Role of Soil, Crop Debris, and a Plant Pathogen in Salmonella enterica Contamination of Tomato Plants.

1. Role of Soil, Crop Debris, and a Plant Pathogen in Salmonella enterica Contamination of Tomato Plants. Barak JD, Liang AS PLoS ONE (2008)

2. Key Terms Phyllosphere - The surface of the plant above ground Rhizosphere/Rhizoplane- The area around the roots of the plant 3-5 leaf period - Tomato plant is approximately 25 to 28 days old Pre-bloom – Tomato plant is approximately 35 to 42 days-old.

3. Introduction There have been 12 outbreaks caused by Salmonella enterica which can be traced back to the consumption of tomatoes, since 1998. Water, soil, animal waste, and/or insects were suspected to be means by which this kind of contamination occurs. It was already known that S. enterica could infect tomatoes, as well as many other plants through a direct route, so this study aimed to determine whether an indirect contamination could result in colonization of the tomato plant.

4. S. enterica It is crucial that more studies reveal the nature of our food production and the dangerous bacteria Salmonella enterica. The experiment was conducted using two strains of the Salmonella enterica that have been associated with outbreaks in California according to the California Health Department, July 2005. http://www.youtube.com/watch?v=GQvrysBDH74

5. Hypothesis Indirect inoculation of S. enterica prior to the developmental stage will result in infection in the phyllosphere and rhizosphere. A plant pathogen, Xanthomonas campestris will promote the infection of S. enterica on the tomato plant.

6. Relevant strains used from actual past outbreaks in California The two S. enterica strains were used in a 1:1 mixture 1) S. enterica serovar Baildon strain 2) S. enterica serovar Enteritidis strain Xanthomonas campestris pathovar vesicatoria was isolated from Lycopersicon esculentum Mill. (scientific name of cultivated tomato) Xanthomonas campestris is a bacterial species that causes a disease which creates spots on tomatoes and weakens them.

7. Xanthomonas campestris This next slide shows the damage Xanthomonas campestris can inflict on the plant. Yellow spots are very apparent and indicate infection by this bacteria

8. Example: Xanthomonas campestris on leaf

9. S. enterica serovar Enteritidis strain

10. Methods Three studies -Each experiment was conducted twice 1)The long term survival assay (contaminated water) 2) The plant debris study 3) The plant pathogen study

11. Tools All bacteria were grown on Luria-Bertani (LB) media and S. enterica populations were enumerated on Salmonella Shigella (SS) media Kanamycin was incorporated into all media at 40 mg/liter.

12. Plant Assay – Study 1 The soil used was an enriched potting soil called Supersoil, which has a pH of 5.5-6.5. The soil was irrigated with 25 ml of S. enterica suspension to simulate an irrigation event of contaminated water. Control for this experiment was soil irrigated with sterile water.

13. Tomato plants were cut in two by a sterile razor blade. This separated the phyllosphere from rhizoplane. Plant parts were put into separate tared microfuge tubes, weighed, and 10 ml of sterile water was added. Following 1 min vortexes for the test tubes, serial dilutions of the suspension were made and aliquots were plated on SS agar. LB broth (with kanamycin) added Incubated overnight at 37 degrees, with shaking at 150 rpm. If colonies were not present on the original SS agar-Kan, one microliter loop of the enrichment was streaked on SS agar-Kan and incubated at 42 degrees for 24 hours to verify the presence of S. enterica in the plant samples.

14. Debris studies – Study 2 Soil was inoculated to simulate contaminated crop debris from a previous crop. Assays for S. enteric populations at the seedling and three to five leaf stages were put forth. Plants grown for 30 days (4 weeks) entire plant cut into 2.5 cm pieces, including the roots

15. After a 24 hour and 1 week period, seeds for the second crop were treated with calcium hypochlorite and sown. The second crop was assayed for S. enterica populations at the seedling and three to five leaf stages. Controls were seeds sown in non-sterile soil with debris from plants grown in soil irrigated with sterile water instead of S. enterica. Pots were irrigated (approximately 25 ml sterile water) every 48 h.

16. Plant Pathogen assays – Study 3 X. campestris pv. Vesicatoria was used to study the effects of a plant pathogen present in conjunction to Salmonella enterica contamination Tomato plants were assayed for S. enterica populations at the seedling stage, three to five leaf stage, and pre-bloom

17. After the calcium hypochlorite treatment and 1 hour of soaking in sterile water, tomato seeds were soaked in the X. campestris pv. vesicatoria suspension for 1 hour, continuously shaking at 40 rpm. Seeds were then sown in the S. enterica contaminated soil as described in the debris study.

18. Revelations of Plant Pathogens and the persistence of S. enterica The tomato plants had Salmonella enterica on their phyllosphere, although over time the colonies began to diminish. The Xanthomonas campestris pv. Vesicatoria strain also promoted the ability of Salmonella enterica to flourish at the three to five leaves and pre-bloom stages on the tomato plants.

19. Results

22. Conclusion The tomato plants had Salmonella enterica on their phyllosphere, although over time the colonies began to diminish. The Xanthomonas campestris pv. Vesicatoria strain promoted the ability of Salmonella enterica to flourish at the three to five leaves and pre-bloom stages on the tomato plants. Contaminated plant debris can serve as inoculums to subsequent crops

23. Significance and Impact of the Study This study is a critical ongoing surveillance of the agricultural industry and the health of the public. Maintenance is an ongoing process, in which harmful bacteria must be understood due to the outbreaks that can devastate a community. There are no references to whether S. enterica could infect the inside of the fruit, but washing the outside of the tomato is recommended before consuming to eliminate possible phyllosphere colonies. Reducing the incidence of this plant pathogen would help to alleviate some of the salmonella enterica contaminations in plants, which is a safety measure planters should take. If plant debris is to be used, soil should be tested for possible contamination of S. enterica, to prevent further outbreaks

24. Questions? Let me know if any aspect of this study was confusing! Thanks for listening!