higher than those of the co-colonized plants. At the three to five leaves and pre-bloom stages, S. enterica populations were significantly higher on those plants which were co-colonized by X. campestris pv. vesicatoria. Discussion Identifying the source of pre-harvest S. enterica contamination of fresh produce has been elusive, yet identification and risk assessment of each contamination route is necessary to control foodborne illness caused by consumption of raw produce in the absence of an available ‘‘kill-step’’ between farm and fork. Irrigation water has been implicated as a source of fresh produce contamination and subsequent human disease [13]. Castillo and colleagues isolated serovars of S. enterica that were the same as the strains which caused three large Salmonellosis outbreaks associ- ated with cantaloupe consumption [14]. These serovars were found in the irrigation water of farms that produced contaminated melons. Drip irrigation is used for melon production in this area; thus, if melon contamination occurred from the irrigation water it was likely via the soil. These reports led us to examine the ability of S. enterica to contaminate tomato plants from irrigation water via soil contamination, in light of the many Salmonellosis outbreaks caused by contaminated fresh tomatoes. Our results reveal the ability of S. enterica to attach to and contaminate both the phyllosphere and rhizoplane of tomato plants via soil following irrigation with contaminated water. Furthermore, contamination of the phyllosphere and rhizoplane occurred for the duration of our six week study, suggesting contaminated soil has the potential to inoculate seeds and subsequently crops weeks after a soil contamination event. Survival of S. enterica in fallow soil has been reported up to five weeks [15,16]; however, the ability to subsequently contaminate agricultural crops has not been addressed. One may assume S. enterica has the capacity to contaminate plants seeded in contaminated soil; however, there are no reported studies to this effect, nor has the affect of a lag period between contamination event and crop seeding been examined. We recovered S. enterica populations in fallow contaminated soil weekly up to six weeks. Furthermore, these cells were capable of attaching to and contaminating the rhizoplane and phyllosphere of tomato plants from seeds sown in these soils. These results suggest any event, i.e., flooding, raw manure or contaminated compost applications, or excretion by infected or carrier animals, which introduce S. enterica into the soil, could lead to subsequent crop contamination; though time may pass between the contamination event and planting. Soil survival by enteric pathogens, i.e., S. enterica, appears dependent on many factors including soil type and cropping system [7,8,17]. In our study and fresh produce fields studied by others, S. enterica populations are not stable and decline over time. Thus, factors which can increase S. enterica persistence are a public health concern. Because plant pathogens can utilize crop debris to colonize subsequent crops [18,19], we examined the capacity of crop debris to act as an inoculum source for S. enterica to a tomato crop. Our results reveal S. enterica contaminated crop debris can lead to contamination of a subsequent crop. The success of subsequent crop colonization appears dependent on the fallow period between crop incorporation and subsequent seeding, as seen by the absence of S. enterica recovery from the phyllosphere of References S. enterica on Tomato Plants plants sown following a one week fallow period between crops. Replanting fields shortly following harvest of a previous crop is a common practice, e.g., lettuce in the Salinas Valley of California [18] and tomato transplants in south Florida. Fields known to have produced crops contaminated with S. enterica may benefit from extended fallow periods between crops. Switching to produce that require cooking before eating would also reduce the risk of foodborne illness. It is well established that the incidence of S. enterica on fresh produce is significantly higher on diseased or injured produce [20,21]. Surprisingly, in our study, the incidence of S. enterica contamination was not significantly different between the plants co-colonized with and without the plant pathogen X. campestris pv. vesicatoria, in the absence of disease. There was no difference between the rhizoplane S. enterica populations of tomato co- colonized or not by the plant pathogen. Whether S. enterica can benefit from the presence of growing roots, since roots are known to release nutrients into the immediate soil available to bacteria [22], remains to be studied further. When S. enterica populations in the contaminated soil were low, the presence of X. campestris pv. vesicatoria had an interesting effect on subsequent colonization of the tomato phyllosphere. At the seedling stage, S. enterica populations in the phyllosphere were lower on the co- colonized plants compared to plants without X. campestris pv. vesicatoria. We hypothesize that the lower S. enterica populations were due to some advantage of X. campestris pv. vesicatoria for plant colonization and thus, excluded or outcompeted S. enterica at the seedling stage. At the three to five leaves and pre-bloom stages, S. enterica populations of the co-colonized plants grew in the phyllo- sphere. Thus, our study shows that in the absence of a plant pathogen, S. enterica could not grow and subsequently colonize the tomato plant. These results suggest a beneficial interaction for S. enterica between the human and plant pathogen prior to plant disease development. Whether the plant pathogen had proceeded to breakdown the plant tissue at a microscopic level was not investigated, but can not be ignored. However, X. campestris pv. vesicatoria populations did not reach populations high enough to cause disease (data not shown) [23]. Several noncompeting hypotheses may explain the advantage afforded S. enterica on tomato plants co-colonized by X. campestris pv. vesicatoria. S. enterica might utilize X. campestris pv. vesicatoria by joining its aggregates or biofilm on the leaf surface, which S. enterica may not produce for itself in the tomato phyllosphere. X. campestris pv. vesicatoria may overcome the innate immune plant response, similar to Pseudomonas syringae pv. tomato [24]; thus, allowing both X. campestris pv. vesicatoria and S. enterica to enter leaf tissue thru open stomata. Further research is needed to support or correct these hypotheses governing the interaction of S. enterica and bacterial plant pathogens on plants in the absence of plant disease. Acknowledgments We would like to thank Dr. Jeffrey Palumbo and Dr. William Miller for helpful critique of this manuscript and Dr. Robert Gilbertson for the gift of the Xanthomonas campestris pv. vesicatoria strain. Author Contributions Conceived and designed the experiments: JB. Performed the experiments: AL. Analyzed the data: JB. Wrote the paper: JB. 1. CDC (2005) Outbreaks of Salmonella infections associated with eating Roma tomatoes–United States and Canada, 2004. MMWR Morb Mortal Wkly Rep 54: 325–328. 2. Cummings K, Barrett E, Mohle-Boetani JC, Brooks JT, Farrar J, et al. (2001) A multistate outbreak of Salmonella enterica serotype Baildon associated with domestic raw tomatoes. Emerg Infect Dis 7: 1046–1048. PLoS ONE | www.plosone.org 4 February 2008 | Volume 3 | Issue 2 | e1657