Water IrrigationWater, Irrigation,and Impacts on E. coli

Steven Koike, Mike Cahn, and Trevor Suslowand Trevor Suslow

University of California

UC Cooperative Extension: L T Obj iLong-Term Objectives

• Conduct practical field studies that• Conduct practical field studies that contribute to an understanding of how E coli and other foodborne pathogensE. coli and other foodborne pathogens exist and survive in agriculture.

• Provide guidance for minimizing risks from foodborne pathogens and forfrom foodborne pathogens and for improving metrics and regulatory guidelines.g

2007 Field Experiments2007 Field Experiments• Examine soil survival of generic E. coliExamine soil survival of generic E. coli

under field conditions.

• Evaluate irrigation methods and soil nutrient levels on generic E. colinutrient levels on generic E. coli survival.

• Develop and refine detection methods for E. coli research.for E. coli research.

Field Trial 1Field Trial 1Objective: Evaluate generic E. coli survival in

il d diff t t f i kl li dsoil under different rates of sprinkler applied water

• Replicated small plots (40-inch bed x 20 feet).• Four E. coli treatments (water, soil, plant, combo.).• Two concentrations (106, 108 cfu/ml) each.• Selected for antibiotic resistance (rif mutant).

Sprinkler irrigated e er 2 da s (5 times total)• Sprinkler irrigated every 2 days (5 times total).• Water volumes were measured.• Monitor E. coli rif survival in soil.

Field Trial 1

5B

4B

3B

2B

1B

88

5A

4A

3A

2A

1A

45B15

54B34

23B43

32B62

11B21

8

7

8

75

5A2

5

4

4A8

2

3

3A3

6

2

2A5

4

1

1A7

8

5B85

4B74

3B53

2B12

1B31

66

5

5A7

4

4A4

3

3A1

2

2A8

1

1A6

B65B

B14B

B83B

B72B

B41B

5

4

5

45A

A3

4A

A6

3A

A7

2A

A2

1A

A5

35B75

64B44

73B13

22B82

51B61

33

5

5A8

6

4

4A7

1

3

3A5

8

2

2A1

7

1

1A3

4

5B25

4B84

3B33

2B52

1B71

2

1

2

1 5

5A1

4

4A3

3

3A4

2

2A6

1

1A2

B5

B2

B6

B4

B8

11

Guard Row Applied Water Applied Water Sprinkler

A4

A5

A2

A3

A1

MUG

Yellow = Neg. Red = Coliform UV = “E. coli”

QuantiTray

ECC

Presumptive ColiformPresumptiveE. coli

Coliform

Field Trial 1 ResultsField Trial 1 Results

• E coli rif recovered from soil up to 8E. coli rif recovered from soil up to 8 days.

• By 14 days E coli rif no longer• By 14 days, E. coli rif no longer detected (w/ exception of two plots).Hi h t i t d ith• Higher recovery rates associated with the larger amounts of applied water.

• Applied strains were never detected in adjacent uninoculated plots.

Field Trial 1

Individual Plots: Field Trial 1

Block A & B differences

5l 4

Block ABlock B

(North)

(South)

r of s

oil

3

g C

FU/g

r

2

log

1

CocktailPlant log 8

Water log 8Soil log 8

0

Treatment

Pl Wa S

4Applied Water at Field Trial 1

ches

)3

4

Wat

er (i

nc

2

S2-5

N2-5

App

lied

W

1S1

Distance from Sprinkler Line (feet)4 6 8 10 12 14 16 18 20

0N1

Distance from Sprinkler Line (feet)

North Side 1st irrigationSouth Side 1st irrigationNorth Side total of subsequent irrigationsNorth Side total of subsequent irrigationsSouth Side total of subsequent irrigations

Field Trial 2: Field SVR 51Objective: Compare soil survival of generic E.

coli under sprinkler/drip and withcoli under sprinkler/drip and with high/standard nutrient inputs.

• Replicated large plots (three 40-inch beds x 145 feet).• Treatments:

Irrigation: drip sprinklers• Irrigation: drip, sprinklers• E. coli rif (107): noninoculated, inoculated• Fertilizer: grower std, grower std + 350 lb N/acre + 250 lb

P/acreP/acre• Plant romaine; follow E. coli survival in soil, in runoff

water (sprinkler plots only), on plants.• Target lettuce harvest: September.

SVR 51 Results A• E. coli rif recovered from soil for only a

short period of time (up to 3 days).short period of time (up to 3 days).• Irrigation methods and nutrient levels

had no effect on E. coli survival in soil.had no effect on E. coli survival in soil.• By 6 d, E. coli rif no longer detected.• No detection of E coli rif on lettuce:• No detection of E. coli rif on lettuce:

– seedling roots and rhizosphere soil– seedling leavesseedling leaves– larger plant leaves– plants of harvestable sizeplants of harvestable size

E. coli survival in soil8 Inoculum concentration

oil

6

Day of inoculation3 days after inoc.6 days after inoc.13 days afer inoc. (5 cm)

CFU

/g d

ry s

o

4

log

C

0

2

D.L. (day 3)

noc.

ntro

l

rient

noc.

ntro

l

rient

-2

D.L. (day 6)

D.L. (day 13)

sprin

kle

unin

prin

kle

inoc

. con

rinkl

e in

oc. N

utri

drip

uni

n

drip

inoc

. con

t

drip

inoc

. Nut

ri

Treatments

sp spri

Day 0, 3 and 6. Samples were collected at the north side of each plot.Day 13. Samples consisted in a composite from 10 sub-samples distributed along the plotData only from Davis analisys

SVR 51 Results BSVR 51 Results B

• Sprinkler irrigation runoff: E coli rifSprinkler irrigation runoff: E. coli rif detected up to 12 days after inoculationinoculation.

• E. coli rif strains were not detected in adjacent uninoculated plots/lettuceadjacent uninoculated plots/lettuce.

• Coliform bacteria were recovered from ff f th d ti f th t i lrunoff for the duration of the trial.

Coliform Bacteria in Sprinkler Run-off from

105

Coliform Bacteria in Sprinkler Run off from SRV 51 field trial

104

105P

N/1

00 m

l

102

103

Control (not inoculated)i l t d / if E li

Detection limit = 24196 MPN/100 ml

MP

101

10 inoculated w/ rif E.coliinoculated w/ rif E. coli + nutrients

Days after Inoculation10 20 30 40 50 60

100

Days after Inoculation

SVR 51 Results CSVR 51 Results C• Starting from 26 days post inoculation, weStarting from 26 days post inoculation, we

recovered presumptive E. coli (growth on rif medium; fluorescence on MUG medium) from plants and runoff from inoculated plots.

• Late in experiment: found presumptive E. coli from uninoculated plots.

• However, all these isolates were later found b f l i i (ID E bto be false positives (ID= Enterobacter

species).

Presumptive E. coli in Sprinkler Run-off from SRV 51 field trial

10000

Control (not inoculated)i l t d / if E li

from SRV 51 field trial0

ml

1000inoculated w/ rif E.coliinoculated w/ rif E. coli + nutrients

MP

N/1

00 100

Detection limit = M 10 1 MPN/100 ml

10 20 30 40 50 60

1

Days after Inoculation

Summary for Generic E. coli in Fi ld S diField Studies

• Simulation of a one-time, high level , gcontamination event (w/ E. coli rif) resulted in very short persistence.I i ti th d/ t i t l l did t ff t• Irrigation method/nutrient level did not affect survival of E. coli in soil or in plant tissue.

• Presumptive E coli was detected in sprinkler• Presumptive E. coli was detected in sprinkler run-off water collected from furrows.

• Water appears to play key role in survival.ate appea s to p ay ey o e su a• Testing issue raised: positives with non-E.

coli on ECC, TSA, QuantiTray assays?

?

E. coli or not E. coli?

E. coli in Irrigation Run-off and Creeks

Presumptive

E. coli in Irrigation Run off and CreeksEast-side Salinas Valley (9/6/07)

Bacterial TMDL proposed for thesite description Coliform

Presumptive Generic E.Coli

MPN/100 ml1 i i ti ff hi h di t l d > 24196 1300

Bacterial TMDL proposed for the Lower Salinas Valley surface water:

1 irrigation run-off high sediment load > 24196 13002 irrigation run-off high sediment load > 24196 7703 creek (downstream from 2) > 24196 4354 irrigation run-off high sediment load > 24196 1120

target for E. coli = 126 MPN/100 ml5 irrigation run-off high sediment basin > 24196 626 road-side run-off (downstream from 5) > 24196 1357 creek > 24196 10468 Irrigation run-off (clear) > 24196 459 road-side run-off (downstream from 8) > 24196 37

10 irrigation run-off high sediment load > 24196 687

Source?Carrier?

Acknowledgements:1. California Lettuce Research Board2. Grower cooperators3. Research team: Patty Ayala, Elena Castro Carol D’lima Riley Hathaway KatCastro, Carol D’lima, Riley Hathaway, Kat Kammeijer, Eric Lauritzen, Salvador Montes, Lisa Quon, Adrian Sbodio, Arnett Young

Effect of Nutrients* on native ColiformEffect of Nutrients on native Coliform Bacteria and Presumptive E.coli levels in

Creek Water (site 3)

120

140

C t l

native E. coli (presumptive)

6000

8000

Control

native coliform

MP

N/1

00 m

l

60

80

100 Control+Nutrients

MP

N/1

00 m

l

4000

6000 Control+ Nutrients

0 2 4 6 8 10

M0

20

40

0 2 4 6 8 10

M

0

2000

days0 2 4 6 8 10

days0 2 4 6 8 10

*50 ppm Nitrate-N, 10 ppm orthophosphatepp pp p p

Effect of Nutrients* on Presumptive pE. coli levels in autoclaved Creek

Water (site 3)

25000

30000autoclaved + inoculated w/ E. coli

N/1

00 m

l

15000

20000

Control

Maximum Detection Limit

MP

N

5000

10000

Control+ Nutrients

d0 2 4 6 8 10

0

days

*50 ppm Nitrate-N, 10 ppm orthophosphate