Results S. enterica survived six weeks in fallow soil with the capacity to contaminate tomato plants To test the longevity of the S. enterica population in soil and whether these cells could contaminate plants, soil was irrigated with S. enterica contaminated water, seeds were sown at weekly intervals, and plants were examined for S. enterica. S. enterica was able to survive in fallow soil and remained capable of plant attachment and contamination at least six weeks following introduction to the soil by irrigation (Fig. 1). S. enterica soil populations declined for three weeks, stabilized for week three to five, and declined again for the sixth week. At six weeks fallow, S. enterica soil populations were approximately three logs lower than their initial levels. Overall, S. enterica populations in the rhizoplane were significantly smaller with each weekly sampling, except between the fourth and fifth week. S. enterica populations in the phyllosphere were stable for five weeks and smaller each week thereafter. S. enterica was not recovered from control plants. Contaminated plant debris can serve as inoculum to subsequent crops To determine the role crop debris may play in the persistence of S. enterica in fields used for continuous tomato cropping, a contaminated tomato crop was produced by irrigating soil with S. enterica, direct seeding tomato into the S. enterica contaminated soil, and allowing the crop to develop for 30 days. The crop was mulched and mixed with soil, and tomato seeds (second crop) were sown in the crop debris soil mixture either 24 h or 7 d later. S. enterica was recovered from the phyllosphere and rhizoplane of the second crop when seeds were sown 24 h following plant debris incorporation (Table 1). At the three to five leaf stage, the S. enterica population in the phyllosphere was below the level of enumeration and required enrichment for detection. S. enterica was not recovered from the phyllosphere of the second crop whose seeds were sown in soil which had lain fallow for seven days. S. enterica rhizoplane populations remained below 100 CFU/g for the duration of the study. S. enterica was not recovered from control plants. S. enterica on Tomato Plants Table 1. Incidence of Salmonella enterica contamination following sowing in non-sterile soil mixed with contaminated plant debris from a previous tomato crop. 1 day fallow* 7 days fallow Phyllosphere Rhizoplane Phyllosphere Rhizoplane Seedling 3–5 Leaf 2/18+ 2/15 14/18 15/15 0/20 0/12 8/20 11/12 *Tomato seeds were sown one or seven days following crop incorporation. +Number of S. enterica positive samples/number of samples tested. These data are combined from two experiments. doi:10.1371/journal.pone.0001657.t001 Plant pathogen causes higher S. enterica populations To investigate whether the presence of a bacterial tomato plant pathogen affects S. enterica in association with tomato, seeds were inoculated with X. campestris pv. vesicatoria, causal agent of bacterial spot of tomato, and sown in S. enterica contaminated autoclaved soil. Immediately proceeding sowing, X. campestris pv. vesicatoria treated seeds contained 4.8610464.26103 CFU/seed and decreased to 1.2610461.46103 CFU/seed after 24 h in soil. At the seedling, 3–5 leaves, and pre-bloom stages, all rhizoplane samples, regardless of the presence of X. campestris pv. vesicatoria, were colonized by S. enterica. There was no statistical difference between the incidence of S. enterica contaminated tomato plants, rhizoplane or phyllosphere, with and without X. campestris pv. vesicatoria plant colonization. To determine whether S. enterica soil contamination levels could affect the subsequent contamination of the tomato plant, tomato seeds with and without X. campestris pv. vesicatoria were seeded in either soil with an initial high (,105 CFU/ml) or low (,103 CFU/ ml) S. enterica inoculum level. There was no difference between the S. enterica populations on plants with or without X. campestris pv. vesicatoria seeded in the high inoculum soil. However, the S. enterica populations on plants seeded in the low inoculum soil differed at each plant development stage between those with or without X. campestris pv. vesicatoria plant colonization (Fig. 2; p#0.005). At the seedling stage, S. enterica populations on plants not co-colonized by X. campestris pv. vesicatoria, were significantly Figure 1. Average Salmonella enterica populations in soil and on leaves, stems, and roots of tomato. Tomato seeds were sown at weekly intervals in S. enterica irrigated soil and S. enterica populations were enumerated at the seedling stage. Phyllosphere and rhizoplane populations were from seedlings planted one week before sampling. Averages from two experiments were calculated from log transformed data and error bars represent standard deviations. T-tests were preformed to determine average population changes over time for each type of sample. Different letters represent significant population changes (p,0.03). doi:10.1371/journal.pone.0001657.g001 Figure 2. Average Salmonella enterica populations in the tomato phyllosphere. S. enterica populations are from plants with (white square) or without (black diamond) inoculation of tomato seed with Xanthomonas campestris pv. vesicatoria. Autoclaved soil was irrigated with S. enterica 24 h prior to seed sowing. Averages from two experiments were calculated from log transformed data and error bars represent standard deviations. doi:10.1371/journal.pone.0001657.g002 PLoS ONE | www.plosone.org 3 February 2008 | Volume 3 | Issue 2 | e1657