The document investigates the internalization of antimicrobials and pathogens into lettuce following wastewater irrigation. It aims to study uptake under different soil textures, lettuce varieties, and stress conditions. Results show antimicrobial uptake did not correlate with soil sorption trends. The rhizosphere and root microbial communities differed, with soil texture having a larger impact than Salmonella in irrigation water. Irrigation reduced rhizosphere but not root microbial diversity.

1. Internalization of Chemical and
Microbial Contaminants in Produce
Following Wastewater Irrigation
Xu Li1, Shannon Bartelt-Hunt1, Daniel Snow2, and
Laurie Hodges3
1Department of Civil Engineering, 2School of Natural
Resources, 3Department of Agronomy and Horticulture
University of Nebraska-Lincoln
USDA PD Meeting
Greensboro, NC, 7/28/2015

2. Outbreak and Illness
 Contamination Pathways
• Preharvest
• Postharvest
(Center for Science in the Public Interest 2014)
(Kroupitski et al 2009) (Klerks et al. 2007)

3. Fate and Transport
Storage
and
treatment
structures
Agricultural
land
Agricultural runoff
Food cropsSludge
digester
Wastewater
treatment
plants

4. 1. Investigate the internalization of antimicrobials and
enteric pathogens into lettuce following wastewater
irrigation.
‒ Normal growth condition vs. drought stressed condition
‒ Soil texture: sand, sandy loam, silt loam, loam
‒ Lettuce cultivars: Green star vs. Green salad bowl
2. Elucidate the proteomic responses of Salmonella after
internalization into lettuce leaves.
3. Determine the effects of wastewater irrigation on the
microbial communities in the rhizosphere and the root
of lettuce plants.
Objectives

5. 0
500
1000
1500
2000
2500
3000
3500
4000
0 200 400 600 800
Lincomycin Cs(ng/g)
Lincomycin Ce (ng/mL)
‐500
0
500
1000
1500
2000
2500
3000
3500
4000
0 500 1000
Sulfamethoxazole Cs
(ng/g)
Sulfamethoxazole Ce (ng/mL)
Linco Sulfa Oxy
Kd R2 Kd R2 Kd R2
Loam
10.2 0.99 2.5 0.98 1050 0.99
Sandy Loam
5.6 0.99 2.4 0.99 470 0.99
Sand
6.3 0.99 2.5 0.98 240 0.98
Soil Adsorption
Characterization
0
50000
100000
150000
200000
0 200 400 600 800
Oxytetracycline Cs(ng/g)
Oxytetracycline Ce (ng/mL)

6. Lettuce Analysis
• Lincomycin: Compared to the other two
antimicrobials, lincomycin had the
highest uptake by lettuce.
• Sulfamethoxazole: Higher uptake was
observed in lettuce grown in sand.
• Oxytetracycline: Even with clear
differences in Kd, uptake in lettuce did
not follow sorption trends.
0
10
20
30
40
50
60
70
Loam Sandy Loam Sand
Lincomycinin
Lettuce (ng/g fw)
0
10
20
30
40
50
60
70
Loam Sandy Loam Sand
Sulfamethoxazolein
Lettuce(ng/gfw)
0
10
20
30
40
50
60
70
Loam Sandy Loam Sand
Oxytetracyclinein
Lettuce(ng/gfw)

7. Salmonella in Lettuce
Soil texture
Negative
(<1
CFU/g)
Positive
1-10 CFU/g
10-100
CFU/g
>100
CFU/g
Total
Sand
17
(71%)
1
(4%)
4
(17%)
2
(8%)
7
(29%) A
Sandy Loam
20
(83%)
2
(8%)
2
(8%)
0
4
(16%) AB
Loam
24
(100%)
0 0 0
0
(0%) B

8. Experimental Design
8
Soil textureIrrigation water Harvest time
Silt loam
(SL)
Lettuce cultivar
Green Star (GS)
Green Salad
Bowl (SB)Salmonella (Trt)
Control (Ctl)
Harvest 2
Harvest 1
Loamy
sand (LS)
……
……
……
…… ……
……
16 treatment combinations; 6 or 12 biological replicates;
2 fractions from each replicate (rhizosphere soil and root);
A total of 288 samples.

9. Experimental Procedure
• Plant growing
• Sampling
Seedling 4‐wk 5‐wk
Sampling Sampling
Repeated irrigation with clean water or water
containing Salmonella
Sonication
Root fraction
Rhizosphere
fraction
Washing
Washing

10. Taxonomic Analyses
C=control, T=Salmonella treatment, SL= silt loam, LS= loamy sand, 1= harvest 1 at week4,
2=harvest 2 at week 5.
Rhizosphere Root

11. Alpha Diversity
11
Ctrl Trt
6789
ShannonIndex
25 75
6789
ShannonIndex
25 75
5.06.07.08.0
ShannonIndex
Ctrl Trt
5.06.07.08.0
ShannonIndex
Loamy SandSilt Loam Loamy SandSilt Loam
Rhizosphere Root
p = 0.04*
p = 0.001*
p = 0.001*
p = 0.282
Salmonella in
irrigation
water
Soil texture

12. Beta Diversity
12

13. Experimental factor Rhizosphere Root
Salmonella
Control vs. Treatment
0.0453
0.0030
0.0078
0.3300
Soil texture
Silt loam vs. Loamy sand
0.2745
0.0010
0.0695
0.0010
Lettuce cultivar
Green star vs. Salad bowl
0.0123
0.1548
0.0273
0.0110
Harvest
4 vs. 5-wk
0.0350
0.0160
0.0224
0.0210
13
R2
p
Beta Diversity

14. Conclusions
 Uptake of the compounds in lettuce did not correspond
well to Kd values, indicating that plant uptake of
antimicrobials is more complex than a purely passive
process
 The microbial community in the root significantly differed
from that in the rhizosphere
 Irrigation with water containing Salmonella reduced the
microbial diversity and changed the microbial
community in the rhizosphere but not in the root
 Compared to lettuce cultivar, harvest time, and
Salmonella in irrigation water, soil texture had more
significant impacts on the microbial communities in the
rhizosphere and the root of lettuce.

15. Acknowledgements
J. Brett Sallach and Yuping Zhang
Funding source USDA 2011-67019-20052

16. 0
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Loam Sandyloam Sand
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Loam Sandyloam Sand
0
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3000
Loam Sandyloam Sand
0
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1000
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3000
Loam Sandyloam Sand
0
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1000
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Loam Sandyloam Sand
0
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1000
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Loam Sandyloam Sand
0
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1000
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Loam Sandyloam Sand
0
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Loam Sandyloam Sand
0
500
1000
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Loam Sandyloam Sand
Lincomycin Sulfamethoxazole Oxytetracycline
Harvest1
(ng/gdw)
Harvest2
(ng/gdw)
Harvest3
(ng/gdw)
Soil Analysis

17.  Antimicrobials:
– Food crops irrigated with contaminated water may
accumulate low levels of antimicrobials (Jones-Lepp
et al., 2010).
 Microbes:
– Through leaf surface: internalization involves
chemotaxis and penetration through open stomata
(Kroupitski et al 2009).
– Through root surface: Salmonella spp. accumulates
on root surface and at emerging lateral roots (Klerks
et al. 2007).
Uptake of Contaminants by Food Crops