This study assessed the contamination of Escherichia coli, in lettuce grown with treated domestic wastewater in four different irrigation configurations: open spray, spray under plastic sheet cover, open drip and drip under plastic sheet cover. Samples of lettuce from each irrigation configuration and irrigating wastewater were collected during the growing season. No E. coli was detected in lettuce from drip irrigated beds. All lettuce samples from spray beds were positive for E. coli, however, no statistical difference (p > 0.05) was detected between lettuces grown in open spray or covered spray beds. The results from the field experiment were also compared to a laboratory experiment which used submersion of lettuce in wastewater of known E. coli concentration as a surrogate method to assess contamination following irrigation. The microbial quality of spray bed lettuces was not significantly different from submersed lettuce when irrigated with wastewater containing 1,299.7 E. coli MPN/100 mL (p > 0.05). This study is significant since it is the first to validate that the microbial contamination of lettuce irrigated with wastewater in the field is comparable with a laboratory technique frequently applied in the quantitative microbial risk assessment of the consumption of wastewater irrigated salad crops.

INTRODUCTION

Water scarcity is emerging as a major problem in many parts of the world, not only in arid areas, but also in other regions where freshwater is extensive (Jiménez & Asano 2008; Steduto et al. 2012). About 700 million people in more than 40 countries live in environments suffering water scarcity (Jiménez & Asano 2008). Moreover, it's estimated that 44% of the world's population will be living in such conditions by the year 2050 (Scheierling et al. 2011). As a result, wastewater reuse is receiving more attention as a component of water resource management. Wastewater reuse applications include industrial and municipal industries; however, reuse in agriculture has the largest share by volume amongst all applications. Agriculture is the largest user of freshwater, accounting for 70% of total freshwater withdrawals globally (Steduto et al. 2012). It is estimated that about 1.5–6.6% of all irrigated area (approximately 301 million hectares worldwide) is irrigated with wastewater, and that 10% of the world's crops are grown using wastewater irrigation (Sato et al. 2013).

Wastewater irrigation in agriculture can be beneficial for farmers because of its nutrient content which is necessary for crops’ growth, resulting in the reduction of chemical fertiliser use, while increasing crop yields (Toze 2006; Drechsel et al. 2009). However, it can also pose public health risks due to the existence of pathogenic microorganisms such as bacteria, viruses and parasites. The occurrence of human health effects from wastewater irrigation is well documented (Shuval et al. 1984; WHO 2006). Not only consumers of wastewater irrigated crops, but also workers and their families and communities in the vicinity of the wastewater irrigated zone may be affected by this practice. However, the critical public health risk is from vegetables eaten raw, such as leafy greens (Beuchat 2002; Scheierling et al. 2010). To manage the public health risk concerns, a guideline for the safe use of wastewater in agriculture has been established by the World Health Organization (WHO 2006). The guidelines propose multiple measures to protect human health together with the log10 reduction in pathogen numbers required for wastewater treatment to meet a health-based target ≤10−6 disability adjusted life years per person per year.

In this study, partially treated domestic wastewater was used to irrigate lettuce. The objective was to assess the contamination of lettuce by applying a widely used microbial indicator of faecal contamination, Escherichia coli at both field and laboratory scales. The microbial contamination of lettuce at different sampling times was also investigated in the field experiment. Submersion ‘irrigation’ as previously used by Shuval et al. (1997) was applied at laboratory scale. This method determines the weight of wastewater retained by lettuce following submersion in the wastewater and calculates the microbial contamination of the lettuce from the initial concentration of E. coli in the wastewater. Shuval et al. (1997) used this method to conduct a risk assessment evaluating wastewater reuse in agriculture by using quantitative microbial risk assessment (QMRA). This QMRA was based on the assumption that any microorganisms contained in the residual wastewater remaining on the vegetable surfaces would cling to the crops, even after the wastewater itself evaporated, and this assumption has subsequently been used for QMRA associated with the consumption of wastewater irrigated crops by many researchers (Hamilton et al. 2006; Mara et al. 2007; Forslund et al. 2010). However, the authors are unaware of published papers which compare the number of E. coli recovered on lettuce using the submersion method with those recovered on lettuce following field irrigation with wastewater. The results presented here will be useful to improve the health risk assessment of the consumption of wastewater irrigated salad crops and to confirm the validity of assumptions used in previous QMRAs.

METHODS

Field experiment

Lettuce plots and irrigation systems

A field experiment was conducted at Mount Barker Wastewater Treatment Plant, District Council of Mount Barker, South Australia during January–March 2014. The fenced field site (14.6 m (L) × 5.4 m (W)) was divided into four beds; open bed with spray irrigation (OS), closed bed (covered with polyethylene sheet) with spray irrigation (CS), open drip irrigation (OD) and closed drip irrigation (CD). Each bed (2.4 m × 2.4 m) was filled with a commercial, agricultural sandy-loam. Combo mixed lettuce seedlings (commercial name), which comprised of Oak leaf, Mignonette and Salad bowl lettuce (Lactuca sativa L.) were bought from a local nursery near the site, and grown in six rows with approximately 30 cm spacing between plants. Forty-two plants were grown in each bed. All beds were irrigated by wastewater pumped directly from the last pond of a waste stabilisation pond series. The wastewater irrigation practice used was adapted from the guidelines of Department of Primary Industries, Queensland Horticulture Institute, Australia (Lovatt & Heisswolf 1997). The practice required the seedlings receive light irrigation (10–15 mm) every other day for the first 2 weeks after transplantation, then 15–20 mm every 2 days for the remaining growing period.

Sample collection

Three replicates of lettuce samples were collected randomly from each irrigation bed within 2 h of irrigation. Sampling commenced on week 4 as the lettuce plants were only then large enough to enable analysis. Subsequent, replicate sampling was conducted on week 5 and week 6 since lettuces sold commercially are normally harvested at age 6–8 weeks. Within the weeks, lettuce samples from dripped beds were collected on the day of irrigation (D0), whilst spray bed lettuces were collected on D0, D1 and D2 (0, 1 and 2 days after irrigation, respectively). This enabled determination of the change in concentration, post irrigation, of E. coli on lettuce spray-irrigated with wastewater. The samples were cut from the stem and three to four outer leaves discarded using aseptic technique. Irrigated wastewaters were also sampled at the same time as crop collection. All samples were transported in cold packs and were analysed within 1 h of sampling.

Meteorological data

Meteorological data; temperature (Temp), rainfall and daily global solar exposure (DGSE) for Mount Barker during growing period were obtained from Bureau of Meteorology, Australian Government (http://www.bom.gov.au/sa/?ref=hdr).

Laboratory scale experiment

Oak leaf lettuces were contaminated with wastewater in the laboratory using the submersion technique (Hawley 2012) which was adapted from Shuval et al. (1997). The whole lettuces were submerged, individually, upside down for 20 s into wastewater (5 L) contained in a bucket. Then, each submersed lettuce was held above the bucket and gently flicked right to left, left to right, and up and down, eight times each way. This procedure was performed four times and after the final submersion the lettuce was held above the water level for about 20 s to drain surplus water. Three samples of lettuce were contaminated using this procedure for each of the three experiments using wastewaters of different E. coli concentrations (102, 103 and 104 MPN/100 mL). These values were selected since the WHO (2006) guidelines identify ≤104E. coli MPN/100 mL for verification monitoring of treated wastewater suitable for unrestricted irrigation (leafy crops). Wastewater with the concentrations of E. coli required were obtained by collecting samples from different points in the wastewater treatment chain, at the Mount Barker wastewater treatment plant, followed by dilution as necessary. The highest E. coli concentration (27,550 MPN/100 mL) was obtained from an aerated lagoon, the intermediate concentration (1,299.7 MPN/100 mL) by dilution of this wastewater with clarified effluent from the on-site dissolved air flotation plant (1:10) and the lowest concentration (75.9 MPN/100 mL E. coli) from the maturation lagoon. Composite samples (inner and outer leaves) of the submersed lettuces were obtained, and analysed for E. coli as described below. In a preliminary experiment, three random samples of Oak leaf lettuce were purchased. They had been grown hydroponically using potable water in a commercial glasshouse. All tested negative for E. coli.

Microbial assay

E. coli numbers in wastewater samples were enumerated using the Colilert®-18 MPN method (IDEXX Laboratories, Maine, USA). Composite lettuce samples were cut into 25 g, added to stomacher bags containing 225 mL, 0.1% sterile buffered peptone water, homogenised by means of a stomacher (Model 2X (IDEXX)) for 1 min. The supernatant from the homogenate was enumerated for E. coli by Colilert®-18 MPN method and expressed as the most probable number (MPN) of E. coli per 100 g of lettuce.

Statistical analysis

The difference in E. coli concentration within weeks between lettuces grown in the open spray bed and the covered spray bed was analysed using the Independent-Sample T-Test. The differences in E. coli concentration of spray bed lettuces between different weeks and different sampling times was analysed using one-way analysis of variance (ANOVA). The difference in E. coli concentration on lettuces between different irrigation methods (open spray, closed spray and submersion) was also analysed using ANOVA. All ANOVA analysis was performed together with Bonferroni post-hoc test. The E. coli data used in the analysis were from D0, except for the analysis of the difference in E. coli concentration of spray bed lettuces between different sampling times (D0, D1 and D2). The correlation of E. coli concentration between irrigated wastewater and lettuces was analysed using Pearson Correlation Test. All statistical analyses were performed using SPSS (PASW Statistics 18) with the confidence level of 95%.

RESULTS AND DISCUSSION

E. coli in irrigated wastewater from the field experimental site ranged from 141 to 962.6 MPN/100 mL (489.8 ± 391.6 MPN/100 mL, mean ± standard deviation) during the growing period. All lettuce samples from both spray beds were positive for E. coli, while no E. coli was detected in drip irrigated lettuces. Irrigation method plays an important role in microbial contamination of crops on site. Drip irrigation can reduce the microbial risks by minimising the exposure of the edible part of crops to irrigated wastewater, when compared to spray or springer irrigation methods. Armon et al. (2002) found that springer irrigation had the highest potential for contamination by Cryptosporidium and Giardia in comparison to drip and subsurface drip irrigation when leafy greens were grown using partially treated wastewater. Fonseca et al. (2011) indicated that lettuces grown in E. coli K-12 spiked water were positive for E. coli K-12 when using sprinkler irrigation, whereas only one sample was found to be positive using subsurface drip and furrow irrigation.

Differences between open spray bed lettuces (OSBL) and closed sprayed bed lettuces (CSBL) were also explored, comparing the E. coli concentration from week 4 to week 6 (W4–W6), at different sampling times; that is the concentration on the day of irrigation (D0), 1 day after irrigation (D1) and 2 days after irrigation (D2). The meteorological data and the result are shown in Tables 1 and 2, respectively.

Table 1

Meteorological data during the sampling times

Sampling week Sampling day DGSEa (mJ/m2Rainfallb (mm) Temperaturec (°C) 
W4 24.8 20.8 
D0 13.9 17.5 
D1 24.4 12.5 
D2 24.6 12.8 
W5 9.7 20.3 
D0 8.9 0.2 22.5 
D1 22.0 3.4 14.5 
D2 21.4 12.5 
W6 14.6 9.2 11.8 
D0 11.9 7.4 16.2 
D1 14.0 2.0 16.9 
D2 21.9 0.2 14.8 
Monthly mean 17.2 29.6 15.5 
Sampling week Sampling day DGSEa (mJ/m2Rainfallb (mm) Temperaturec (°C) 
W4 24.8 20.8 
D0 13.9 17.5 
D1 24.4 12.5 
D2 24.6 12.8 
W5 9.7 20.3 
D0 8.9 0.2 22.5 
D1 22.0 3.4 14.5 
D2 21.4 12.5 
W6 14.6 9.2 11.8 
D0 11.9 7.4 16.2 
D1 14.0 2.0 16.9 
D2 21.9 0.2 14.8 
Monthly mean 17.2 29.6 15.5 

aDGSE: daily global solar exposure.

bMonthly mean of rainfall is not provided, the data shown in this table is the total amount within a month.

cTemperature was the value at 9.00 a.m.

D: The day before irrigation; D0 = Irrigation day; D1: 1 day after irrigation; D2: 2 days after irrigation.

Table 2

E. coli concentration (mean ± standard deviation) of the irrigating wastewater (E. coli MPN/100 mL), and spray bed lettuces (E. coli MPN/100 g) sampled within different weeks of the irrigation period on the day of irrigation (D0) and 1 (D1) and 2 days (D2) after irrigation

Sampling week   E. coli concentration
 
Irrigating wastewater (MPN/ 100 mL) OSBL (MPN/100 g)
 
CSBL (MPN/100 g)
 
D0 D1 D2 D0 D1 D2 
W4 210.0 ± 99.8 94 ± 36.3 37.7 ± 5.8 ND 93.7 ± 26.6 70.7 ± 23.5 ND 
W5 177.8 ± 27.8 74.3 ± 11.5 244.3 ± 190.2 93.7 ± 17.2 83.7 ± 12.5 141.7 ± 18.3 63.3 ± 11.5 
W6 962.6 ± 72.4 235.7 ± 100.9 727.3 ± 670.9 ND 249.9 ± 68.3 770.7 ± 730.5 103.3 ± 62.5 
Sampling week   E. coli concentration
 
Irrigating wastewater (MPN/ 100 mL) OSBL (MPN/100 g)
 
CSBL (MPN/100 g)
 
D0 D1 D2 D0 D1 D2 
W4 210.0 ± 99.8 94 ± 36.3 37.7 ± 5.8 ND 93.7 ± 26.6 70.7 ± 23.5 ND 
W5 177.8 ± 27.8 74.3 ± 11.5 244.3 ± 190.2 93.7 ± 17.2 83.7 ± 12.5 141.7 ± 18.3 63.3 ± 11.5 
W6 962.6 ± 72.4 235.7 ± 100.9 727.3 ± 670.9 ND 249.9 ± 68.3 770.7 ± 730.5 103.3 ± 62.5 

ND: not detected (<10 MPN/100 g); OSBL: open spray bed lettuces; CSBL: closed spray bed lettuces.

There was no statistical difference in E. coli concentration (mean ± standard deviation) between OSBL (53.87 ± 37.36) and CSBL (56.93 ± 35.51) within each week or over the entire experimental period (t(16) = 0.179, p > 0.05). However, there were statistical differences in E. coli concentration between weeks in both OSBL (W5 & W6) (F2, 6 = 6.70, p < 0.05) and CSBL (W4 & W6 and W5 & W6) (F2, 6 = 14.11, p < 0.05). The highest contamination was found at W6 for both OSBL and CSBL, the lettuce contamination was significantly correlated with the microbial quality of irrigated wastewater (r2 = 0.99, p < 0.05, n = 3). The concentration of E. coli in irrigated wastewater at W6 was 962.6 ± 72.4 MPN/100 mL which was the highest concentration during the growing period, while the concentrations at W4 and W5 were 210.0 ± 99.8 and 177.8 ± 27.8 MPN/100 mL, respectively. It can be seen that the degree of microbial contamination of the irrigating wastewater was another important factor influencing the level of pathogen contamination of produce at harvest. These data are comparable with research by Solomon et al. (2003), where lettuces irrigated with water inoculated with 104 CFU E. coli O157: H7/mL were more contaminated at harvest time when compared with those irrigated with water containing 102 CFU E. coli O157: H7/mL.

The time elapsed after irrigation influenced the degree of E. coli contamination of the lettuce. In week 4 there was a statistically significant difference in E. coli concentration of lettuce sampled on the day of irrigation (D0) and 1 (D1) and 2 (D2) days after irrigation for both OSBL (F2, 6 = 12.43, p < 0.05) and CSBL (F2, 6 = 13.74, p < 0.05). The numbers of E. coli decreased 1 day after irrigation, no E. coli was detected on the lettuce 2 days after irrigation. This may be explained by the daily solar exposure data during W4, which was very high, greater than monthly mean (17.2 mJ/m2), 1 (D1) and 2 (D2) days after irrigation were 24.4 and 24.6 mJ/m2, respectively. Although the effect of sunlight on the survival of microorganisms on fresh produce surface has not been well described, sunlight is an important factor which inactivates microorganisms in contaminated water and wastewater (Bolton et al. 2010). It is not only sunlight that could influence the microbial contamination and pathogens' persistence on fresh produce pre-harvest, rainfall events may also be potential factors. There was rain during sampling times at W5 (0.2 and 3.4 mm on D0 and D1, respectively) and W6 (7.4, 2.0 and 0.2 mm on D0, D1 and D2, respectively). Also, there was 9.2 mm on the day prior to irrigation day (D). It has been reported that resuspension of sediments during or after rainfall events may result in the increasing numbers of E. coli in stream water (Hunter et al. 1992). There is no evidence regarding this effect in wastewater pond systems. However, in this study, the concentration of E. coli in irrigating wastewater at W6 was highest of the growing season and could be a consequence of rainfall. The high concentration of E. coli in irrigating wastewater may result in the recovery of high numbers of E. coli in lettuces. In addition, Monaghan & Hutchison (2012) noted that rainfall created soil splash which could transfer microorganisms from soil to vegetables. Soil splash created by heavy rainfall on D1 could have also affected the E. coli concentrations on the lettuce which were higher in OSBL and CSBL than on D0 in W5 and W6. However, at W6, no E. coli was detected on open dripped bed lettuces, although there was heavy rain both on D and D0. The geometry of the irrigation systems may explain this observation. The surface area of irrigated soil in the dripped beds was much smaller than sprayed beds. The wetted zone, following irrigation of drip beds, extended only around the drippers, furthermore, the canopy of the mature lettuce protected this area from the direct impact of rain, which may minimise contamination of the lettuce by soil splash. In contrast, the entire area of the spray beds was contaminated with irrigated wastewater, providing greater opportunity for lettuce in spray irrigated beds to be contaminated by soil splash. Further studies, determining the relative concentration of E. coli in soil samples taken from transects across irrigation beds, are required to confirm the potential effect of irrigation geometry on lettuce contamination by soil splash. It was observed from the meteorological data in Table 1 that DGSE was relatively high even on the day of maximum rainfall (D at week 6, DGSE was 14.6 mJ/ m2 and rainfall was 9.2 mm). UVA and UVB are recognised as the main disinfection wavelengths from sunlight (Bolton et al. 2010). While they were not measured directly in this study, the measurement of DGSE was considered a surrogate measurement for likely ultraviolet (UV) exposure. Cloud cover will obviously moderate surface exposure to UV and it might be expected that DGSE and cloud cover are inversely related. However, low DGSE was not always associated with rainfall; there were low values of DGSE recorded in W4 and W5 on days when there was no rain – presumably due to cloud cover moderating DGSE but not resulting in precipitation. It is important to note that the rainfall and DGSE data are daily total values; no hourly data were available. The high rainfall could have occurred at night in W6 resulting in high rainfall and maintenance of a high DGSE, alternatively, as occurs frequently in this location the rainfall event (and cloud cover) could have been of high intensity but short duration, which again would have had a minor effect on the DGSE value. These factors may explain apparent contradiction for the results obtained for W6 whereby the rainfall was the maximum recorded for the study while the DGSE was also high, although slightly less than the monthly mean.

Strong wind during rainfall in W6 also blew off the plastic sheet cover of CS, which also resulted in the contamination of the produce in CSBL. At week 6 (W6) of the growing season, there was no E. coli detected in OSBL on D2, while there was some still detected in CSBL. The polyethylene film use to cover the CBSL has the ability to absorb UV and visible light (Kamweru et al. 2014) which could minimise the exposure of E. coli on the lettuce leaf surfaces to UV and visible light, resulting in the numbers of E. coli still retained on CSBL. This is supported by the observation that, on D0 at W4–W6, E. coli was only detected on a third of the OSBL, whereas E. coli was detected on two-thirds of the CSBL sampled, suggesting that the UV disinfection in open spray bed was greater than the closed bed covered by polyethylene film. It may have been desirable to include in the study control plots irrigated with potable water, however, potable water was unavailable at the site. It can, however, be concluded from the absence of E. coli on lettuces from the drip-irrigated plots that the E. coli contamination was derived from the wastewater. Two mechanisms for the contamination are considered: firstly, direct from the wastewater following spray irrigation and secondly, indirectly by E. coli present on the spray irrigated soil surface subsequently contaminating lettuce via ‘soil splash’ during rainfall events.

Regarding the laboratory scale experiment, the data on the concentration of E. coli retained on lettuces following submersion in wastewaters of differing microbial quality are shown in Table 3. The commercially produced Oak leaf lettuce used in this study were grown hydroponically using water of potable quality to ensure compliance with the guideline value of <3 E. coli MPN/g (FSANZ 2001). Sourcing commercially produced lettuce minimised the likelihood that the E. coli contamination originated from the lettuce. Significantly, E. coli was shown to be absent in all three control lettuces tested immediately post-purchase, confirming that the E. coli contamination in the wastewater-exposed lettuce originated from the wastewater.

Table 3

The number (mean ± standard deviation; n = number of samples analysed) and percentage of E. coli recovered from lettuce following submersion in wastewater

E. coli concentration of wastewater (MPN/100 mL) Recovered E. coli in lettuces (MPN/100 g) Percentage recovery of wastewater E. coli on lettuce 
75.9 20.3 ± 10.5 (n = 3) 26.74 
1,299.7 405.0 ± 29.8 (n = 3) 31.16 
27,550 9,424.0 ± 658.2 (n = 3) 34.20 
E. coli concentration of wastewater (MPN/100 mL) Recovered E. coli in lettuces (MPN/100 g) Percentage recovery of wastewater E. coli on lettuce 
75.9 20.3 ± 10.5 (n = 3) 26.74 
1,299.7 405.0 ± 29.8 (n = 3) 31.16 
27,550 9,424.0 ± 658.2 (n = 3) 34.20 

The number of E. coli on lettuces was significantly, positively correlated with the E. coli concentration in the irrigating wastewater (r2 = 1.00, p < 0.05, n = 3), which was consistent with the results of the field experiment. Moreover, approximately 30% of the E. coli present in the wastewater were recovered on the lettuce, independent of the initial wastewater concentration of E. coli. This was likely dependent upon the volume of wastewater retained by the lettuce, which may be influenced by lettuce morphology. The E. coli concentration of wastewater irrigated lettuce following submersion in wastewater containing 1,299.7 E. coli MPN/100 mL was compared with the results from lettuce harvested in W6 of the field experiment, which had a similar E. coli concentration (962.6 MPN/100 mL) in the irrigating wastewater. It was found that there was no difference in E. coli concentration between W6 field experiment lettuces (both CSBL and OSBL, using E. coli data on D0 and D1) and lettuces submersed with wastewater containing 1,299.7 E. coli MPN/100 mL (F2, 6 = 0.366, p > 0.05). However, as it was discussed above, the microbial quality of crops at harvest was also dependent on the holding period after the last irrigation. Therefore, the numbers of E. coli in wastewater submersed lettuces were different from OSBL on D2, since there was no E. coli detected at that harvesting time. From this result, it could concluded that the microbial contamination by submersion technique in the laboratory was likely equivalent to those from experimental sites on the day of irrigation. Hence, the submersion technique is an acceptable surrogate method to assess initial contamination from spray irrigation practiced on farms, and it is reasonably used to conduct a conservative risk assessment associated with consuming wastewater irrigated salad crops.

QMRA has often been applied to the consumption of wastewater irrigated salad crops. The concentration of E. coli in the wastewater together with the volume of wastewater retained by the lettuce following submersion in that wastewater has commonly been used, including in WHO guidelines (Shuval et al. 1997; WHO 2006), to determine the initial E. coli contamination of the lettuce. This study is the first to confirm that the results from laboratory submersion of lettuce in wastewater are comparable with those obtained from field studies of wastewater irrigated lettuce. Consequently, this study validates the inclusion of E. coli data derived from laboratory submersion experiments into QMRA of salad crops. The study reported here was based on only one type of lettuce, Oak leaf; other types should be further investigated to ensure the general applicability of the surrogate emersion method.

CONCLUSIONS

This study examined the degree of E. coli contamination on lettuces following spray and drip irrigation in the field experiment, and submersion irrigation in the laboratory, using partially treated domestic wastewater. Spray irrigation method implied a higher risk than drip irrigation, since there was no E. coli detected in any of the drip-irrigated lettuce samples in this study. Therefore, in order to minimise public health risk, drip irrigation should be the recommended irrigation method when wastewater is applied to crops. However, when drip irrigation is not applied and spray irrigation is used, the microbial quality of irrigating water, the time of harvest following the last irrigation and climate conditions such as rainfall and sunlight all have the potential to influence the degree of contamination of the harvested lettuce. The results from the laboratory scale support the evidence from the field experiment that the microbial quality of irrigating wastewater was a significant factor affecting the microbial safety of crops at harvest. Uniquely, this study confirmed that the laboratory submersion method was a valid surrogate representing the effect of spray irrigation in the field and was suitable for inclusion into wastewater-irrigated salad crop risk assessment. However, the potential influence of lettuce leaf morphology should be considered in further studies.

ACKNOWLEDGEMENTS

The authors are grateful to Flinders–Hunan Seed Funding Project and School of the Environment, Flinders University, South Australia for support.

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