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

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.

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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*

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

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.

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

• 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 thenthese human pathogens were analyzed

Detection of human pathogens on

strawberries (juice) after long-term storage

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.

1 2

75Kda

10 Kda

Lane 1: Strawberry sample with E. coli/Listeria

Lane 2: Bacterial Control of E. Coli/Listeria

66.2Kda66 Kda

1 2

Western blot analysis

E.coli Listeria sppFig 5: Fig 6:

Western Blot Analysis of Salmonella Typhimurium

Lanes 1 and 3: Strawberry extract with S.typhimuriumLanes 2 and 4: Bacterial Control of S.typhimurium

1 2 34

Monoclonal studyPolyclonal study

Results Cont…

Fig 7:Fig 8:

Results:

Bacteria were

died, but their

proteins were

still intact in

strawberry

juice.

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

Growth of bacteria on fresh strawberries

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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

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1

2

3

4

5

6

Day One Day Three Day Five

Listeria

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1

2

3

4

5

Day One Day Three Day Five

Salmonella

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.

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

Results

Dip stick analysis of Listeria

Single bacterial colony of Listeria Dip stick analysis of Listeria

Results

Dip stick analysis of Salmonella

Single bacterial colony of Salmonella Dip stick analysis of Salmonella

Results

Single bacterial

colony of E. coli

Agglutination test showing

the presence of E. coli

Dip stick analysis of E. coli 0157:H7

Conclusion

The Dipstick assay can produce reliabledetection result for single colony inoculationof Salmonella and Listeria on freshstrawberries;

Detection of E. coli 0157:H7 through thecommercially available dip sticks isn’tsuccessful. So, there is a need to develop animprovised tool to detect the presence ofE.coli on contaminated food products.

Objectives: Three and Fourth

Develop science based modules for

sustainable organic, human-pathogen- free

strawberry production through outreach

activities

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:

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

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

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)

BioVam’s Advantage

Without BioVam With BioVam

Identification of human pathogen contamination points

• Irrigation water;

• Domestic animals;

• Humans on U-pick

farms.

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.

Portland Strawberry FestivalsMay 10, 2014

Dr. Zhou’s Laboratory Website

http://www.tnstate.edu/zhoulab

Next activity

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