Internal Colonization of Salmonella enterica Serovar Typhimurium in Tomato Plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 109 CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 104 CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 106 CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth

Percolation and Survival of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Soil Amended with Contaminated Dairy Manure or Slurry▿

The effect of cattle manure and slurry application on percolation and survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was investigated for different soil depths after the addition of water. Four treatments were chosen for the first set of experiments: (i) addition of inoculated farmyard manure on the soil surface, (ii) mixing of inoculated farmyard manure with the top 10 cm of soil, (iii) addition of inoculated slurry on the soil surface, and (iv) injection of inoculated slurry into the top 10 cm of the soil. Homogeneity of water distribution in the soil profile was confirmed by a nondestructive nuclear magnetic resonance method. Survival data were fitted to a modified logistic model, and estimated survival times were compared. In the second set of experiments, pathogen-inoculated farmyard manure or slurry was applied to soil columns with 1-month-old lettuce plants. More pathogen cells percolated to greater depths after slurry than after manure application. Survival of E. coli O157:H7 was significantly longer in soil with slurry than in that with manure, while survival of Salmonella serovar Typhimurium was equally high with manure and slurry. The densities of the pathogens were not different in the rhizosphere compared to the bulk soil with manure, while the densities were higher by 0.88 ± 0.11 and 0.71 ± 0.23 log CFU per g (dry weight), respectively, in the rhizosphere than in bulk soil after slurry application. Our results suggest that surface application of manure may decrease the risk of contamination of groundwater and lettuce roots compared to injection of slurry

COLIWAVE a simulation model for survival of E. coli O157:H7 in dairy manure and manure-amended soil

A simulation model was developed to investigate the relative effects of temperature, oxygen concentration, substrate content and competition by autochthonous microbial community on the oscillatory behaviour and survival of Escherichia coli O157:H7 in manure and manure-amended soil. The overall decline in E. coli O157:H7 was primarily determined by competition with autochthonous copiotrophic bacteria simulated by an inter-specific competition term according to Lotka-Volterra. Oscillations of bacterial populations were attained by the relationships between relative growth and death rates with readily available substrate content. The model contains a logistic and exponential relation of relative growth and death rates, respectively, of E. coli O157:H7 and copiotrophic bacteria with temperature, resulting in optimum curves for net growth rates similar to the curves reported in the literature. The model has been both calibrated and validated on experimental data. The model was used to perform sensitivity analysis and to evaluate different manure and soil management scenarios in terms of survival of E. coli O157:H7. The relative effects of changes in temperature on simulated survival time of E. coli O157:H7 were more pronounced than changes in oxygen condition. Testing manure storage scenarios with realistic data revealed that manure stored in a heap that was turned every week resulted in almost 70% reduction of E. coli O157:H7 survival compared to unturned manure. At the surface of a heap with unturned manure, simulated survival time was the longest (2.4 times longer than inside the same heap). The simulation model provides a new approach to investigating dynamic changes of invasive microorganisms in natural substrates such as manure or manure-amended soil. (C) 2009 Elsevier B.V. All rights reserved

Corky Root of Lettuce Caused by Strains of a Gram-Negative Bacterium from Muck Soils of Florida, New York, and Wisconsin

Slow-growing bacteria similar to the bacterium causing lettuce corky root (CR) in California (strain CA1) were isolated from muck soils of Florida, New York, and Wisconsin, using lettuce seedlings as bait. All strains were tested for reaction with polyclonal antibodies produced against strain CA1 and for pathogenicity on CR-susceptible (Salinas) and CR-resistant (Green Lake) lettuce cultivars in a greenhouse. Five strains from Florida, three from New York, and three from Wisconsin induced severe CR symptoms on Salinas and mild symptoms on Green Lake. All strains were gram-negative, aerobic, oxidase positive, and catalase positive and reduced nitrate to ammonia. Whole-cell fatty acid compositions were similar for all strains and resembled that of Pseudomonas paucimobilis. Since this fatty acid pattern is unique, it is suggested that CR of lettuce is caused by strains of the same bacterium in Florida, New York, Wisconsin, and California

Reclassification of rhizosphere bacteria including strains causing corky root of lettuce and proposal of Rhizorhapis suberifaciens gen. nov., comb. nov., Sphingobium mellinum sp nov., Sphingobium xanthum sp nov and Rhizorhabdus argentea gen. nov., sp nov.

The genus Rhizorhapis gen. nov. (to replace the illegitimate genus name Rhizomonas) is proposed for strains of Gram-negative bacteria causing corky root of lettuce, a widespread and important lettuce disease worldwide. Only one species of the genus Rhizomonas was described, Rhizomonas suberifaciens, which was subsequently reclassified as Sphingomonas suberifaciens based on 16S rRNA gene sequences and the presence of sphingoglycolipid in the cell envelope. However, the genus Sphingomonas is so diverse that further reclassification was deemed necessary. Twenty new Rhizorhapis gen. nov.- and Sphingomonas-like isolates were obtained from lettuce or sow thistle roots, or from soil using lettuce seedlings as bait. These and previously reported isolates were characterized in a polyphasic study including 16S rRNA gene sequencing, DNA-DNA hybridization, DNA G+C content, whole-cell fatty acid composition, morphology, substrate oxidation, temperature and pH sensitivity, and pathogenicity to lettuce. The isolates causing lettuce corky root belonged to the genera Rhizorhapis gen. nov., Sphingobium, Sphingopyxis and Rhizorhabdus gen. nov. More specifically, we propose to reclassify Rhizomonas suberifaciens as Rhizorhapis suberifaciens gen. nov., comb. nov. (type strain, CA1(T)=LMG 17323(T)=ATCC 49355(T)), and also propose the novel species Sphingobium xanthum sp. nov., Sphingobium meflinum sp. nov. and Rhizorhabdus argen tea gen. nov., sp. nov. with the type strains NL9(T) (=LMG 12560(T)=ATCC 51296(T)), WI4(T) (=LMG 11032(T)=ATCC 51292(T)) and SP1(T) (=LMG 12581(T)=ATCC 51289(T)), respectively. Several strains isolated from lettuce roots belonged to the genus Sphingomonas, but none of them were pathogenic

Ingress of <em>Salmonella enterica</em> Typhimurium into Tomato Leaves through Hydathodes

<div><p>Internal contamination of <em>Salmonella</em> in plants is attracting increasing attention for food safety reasons. In this study, three different tomato cultivars “Florida Lanai”, “Crown Jewel”, “Ailsa Craig” and the transgenic line Sp5 of “Ailsa Craig” were inoculated with 1 µl GFP-labeled <em>Salmonella</em> Typhimurium through guttation droplets at concentrations of 10⁹ or 10⁷ CFU/ml. Survival of <em>Salmonella</em> on/in tomato leaves was detected by both direct plating and enrichment methods. <em>Salmonella</em> cells survived best on/in the inoculated leaves of cultivar “Ailsa Craig” and decreased fastest on/in “Florida Lanai” leaves. Increased guttation in the abscisic acid over-expressing Sp5 plants may have facilitated the entrance of <em>Salmonella</em> into leaves and the colonization on the surface of tomato leaves. Internalization of <em>Salmonella</em> Typhimurium in tomato leaves through guttation drop inoculation was confirmed by confocal laser microscopy. For the first time, convincing evidence is presented that <em>S. enterica</em> can enter tomato leaves through hydathodes and move into the vascular system, which may result in the internal translocation of the bacteria inside plants.</p> </div

<i>Salmonella</i> Typhimurium population density (mean ± standard deviation) on/in tomato leaves 1 day after inoculation through guttation droplets (1 droplet per leaf).

a<p>and ^bindicate significant differences among cultivars using the least significant difference test of ANOVA (P = 0.05).</p

Confocal laser microscope images of tomato leaf tissue sections colonized by <i>Salmonella</i> Typhimurium after inoculation (10⁹ CFU/ml) through guttation droplets.

<p>White arrows point out the GFP-tagged <i>Salmonella</i> cells (green) which entered into the vascular system of tomato leaves. Red fluorescence is the autofluorescence of plant chloroplasts. Images A2, B2, C2 and D2 are merged images under GFP and TRITC filters obtained by projecting 20 Z section overlaid fluorescence images of different layers with 1 µm interval into one combined image <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053470#pone.0053470-Gu1" target="_blank">[11]</a>.</p