSG626 whole culture for 48 h at 28°C. The Salmonella whole culture inhibited Cmm and Xg growth, with maximum inhibition zone radii of ca. 5 and 8 mm for Xg and Cmm, respectively. Similar trends were observed with the Salmonella Typhimurium JSG626 supernatant but at lower magnitude, with maximum inhibition zone radii of ca. 3 and 5 mm for Xg and Cmm, respectively. The pH of both Salmonella Typhimurium JSG626 whole culture and its supernatant was 6.1 to 6.5. No inhibition was observed when Salmonella Typhimurium JSG626 was challenged on YDC agar with Xg and Cmm supernatants for 48 h at 28°C. Cmm supernatant did not inhibit Xg growth on YDC agar and vice versa.

Numerous factors affect the persistence of foodborne pathogens in plant tissues during preharvest procedures (4, 5, 7, 32, 49, 53, 60, 70). Therefore, an understanding of the nature of the interactions among the plant host, its phytobiome, and foodborne pathogens would help food producers develop new guidelines and novel control methods, thus reducing the food safety risks related to foodborne pathogens in fresh produce. We hypothesized that the presence of Salmonella Typhimurium in plant tissues modulates directly (through the production of antimicrobial molecules or competition for space and nutrients) or indirectly (through alteration of the phytobiome or activation of host defenses) the colonization of tomato plant tissues by coinoculated Xg and Cmm during the early stages of the interaction and vice versa. The presence of phytopathogens such as X. perforans, X. campestris, X. vescicatoria, P. syringae, E. herbicola, and bacteria causing soft rot (e.g., P. carotovorum) increases the persistence of S. enterica in plant tissues (1, 6, 27, 60, 72). We obtained similar results when Salmonella Typhimurium JSG626 and Cmm were coinoculated into tomato cotyledons. Cmm secretes a broad diversity of serine proteases and cell wall degrading enzymes (endocellulase, polygalacturonase, pectinethylesterase, and xylanase), resulting in the maceration of plant tissues and release of their cellular contents (65). A transcriptomic analysis revealed that Salmonella benefits from the nutrients released by soft rot bacteria by upregulating specific nutritional and metabolic pathways, which enhance Salmonella persistence in plant tissues (29). Gammaproteobacterial phytopathogens use the KdgR transcriptional regulator to degrade the plant cell wall and take up pectin degradation products and other carbon sources (28, 58, 63). S. enterica cannot degrade pectin; however, this pathogen uses an orthologous version of KdgR to enhance its persistence in soft rotted tissues by assimilating products released by the pectinolytic activity of phytopathogens (28). However, whether pectin degradation by Xg or Cmm influences Salmonella persistence by allowing Salmonella to use these carbon sources is unknown. Several studies have revealed the importance of ethylene in Cmm virulence and disease development and in Salmonella persistence in tomato plant tissues (1, 2, 54). Thus, we hypothesized that the modifications in ethylene metabolism in plant tissues potentially caused by Cmm will also enhance Salmonella Typhimurium JSG626 persistence in tomato tissues. In contrast, the coexistence of both Salmonella Typhimurium JSG626 and Cmm or Xg in the same tissues was detrimental to the growth of the phytopathogens, which suggests that these pathogens compete for space and nutrients in the inoculated plant tissues. However, the results of our study also revealed that Salmonella inhibits Cmm and Xg growth in vitro, which might explain part of the reduction in Cmm and Xg populations observed in the plant tissues. Salmonella Typhimurium JSG626 also may trigger plant metabolic pathways detrimental to Cmm and Xg colonization of tomato plants. Salmonella flagellin proteins Flg22 and FlgII-28 can trigger tomato plant defenses (reactive oxygen species (25, 67), callose accumulation (25), stomatal closure (42, 57, 64), and plant hormone responses (34)), which could be detrimental to Cmm and Xg proliferation in plant tissues. In contrast, Xg had no effect on the persistence of Salmonella Typhimurium JSG626 in inoculated foliage. These results were contradictory to those of a previous study, which might be the result of differences in bacterial strains, tomato varieties, or the protocol used (60). Our study also revealed different trends when plants were inoculated with Salmonella Typhimurium JSG626 7 days after infection with Cmm or Xg, suggesting that the timing between inoculation with the phytopathogen and inoculation with Salmonella influences the ability of both pathogens to colonize tomato tissues. Salmonella Typhimurium JSG626 did not affect Cmm populations but increased Xg populations in plant tissues when introduced after phytopathogen inoculation. Therefore, the presence of Salmonella Typhimurium JSG626 in the plant might affect the host and/or the phytobiome in favor of Xg. Our in vitro agar well diffusion results suggest that Salmonella produces antimicrobial molecules that are excreted in the environment. These molecules might be associated with the reduction of Xg and Cmm populations observed in the plant tissues when the phytopathogens were coinoculated with Salmonella. However, these observations also can be explained by other alterations associated with the phytobiome or the plant host. Additional experiments are required to confirm this hypothesis. The location (in the phyllosphere versus inside the inoculated tissues) of the three pathogens in plant tissues was not analyzed in this study; therefore, the phytopathogens may have modulated the internalization of Salmonella inside the inoculated plant tissues and vice versa.

In conclusion, our study revealed that the timing of inoculation and the type of phytopathogens present in plant tissues are decisive factors affecting the persistence of Salmonella Typhimurium in these tissues. Modifications in the plant host during Cmm colonization (e.g., release of water and nutrients, alteration of the phytobiome, and the modulation of pathways associated with free oxygen radicals, ethylene, and plant hormones) might facilitate the persistence of Salmonella Typhimurium in plant tissues (1, 2). The timing of inoculation of Salmonella Typhimurium and each phytopathogen impacted the growth of these phytopathogens in plant tissues. When coinoculated with Cmm and Xg, Salmonella Typhimurium might alter the phytobiome composition and compete for space and nutrients with these phytopathogen, thus reducing their colonization of tomato tissues. When inoculated after inoculation of the phytopathogen, Salmonella Typhimurium seem to modulate different plant pathways, aiding the growth of the phytopathogens in the plant tissues. Additional experiments are required to elucidate the mechanisms behind Salmonella-phytopathogen interactions in tomato plants at the community and molecular levels. Several studies have highlighted the sequence homology of TTSS-associated proteins (FliT and AvrA) present in S. enterica and their role in the virulence of specific phytopathogens in plant tissues (i.e., Pseudomonas fluorescens, Xanthomonas oryzae pv. oryzae, and X. campestris pv. vescicatoria) (33, 74). However, these studies also indicated that FliT and TTSS are not involved in the virulence and persistence of Salmonella in plant tissues. These phenotypes might be associated with the expendable nature of the TTSS depending on the Salmonella strain and plant host (18, 57). Additional comparative whole genome studies are required to better understand the complex interactions among Salmonella, phytopathogens, and their hosts.

We thank Claudio Vrisman and Rosario A. Candelero for the technical support. This research was supported by the U.S. Department of Agriculture, National Institute for Food and Agriculture, Agriculture and Food Research Initiative (grant 2013-67018-21240) and by state and federal funds appropriated to the Ohio Agricultural Research and Development Center (The Ohio State University).

1.
Balaji,
V.,
Mayrose
M.,
Sherf
O.,
Jacob-Hirsch
J.,
Eichenlaub
R.,
Iraki
N.,
Manulis-Sasson
S.,
Rechavi
G.,
Barash
I.,
and
Sessa
G.
2008
.
Tomato transcriptional changes in response to Clavibacter michiganensis subsp. michiganensis reveal a role for ethylene in disease development
.
Plant Physiol
.
146
:
1797
1809
.
2.
Balaji,
V.,
and
Sessa
G.
2008
.
Activation and manipulation of host responses by a gram-positive bacterium
.
Plant Signal. Behav
.
3
:
839
841
.
3.
Balouiri,
M.,
Sadiki
M.,
and
Ibnsouda
S. K.
2016
.
Methods for in vitro evaluating antimicrobial activity: a review
.
J. Pharm. Anal
.
6
:
71
79
.
4.
Barak,
J. D.,
Jahn
C. E.,
Gibson
D. L.,
and
Charkowski
A. O.
2007
.
The role of cellulose and O-antigen capsule in the colonization of plants by Salmonella enterica
.
Mol. Plant Microbe Interact
.
20
:
1083
1091
.
5.
Barak,
J. D.,
Kramer
L. C.,
and
Hao
L.
2011
.
Colonization of tomato plants by Salmonella enterica is cultivar dependent, and type 1 trichomes are preferred colonization sites
.
Appl. Environ. Microbiol
.
77
:
498
504
.
6.
Barak,
J. D.,
and
Liang
A. S.
2008
.
Role of soil, crop debris, and a plant pathogen in Salmonella enterica contamination of tomato plants
.
PLoS One
3
:
e1657
.
7.
Barak,
J. D.,
and
Schroeder
B. K.
2012
.
Interrelationships of food safety and plant pathology: the life cycle of human pathogens on plants
.
Annu. Rev. Phytopathol
.
50
:
241
266
.
8.
Batz,
M.,
Hoffmann
S.,
and
Morris,
J. G.
Jr.
2014
.
Disease-outcome trees, EQ-5D scores, and estimated annual losses of quality-adjusted life years (QALYs) for 14 foodborne pathogens in the United States
.
Foodborne Pathog. Dis
.
11
:
395
402
.
9.
Batz,
M. B.,
Hoffmann
S.,
and
Morris,
J. G.
Jr.
2011
.
Ranking the risks: the 10 pathogen-food combinations with the greatest burden on public health
.
Emerging Pathogens Institute, University of Florida
,
Gainesville
.
10.
Behravesh,
C.,
Williams
I.,
and
Tauxe
R.
2012
.
Emerging foodborne pathogens and problems: expanding prevention efforts before slaughter or harvest, A14
.
In
Improving food safety through a One Health approach: workshop summary
.
National Academies Press
,
Washington, DC.
11.
Bernstein,
N.,
Sela (Saldinger)
S.,
Dudai
N.,
and
Gorbatsevich
E.
2017
.
Salinity stress does not affect root uptake, dissemination and persistence of Salmonella in sweet-basil (Ocimum basilicum)
.
Front. Plant Sci
.
8
:
675
.
12.
Bertoia,
M. L.,
Mukamal
K. J.,
Cahill
L. E.,
Hou
T.,
Ludwig
D. S.,
Mozaffarian
D.,
Willett
W. C.,
Hu
F. B.,
and
Rimm
E. B.
2015
.
Changes in intake of fruits and vegetables and weight change in United States men and women followed for up to 24 years: analysis from three prospective cohort studies
.
PLoS Med
.
12
:
e1001878
.
13.
Brandl,
M. T.
2006
.
Fitness of human enteric pathogens on plants and implications for food safety
.
Annu. Rev. Phytopathol
.
44
:
367
392
.
14.
Brandl,
M. T.,
Cox
C. E.,
and
Teplitski
M.
2013
.
Salmonella interactions with plants and their associated microbiota
.
Phytopathology
103
:
316
325
.
15.
Carstens,
C. K.,
Salazar
J. K.,
and
Darkoh
C.
2019
.
Multistate outbreaks of foodborne illness in the United States associated with fresh produce from 2010 to 2017
.
Front. Microbiol
.
10
:
2667
.
16.
Centers for Disease Control and Prevention.
2020
.
List of selected multistate foodborne outbreak investigations
.
Centers for Disease Control and Prevention
,
Atlanta
.
17.
Cevallos-Cevallos,
J. M.,
Gu
G.,
Danyluk
M. D.,
and
van Bruggen
A. H. C.
2012
.
Adhesion and splash dispersal of Salmonella enterica Typhimurium on tomato leaflets: effects of rdar morphotype and trichome density
.
Int. J. Food Microbiol
.
160
:
58
64
.
18.
Chalupowicz,
L.,
Nissan
G.,
Brandl
M. T.,
McClelland
M.,
Sessa
G.,
Popov
G.,
Barash
I.,
and
Manulis-Sasson
S.
2017
.
Assessing the ability of Salmonella enterica to translocate type III effectors into plant cells
.
Mol. Plant Microbe Interact
.
31
:
233
239
.
19.
Deblais,
L.,
Helmy
Y. A.,
Kathayat
D.,
Huang
H.,
Miller
S. A.,
and
Rajashekara
G.
2018
.
Novel imidazole and methoxybenzylamine growth inhibitors affecting Salmonella cell envelope integrity and its persistence in chickens
.
Sci. Rep
.
8
:
13381
.
20.
Deblais,
L.,
Helmy
Y. A.,
Testen
A.,
Vrisman
C.,
Madrid
A. M. J.,
Kathayat
D.,
Miller
S. A.,
and
Rajashekara
G.
2019
.
Specific environmental temperature and relative humidity conditions and grafting affect the persistence and dissemination of Salmonella in tomato plant tissues
.
Appl. Environ. Microbiol
.
85
:
e00403
-
19
.
21.
Deblais,
L.,
Vrisman
C.,
Kathayat
D.,
Helmy
Y. A.,
Miller
S. A.,
and
Rajashekara
G.
2019
.
Imidazole and methoxybenzylamine growth inhibitors reduce Salmonella persistence in tomato plant tissues
.
J. Food Prot
.
82
:
997
1006
.
22.
de Moraes,
M. H.,
Desai
P.,
Porwollik
S.,
Canals
R.,
Perez
D. R.,
Chu
W.,
McClelland
M.,
and
Teplitski
M.
2017
.
Salmonella persistence in tomatoes requires a distinct set of metabolic functions identified by transposon insertion sequencing
.
Appl. Environ. Microbiol
.
83
:
e03028
-
16
.
23.
Devleesschauwer,
B.,
Marvasi
M.,
Giurcanu
M. C.,
Hochmuth
G. J.,
Speybroeck
N.,
Havelaar
A. H.,
and
Teplitski
M.
2017
.
High relative humidity pre-harvest reduces post-harvest proliferation of Salmonella in tomatoes
.
Food Microbiol
.
66
:
55
63
.
24.
Fornefeld,
E.,
Schierstaedt
J.,
Jechalke
S.,
Grosch
R.,
Schikora
A.,
and
Smalla
K.
2017
.
Persistence of Salmonella Typhimurium LT2 in soil enhanced after growth in lettuce medium
.
Front. Microbiol
.
8
:
757
.
25.
García,
A. V.,
and
Hirt
H.
2014
.
Salmonella enterica induces and subverts the plant immune system
.
Front. Microbiol
.
5
:
141
.
26.
Ge,
C.,
Lee
C.,
Nangle
E.,
Li
J.,
Gardner
D.,
Kleinhenz
M.,
and
Lee
J.
2013
.
Impact of phytopathogen infection and extreme weather stress on internalization of Salmonella Typhimurium in lettuce
.
Int. J. Food Microbiol
.
168–169
:
24
31
.
27.
George,
A. S.,
Cox
C. E.,
Desai
P.,
Porwolik
S.,
Chu
W.,
de Moraes
M. H.,
McClelland
M.,
Brandl
M. T.,
and
Teplitski
M.
2017
.
Interactions of Salmonella enterica sv. Typhimurium and Pectobacterium carotovorum within a tomato soft rot
.
Appl. Environ. Microbiol.
28.
George,
A. S.,
Salas González
I.,
Lorca
G. L.,
and
Teplitski
M.
2016
.
Contribution of the Salmonella enterica KdgR regulon to persistence of the pathogen in vegetable soft rots
.
Appl. Environ. Microbiol
.
82
:
1353
1360
.
29.
Goudeau,
D. M.,
Parker
C. T.,
Zhou
Y.,
Sela
S.,
Kroupitski
Y.,
and
Brandl
M. T.
2013
.
The Salmonella transcriptome in lettuce and cilantro soft rot reveals a niche overlap with the animal host intestine
.
Appl. Environ. Microbiol
.
79
:
250
262
.
30.
Gould,
L. H.,
Walsh
K. A.,
Vieira
A. R.,
Herman
K.,
Williams
I. T.,
Hall
A. J.,
Cole
D.,
and
Centers for Disease Control and Prevention
.
2013
.
Surveillance for foodborne disease outbreaks—United States, 1998–2008
.
Morb. Mortal. Wkly. Rep. Surveill. Summ
.
62
:
1
34
.
31.
Gu,
G.,
Cevallos-Cevallos
J. M.,
and
van Bruggen
A. H. C.
2013
.
Ingress of Salmonella enterica Typhimurium into tomato leaves through hydathodes
.
PLoS One
8
:
e53470
.
32.
Gurtler,
J. B.,
Harlee
N. A.,
Smelser
A. M.,
and
Schneider
K. R.
2018
.
Salmonella enterica contamination of market fresh tomatoes: a review
.
J. Food Prot
.
81
:
1193
1213
.
33.
Hardt,
W.-D.,
and
Galán
J. E.
1997
.
A secreted Salmonella protein with homology to an avirulence determinant of plant pathogenic bacteria
.
Proc. Natl. Acad. Sci. USA
94
:
9887
9892
.
34.
Iniguez,
A. L.,
Dong
Y.,
Carter
H. D.,
Ahmer
B. M. M.,
Stone
J. M.,
and
Triplett
E. W.
2005
.
Regulation of enteric endophytic bacterial colonization by plant defenses
.
Mol. Plant. Microbe Interact
.
18
:
169
178
.
35.
Islam,
M.,
Morgan
J.,
Doyle
M. P.,
Phatak
S. C.,
Millner
P.,
and
Jiang
X.
2004
.
Persistence of Salmonella enterica serovar Typhimurium on lettuce and parsley and in soils on which they were grown in fields treated with contaminated manure composts or irrigation water
.
Foodborne Pathog. Dis
.
1
:
27
35
.
36.
Jacobsen,
C. S.,
and
Bech
T. B.
2012
.
Soil survival of Salmonella and transfer to freshwater and fresh produce
.
Food Res. Int
.
45
:
557
566
.
37.
Jechalke,
S.,
Schierstaedt
J.,
Becker
M.,
Flemer
B.,
Grosch
R.,
Smalla
K.,
and
Schikora
A.
2019
.
Salmonella establishment in agricultural soil and colonization of crop plants depend on soil type and plant species
.
Front. Microbiol
.
10
:
967
.
38.
Karmakar,
K.,
Nath
U.,
Nataraja
K. N.,
and
Chakravortty
D.
2018
.
Root mediated uptake of Salmonella is different from phyto-pathogen and associated with the colonization of edible organs
.
BMC Plant Biol
.
18
:
344
.
39.
Kisluk,
G.,
and
Yaron
S.
2012
.
Presence and persistence of Salmonella enterica serotype Typhimurium in the phyllosphere and rhizosphere of spray-irrigated parsley
.
Appl. Environ. Microbiol
.
78
:
4030
4036
.
40.
Klerks,
M. M.,
Franz
E.,
van Gent-Pelzer
M.,
Zijlstra
C.,
and
van Bruggen
A. H. C.
2007
.
Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-microbe factors influencing the colonization efficiency
.
ISME J
.
1
:
620
631
.
41.
Kozak,
G. K.,
MacDonald
D.,
Landry
L.,
and
Farber
J. M.
2013
.
Foodborne outbreaks in Canada linked to produce: 2001 through 2009
.
J. Food Prot
.
76
:
173
183
.
42.
Kroupitski,
Y.,
Golberg
D.,
Belausov
E.,
Pinto
R.,
Swartzberg
D.,
Granot
D.,
and
Sela
S.
2009
.
Internalization of Salmonella enterica in leaves is induced by light and involves chemotaxis and penetration through open stomata
.
Appl. Environ. Microbiol
.
75
:
6076
6086
.
43.
Lake,
I. R.
2017
.
Food-borne disease and climate change in the United Kingdom
.
Environ. Health
16
(1)
:
117
.
44.
Lapidot,
A.,
and
Yaron
S.
2009
.
Transfer of Salmonella enterica serovar Typhimurium from contaminated irrigation water to parsley is dependent on curli and cellulose, the biofilm matrix components
.
J. Food Prot
.
72
:
618
623
.
45.
Lee,
S.-Y.,
and
Baek
S.-Y.
2008
.
Effect of chemical sanitizer combined with modified atmosphere packaging on inhibiting Escherichia coli O157:H7 in commercial spinach
.
Food Microbiol
.
25
:
582
587
.
46.
Lemunier,
M.,
Francou
C.,
Rousseaux
S.,
Houot
S.,
Dantigny
P.,
Piveteau
P.,
and
Guzzo
J.
2005
.
Long-term survival of pathogenic and sanitation indicator bacteria in experimental biowaste composts
.
Appl. Environ. Microbiol
.
71
:
5779
5786
.
47.
Lewis Ivey,
M. L.,
and
Miller
S. A.
2005
.
Evaluation of hot water seed treatment for the control of bacterial leaf spot and bacterial canker on fresh market and processing tomatoes
.
Acta Hortic
.
695
:
197
204
.
48.
Lewis Ivey,
M. L.,
Strayer
A.,
Sidhu
J. K.,
and
Minsavage
G. V.
2016
.
Bacterial leaf spot of tomato (Solanum lycopersicum) in Louisiana is caused by Xanthomonas perforans, tomato race 4
.
Plant Dis.
100
.
49.
Lim,
J.-A.,
Lee
D. H.,
and
Heu
S.
2014
.
The interaction of human enteric pathogens with plants
.
Plant Pathol. J
.
30
:
109
116
.
50.
Liu,
H.,
Whitehouse
C. A.,
and
Li
B.
2018
.
Presence and persistence of Salmonella in water: the impact on microbial quality of water and food safety
.
Front. Public Health
6
:
159
.
51.
Ma,
X.
2015
.
Characterization and management of bacterial leaf spot of processing tomato in Ohio
.
Ph.D. dissertation.
The Ohio State University
,
Wooster
.
52.
Major,
N.,
Schierstaedt
J.,
Jechalke
S.,
Nesme
J.,
Ban
S. G.,
Černe
M.,
Sørensen
S. J.,
Ban
D.,
and
Schikora
A.
2020
.
Composted sewage sludge influences the microbiome and persistence of human pathogens in soil
.
Microorganisms
8
:
1020
.
53.
Martínez-Vaz,
B. M.,
Fink
R. C.,
Diez-Gonzalez
F.,
and
Sadowsky
M. J.
2014
.
Enteric pathogen-plant interactions: molecular connections leading to colonization and growth and implications for food safety
.
Microbes Environ
.
29
:
123
135
.
54.
Marvasi,
M.,
Noel
J. T.,
George
A. S.,
Farias
M. A.,
Jenkins
K. T.,
Hochmuth
G.,
Xu
Y.,
Giovanonni
J. J.,
and
Teplitski
M.
2014
.
Ethylene signalling affects susceptibility of tomatoes to Salmonella
.
Microb. Biotechnol
.
7
:
545
555
.
55.
Matos,
A.,
and
Garland
J. L.
2005
.
Effects of community versus single strain inoculants on the biocontrol of Salmonella and microbial community dynamics in alfalfa sprouts
.
J. Food Prot
.
68
:
40
48
.
56.
McClelland,
M.,
Sanderson
K. E.,
Spieth
J.,
Clifton
S. W.,
Latreille
P.,
Courtney
L.,
Porwollik
S.,
Ali
J.,
Dante
M.,
Du
F.,
Hou
S.,
Layman
D.,
Leonard
S.,
Nguyen
C.,
Scott
K.,
Holmes
A.,
Grewal
N.,
Mulvaney
E.,
Ryan
E.,
Sun
H.,
Florea
L.,
Miller
W.,
Stoneking
T.,
Nhan
M.,
Waterston
R.,
and
Wilson
R. K.
2001
.
Complete genome sequence of Salmonella enterica serovar Typhimurium LT2
.
Nature
413
:
852
856
.
57.
Meng,
F.,
Altier
C.,
and
Martin
G. B.
2013
.
Salmonella colonization activates the plant immune system and benefits from association with plant pathogenic bacteria
.
Environ. Microbiol
.
15
:
2418
2430
.
58.
Nasser,
W.,
Reverchon
S.,
Condemine
G.,
and
Robert-Baudouy
J.
1994
.
Specific interactions of Erwinia chrysanthemi KdgR repressor with different operators of genes involved in pectinolysis
.
J. Mol. Biol
.
236
:
427
440
.
59.
Pollard,
S.,
Barak
J.,
Boyer
R.,
Reiter
M.,
Gu
G.,
and
Rideout
S.
2014
.
Potential interactions between Salmonella enterica and Ralstonia solanacearum in tomato plants
.
J. Food Prot
.
77
:
320
324
.
60.
Potnis,
N.,
Colee
J.,
Jones
J. B.,
and
Barak
J. D.
2015
.
Plant pathogen–induced water-soaking promotes Salmonella enterica growth on tomato leaves
.
Appl. Environ. Microbiol
.
81
:
8126
8134
.
61.
Poza-Carrion,
C.,
Suslow
T.,
and
Lindow
S.
2013
.
Resident bacteria on leaves enhance survival of immigrant cells of Salmonella enterica
.
Phytopathology
103
:
341
351
.
62.
Rebuffat,
S.
2011
.
Bacteriocins from gram-negative bacteria: a classification?
p.
55
72
.
In
Drider
D.
and
Rebuffat
S.
(ed.),
Prokaryotic antimicrobial peptides
.
Springer
,
New York
.
63.
Rodionov,
D. A.,
Gelfand
M. S.,
and
Hugouvieux-Cotte-Pattat
N.
2004
.
Comparative genomics of the KdgR regulon in Erwinia chrysanthemi 3937 and other gamma-proteobacteria
.
Microbiology (Reading)
150
:
3571
3590
.
64.
Roy,
D.,
Panchal
S.,
Rosa
B. A.,
and
Melotto
M.
2013
.
Escherichia coli O157:H7 induces stronger plant immunity than Salmonella enterica Typhimurium SL1344
.
Phytopathology
103
:
326
332
.
65.
Savidor,
A.,
Teper
D.,
Gartemann
K.-H.,
Eichenlaub
R.,
Chalupowicz
L.,
Manulis-Sasson
S.,
Barash
I.,
Tews
H.,
Mayer
K.,
Giannone
R. J.,
Hettich
R. L.,
and
Sessa
G.
2012
.
The Clavibacter michiganensis subsp. michiganensis–tomato interactome reveals the perception of pathogen by the host and suggests mechanisms of infection
.
J. Proteome Res
.
11
:
736
750
.
66.
Schierstaedt,
J.,
Jechalke
S.,
Nesme
J.,
Neuhaus
K.,
Sørensen
S. J.,
Grosch
R.,
Smalla
K.,
and
Schikora
A.
2020
.
Salmonella persistence in soil depends on reciprocal interactions with indigenous microorganisms
.
Environ. Microbiol
.
22
:
2639
2652
.
67.
Shirron,
N.,
and
Yaron
S.
2011
.
Active suppression of early immune response in tobacco by the human pathogen Salmonella Typhimurium
.
PLoS One
6
:
e18855
.
68.
Sivapalasingam,
S.,
Friedman
C. R.,
Cohen
L.,
and
Tauxe
R. V.
2004
.
Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997
.
J. Food Prot
.
67
:
2342
2353
.
69.
Slavin,
J. L.,
and
Lloyd
B.
2012
.
Health benefits of fruits and vegetables
.
Adv. Nutr
.
3
:
506
516
.
70.
Soto-Arias,
J. P.,
Groves
R.,
and
Barak
J. D.
2013
.
Interaction of phytophagous insects with Salmonella enterica on plants and enhanced persistence of the pathogen with Macrosteles quadrilineatus infestation or Frankliniella occidentalis feeding
.
PLoS One
8
:
e79404
.
71.
Tauxe,
R. V.
1997
.
Emerging foodborne diseases: an evolving public health challenge
.
Emerg. Infect. Dis
.
3
:
425
434
.
72.
Wells,
J. M.,
and
Butterfield
J. E.
1997
.
Salmonella contamination associated with bacterial soft rot of fresh fruits and vegetables in the marketplace
.
Plant Dis
.
81
:
867
872
.
73.
Xu,
X.,
Miller
S. A.,
Baysal-Gurel
F.,
Gartemann
K.-H.,
Eichenlaub
R.,
and
Rajashekara
G.
2010
.
Bioluminescence imaging of Clavibacter michiganensis subsp. michiganensis infection of tomato seeds and plants
.
Appl. Environ. Microbiol
.
76
:
3978
3988
.
74.
Yu,
C.,
Chen
H.,
Tian
F.,
Yang
F.,
and
He
C.
2017
.
RpoN2- and FliA-regulated fliTX is indispensible for flagellar motility and virulence in Xanthomonas oryzae pv. oryzae
.
BMC Microbiol
.
17
:
171
.