Effects of Cover Crop Species and Season on Population Dynamics ofEscherichia coli and Listeria innocua in Soil

Neiunna L. Reed-Jones,a Sasha Cahn Marine,b Kathryne L. Everts,b,c Shirley A. Micallefa,d

Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland, USAa; Department of Plant Science and LandscapeArchitecture, University of Maryland, Lower Eastern Shore Research and Education Center, Salisbury, Maryland, USAb; University of Delaware, Carvel Research andEducation Center, Georgetown, Delaware, USAc; Center for Food Safety and Security Systems, University of Maryland, College Park, Maryland, USAd

Cover crops provide several ecosystem services, but their impact on enteric bacterial survival remains unexplored. The influenceof cover cropping on foodborne pathogen indicator bacteria was assessed in five cover crop/green manure systems: cereal rye,hairy vetch, crimson clover, hairy vetch-rye and crimson clover-rye mixtures, and bare ground. Cover crop plots were inoculatedwith Escherichia coli and Listeria innocua in the fall of 2013 and 2014 and tilled into the soil in the spring to form green manure.Soil samples were collected and the bacteria enumerated. Time was a factor for all bacterial populations studied in all fields (P <0.001). E. coli levels declined when soil temperatures dipped to <5°C and were detected only sporadically the following spring. L.innocua diminished somewhat but persisted, independently of season. In an organic field, the cover crop was a factor for E. coliin year 1 (P � 0.004) and for L. innocua in year 2 (P � 0.011). In year 1, E. coli levels were highest in the rye and hairy vetch-ryeplots. In year 2, L. innocua levels were higher in hairy vetch-rye (P � 0.01) and hairy vetch (P � 0.03) plots than in the rye plot.Bacterial populations grew (P < 0.05) or remained the same 4 weeks after green manure incorporation, although initial reduc-tions in L. innocua numbers were observed after tilling (P < 0.05). Green manure type was a factor only for L. innocua abun-dance in a transitional field (P < 0.05). Overall, the impacts of cover crops/green manures on bacterial population dynamics insoil varied, being influenced by bacterial species, time from inoculation, soil temperature, rainfall, and tillage; this reveals theneed for long-term studies.

In agricultural environments, soil can serve as a reservoir androute of transmission for foodborne pathogens (1). Since fresh

produce is often consumed raw without a “kill step” to inactivatehuman-pathogenic microorganisms, it is important to preventthe contamination of these foods during production (2). Growersmay implement good agricultural practices (GAPs) to minimizethe risk of produce contamination at the preharvest level (3), asthis program focuses on on-farm risk factors, including animal-based fertilizers, irrigation water quality, farm worker trainingand hygiene, and wildlife exclusion. Outbreaks and contamina-tion events, however, continue to occur, emphasizing the need toevaluate the role of other agricultural practices, which thus farhave received less attention, on enteric pathogen dynamics on afarm.

One such practice is cover cropping, the establishment of acrop, typically a small grain or legume, in between cultivations ofa cash crop. In recent years, economic and environmental consid-erations have renewed interest in this old practice for improvingcrop productivity and soil health and maintaining the sustainabil-ity of agroecosystems (4). Cover cropping brings a variety of eco-system services to agricultural systems, including seasonal protec-tion of soil from erosion, weed suppression, soil improvement,and nutrient management though nitrogen fixation and carbonaccumulation (5). From a microbial perspective, the establish-ment of a cover crop provides a rhizosphere effect (6), wherebyplant roots modify the soil habitat as they grow, improving aera-tion and serving as a source of nutrients to microorganisms, lead-ing to enhanced microbial growth and activity (7). Consequently,cover cropping has a positive effect on soil microbial populations,processes, and activities (8). In comparison to the biomass andactivities of microorganisms in bulk soil, those of soilborne mi-croorganisms are boosted as a result of the exudation of phyto-

compounds by plant roots (9). In long-term field trials in whichdifferent agricultural systems were compared, soils from organi-cally managed plots had increased microbial biomass and biodi-versity, enhanced soil fertility, reduced soil erosion, and improvedsoil quality, cycling efficiency, and overall crop productivity (10,11). Whether there is a positive rhizosphere effect from a variety ofcover crop species on the growth, persistence, and activity of food-borne pathogens in soil, however, has not been evaluated.

Green manure, which is formed by the incorporation of a fall-planted cover crop into the soil in the spring, is employed to in-crease soil organic matter (reviewed in reference 12) and soil qual-ity (13), stimulate soil microbial growth and activity, whichenhances the subsequent mineralization of plant nutrients (14),and increase soil fertility (15). Cover crops and green manureshave also been used to suppress soilborne and foliar plant diseases(16), plant-parasitic nematodes (17), and insect pests (reviewed inreference 18). For example, a hairy vetch (HV) cover crop (Viciavillosa Roth) suppressed multiple plant diseases on watermelon(19, 20) and pumpkin (21) and reduced foliar necrosis in process-ing tomato (22). Ryegrass (Lolium multiflorum Lam) green ma-nures reduced powdery scab in potatoes (23) and Verticillium mi-

Received 14 November 2015 Accepted 30 December 2015

Accepted manuscript posted online 4 January 2016

Citation Reed-Jones NL, Marine SC, Everts KL, Micallef SA. 2016. Effects of covercrop species and season on population dynamics of Escherichia coli and Listeriainnocua in soil. Appl Environ Microbiol 82:1767–1777. doi:10.1128/AEM.03712-15.

Editor: J. Björkroth, University of Helsinki

Address correspondence to Shirley A. Micallef, smicall@umd.edu.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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crosclerotia density in soil in cauliflower fields (24). While thereare many instances in which cover crop-suppressive effects onplant pathogens have been documented, their potential biocon-trol activity on enteric foodborne pathogens has not been ex-plored.

The objective of this study was to evaluate the impact of single-species and multispecies cover crop mixtures on bacteria relevantto food safety. The influence of cover cropping on foodbornepathogen indicators was assessed in five cover crop/green manuresystems: cereal rye (R), hairy vetch, crimson clover (C), a hairyvetch-rye mixture (HVR), a crimson clover-rye mixture (CR),and a no-cover crop bare-ground (BG) control. The study wasconducted over a 2-year period in an organic field and over a1-year period in a conventional field that was transitioning toorganic production.

MATERIALS AND METHODSField preparation. Field experiments were conducted on certified organicand transitional (previously conventional) land at the University of Mary-land (UMD) Lower Eastern Shore Research and Education Center(LESREC) in Salisbury, MD (about 38°N and 75°W). Both fields had loworganic matter (�1%) and dimensions of 107 m long by 27 m wide (�0.3ha in size). The organic field was composed of Fort Mott and Rosedaleloamy sand soils (0 to 5% slope and pH 6.8), was within 40 m of a wood-land conservation buffer, and had a history of organic mixed-vegetableproduction. The transitional field was composed of Fort Mott loamy sandsoils (0 to 2% slope and pH 5.9), surrounded by other fields, and had ahistory of conventional agronomic crop (primarily field corn and soy-bean) production. The trial was conducted three times: in 2013 to 2014and 2014 to 2015 in the organic field and in 2014 to 2015 in the transitionalfield. Cover crops were sown using a grain drill on 22 October 2013 (year1) and 23 September 2014 (year 2), and overhead irrigation was appliedbriefly to improve seed germination and establishment. Bare-ground(BG) plots served as the control and remained fallow throughout the fall,winter, and spring seasons. The field experiments were arranged in a ran-domized complete block design with four replications in year 1 (organicfield only), with an additional replication (n � 5) in year 2 (organic andtransitional fields). The individual plots were 27 m long by 3 m wide. Deerfencing was installed after cover crop seeding to deter wildlife intrusion.At the flowering stage, cover crops were tilled into the soil to a depth of 15to 20 cm using a moldboard-type plow (15 May 2014 for year 1 and 6 May2015 for year 2). The fields were plowed again on 3 June 2014 (year 1) and22 May 2015 (year 2) to further incorporate the green manure into thesoil.

Cover crops. Cover crop treatments included hairy vetch (V. villosaRoth) (HV) at 18.14 kg/ha, crimson clover (Trifolium incarnatum) (C) at9.07 kg/ha, cereal rye (Secale cereale L.) (R) at 31.75 kg/ha, a 2:3 (wt/wt)mixture of hairy vetch (9.07 kg/ha) and rye (13.60 kg/ha) (HVR), and a 2:1(wt/wt) mixture of crimson clover (9.07 kg/ha) and rye (4.54 kg/ha) (CR).The C and CR plots were planted in the second year only. Certified organic(when available) or non-genetically modified organism (GMO)-treatedcover crop seed was purchased from the following suppliers: Johnny’sSelected Seeds (Winslow, ME), High Mowing Organic Seeds (Wolcott,VT), Fedco Seeds (Waterville, ME), and Territorial Seed Company (Cot-tage Grove, OR). Legume cover crop seed (hairy vetch and crimson clo-ver) was mixed with Organic Materials Review Institute (OMRI)-listedN-Dure inoculant (INTX Microbials, LLC, Cary, NC) prior to beingseeded to encourage nitrogen-fixing nodule formation.

Bacterial indicators. Nonpathogenic strains were used for introduc-tion into field plots as bacterial indices for enteric pathogens. GenericEscherichia coli strains isolated from liquid dairy manure collected from adairy farm in Clarksville, MD, and Listeria innocua (ATCC 33090) wereused. E. coli was incubated in Trypticase soy broth (TSB) (BD DiagnosticSystems, Franklin Lakes, NJ) for 24 h at 37°C. L. innocua was incubated in

brain heart infusion (BHI) broth (BD) for 48 h at 30°C. The cells werecentrifuged for 10 min at 2,000 � g, the supernatant was discarded, andpellets were resuspended and washed with phosphate-buffered saline(PBS). After the pellets were washed, 10 ml of 0.1% peptone water (PW)was added to the pellets and the suspension was vortexed. The concentra-tions of the stock suspensions were confirmed by serial dilutions in 0.1%PW. The dilutions were plated onto Trypticase soy agar (TSA) (BD) platesfor generic E. coli and brain heart infusion agar (BHIA) (BD) for L. in-nocua. A cocktail of the two bacteria (�106 CFU/ml) was prepared forfield inoculations. The inoculum was kept on ice until application to avoidany growth while in transit.

Field inoculation. The bacterial cocktail was applied to the field 6 daysafter cover crop seeding in year 1 and 8 days after cover crop seeding inyear 2. In year 1, the bacterial suspension was adjusted to a final concen-tration of �106 CFU/ml; this was sprayed evenly on the soil surface of thetreatment plots using a handheld sprayer boom with four nozzles, with ahorizontal spray width of 1.8 m and application at a rate of �8 ml/m2. Inyear 2, the applied concentration was adjusted to account for levels al-ready present in the soil. The applied concentrations were 104 CFU/ml E.coli and 103 CFU/ml L. innocua.

Field sample collection. Soil samples were collected immediatelyprior to field inoculation and within 2 h following inoculation. Samplingcontinued every 2 weeks postinoculation until frost, monthly thereafteruntil cover crop incorporation into the soil, and then biweekly for 4 weeks.Field soil samples (�100 g of composite soil) consisted of four soil sub-samples per cover crop treatment per replicate per field and were collectedin sterile Whirl-Pak bags (Nasco, Ft. Atkinson, WI) using 60-ml sterilescoops (Fisher Scientific, Hampton, NH). In the cover crop treatments,samples were collected to a 7-cm depth from the root zone and werenondestructive to the cover crops. In the bare-ground treatments, soilsamples were collected in areas devoid of weeds to a 7-cm depth. Latexgloves and plastic boot covers were worn for sample collection, changedbetween fields, and disinfected with 70% ethanol between samples. Thesamples were sealed, transported in coolers with ice packs, and deliveredthe same day or shipped overnight to the laboratory for microbiologicalanalysis within 24 h. A total of 224 field soil samples were collected in year1 (56 composite samples for each of the four cover crop treatments). Inyear 2, a total of 780 soil samples were collected (65 composite samples foreach of the six cover crop treatments in each of the two field locations).

Sample preparation and bacterial enumeration. Thirty grams of soilwas mixed with buffered peptone water (BPW) (1:5 [wt/vol]), stomachedat 200 rpm for 1 min, and allowed to recover for 1 h to revive injured cells.Two 96-well plates for a 3-tube most-probable-number (MPN) analysiswere prepared, one for E. coli with brilliant green bile broth with 4-methyl-umbelliferyl-�-D-glucuronide (BRILA-MUG) (Criterion, Santa MariaCA) and one for Listeria using buffered Listeria enrichment broth (BLEB)(BD). The samples were shaken by hand for 10 s to homogenize them,serial dilutions were then prepared in the respective medium, and E. coliplates were incubated at 42°C for 24 h and Listeria plates at 30°C for 48 h.Following incubation, 10 �l of culture was either plated on Tryptone bileglucuronic agar (TBX) (Hi Media, Mumbai, India) and incubated at 42°Cfor 24 h for E. coli or plated on Oxford Listeria agar (OXA) (EMD Milli-pore, Billerica, MA) and incubated at 30°C for 24 to 48 h for L. innocua.The MPN protocol was modified from that used by Ingram et al. (25).

Meteorological data. On-site weather data were recorded every 60min using a CR1000 data logger (Campbell Scientific, Logan, UT)equipped with the following instruments: reflectometer (model CS650),anemometer (model 034B), pyranometer (model LI-COR), rain gauge(model TE525WS), and temperature and humidity probes (models 107and HMP60, respectively). Soil temperature was evaluated on the day ofsampling and recorded as the average of the maximum and minimumtemperature readings.

Statistical analysis. The MPN was calculated using the MPN Calcula-tor, version VB6 (http://www.i2workout.com/mcuriale/mpn/). The datawere log transformed and analyzed using a mixed model with repeated

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measures. Fixed effects were cover crop species and day postinoculation,while subject was treated as a random effect, due to repeated measures andnonindependence of the observations and residuals. Least-squares meanswere calculated, along with the standard error. Statistically significanteffects at a P value of �0.05 were further analyzed with Tukey’s honestlysignificant difference (HSD) test. All analyses were performed using JMPPro version 11 (SAS Institute, Inc., Cary, NC).

RESULTSBacterial population dynamics in organic and transitionalfields. In the first year of the study from October 2013 to May2014, cover crops were grown using organic practices, and soilsamples were collected over a 27-week time period, at which timecover crops were incorporated into the soil. In year 2, cover cropswere grown in two fields (one organic and one transitional) fromOctober 2014 to June 2015, and soil sampling occurred over a30-week period before the tilling. In both years, sampling of thegreen manures continued for an additional 4 weeks. Prior to fieldinoculation with the bacterial cocktail in the fall of year 1, E. coliwas found to be mostly undetectable in the soil in the organic fieldbut sporadically present at a mean of 0.4 log MPN/g of soil in twoplots. L. innocua was undetectable in all plots. Following inocula-tion, the mean levels of bacteria in the organic field were approx-imately 6 log MPN/g of soil in year 1 and 4 log MPN/g of soil inyear 2 (Fig. 1 and 2 and Table 1). In the transitional field in year 2,the mean bacterial levels following inoculation were 3 log MPN/gof soil for E. coli and 7 log MPN/g of soil for L. innocua (Fig. 1C and2C and Table 1). In all cases, bacterial levels declined from fall tolate spring (Table 1). E. coli levels declined to below the detectionlimit by weeks 5 and 9 in the organic field in the two consecutiveyears, respectively (Fig. 1A and B), and by week 5 in the transi-tional field (Fig. 1C). Although L. innocua levels diminished over-all, this species persisted throughout the study period, dippingbelow the detection limit only in week 23 in the organic field inyear 2 (Fig. 2B) and in week 30 in the transitional field (Fig. 2C).

Effect of cover crop species on bacterial population dynamicsin an organic field. (i) E. coli. In the organic field in year 1, bothcover crop treatment (P � 0.004) and time (P � 0.001) weresignificant factors for E. coli survival. A statistically significant in-teraction between cover crop and time (P � 0.001) denoted thatthe effect of cover crop was most significant in the first 3 weeks ofthe study (Fig. 1A). E. coli populations in the bare-ground and HVplots displayed sharp declines in the survival of E. coli after week 1,while E. coli levels in R and HVR plots persisted at around 4 to 5 logMPN/g of soil until week 3 before declining. HVR and R plotssupported the highest mean E. coli populations at 1.85 � 0.15 and1.82 � 0.15 log MPN/g of soil, respectively, and were both signif-icantly different (P � 0.007 and �0.01, respectively) from thelowest levels detected in bare-ground plots at 1.01 � 0.15 logMPN/g of soil. In year 2, time (P � 0.001) was a significant factorin E. coli survival (Fig. 1B and C), while no significant differencesamong cover crops were detected. An interaction (P � 0.001)between the two factors revealed that the C, CR, and HV plots hadhigher E. coli levels in the fall than in other seasons, with overallmeans of 1.31 � 0.23, 1.14 � 0.23, and 1.10 � 0.23 log MPN/g ofsoil. The lowest levels were recorded from the R plots (mean of0.70 log MPN/g of soil).

(ii) L. innocua. Time was also a factor for L. innocua persis-tence (P � 0.001) in year 1, while cover crop was not (Fig. 2).However, an interaction between cover crop and time (P � 0.001)

revealed increased L. innocua persistence in the first few weeks ofthe study in the R and HVR plots (Fig. 2A). The HV plots exhibitedthe sharpest declines. The highest mean L. innocua levels wererecovered from the R plots (4.56 � 0.20 log MPN/g of soil), andthe lowest were recovered from the HV plots (3.85 � 0.20 logMPN/g of soil). In year 2, both cover crop (P � 0.011) and time (P� 0.001) had a significant impact on L. innocua survival (Fig. 2A).A significant interaction (P � 0.013) highlighted the dependencyof the two factors. The levels of L. innocua cells were significantlyhigher in HVR (P � 0.010) and HV (P � 0.029) plots than in Rplots (Fig. 2B). The mean levels were determined to be 3.04, 2.91,and 1.93 � 0.21 log MPN/g of soil, respectively.

Effect of field management on bacterial population dynam-ics. To determine whether field management (i.e., transition fromconventional to organic production) would influence indicatorbacterial population dynamics, a field previously under conven-tional management was included in the study in year 2. Prior tocover crop seeding in the fall of 2014, this field had been in a 3-yearagronomic crop rotation (corn followed by wheat in 2011, dou-ble-crop soybeans in 2012, and full-season soybeans in 2013).Time (P � 0.001), but not cover crop, was a factor for E. coli and L.innocua survival (Fig. 1C and 2C). A significant interaction of timeand cover crop (P � 0.001) indicated higher E. coli levels in theHVR and CR plots at certain time points. The HVR plots sup-ported the highest mean population, at 1.05 � 0.21 log MPN/g ofsoil, while the R plots had the lowest, at 0.39 � 0.21 log MPN/gof soil. Survival of L. innocua was higher in bare-ground plots(4.11 � 0.22 mean log MPN/g of soil) than in other cover croptreatments. The lowest population was detected in the R plots(3.20 � 0.22 mean log MPN/g of soil). Differences were not sta-tistically significant.

Effect of tillage and green manure on bacterial populationdynamics. (i) E. coli. Following week 27 in year 1 and week 31 inyear two, the cover crops were tilled into the soil, resulting in agreen manure. A comparison of bare-ground and cover crop orgreen manure soil in the organic field before and after tillage re-vealed no significant difference in the persistence of E. coli thatcould be attributed to cover crop treatment. In the transitionalfield, a very weak effect was detected, with the highest populationsrecorded in CR, HVR, and HV green manures, in decreasing order(P � 0.071) (Table 2). In the organic field during year 2, a statis-tically significant increase in E. coli populations was observed fol-lowing tillage (P � 0.001) (Table 2).

(ii) L. innocua. In the organic field, tillage negatively impactedL. innocua populations. Declines of about 1 log MPN/g of soil wererecorded in both years, with a subsequent recovery to pretillagelevels by week 4 posttillage (P � 0.05) (Table 2). In the transitionalfield, posttillage levels were significantly higher than pretillage lev-els (P � 0.001). Cover crop treatment was a significant factor onlyin the transitional field (P � 0.027) (Table 2), with mean R pop-ulations being 1.71 log MPN/g of soil higher than HVR popula-tions (P � 0.011).

Effect of soil temperature on bacterial persistence. (i) E. coli.In general, declines in the E. coli population appeared to coincidewith decreasing soil temperatures as the fall season progressed,with resurgence observed in the spring when soil temperaturesclimbed above freezing. Following inoculation in year 1, E. colipopulations persisted at 3 log MPN/g of soil in all cover cropplots at soil temperatures of 10°C (Fig. 3A). However, in bare-ground plots, a decline of 4.5 log MPN/g of soil by week 2 was

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observed despite temperatures being �10°C. As temperatures fellto �5°C, a 5- to 6-log MPN/g of soil decrease was observed afterweek 3, even in the cover crop plots. When temperatures warmedup again in the spring of the following year, E. coli resurged, but at

low levels, never exceeding 1.72-log MPN/g of soil. In year 2, asimilar decline in E. coli populations was seen in both bare-groundand all cover crop plots. E. coli levels hovered between 1.5 and 2mean log MPN/g of soil at temperatures around 10°C during

FIG 1 Population dynamics of E. coli in the organic field (A and B) and the transitional field (C) as impacted by cover crop, namely, hairy vetch (HV), crimsonclover (C), rye (R), hairy vetch-rye (HVR), crimson clover-rye (CR), and a bare-ground no-cover-crop control (BG) in years 1 (A) and 2 (B and C) of the study.The data are expressed as the mean log MPN/g of soil from independent replicates. LOD, limit of detection.

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FIG 2 Population dynamics of L. innocua in the organic field (A and B) and the transitional field (C) as impacted by cover crop, namely, hairy vetch (HV),crimson clover (C), rye (R), hairy vetch-rye (HVR), crimson clover-rye (CR), and a bare-ground (BG) no-cover-crop control in years 1 (A) and 2 (B and C) ofthe study. The data are expressed as the mean log MPN/g of soil from independent replicates. LOD, limit of detection.

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weeks 5 and 7 (Fig. 3B). The populations declined further as tem-peratures fell to �10°C. E. coli levels increased when temperatureswere 20°C in the bare-ground and cover crop plots in week 34.Although the E. coli population in the transitional field exhibited atrend similar to that of the organic field, no significant increase inbacterial populations was observed in the spring in either the bare-ground or cover crop plots.

(ii) L. innocua. L. innocua persistence was independent of soiltemperature, and bacterial populations continued to flourishthroughout the study, regardless of season (Fig. 3D to F). Ataround 10°C, L. innocua levels persisted at around 4 to 5 logMPN/g of soil. In year 1, bacterial populations were highest whentemperatures fell to 5°C in week 11 (Fig. 3D), when mean in-creases of 3.03 and a 4.74 log MPN/g of soil were observed inbare-ground and cover crop soil, respectively. Bacteria persisted at1.4 to 4 mean log MPN/g of soil in year 2 at the lowest soil tem-peratures (Fig. 3E and F). Warming temperatures did not coincidewith the increases in L. innocua. Populations either remained con-stant (Fig. 3D) or declined (Fig. 3E and F) with increasing temper-atures in spring.

Effect of total precipitation on bacterial persistence. The totalprecipitation for the 3 days prior to each sampling was summatedto assess the effect rainfall had on bacterial population dynamics.In year 1, E. coli levels were higher in the absence of precipitationearly in the sampling period (weeks 1 to 3), but declines coincidedwith a temperature dip rather than rainfall (week 5) (Fig. 3A).Population declines coincided directly with precipitation only inyear 2 in sampling week 1 (4.8 mm of rain) and week 23 (9.4 mm

of rain) (Fig. 3B). A resurgence of E. coli was observed only afterthe week 1 rain event. Similarly, L. innocua populations persistedregardless of precipitation in year 1, despite a 26.7-mm depth ofrain recorded in week 7 and 36.6 mm in week 29 (Fig. 3C). Incontrast, declines in the populations coincided with specific rainevents in year 2 in weeks 1 and 23 but not week 30 (Fig. 3D).Population levels increased following the week 1 and week 23events. In the transitional field, lower levels of E. coli and L. in-nocua were also recorded following the rain events of samplingweeks 1, 23, and 30, followed by bacterial population increases(Fig. 4).

DISCUSSION

This study examined the influence of cover crops on the dynamicsof foodborne pathogen indicator bacteria in soil in a vegetable-producing area. The results from this study reveal that cover cropsmay influence E. coli and L. innocua population dynamics. Thehairy vetch-rye cover crop mixture tended to support the highestbacterial populations, although the levels were sensitive to sam-pling date, and increases were not always statistically supported.The other multispecies cover crop treatment of crimson cloverand rye also appeared to have a weak stimulatory effect on E. colibut not L. innocua. Monoculture cover crop treatments of hairyvetch, a legume that boosts nitrogen levels in soil (reviewed inreference 4), gave varied results but appeared to have an impact onbacterial population levels equivalent to those of the bare-groundplots in the first year. Rye monocultures, which are known toaccrue soil carbon over time (26, 27), supported larger popula-tions of both E. coli and L. innocua in year 1 while being the covercrop treatment harboring the lowest bacterial levels in year 2. Bio-mass variability has been reported for monocultures of rye com-pared to mixtures with hairy vetch (28). This might account forthe variability in ecosystem services linked to nutrient availability,which in turn impacts bacterial population dynamics. Likely, theimpact of the cover crop treatment on the indicator bacterial pop-ulations was further complicated by the interaction between thecover crop plant species, local environment, and timing of man-agement practices (reviewed in reference 29).

Although one of the objectives of this study was to explore thebiocontrol potential of cover crops against enteric bacteria, thehairy vetch-rye mixture favored the persistence of bacterial pop-ulations. Recalcitrance of enteric pathogens as a result of covercropping is undesirable from a food safety perspective, althoughmaybe not surprising, since a robust rhizosphere is expected toexert a strong rhizosphere effect. Understanding the physico-

TABLE 1 Mean differences in E. coli and L. innocua populations inorganic and transitional fields between the time of inoculation and thetime just prior to tillagea

Yr and field Wk

Log MPN/g of soil (mean � SE)a

E. coli L. innocua

2014Organic 0 6.15 � 0.21 A 6.47 � 0.23 A

27 0.57 � 0.2 B 4.61 � 0.23 B2015

Organic 0 3.62 � 0.22 A 4.79 � 0.28 A30 0.000 B 1.82 � 0.29 B

Transitional 0 3.20 � 0.21 A 7.55 � 0.24 A30 0.000 B 0.22 � 0.24 B

a Different letters between the first and last week denote a statistically significantdifference (P � 0.001) for that field.

TABLE 2 Impact of tilling in green manure on bacterial population levels in soil

Organism Yr Field

Log MPN/g of soil (mean � SE)a

P valueBefore tillage 2 wk after tillage 4 wk after tillage

E. coli 1 Organic 0.57 � 0.30 0.23 � 0.30 0.59 � 0.30 0.6992 Organic 0.00 � 0.21 A 0.29 � 0.21 A 1.23 � 0.21 B 0.0012 Transitionalb 0.00 � 0.13 0.20 � 0.13 0.42 � 0.13 0.117

L. innocua 1 Organic 4.61 � 0.15 A 3.57 � 0.15 B 4.14 � 0.15 A 0.0012 Organic 1.82 � 0.29 A 0.73 � 0.29 B 1.82 � 0.29 A 0.0162 Transitionalc 0.22 � 0.23 A 0.62 � 0.23 A 1.97 � 0.23 B �0.001

a Different letters denote statistically significant differences among time points.b The cover crop treatment was a significant factor (P � 0.1).c The cover crop treatment was a significant factor (P � 0.05).

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chemical niche established in various cover crop rhizospheres willbe critical for devising effective cover crop application and man-agement practices that augment their biocontrol potential or at-tenuate the promotion of undesirable plant and human patho-gens. Additionally, although this study did not consider rotations,the long-term benefits of certain cover crop species, such as car-

bon accumulation from rye, might also contribute to bacterialpopulation fluxes. The favorable environment offered by therhizosphere is expected to preferentially benefit microorganismsthat are better adapted to soil than most enteric bacteria. Thiswould explain the rapid declines seen for E. coli, as opposed to themore ubiquitous and persistent L. innocua levels. Ultimately, an

FIG 3 Soil temperatures, three previous days’ rainfall sums, and population levels of E. coli (A and B) and L. innocua (C and D) in soil from bare-ground (BG)or cover crop plots in year 1 (A and C) and year 2 (B and D) in an organic field. The population data are expressed as the mean log MPN/g of soil. The cover cropdata are the combined log MPN values for all treatments.

FIG 4 Soil temperatures, three previous days’ rainfall sums, and population levels of E. coli (Ec) and L. innocua (Li) in soil from bare-ground (BG) or cover cropplots in year 2 in a transitional field. The population data are expressed as the mean log MPN/g of soil. The cover crop data are the combined log MPN values forall treatments.

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understanding of the functions and interactions of various micro-organisms in cover crop rhizospheres, be they antagonistic, com-petitively exclusive, or mutualistic, is needed for the optimizationof cover crops for biocontrol purposes, although such data forenteric pathogens do not currently exist.

Cover crop treatment effects were inconsistent over the 2 years,exhibiting interactions with time and suggesting that other factorsmay be stronger determinants. The variability may have been af-fected by cover crop effects from the previous year, since the sameorganic field was used, and randomization was implemented. Fewlong-term cover crop studies have been published, but signifi-cantly higher invertebrate and nematode biomass in soil frommaize plots intercropped with a legume cover crop over a 10-yearperiod was measured compared to monoculture maize plots withor without mineral fertilizer application (30). Interestingly,Schutter et al. (31) found that season, not cover cropping system,was the most influential determinant of microbial communitystructure in vegetable fields with a 5-year history of winter covercrop rotations. The bacterial population dynamics that we ob-served in soil were specific to the temperature requirements of thetwo indicator taxa used. Both E. coli and L. innocua exhibitedpopulation declines with increasing time from inoculation. How-ever, E. coli populations waned when temperatures dipped to�5°C and reappeared only sporadically in the spring and summer,while L. innocua persisted throughout the study. Jiang et al. (32)reported more-rapid declines in E. coli O157:H7 numbers in ma-nured soil at 5°C than at 15°C and 21°C. Moreover, the carbonstatus of soil may interact with temperature, resulting in moredetrimental cold and starvation stresses on E. coli O157:H7 (33).This raises the question of whether cover crop species that accruecarbon over time might favor temperature-sensitive bacteria. Thefailure of E. coli to resurge when temperatures warmed the follow-ing spring might be attributed to the significant die-off. Addition-ally, any recovery attained during the milder spring period mightagain be restricted by soaring temperatures in the summer. E. coliO157:H7-contaminated agricultural soil exhibited shorter sur-vival duration in tropical soil than in temperate soil, partly attrib-uted to temperature and moisture levels, combined with UV ra-diation (34). E. coli is not ubiquitous in agricultural environments,as it is typically introduced through manure or animal feces. E. colidetected on deer pellets grew at maximal rates at 20°C rather thanat 4°C or 35°C, with minimal growth observed at 4°C (35); this isconsistent with the results of E. coli survival studies in dairy ma-nure (36) and cattle feces (37). Higher water content has also beenassociated with a decrease in microbial die-off rates in soil (38).Soil type greatly determines the soil water content. The extendedsurvival of nonpathogenic E. coli and E. coli O157:H7 has beenattributed to higher water availability in different soil types (39)and to moisture content (40), while the long-term survival of Lis-teria monocytogenes in soil has been attributed to soil texture andclay content (41, 42). Sandy soils, like the soil in our study, retainless water and have been associated with faster declines in E. coli(43). We also observed declines in the populations of both E. coliand L. innocua following moderate rain events, suggesting thatbacteria were removed from upper soil layers through infiltration.These rain events were generally succeeded by population resur-gence. On the other hand, heavier rain (25 mm) did not appearto affect the populations, probably as a result of larger amounts ofrainfall contributing to the formation of a soil surface seal, result-ing in a reduction in water infiltration (44) and bacteria remaining

in surface layers. Cover crop root systems are expected to improvewater infiltration and soil water retention (45).

While mesophilic E. coli was outcompeted during the coldermonths, exhibiting very weak resurgence in the spring, L. innocuapersisted throughout the winter, allowing it to prevail at high lev-els in the spring. Both soil and decaying vegetation can serve asreservoirs of Listeria spp., and the bacteria have resilience underharsh or nutrient-scarce environmental conditions (46, 47). Tem-perature has been shown to play an important role in Listeriasurvival in soil, and it has a competitive advantage at lower tem-peratures. During the course of this study, Arctic air carried by thejet stream brought record cold temperatures to the eastern UnitedStates. In Maryland, January 2014 (year 1) and February 2015(year 2) were the 12th (48) and 6th (49) coldest on record, respec-tively. Previous research has shown that L. monocytogenes survivesbetter at 5°C than at 15 to 20°C (50, 51), and it is able to survive insoil for extended periods at low temperatures (52, 53). The persis-tence of pathogenic Listeria spp. over the winter is a worryingprospect. Genes associated with nutrient acquisition mechanismsare overrepresented in L. monocytogenes, enabling the pathogen togenerate energy from a wide range of substrates (54), and long-term persistence in stationary phase was reported not to compro-mise its ability to cause infection (55). L. monocytogenes is alsofrequently associated with manure (56) and the presence of wild-life (57), two common on-farm risk factors. Due to its recalci-trance to inactivation and adaptability to the soil niche, allowinglonger time intervals between manure application and harvestmight not be sufficient to reduce the likelihood of disseminationonto crops. However, employing strategies that improve soil mi-crobial diversity, such as varying the plant genotypes within a field(reviewed in reference 58), may provide a biological barrieragainst the establishment of L. monocytogenes (59).

In the organic field, tillage, but not green manure type, ap-peared to correlate with larger bacterial populations in the soil. Nodifferences among cover crop and bare-ground treatments wereobserved. As temperatures warm up, spring tillage and green ma-nure incorporation may contribute to the resurgence of bacterialpopulations in the soil as a result of added carbon and enhancedsoil porosity and oxygen diffusion (60). In our study, tillage didnot diminish E. coli populations but appeared to be detrimental toL. innocua in the organic field. Subsequent recovery was eventu-ally observed, although it is not possible to determine whetherother factors, such as temperature, contributed to this effect, sincea no-till control trial was not conducted. Conversely, in the tran-sitional field, green manure type did indeed have an effect. E. coliwas highest in the crimson clover-rye mixtures, and the bacterialpopulation within this treatment was different than that in eithercover crop (crimson clover or rye) in monoculture and bare-ground plots. The effects of green manure type on L. innocua werenoted, with the hairy vetch-rye mixture harboring the smallestbacterial populations and rye the largest. In spite of the benefits,tillage events can cause temporary stress conditions for soil mi-crobes, decreasing their ability to assimilate nutrients, altering mi-crobial community structure, and increasing the potential for theloss of carbon and nitrogen from the soil and degradation of or-ganic matter (61). The incorporation of green manure may tem-per these effects as a result of nutrient inputs and reduced soilcompaction. Conversely, in a no-till setting, crop residues canserve as insulators, attenuating soil temperature fluctuations, con-serving soil moisture, and building up organic matter and nutri-

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ents (62, 63). Soil organic matter has been reported to be corre-lated with a prolonged persistence of E. coli O157:H7 (40). There istherefore a need to assess the long-term effects of green manure onfoodborne pathogen populations.

Although cover crops may have an impact on bacterial popu-lation dynamics, they were not the only or most influential drivingforce. The utilization of cover crops and green manures as possiblebiocontrol strategies against foodborne pathogens does not ap-pear to be promising. More problematic is the potential of thecover crop rhizosphere or green manure to promote microbialgrowth, including that of foodborne pathogens, since prolongedpresence in the soil increases the likelihood of transmission ontocrops (64). This is an important consideration for farmers whoproduce crops that are considered high risk from a food safetystandpoint, especially if their farm management program includesthe use of multispecies cover crop cocktails in which high biomassis attained. E. coli and L. innocua were used in this study as surro-gates for pathogenic enteric bacteria. Generic E. coli has been re-ported to behave in parallel with pathogenic strains (39) and withSalmonella enterica serotype Typhimurium (65) in the soil. L. in-nocua was used in this study as a surrogate for the pathogenicspecies L. monocytogenes, as the two species exhibit similar behav-iors in soil, although L. innocua may be better adapted (66). Ourfindings therefore suggest that in regions with cold winters wheredie-off of enteric bacteria is expected, cover cropping may prolongthe survival of mesophilic pathogenic bacteria, as seen in our studywith E. coli in the hairy vetch-rye and rye plots in the first year. Onthe other hand, psychrotrophic Listeria spp. are already welladapted to persist in soil, and the benefits of a rhizosphere effectmay be less significant in view of their robust competitiveness. Soiltype is another important factor influencing bacterial infiltrationand survival, with extended pathogen survival observed in subsur-face loamy and clay soils (67). Future studies should evaluate howsoil type interacts with cover crop rhizospheres to influence en-teric pathogen population dynamics. Ultimately, preventing theintroduction of enteric bacteria into production areas remains themost important step that growers can take to prevent them frombecoming established there. The application of pathogen-freecomposted manure is an important recommendation for fresh-produce growers, although wildlife exclusion continues to be achallenge. The Food Safety Modernization Act (68) has deferredrecommendations for soil amendments until such a time thatmore data can be acquired. Considering the important ecosystemservices provided by cover crops and the benefits of organic fertil-ization on soil fertility and health, future studies should assess theshort-term and long-term impacts of cover cropping on entericbacterial population dynamics in various soil types, includingpathogens which may be introduced to the soil via manureamendments or by wildlife.

ACKNOWLEDGMENTS

We thank Louisa Martinez, Adriana Echalar, Seun Agbaje, Nicole Lee,Nazleen Khan, Marie Pham, Mary Theresa Callahan, Robert Korir, andDavid Armentrout for their assistance with this project.

FUNDING INFORMATIONThis work was supported by the U.S. Department of Agriculture’s Na-tional Institute of Food and Agriculture through the Organic TransitionsProgram under grant number 2014-51106-22090 to Shirley A. Micallef.Any opinions, findings, and conclusions expressed in this material are

those of the authors and do not necessarily reflect the views of the USDANIFA.

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