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Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee

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2014 National Sustainable Strawberry Initiative Project Leader Meeting

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Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee

  1. 1. Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee A project funded by a grant from the Walmart Foundation and administered by the University of Arkansas System Division of Agriculture Center for Agricultural and Rural Sustainability
  2. 2. Participants PIs: Drs: Suping Zhou, Fur-chi Chen, Roy-Bullock, Theodore W Thannhauser. Research associates and assistants: Long Zhang, Christine Moore, Kathyleen Yan, Himabindu Gazula (graduate student). Eight local farms in Middle Tennessee.
  3. 3. 0 2000 4000 6000 8000 10000 12000 14000 16000 1950'S 1970'S 2010'S Acreage Acreage in Strawberries over the years • 1950’s: Tennessee was the #1 state in the United States for growing strawberries using a matted row system with about 15,000 acres • 1970’s: Acreage dropped to about 200 acres due to labor, disease (anthracnose), and other factors • 2010’s: Current growing practices utilize around 575 acres • Plasticulture with approximately 500 acres • Matted Row approximately 50-75 acres • Organic production approximately 10 acres *Reference: Bob Ary, County Extension Agent for Sumner County, TN Background information of the project*
  4. 4. Outbreaks of human pathogen on fresh and frozen strawberries have been reported on E. coli O157:H7, Salmonella, hepatitis A, Listeria monocytogenes Incidences of human pathogen contamination of fresh strawberries
  5. 5. Project Goal and Priority •Goal is to promote sustainable fresh strawberry production in Middle Tennessee •Two priority areas in this project: 1) Reduce the risk of human pathogens on fresh berries; 2) Implement meaningful and constructive metrics for strawberry production sustainability.
  6. 6. Objective 1. Developing detection method for human pathogens on fresh strawberries •Identification of microbial populations on organic and inorganic fresh strawberries. •Survival analysis of the human pathogens in fresh strawberry. •Developing dipstick assay method to detect the human pathogens on fresh strawberries
  7. 7. • In July, 2013, strawberry samples collected from local farms, and immediately inoculated with bacterial strains of •Escherichia coli 0157:H7 •Solmonella typhimurium •Listeria monocytogenes. •Strawberries were stored at 4 and 25 oC for two months and then these human pathogens were analyzed Detection of human pathogens on strawberries (juice) after long-term storage
  8. 8. 1.Direct spreading and 2.Enrichment method Identification of pathogenic bacteria Fig 3:Direct spreading Fig 4:Enrichment method Results: Non-bacterial colony was recovered, indicating all bacteria were died.
  9. 9. 1 2 75Kda 10 Kda Lane 1: Strawberry sample with E. coli/Listeria Lane 2: Bacterial Control of E. Coli/Listeria 66.2Kda 66 Kda 1 2 Western blot analysis E.coli Listeria sppFig 5: Fig 6:
  10. 10. Western Blot Analysis of Salmonella Typhimurium Lanes 1 and 3: Strawberry extract with S.typhimurium Lanes 2 and 4: Bacterial Control of S.typhimurium 1 2 3 4 Monoclonal study Polyclonal study Results Cont… Fig 7: Fig 8: Results: Bacteria were died, but their proteins were still intact in strawberry juice.
  11. 11. Procedure of detection Growing of isolated cultures of bacteria in tryptic soy broth Spot inoculation of strawberry with 10µl of suspended bacteria Storage of strawberries at 4˚C and 25˚C Homogenization in 50 mL butterfield buffer by stomacher Serial dilution of homogenate in 9 mL butterfield buffer Spread plating on selective media plate Analysis of bacterial colonies Fig 9: Spot inoculation of strawberry with bacteria
  12. 12. Growth of bacteria on fresh strawberries 0 2 4 6 8 10 Day One Day Three Day Five E.coli X-axis: Day of culture Y-axis: Log CFU/berry 4°C 25°C 0 1 2 3 4 5 6 Day One Day Three Day Five Listeria 0 1 2 3 4 5 Day One Day Three Day Five Salmonella
  13. 13. Objective 2 Developing Dip-Stick Detection of Single Bacterial Colony on Fresh strawberries Detection methods: E. coli 0157:H7 Reveal 2.0 medium, Salmonella: Rappaport-vassiliadis (RV) medium, Listeria: Listeria Enrichment Single Step (LESS) medium Fig.20 Dip Sticks “Dip stick analysis” method has been followed to detect human pathogens viz. E. coli (0157:H7), Salmonella typhimurium and Listeria monocytogenes in fresh strawberry.
  14. 14. Dip stick analysis procedure Serial dilutions of bacterial culture Inoculation of strawberry with 100µl diluent from each dilution. Incubation at selective temperature for 24-48 hrs. Observation of (single) colonies in the selective media plates Dip stick analysis
  15. 15. Results Dip stick analysis of Listeria Single bacterial colony of Listeria Dip stick analysis of Listeria
  16. 16. Results Dip stick analysis of Salmonella Single bacterial colony of Salmonella Dip stick analysis of Salmonella
  17. 17. Results Single bacterial colony of E. coli Agglutination test showing the presence of E. coli Dip stick analysis of E. coli 0157:H7
  18. 18. Conclusion  The Dipstick assay can produce reliable detection result for single colony inoculation of Salmonella and Listeria on fresh strawberries; Detection of E. coli 0157:H7 through the commercially available dip sticks isn’t successful. So, there is a need to develop an improvised tool to detect the presence of E.coli on contaminated food products.
  19. 19. Objectives: Three and Fourth Develop science based modules for sustainable organic, human-pathogen- free strawberry production through outreach activities
  20. 20. 1. McCraw’s Farms 2. Head Farms 3. Madison Creek Farms 4. Wade Farmz 5. Southland Farms 6. Mountain View Farms 7. Ferrell Farms 8. Pickett Farms 8 farms located in the Middle Tennessee:
  21. 21. Ground Breaking Soil Sampling Planted Strawberries Soil analysis: •Five farms were very high in phosphorus and potassium •One farm was low in phosphorus, but a different farm was low in potassium •Other farms were high in phosphorus and contained a medium amount of potassium. • Calcium and magnesium were sufficient for strawberry growth *Analysis completed by the UT Extension Soil, Plant, and Pest Center in Nashville, TN
  22. 22. Farm Management • Fungal and Bacterial Controls* • Oxidate • Hydrogen Dioxide – 27.1% • Peroxyacetic Acid – 2.0% • Acitovate AG • Streptomyces lydicus WYEC 108 • Pest Control* • Trilogy • Clarified Hydrophobic Extract of Neem Oil – 70% • Weed Control* • Hand Weed • AXXE • Broad Spectrum Herbicide • Ammonium Nananoate – 40% • Soil Analysis from each plot/field • Soil Amendments* • BioVam – T&J Enterprises • Endomycorrhizae, Ectomycorrhizae, and additional Biological community • SeaFish Liquid Fertilizer • 5-1-1 • whole Menhaden (ocean) fish • Green Potash • Soluble Potash (K2O) 0-0-15 (without sulfur) • Derived from Greensand and Kelp * All amendments and control additives OMRI approved * Amendment selection through consultation with extension agents and purchasing entities
  23. 23. A booming production 3 of the 5 farms with 2 other farms in question McCraw’s Farm Head’s Farm 2 gallons picked from 1 demonstration plot inoculated with BioVam Wade Farmz 2 ½ gallons picked from 1 demonstration plot on the most eastern edge of the fieldAlmost 3 gallons with largest amount harvested from the BioVam plot (Back Plot)
  24. 24. BioVam’s Advantage Without BioVam With BioVam
  25. 25. Identification of human pathogen contamination points • Irrigation water; • Domestic animals; • Humans on U-pick farms.
  26. 26. Outreach activities 1. Workshop: One at TSU; 2. Blind taste survey, >200; 3. Portland (Tennessee) Strawberry Festival, >500 visitors; 4. Demonstrations in public events, four times; 5. Social media and websites, >2000 visitors.
  27. 27. Portland Strawberry Festivals May 10, 2014
  28. 28. Dr. Zhou’s Laboratory Website http://www.tnstate.edu/zhoulab
  29. 29. Next activity Strawberry Variety comparison test Six strawberry varieties harvested from the project demonstration plots will be compared in a blind taste test

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