2014 National Sustainable Strawberry Initiative Project Leader Meeting

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. 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. 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. 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. 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. 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. • 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. 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. 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. 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. 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. 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. 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. 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. Results
Dip stick analysis of Listeria
Single bacterial colony of Listeria Dip stick analysis of Listeria

16. Results
Dip stick analysis of Salmonella
Single bacterial colony of Salmonella Dip stick analysis of Salmonella

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. 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. Objectives: Three and Fourth
Develop science based modules for
sustainable organic, human-pathogen- free
strawberry production through outreach
activities

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. 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. 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. 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. BioVam’s Advantage
Without BioVam With BioVam

25. Identification of human pathogen
contamination points
• Irrigation water;
• Domestic animals;
• Humans on U-pick
farms.

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.

29. Portland Strawberry Festivals
May 10, 2014

31. Dr. Zhou’s Laboratory Website
http://www.tnstate.edu/zhoulab

33. Next activity
Strawberry Variety comparison test Six
strawberry varieties harvested from the
project demonstration plots will be
compared in a blind taste test