Impact of reused or new plastic mulch on survival of Escherichia coli and transfer to tomato and bell pepper fruit

This study determined survival of the surrogate Escherichia coli TVS 353 GFP on reused plastic mulch (double-cropped) versus new plastic mulch (single-cropped) and transfer to fruit with ground contact in a field setting. Fruit ground contact treatments included dropped fruit treatments from various heights (30, 60, and 120 cm) and “drooping” treatments (fruit that is still attached to the plant, but touches the ground) for a duration of 1 or 24 h. When comparing survival on shaded versus unshaded locations on plastic mulch, survival over 24 h of E. coli on all locations and mulch types was reduced greater than 3.5 log CFU/64 cm². At 4 h after inoculation, reused plastic mulch retained significantly less E. coli than new mulch at both locations under canopy (shaded) and on the exterior (unshaded) (p < 0.05). Plastic mulch in drooping fruit treatments transferred low amounts of E. coli to fruit (0.01–0.03% of dried inoculum) with no significant differences between reused or new plastic mulch or duration of contact (p < 0.05). Likewise, there was low transfer (0.02–1.87%) from plastic mulch to dropped fruit. Transfer to dropped fruit was affected by treatment including reused or new type of mulch, crop, and height. These results provide information to shape future regulations and risk assessments in preharvest foodborne pathogen contamination, as well as indicate food safety implications for the sustainable practice of reusing plastic mulch.

1 Introduction

Crops such as tomatoes (Solanum lycopersicum) and bell peppers (Capsicum annuum) are often consumed fresh without cooking, making food safety a concern in preharvest production practices. Routes of contamination in the preharvest field environment can occur by the presence of organisms causing foodborne illness through irrigation water (Greene et al., 2008), wild animal feces [United States Centers for Disease Control and Prevention (CDC), 2007], and runoff from excess rainfall (Bennett et al., 2015). Produce contamination at the preharvest stage can also occur through direct contact with soil, from indirect soil contact via rain or irrigation splash, or even from contaminated seed (Thomas et al., 2024). Conditions that have been demonstrated to affect foodborne pathogen survival in the field include air temperature (Cabrera-Díaz et al., 2022; Deblais et al., 2019; Park et al., 2015), soil moisture and rainfall events (Allard et al., 2020; Guo et al., 2002; Micallef et al., 2016; Xu et al., 2016), relative humidity (RH) (Iturriaga et al., 2007; Jung and Schaffner, 2021; Stine et al., 2005), and ultraviolet (UV) radiation (Jeon and Ha, 2018; Sandri et al., 2023). Tomatoes and bell peppers grown in the Southeastern US are typically trellised using wooden stakes and nylon string which suspends plants and harvestable fruit off the ground. However, in these production systems, fruit may still contact the ground by “drooping” (fruit growing on the ground that is still attached to the plant) throughout the growing season or by being dropped to the ground (fruit that falls off the plant) near harvest. Fruit with ground contact prior to harvest may have direct contact with pathogens through contacting contaminated ground surfaces such as polyethylene plastic mulch (Gu et al., 2018; Pagadala et al., 2015).

In commercial vegetable production systems such as tomatoes and bell peppers, plastic mulch is often used due to its ability to improve weed control, reduce the incidence of plant diseases, and retain water in the root zone, resulting in higher yields and improved crop quality (Shah and Wu, 2020). Different colors of plastic mulch can directly enhance plant growth due to their ability to moderate soil temperatures (Lamont, 2005). This is illustrated by growers often utilizing black plastic mulches in the spring to warm cool soils, then switching to white plastic mulch when temperatures increase where contact with black plastic may damage plants (Decoteau et al., 1989; Díaz-Pérez and Batal, 2002; Ham et al., 1993; Parish et al., 2002). Plastic mulch is routinely used for vegetable crop production in the Southeastern US, however it presents a sustainability problem as disposal is typically done by landfill, burning, or burying; all which have the potential to pollute the environment (Shah and Wu, 2020). In order to reduce waste and increase sustainability, growers in the southeastern US have been known to reuse plastic mulches for two or more successive crops, which has been termed “double-cropping.” Reusing plastic mulch has been shown to have comparable yields to using new mulches in several vegetable crops, while reducing costs and increasing sustainability (Hanna, 2000; Hanna et al., 2003; Nyoike and Liburd, 2014; Parish et al., 2002; Waterer et al., 2008). When black plastic mulch is reused for a second crop, growers will often paint plastic mulch white prior to transplanting using water-diluted white paint or calcium carbonate mixture to reduce heat stress on transplants. This paint treatment lightens the mulch surface, increasing sunlight reflectance and reducing heat absorption to allow plants to establish at cooler soil temperatures. As the season progresses, weathering allows the paint on the mulch surface to gradually fade (Figures 1A,C). By the time the white color is faded plants generally are large enough to shade the mulch, further reducing any negative impacts from excessive temperatures at the mulch surface. While the benefits of plastic mulch reuse are known, potential food safety concerns between painted reused mulch and new mulch are not well understood.

Figure 1

Figure 1. Rows were covered with two polyethylene plastic mulches: (A) reused mulch that had a crop grown and removed from it, painted, then planted in for a second time; and (B) new mulch which had been applied shortly before transplanting. (A–D) Represent inoculation locations during the survival experiment: (A) reused plastic mulch on the exterior of the row (RE), (B) new plastic mulch on the exterior of the row (NE), (C) reused plastic mulch under the plant canopy (RC), and (D) new plastic mulch under plant canopy (NC). During fruit contact experiments, inoculation locations were restricted to the exterior of the row (A,B) with both tomatoes and peppers.

Contamination of Salmonella or Shiga-toxigenic E. coli (STEC) onto plastic mulch may affect fruit with ground contact if the mulches act as a source for spreading pathogens to crops prior to harvest (Doren et al., 2022). The US Food & Drug Administration’s Food Safety Modernization Act (FSMA): Standards for the Growing, Harvesting, Packing, and Holding of Produce for Human Consumption (Produce Safety Rule, PSR) 21 CFR Part 112.114 does not allow dropped produce or produce that contacts the ground via “drooping” to be distributed (USFDA, 2019). Trellised crops such as tomatoes and bell peppers are covered by this regulation. Despite being trellised, these crops often have drooping fruit that mature while having ground contact. If drooping fruit were required to be left on the plant in the field, it could result in critical yield losses in the field and a reduction in profitability and sustainability. This study aims to provide data on the survival and transfer of E. coli on reused and new plastic mulch under field conditions which may inform future regulatory decisions, in addition to supporting sustainability by reducing fruit and plastic waste.

The objectives of this study were to (1) determine survival over time comparing reused and new plastic mulch using nonpathogenic Escherichia coli as a surrogate for Salmonella and Shiga-toxigenic E. coli (STEC) during the fall production season, and (2) determine if surrogate E. coli transfer from plastic mulch to peppers and tomatoes by drooping or dropping was affected by using reused or new plastic mulches during the fall production season in the humid subtropical climate of Georgia, United States.

2 Materials and methods

2.1 Field design and crop production practices

Experiments were conducted in a research field at the University of Georgia Horticulture Farm in Watkinsville, Georgia, United States (lat. 33° 5′N, long. 83° 3′W) during the months of August to November 2023. Two mulch treatments were evaluated: new and reused polyethylene plastic mulch. Both new and reused plastic mulch were 31.75 μm white-on-black polyethylene plastic mulch (TriEst Ag. Group, Tifton, GA, United States). The reused plastic mulch was placed in the field in Spring 2023 and had been previously utilized for a spring crop of tomatoes and peppers. Reused mulch was initially placed in the field in the spring with the black side facing upward. Plants from the spring crop were removed after harvest (July), any weeds were killed with glyphosate herbicide (a.i. 0.11 g/m², Ranger Pro, Bayer Chemical, St. Louis, MO, United States) and removed, and the plastic was painted white using a 1:10 (paint:water) dilution of paint (Valspar latex barn and fence paint, Lowes, Mooresville, NC, United States). The paint:water mixture was applied to the plastic mulch using a backpack sprayer at a volume of approximately 0.05 L/m² approximately 1 week prior to transplanting the second crop. The plastic was painted white to encourage cooler mulch surfaces and soil temperatures for transplants (Figure 1A). This is a routine practice by some growers when reusing plastic mulch in the fall in Georgia, United States. The new mulch was applied in August with the white side facing upward (Figure 1B). Both mulches covered raised beds (rows) spaced 1.8 m center to center with row lengths 45 m long with four rows total for each type of mulch. Rows were approximately oriented east to west. The plot was irrigated using drip tubing (Rivulus United States, San Diego, CA, United States) which was placed underneath plastic mulch prior to application. Tomato “Grand Marshall” (Sakata Seed, Morgan Hill, CA, United States) and bell pepper “PS 09979325” (Seminis Vegetable Seed, St. Louis, MO, United States) were grown by a commercial greenhouse (LTF Greenhouses, TyTy, GA, United States). Tomato and bell pepper seedlings were 5 and 7 weeks old, respectively, when transplanted into new and reused plastic mulch on 15 August 2023. Tomatoes were transplanted in single rows in the center of each bed with 0.6 m in-row spacing while bell peppers were transplanted in double rows on each bed with 0.3 m in-row spacing and approximately 0.4 m between-row spacing for each bed. All plants were grown using a trellis supported by wooden stakes with nylon string. Plants were grown according to typical commercial growing practices for the Southeastern United States for fertilization and pest control (Kemble et al., 2023).

2.2 Inoculum preparation

Escherichia coli TVS 353 with a green fluorescent protein (GFP) tag was used as a nonpathogenic surrogate organism for field trials. This organism has been fully sequenced (Harrand et al., 2019) and was GFP transformed by methods described in Oguadinma et al. (2023). Escherichia coli TVS 353 has been used by others as a surrogate for Salmonella and STEC in field trials where use of BSL-2 organisms is inappropriate (Burnett et al., 2025; Belias et al., 2020; Gutiérrez-Rodríguez et al., 2012; Murphy et al., 2023; Murphy et al., 2024; Tomás-Callejas et al., 2011; Weller et al., 2017; Wright et al., 2018). The E. coli GFP used in this study was resistant to rifampicin and ampicillin, and to ensure proper identification when quantifying in the laboratory, was verified under UV fluorescence using a UV viewing cabinet containing a long-wave UV lamp with bulb 365 nm (25.4 cm × 7.5 cm × 6 cm) (IDEXX Laboratories, Inc., Westbrook, ME). Preliminary trials were performed to assess the accuracy of the differential and selective media used to inhibit the growth of background microbiota on the reused mulch. These methods were previously validated on plastic mulch in an environmental chamber (Burnett et al., 2025). Five days before the experiment, the bacteria were grown in tryptic soy broth (TSB) (Difco, Becton Dickinson Co., Sparks, MD, United States) with 80 μg/mL rifampicin (TCI America™, Portland, OR, United States) and 100 μg/mL ampicillin (Fisher BioReagents™, Fair Lawn, NJ, United States) (TSBRA) for 24 h at 37 °C with two successive transfers. Then a 250 μL aliquot of the overnight culture was spread plated on tryptic soy agar (TSA) (Difco, Becton Dickinson Co.) with rifampicin and ampicillin at previously mentioned concentrations (TSARA) to create a bacterial lawn, then incubated for 24 h at 37 °C. After incubation, the plate was filled with 10 mL of buffered peptone water (BPW) (Difco, Becton Dickinson Co.) and a cell spreader was used to displace cells from plate. The inoculum culture was placed in a 50-mL centrifuge tube at an approximate population of 10¹⁰ CFU/mL. The inoculum was diluted in 0.1% w/v peptone (Difco, Becton Dickinson Co.) to obtain a target population of 10⁷ CFU/mL. The concentration of inoculum was verified by making 10-fold serial dilutions in 0.1% peptone, plating in duplicate on TSARA, incubating for 24 h at 37 °C, and enumerating under UV light.

2.3 Field inoculation

Prior to inoculation, locations were marked by using a petri dish as a template to draw circles that were approximately 64 cm² on both types of mulch (Figure 1) for both the survival and transfer experiments. Inoculation locations were randomly assigned within each mulch type (reused or new) using a completely randomized design. Interior rows were selected for both survival and transfer experiments on reused or new mulch. Except for sites under the plant canopy for the survival experiment, each row was inoculated only on the south-facing side of the row, which had the most sunlight exposure. On the day of the experiment, mulches were spot inoculated using 10 drops of 10 μL each (100 μL) of the E. coli strain uniformly distributed within each inoculation site. Due to time limitations of sample analysis and differences in harvest dates for tomatoes and peppers, three separate inoculation experiments were performed on three different days within a 2 week period. As a result, separate inoculum was produced for each experiment. All field inoculations for survival and transfer experiments were conducted in the morning, between 8 and 9 a.m.

2.4 Bacterial survival on mulch over time

Bacterial survival over time was determined in the field beginning on 25 Oct 2023. The average inoculum population at inoculation (time point 0) on plastic mulch was approximately 6.1 log CFU/64 cm². Reused and new plastic mulch rows in bell peppers were inoculated at four different locations: reused plastic mulch under the plant canopy (RC), reused plastic mulch on the exterior of the row (RE), new plastic mulch under plant canopy (NC), and new plastic mulch on the exterior of the row (NE) (Figures 1A–D). At 1, 4, 8, and 24 h after inoculation, inoculation circles on all treatment locations were sampled using a sponge sampler with 10 mL D/E neutralizing broth (EZ Reach™, World Bioproducts, Woodinville, WA, United States) to quantify surviving bacteria. Sponge sampling followed the U. S. Food & Drug Administration’s Bacteriological Analytical Manual (BAM) protocol, which was previously validated for detecting Salmonella and Listeria monocytogenes (Andrews et al., 2024; Hitchins et al., 2022). Sponges were squeezed prior to sampling to remove excess liquid, then used to swab each mulch surface with 10 vertical, 10 horizontal, and 10 diagonal passes before being returned to the bag and submerged in the D/E neutralizing broth. The use of D/E broth for environmental sampling and recovery of different bacteria is supported by multiple research studies (Brauge et al., 2025; Li et al., 2020; Limoges et al., 2020; Zhu et al., 2012). Polyurethane foam sponges, used in this study, have been proven to improve recovery and release of collected microorganisms (Jones and Gibson, 2021). In addition, preliminary studies in the field were conducted where plastic mulch was inoculated with a known inoculum of E. coli GFP and E. coli populations consistent with inoculum levels were successfully recovered after sponge sampling. In the present study, after sampling, sponges were immediately placed in sterile sampling bags, then placed in a cooler with ice packs for transportation from the field to the laboratory. Inoculation circles were randomly assigned a number which correlated to sampling time and were only sampled once. Once all sponge samples were collected, transportation from field to the laboratory took approximately 20 min. On arrival, each sponge sample was diluted 10-fold in 0.1% w/v peptone, plated on TSARA, incubated for 24 h at 37 °C, and enumerated. All plates were verified to be E. coli GFP by green fluorescence under UV light. If samples obtained from plastic mulch at various time points were below the limit of detection for the plating method used, the samples were assigned a value of 1.96 log CFU/64 cm², which corresponds to the limit of detection for the plating method. Enrichment was not performed on the samples. Surface temperatures of reused and new plastic mulch were measured at 0, 1, 4, 8, and 24 h after inoculation by using a handheld infrared thermometer (Traceable®, Thomas Scientific, Swedesboro, NJ, United States) held at a distance of 0.9 m from the mulch surface. Ultraviolet (UV) radiation intensity was measured at inoculation sites at 0, 1, 4, 8, and 24 h after inoculation using a handheld UV_AB meter (EXTECH, FLIR Systems Inc., Wilsonville, OR, United States) that measures wavelengths between 290 ~ 390 nm. Ambient air and RH data were recorded every 15 min and were obtained from a weather station at the site location (Durham Horticulture Farm) from the University of Georgia Weather Network (University of Georgia, 2023). No rainfall events occurred during the entirety of the experiment.

2.5 Bacterial transfer from plastic mulch to fruit

Inoculum was prepared as described previously. Inoculation locations for fruit transfer experiments were on south-facing exterior of rows in tomato and bell pepper on reused and new plastic mulch (Figures 1A,B). These locations were selected to eliminate variability that might occur due to shading from plants. Sampling was conducted when tomato fruit were mature-green on 3 Nov 2023 and bell pepper fruit were mature-green on 1 Nov 2023. Fruit was weighed for uniformity prior to dropping (tomatoes 183 ± 30 g and bell peppers 165 ± 18 g). At the time of inoculation, populations on plastic mulch were approximately 5.93 log CFU/64 cm² for tomatoes and 5.87 log CFU/64 cm² for bell peppers. The inoculum was allowed to dry on the mulch prior to fruit sampling, which took approximately 60 and 90 min for bell pepper and tomato, respectively. After drying, control sponge samples with 10 mL D/E neutralizing broth (EZ Reach™, World Bioproducts) were obtained using the same methods described previously to quantify bacterial populations after drying but prior to fruit contact on plastic mulch. These were taken to the laboratory and plated, incubated 24 h at 37 °C, and enumerated.

Drop height and drooping treatments were conducted on the same day for each crop. Immediately after inoculum was visually dry, bell peppers and tomatoes that had been harvested from the plant the morning of the experiment were dropped through pre-cut PVC pipes (10.2 cm in diameter) from heights of 30, 60, and 120 cm. Dropped fruit had contact with inoculated sites for 1–2 s before being picked up. Drooping treatments began approximately the same time as the drop treatments. Drooping fruit that was still attached to the plant was gently placed on the inoculation site for contact times of either 1 h or 24 h. Immediately after drooping or drop treatments, the fruit samples were aseptically placed in a 30 × 18 cm sterile sampling bag (Fisher Scientific, Pittsburg, PA, United States) then placed in a cooler with ice packs to be transported from the field to the laboratory for bacterial quantification. Transportation from the field to the laboratory took approximately 20 min. Control fruit samples from throughout the plant canopy were taken from each crop and mulch type combination and placed into a sterile bag to ensure that naturally ampicillin-resistant E. coli was not present on fruit in the field.

Environmental conditions were monitored as well. Surface temperatures of plastic mulch and UV_AB radiation were measured as described for the survival experiment. Air temperature and RH were measured in the center of plastic mulch beds with sensors placed approximately 50 cm above the mulch surface (VP3; Meter Group, Pullman, WA, United States). Sensors were connected to a data logger (EM 50G, Meter Group) that recorded temperature and RH every 60 s and reported an average value every 30 min. Rainfall events did not occur during the 24 h of ground contact experiments.

2.6 Quantification of Escherichia coli GFP on tomatoes and bell peppers

After transport from field to laboratory, 100 mL of BPW plus ampicillin (100 μg/mL) was added to each fruit sample bag, the bag was sealed, and the fruit was firmly rubbed by hand for 30 s to release cells from the fruit surface (Burnett and Beuchat, 2001). Colilert reagent (IDEXX Laboratories Inc., Westbrook, ME, United States) was then added to bags until it dissolved completely to allow rapid identification of total coliforms and E. coli. To allow detection to range from 1 MPN to 241,960 MPN (5.38 log MPN/fruit), the fruit wash was further diluted (1:100) by adding 1 mL of the mixture into a second sterile sampling bag with 99 mL of BPW + 100 μg/mL ampicillin. Each 100 mL aliquot was transferred to a Quanti-tray 2000 (IDEXX Laboratories Inc.), sealed, and incubated at 35 °C for 24 h. After incubation, Quanti-tray cells with fluorescence under UV light were counted as positive for E. coli.

2.7 Statistical analysis

For all experiments, treatment combinations had three replicates, with three samples each. Bacterial populations from sponge samplers from the field survival experiment were transformed by logarithmic base 10 transformation before statistically analyzing to confirm normality and equal variance.

For the fruit transfer experiment, when Quanti-trays obtained no positive cells at the lowest dilution, the sample was set at half the limit of detection (0.5 MPN/fruit) for statistical analysis. Percent transfer from surface of new and reused plastic mulch was calculated by the following formula:

$\begin{array}{l}\text{Percent Transfer from mulch to produce}\\ =\left(\frac{\mathrm{MPN}/\text{fruit}}{\mathrm{CFU}/64{\mathrm{cm}}^2\mathrm{on}\text{mulch after drying}}\right)\times 100\end{array}$

The denominator of this equation was obtained from the sponge samples of the inoculum populations remaining on reused or new plastic mulch after drying (Table 1). Similar approaches of averaging CFU and MPN values to report results has been used in previous studies on onions during curing (Moyne et al., 2022) and in biological amendments in soil (Kharel et al., 2025). In addition, correlations between MPN and CFU measurements have been evaluated and equivalent results have been found (Chen et al., 2017; Katase and Tsumura, 2011). Before statistical analysis, percent transfer values were log₁₀ transformed to ensure normality and equal variances. Statistical analysis for both experiments were conducted using JMP (SAS Institute Inc. Cary, NC, United States). Analysis of means (ANOVA) tests were conducted and when significant, mean separations were determined by Tukey–Kramer Honestly Significant Difference test (p < 0.05). When reporting log₁₀ percent transfer values, back transformed percentages will follow in parentheses for ease of interpretation.

Table 1

Table 1. Population of E. coli GFP remaining on reused and new plastic mulch during fruit contact experiments after inoculum drying for 90 min on tomato and for 60 min on bell pepper sampling days.

3 Results and discussion

3.1 Bacterial survival on new or reused plastic mulch

Previously, E. coli GFP has been found to survive similarly to Salmonella on plastic mulch, allowing it to be used as a surrogate for field research (Burnett et al., 2025). In the present study, the survival behavior of E. coli GFP provides insight into how Salmonella may behave under field conditions. On plastic mulch in the field environment, E. coli GFP populations in all locations decreased rapidly, falling more than 3.5 log CFU/64 cm² after 24 h of sampling (Table 2). All treatment combinations of mulch type (new and reused) and location at 1 h had significantly greater (p < 0.05) bacterial populations than the same treatment combinations sampled at 8 h and 24 h. At 4 h post inoculation, E. coli GFP populations differed by sampling location and mulch type. At 24 h after inoculation, there were no significant differences between treatments, but it is notable that at 24 h after inoculation NC had detectable levels of bacteria while NE, RE, and RC fell below the limit of detection. This indicated that certain mulch types and locations, particularly new mulch which is shaded under the plant canopy, may support inoculated bacterial populations beyond 24 h. Typically, studies show a decline in inoculated bacterial populations as time increases post inoculation, but it has been found that inoculated bacteria can persist beyond 24 h of sampling on plant phyllosphere (Park et al., 2015; Xu et al., 2016), fruit (Cabrera-Díaz et al., 2022; Iturriaga et al., 2007; Jung and Schaffner, 2021; Lang et al., 2004; Stine et al., 2005; Tokarskyy et al., 2018), and in soil covered with plastic mulch (Honjoh et al., 2014; Litt et al., 2021; Topalcengiz and Danyluk, 2022). Rosenbaum et al. (2024) found Salmonella rapidly decreased 1.44 log CFU/cm² on plastic mulch at 24 h after inoculation but populations had a tailing effect, as it was found to persist in small amounts on the surface of plastic mulch up to 140 days after inoculation. It must be noted their study was conducted in a dark environmental chamber set at constant temperature and RH (23 °C and 55% RH) which would not necessarily represent environmental conditions in the field which include sunlight and diurnal temperature and RH variations as stress factors. In further research comparing field versus controlled environments, Rosenbaum et al. (2025) confirmed that controlled environments often overestimate persistence of foodborne pathogens as E. coli persisted significantly longer over time in greenhouse and growth chamber versus open-field experiments.

Table 2

Table 2. E. coli GFP survival (log CFU/64 cm² ± standard deviation) in a vegetable field over time on plastic mulch (new or reused) at two locations (under plant canopy or exterior of row) at 1, 4, 8, and 24 h after inoculation.

Escherichia coli GFP survival was affected by type of mulch and location in the planted row over 24 h of sampling (Table 2). At 4 h after inoculation, populations on NE were significantly higher (p < 0.05) than those sampled on RE. Similarly, at 4 h and 8 h, bacterial populations at NC were significantly greater than at RC, indicating that populations may decline faster on reused mulch compared to new mulch. Location relative to the crop also affected bacterial survival, as bacterial populations at RC were significantly greater (p < 0.05) than RE at 4 h after inoculation. After 8 h, both RC and RE had similar reductions in bacterial populations with no significant differences between them, and after 24 h, both locations decreased to below the limit of detection (1.96 log CFU/64 cm²). New plastic mulch showed similar trends to reused mulch, with significantly greater populations at 4 and 8 h after inoculation on NC compared to NE. These results are consistent with Van Kessel et al. (2007), who reported that in a pasture, E. coli populations inoculated inside cattle fecal pats decreased significantly faster in unshaded locations than shaded areas in Maryland, United States. In contrast, in a field tomato planting on plastic mulch in Florida, United States, Topalcengiz and Danyluk (2022) reported no differences in E. coli populations in cattle fecal pats on plastic mulches between shaded and non-shaded areas. In the present study, inoculum was not protected inside a fecal pat and direct exposure to harsh environmental conditions such as UV radiation was occurring in unshaded locations. This may have contributed to the finding that, under daytime conditions, shaded inoculation sites beneath the plant canopy supported higher populations of E. coli GFP compared to exposed sites on the exterior of the mulch bed.

Surface temperatures of plastic mulch likely had an influence on bacterial survival due to location (Figure 2). At 4 h after inoculation (midday), both new and reused plastic mulch locations under the canopy had significantly lower (p < 0.05) surface temperatures than locations on the exterior of the row. These surface temperatures at 4 h after inoculation at RC were 23 °C lower than RE, and at NC were 13 °C less than NE. Morning measurements, which were at 0 and 24 h after inoculation, displayed similar surface temperatures at all locations and mulch types. At 4 h and 8 h after inoculation, locations under plant canopy were associated with lower mulch surface temperatures than exterior locations, which could be attributed to shading. Shading also impacted UV_AB radiation, measured at the surface of the plastic mulch (Figure 3). Most notably, exterior locations on both types of mulch at measured time points had consistently higher UV_AB intensity compared to under canopy locations. The greatest difference between locations occurred at 4 h after inoculation, where UV_AB intensity in reused mulch at RE was 2.37 mW/cm² higher than RC, and in new mulch at NE was 2.01 mW/cm² higher than NC. The fluctuations of both surface temperature and UV intensity are typical of diurnal variations during day and night conditions. Diurnal variations were also observed during the survival experiment with ambient air temperatures ranging from 11 °C to 24 °C as well as RH from 50 to 99% (Figure 4). During exposure to natural sunlight conditions, Boyle et al. (2008) found complete inactivation of E. coli O157 suspended in water within 90 m when sunlight was combined with increasing temperatures (beginning at 22 °C and reaching 38 °C after 4 h of sun exposure). In addition, 90% of E. coli in water was inactivated within 33 min of exposure (Boyle et al., 2008). Similarly, Berney et al. (2006) found that when E. coli was suspended in mineral water and held constant at a temperature of 37 °C, inactivation occurred after 4 h of natural sunlight exposure during a sunny day. Temperatures over 47 °C could enhance inactivation, allowing synergy to occur between heightened temperatures and UV radiation from sunlight (Berney et al., 2006). In the present study, surface temperatures of RE and NE at 4 h post-inoculation were greater than 35 °C. Most notable, the surface temperature of RE after 4 h was over 50 °C, which is consistent with inactivation due to sunlight and temperature that occurred in Boyle et al. (2008) and Berney et al. (2006). However, despite surface temperature for locations NC and RC remaining lower than 30 °C, E. coli populations were declining rapidly in all locations. We hypothesize that E. coli populations were reducing on plastic mulch over 24 h in the present study due to a reduction effect on bacterial populations caused by field conditions such as mulch surface temperatures and environmental conditions including changes in surface texture, microbial diversity, and the presence of soil and plant matter on plastic mulch.

Figure 2

Figure 2. Mean mulch surface temperatures (°C) during the survival experiment sampled over time at 0, 1, 4, 8, and 24 h after inoculation at four locations: new plastic mulch under plant canopy (NC), new plastic mulch on exterior of row (NE), reused plastic mulch under plant canopy (RC), and reused plastic mulch on exterior of row (RE).

Figure 3

Figure 3. Mean UV_AB intensity (mW/cm²) during the survival experiment sampled over time at 1, 4, 8, and 24 h after inoculation on four locations: new plastic mulch under plant canopy (NC), new plastic mulch on exterior of row (NE), reused plastic mulch under plant canopy (RC), and reused plastic mulch on exterior of row (RE).

Figure 4

Figure 4. Mean ambient air temperature (°C) and relative humidity (RH) (%) from 0 to 24 h post-inoculation during the field survival, bell pepper transfer, and tomato transfer studies.

While some field survival studies track bacterial populations over several days or weeks, this study sought to understand what was happening during the first 24 h post-inoculation. This time period aligns with the time frame relevant to the fruit contact experiment. Although this could appear to be a limitation of the study, it was intentionally chosen to reflect field conditions during harvest when fruit may directly contact the ground. By focusing efforts on a 24 h period, we were able to better understand the survival dynamics in the field on new and reused plastic mulch and apply this knowledge to the fruit contact experiment.

3.2 Escherichia coli transfer from plastic mulch to fruit with ground contact

During the bell pepper and tomato fruit contact experiments, inoculum concentrations were reduced approximately 1 log CFU/64 cm² during drying, but prior to fruit contact, in new and reused plastic mulch (Table 1). All control fruit samples were negative for E. coli, confirming that no ampicillin-resistant background microbiota were present on fruit or mulch. Fruit that contacted reused or new plastic mulch by being dropped transferred no greater than 0.27 log percent (1.87%) of inoculated E. coli to tomatoes and peppers (Table 3). All dropped tomatoes and peppers were positive for bacterial transfer from heights of 30–120 cm on both types of mulch. Dropped tomatoes had no significant differences (p < 0.05) between treatments of plastic mulch type or drop height. In pepper, when main effects were compared, new plastic mulch transferred significantly higher amounts of E. coli than reused plastic mulch. This was consistent within treatments as shown in Table 3, as dropped peppers from heights of 30 cm and 60 cm onto new plastic mulch (0.58 and 1.87%) had significantly higher percent transfer than on reused mulch (0.08 and 0.21%).

Table 3

Table 3. Percent transfer of E. coli GFP from plastic mulch to fruit dropped from 30, 60, and 120 cm heights comparing bacterial transfer rates from mulch to fruit with two types of crops (bell pepper and tomato) and two types of plastic mulch (new and reused).

In drooping fruit treatments in tomatoes and peppers, there were no significant differences (p < 0.05) between mulch type or contact duration (Table 4). Although both drop and drooping treatments were conducted almost simultaneously, all dropped fruit samples were positive for bacterial transfer while some drooping fruit samples were non-detectable for transfer. Overall rates of transfer to drooping fruit for both bell pepper and tomato were low, ranging from −2.35 to −1.48 log percent transfer (0.01–0.03%) to fruit from inoculated plastic mulch. Drooping fruit results are similar to previous work which found that there were no significant differences in bacterial transfer between drooping durations for fruit contacting plastic mulch (Burnett et al., 2025). In addition, Tokarskyy et al. (2020) found that allowing E. coli inoculum to dry for 90 min on polyethylene plastic and then touching tomatoes to the inoculation area resulted in no significant differences between bacterial populations on fruit quantified immediately after contact or after 24 h of contact. Similarly, Todd-Searle et al. (2020) found no significant differences in bacterial transfer from new versus reused plastic mulch to fruit at contact durations of touch (1–5 s) or 24 h. However, in their study, new mulch indicated plastic mulch taken directly from the application roll while used mulch was in the field for one growing season. In our study, reused mulch had been in the field for two growing seasons while new mulch was in the field for one growing season.

Table 4

Table 4. Percent transfer of E. coli GFP from inoculum on plastic mulch after drying to fruit drooped with contact times of 1 h or 24 h, comparing two types of crops (bell pepper and tomato) and two colors of plastic mulch (new and reused).

One novel finding of the present study are the results comparing reused (double-cropped) versus new (single-cropped) plastic mulch in the field. In the survival experiment, inoculum survived significantly better on new mulch than reused mulch at 4 h and 8 h. In addition, significantly greater log percent transfer occurred to dropped peppers on new mulch versus reused mulch. These findings of survival and transfer may be due to multiple factors on reused mulch including changes in microbial diversity, differences in surface roughness, and accumulated soil and crop residue. Microbial diversity may increase the longer plastic mulch is in the field as noted in Yu et al. (2025), where the authors showed that microbial diversity increased over time on polyethylene plastic mulch in the field. This diversity may result in increased competition for resources for inoculated bacteria, as native microflora may have colonized the reused mulch heavily, potentially decreasing inoculated bacterial persistence. However, it has been reported that increases in coarseness of the surface texture on fruit or other surfaces can lead to increased bacterial survival and retention (Wang et al., 2009). This can lead to attachment and formation of bacterial biofilm structures on polyethylene, which can protect bacteria from environmental stresses (Liu et al., 2023). Previous studies have reported that there is a greater likelihood of bacterial transfer from fruit to mulch than from mulch to fruit (Todd-Searle et al., 2020; Tokarskyy et al., 2020), possibly due to bacterial attachment on plastic mulch. Weathering of plastic mulch over time changes the surface texture of plastic mulch from a relatively smooth surface to a surface that includes pits, flakes, grooves, and attached particles (Zhang et al., 2019). Likewise, the paint application on reused mulch which was used to reflect light and cool the surface, likely affected surface texture and porosity on reused mulch. The potential increase in surface irregularities of the reused plastic mulch compared to the new plastic mulch may have provided more locations for microbial colonization and biofilm formation, leading to the decrease in bacterial transfer to fruit in the present study. In addition, Waterer et al. (2008) found that there were high amounts of soil and crop residue on double-cropped plastic mulch, even though it could be removed through washing. Despite uniform exposure to field conditions, reused mulch was not washed prior to initiating our study and may have contributed to non-uniform soil depositions and weathering on the surface which may have influenced surface texture and bacterial survival. Although the interactions that occurred in this study between plastic mulch surface textures, native microbial populations, and result of biofilms (native and inoculated) were outside the scope of this study and may be explored in future research, our results provide evidence that reused plastic mulch is less conducive to the persistence of foodborne pathogens under field conditions.

3.3 Field conditions during fruit ground contact experiments

Plastic mulch surface temperatures reached ~30 °C on reused mulch on both crop sample days (Table 5). In bell peppers, new mulch surface temperatures were less than on reused mulch in bell peppers, only reaching 16 °C on new mulch while reaching 31 °C on reused. In tomatoes, surface temperatures on new mulch reached 26 °C and on reused mulch reached 32 °C. The reused mulch was painted white to reduce temperatures at planting, while the new mulch was placed with the white plastic surface facing upward. In comparison to transplanting, after 2 months of plant growth some of the paint had faded from the plastic resulting in a darker surface and greater subsequent temperatures compared to the white plastic (Figure 1A; Table 5). Ambient air temperatures and RH levels were similar between tomato and pepper sampling days, with the bell pepper sampling day being cooler on average than the tomato sampling day (Figure 4). RH levels were very low during both tomato and pepper sampling days, staying below 30% RH from 4 h until 12 h after inoculation. UV_AB intensity stayed below 2 mW/cm², which was likely due to time of year (fall) and orientation of the rows (Table 6). UV_AB intensity between bell pepper versus tomato was significantly different at each time point, which may be the result of the taller trellised height of tomato plants, which provided partial shading on both sides of the row. As described above, both UV_A and UV_B radiation have been shown to injure bacteria in various ways and attribute to the complexity of bacteria survival in the field environment (Jeon and Ha, 2018; Sandri et al., 2023).

Table 5

Table 5. Surface temperature (°C) measured on reused and new plastic mulch during bell pepper and tomato sampling days at 0, 1, 2, and 3 h after inoculation. Bell pepper and tomato measurements were conducted on separate days.

Table 6

Table 6. UV_AB intensity (mW/cm²) measured on reused and new plastic mulch during bell pepper and tomato sampling days at 0, 1, 2, and 3 h after inoculation. Bell pepper and tomato measurements were conducted on separate days.

Weather conditions during different times of year may have an impact on survival and transfer from plastic mulch to fruit. Building upon previous research in Burnett et al. (2025), inoculated E. coli GFP bacteria during the summer season decreased after drying in greater amounts (2–3 log CFU/64 cm²) than in the current study (~1 log CFU/64 cm²) which was conducted in the fall season. Ambient air conditions are typically cooler in the fall in Georgia, United States than they are in the summer growing season, and RH levels are often lower. Ambient air temperatures during the fall transfer studies (Figure 4) ranged from −3 to 17 °C and were cooler than those recorded during the summer growing season in Burnett et al. (2025), which ranged between 18 and 36 °C. Relative humidity during the present study varied but was lower during the day (20–45% RH, Figure 4), compared with higher humidity conditions observed during the day in Burnett et al. (2025) which was conducted during the summer (45–80% RH). Despite these seasonal differences, bacteria transferred no greater than 1.87% of dried inoculum on plastic mulch to dropped or drooping fruit in our study, and bacterial transfer from plastic mulch was no greater than 2.88% in dropped and drooping fruit in Burnett et al. (2025). This suggests that even though bacterial populations after drying in Burnett et al. (2025) were between 2.53 to 3.67 log CFU/64 cm² and in this study was between 4.89 to 5.14 log CFU/64 cm², only a small percentage of bacteria transferred to fruit upon ground contact. Seasonality has been reported to affect bacterial contamination in some leafy greens and herbs in field environments, with higher incidence of Salmonella or E. coli reported in crops grown during the fall production season (cooler weather) compared to summer (warmer weather) (Ailes et al., 2008; Marine et al., 2015). In contrast, Strawn et al. (2013) found no difference in bacterial incidence when conducting preharvest sampling on New York, United States vegetable farms throughout four seasons. In combination with previous studies, our findings suggest that while bacterial survival on plastic mulch may fluctuate with season, the percentage transferring from plastic mulch to fruit with ground contact remains low.

4 Conclusion

E. coli GFP declined in the field environment over 24 h on both new and reused plastic mulch. Increased shading from the plant canopy allowed bacteria to persist longer compared to inoculation sites on the exterior of the row, with the longest persistence occurring on new mulch under the plant canopy. Harsh environmental conditions, including heightened surface temperatures, fluctuations in ambient temperature and relative humidity, and UV_AB exposure from sunlight, likely influenced bacterial survival on plastic mulch and subsequent transfer to fruit. In comparing new versus reused plastic mulch, inoculum survived significantly better on new mulch than reused mulch over time, suggesting that future research could explore the potential for increased bacterial retention and transfer on new mulch compared to reused mulch. Overall, both reused and new plastic mulch transferred very low amounts of bacteria to drooping fruit (0.01–0.03%) and low amounts to dropped fruit (0.02–1.87%). While seasonal conditions may affect overall bacterial survival rates, transfer rates from plastic mulch to fruit in the field environment were consistently low. This study identifies reduced persistence of foodborne pathogens on reused (double-cropped) plastic mulch, and suggests future research to understanding microscopic differences at the plastic mulch interface, comparing single-cropped (new) and double-cropped (reused) mulches. In summary, these results provide data to support future risk assessments and inform regulatory agencies, particularly regarding the food safety implications of the sustainable reuse of plastic mulch across multiple cropping seasons.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

AB: Data curation, Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review & editing. BR-L: Investigation, Methodology, Project administration, Writing – review & editing. FC: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing. TC: Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This work is supported by Specialty Crops Research Initiative [2020-51181-32157] from the USDA National Institute of Food and Agriculture. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U. S. Department of Agriculture.

Acknowledgments

The authors would like to thank Rawane Raad, Halle Greenbaum, Jia Yan Hiew, Justin Daniel, and Charles Appolon for their assistance with sample analysis.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The authors declare that no Gen AI was used in the creation of this manuscript.

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