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a Departments of Agronomy and Microbiology, 2537 Agronomy Hall, Iowa State University, Ames, IA 50011-1010
b J.C. Lorimer, Agricultural Biosystems Engineering, 3224 NSRIC, Iowa State University, Ames, IA 50011-1010
* Corresponding author (larryh{at}iastate.edu).
Received for publication July 10, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: CFU, colony forming units • RS2G, E. coli strain RS2 expressing the green fluorescent protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unlike human sewage sludge, animal manures are not treated to reduce the pathogen content before land application. Strategies for applying swine manure to soil generally incorporate best management practices that are designed to limit nitrogen to amounts that crops can readily use, which may or may not reflect the amount of manure that a soil can accommodate to successfully reduce contamination of water by pathogenic bacteria. The greatest risk of contamination by pathogenic bacteria occurs when high-intensity and/or long-duration rainfall causes runoff or subsurface drainage on agricultural land that recently received manure (Joy et al., 1998). Contamination of well water is of particular concern since it can expose humans and farm animals to pathogenic bacteria.
The ability of manure-derived pathogenic bacteria to survive in soil following manure application increases the probability of water contamination after rainfall or irrigation events (Joy et al., 1998). Pathogen survival will initially reflect the ability to tolerate the sudden change in habitat and subsequent ability to tolerate possibly adverse environmental conditions such as extremes in temperature or desiccation. Soil moisture is one of the more important factors influencing pathogen survival, and survival is more likely when soils are moist (Crane and Moore, 1986; Entry et al., 2000b; Gagliardi and Karns, 2000; Mubiru et al., 2000). Soil temperature also influences survival since warm temperatures along with drying decrease survival rates (Entry et al., 2000b; Kemp et al., 1992; Sjogren, 1994) while cooler temperatures promote survival (Kibbey et al., 1978; Ogden et al., 2001). Nutrient availability will also influence survival, and manure undoubtedly provides a supply of potentially utilizable nutrients that support the survival and growth of bacteria, at least for a while, once they are introduced into soil. Several recent studies have shown that Escherichia coli O157:H7 can survive for extended periods of time in manure-amended and unamended soils (Fenlon et al., 2000; Gagliardi and Karns, 2000; Gagliardi and Karns, 2002) and that vegetation promotes survival (Bolton et al., 1999; Fenlon et al., 2000; Gagliardi and Karns, 2002; Sjogren, 1995).
Survival may also require colonization of specific microhabitats, such as within the dispersible clay fraction, which protects pathogenic organisms from abiotic stresses (Recorbet et al., 1995). Survival in soil is probably a dynamic process were the majority of cells may die off quickly once introduced into the soil environment, but a subpopulation may be better suited for survival and this subpopulation may die off more slowly possibly because of colonization of favorable sites or because of its physiological properties (Ogden et al., 2001).
Movement of bacteria through soil to ground water will in part depend on soil type, climatic and soil properties, manure properties and the method and amount applied, and the amount and type of vegetation (Entry et al., 2000a, b; McMurry et al., 1998). The timing and frequency of rainfall following manure application to soil will strongly influence both vertical and lateral movement of pathogenic bacteria through soil. The potential for ground water contamination depends on the depth of soil to the water table as well as the properties of the soil. Preferential water movement, due to the presence of old root channels, insect and animal burrows, and natural soil features, is probably the primary means by which bacteria move through soil (Abu-Ashour et al., 1998; McMurry et al., 1998; Smith et al., 1985). Tillage disrupts soil structure and pores and can reduce fecal coliform (McMurry et al., 1998) and E. coli O157:H7 (Gagliardi and Karns, 2000) transport compared with transport through similar intact soils. Rain can promote survival of pathogenic bacteria by keeping the soil moist, and it can redistribute bacteria through the profile to more or less favorable sites in addition to potentially contaminating ground water.
Most often bacterial transport is investigated following one or more simulated rainfalls immediately following manure application and less is known on how periodic and/or intermittent rainfalls influence pathogen transport or survival in soil (Stoddard et al., 1998). In the present study, we evaluated the effect of time between manure application and the first rainfall and the frequency of rainfall events on the leaching of a swine manure E. coli isolate through intact soil cores and on its survival in those cores. Since many Iowa soils have had an extensive history of manure application, we chose to examine the fate of a green fluorescent protein-marked E. coli strain that we isolated from swine manure to facilitate discrimination between manure-derived and soil-derived pathogenic bacteria. The E. coli was isolated from the source of manure that we used in these studies. Additionally, we evaluated the role of different manure application procedures on E. coli leaching and survival in soil.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Amplification of 16S rDNA gene
Genomic DNA was extracted using a QIAamp DNA mini kit (QIAGEN, Valencia, CA) and the DNA was eluted from the spin column with water. For PCR amplification of a nearly full-length 16S rDNA gene, we used domain bacteria primers 27F (5'-AGAGTTTGATCMTGGCTC-3'; M = A or C) that corresponds to positions 8 to 25 in the E. coli numbering system (Weisburg et al., 1991) and 1387R (5'-GGGCGGWGTGTACAAGGC-3'; W = A or T) that corresponds to positions 1387 to 1404 (Marchesi et al., 1998). The PCR amplification was performed using reaction mixtures (final volume, 50 µL) containing 100 ng of sample DNA, 2 U of Recombinant Taq DNA polymerase (Gibco/BRL), 1X PCR buffer, 1.5 mM MgCl2, each deoxynucleotide triphosphate at a concentration of 0.2 mM, and each primer at a concentration of 2 µM. The thermocycling program used was as follows: initial denaturation at 94°C for 1 min; 35 cycles consisting of a denaturation at 95°C for 30 s, an annealing temperature of 45°C for 30 s, and an extension step at 72°C for 1 min; and a final extension step at 72°C for 5 min. The product obtained from the amplification reaction was purified using a QIAquick gel-extraction kit (QIAGEN). The amplified product was sequenced using primers 27F, 1387R, and 533F (5'-GTGCCAGCMGCCGCGGTAA-3'; M = A or C). The sequences were determined by using an Applied Biosystems Model 377 Prism DNA sequencer (PerkinElmer, Wellesley, MA) at the Iowa State University DNA Sequencing and Synthesis Facility. For the nearly full-length 16S rDNA sequence, we queried the BLAST-nt search program (Altschul et al., 1990) of the GenBank database maintained by the National Center for Biotechnology Information (Bethesda, MD).
Selective Media and Enumeration of Bacteria
Leachate samples were serially diluted in saline-phosphate buffer and plated onto Luria–Bertani agar supplemented with 100 µg mL-1 kanamycin and 10 µg mL-1 rifampicin, respectively, for enumeration of RS2G. The membrane filtration technique was used to enumerate fecal streptococci by plating onto KFS agar (Difco, Detroit, MI), and E. coli and total (non-E. coli) coliforms by plating on m-Coliblue24 (Hach Company) using the procedures provided by the manufacturer. Plates were inverted and incubated at 37°C for 24 h. RS2G colony counts were confirmed by placing the plates in a long-wavelength UV light box and counting only those colonies that fluoresced green. Green fluorescent protein fluoresces green when excited with ultraviolet light (365 nm) or optimally when excited at 488 nm and the emission optimum is 510 nm. All red and light red colonies growing on KFS agar were counted and assumed to be presumptive fecal streptococci. All light red to red and light blue to blue colonies growing on m-Coliblue24 were counted and assumed to be presumptive total coliforms (non-E. coli) and E. coli, respectively. Soil samples (10 g) were resuspended in sterile buffered saline, sonicated in an ultrasonic cleaning bath for 5 min, and then shaken for 15 min on a gyratory shaker before enumeration of bacteria. The number of colony forming units (CFU) per sample was log10–transformed and expressed as log CFU 100 mL-1 or total log CFU in the leachate or soil sample. For purposes of statistical analysis, in samples that had no colonies at our detection limit we assigned a value that was 10-fold less than the detection limits, which was 10 CFU mL-1 leachate and 100 CFU g-1 of soil for RS2G and 100 CFU mL-1 of leachate for total coliforms and fecal streptococci.
Inoculum Preparation
All soil cores were inoculated with manure spiked with E. coli isolate RS2G grown overnight in Luria–Bertani broth (Gibco/BRL) on a gyratory shaker (200 rpm) at room temperature. The culture was pelleted by centrifugation (5000 x g) and resuspended to the original volume in sterile distilled water. Fresh manure was obtained from Iowa State University's Swine Nutrition Management and Research Center near Ames, IA. For each core that received manure, 15 mL of inoculum was mixed with 152 mL of liquid manure slurry, which resulted in each core receiving 8.02 x 109 CFU (4.8 x 107 CFU mL-1) of RS2G with 100% of the population expressing green fluorescent protein as determined by visual inspection of the colonies.
Soil Cores and Experimental Treatments
Thirty soil cores were collected from the Kelly Farm located near the Iowa State University Agronomy and Agricultural Engineering Research Center near Ames, IA. The soil was Clarion loam (fine-loamy, mixed, superactive, mesic Typic Hapludoll) (bulk density = 1.42 g cm-3, pH = 6.8, and 3.5% organic matter) and was previously cropped under an annual corn (Zea mays L.) and soybean [Glycine max (L.) Merr.] rotation. Soil cores were extracted in late summer 2000, before the soybean harvest, using a Giddings probe. The 20-cm-wide x 30-cm-long cores were extracted in a 38-cm-long autoclaved galvanized tube that had been sharpened on the down-facing edge; this provided a holding area above the soil surface for the liquid manure and simulated rain. Autoclaved screens were installed on the bottom of each core to prevent soil loss. Wax was poured into crevices and spaces visible between the soil and the wall of the galvanized tube. The cores were then arranged in a random block design in a leachate collection apparatus comprised of 25-cm-diameter autoclaved funnels and a guide table that held the cores upright over the funnel. The cores were kept at room temperature (approximately 21°C) and normal building relative humidity (approximately 60%). The cores were saturated to field capacity by placing them in buckets of water for 48 h and allowing saturation to take place from bottom to top, and then the soil water was allowed to drain for 2 d. Three manure application methods were simulated in this study. The manure was poured directly over the soil column to simulate a no-till broadcast manure application. The top 2.5 cm of the soil column was disrupted with a sterile spatula before the manure application to simulate a surface-tilled broadcast manure application. The manure was poured directly over the soil column and then the top 2.5 cm of soil was disturbed with a sterile spatula to simulate an incorporated broadcast manure application. Manure was applied to cores once at the beginning of the experiment at a rate consistent with best management practices for optimal nitrogen utilization in a corn–soybean rotation in these soils.
Rainfall and Leachate Collection
We chose to simulate intense convective rains (thunderstorms), which are common rainfall events in Iowa during spring and early summer (April–June). A rainfall rate of 50.8 mm over a 4-h period was simulated by applying five 330-mL aliquots of sterile distilled water at hourly intervals (total of 1650 mL) to each core surface. A subset of cores received one rainfall event on either the 4th, 8th, or 16th day after manure application. A second subset of cores received two rainfall events on the 8th and 16th day after manure application. A third subset of cores received three rainfall events on Days 4, 8, and 16 following manure application. A fourth set of cores did not receive any manure application, but they did have three simulated rainfall events on Days 4, 8, and 16. After each rain, leachate was collected at the bottom of each soil core in sterile plastic sample bottles until the cores stopped draining. Leachate samples were stored at 4°C for no longer than 24 h before enumeration of bacteria. At the end of the experiment, we assessed RS2G survival in the soil and its distribution throughout the core as well as soil moisture content at each depth. Soil was sampled at 0- to 2.54-, 0- to 10-, 10- to 20-, and 20- to 30-cm depths. Three 1-cm-diameter subcores were taken from each depth and each subcore was homogenized in a plastic bag by hand before determining RS2G concentrations.
Statistical Analysis
Statistical analyses were performed by using JMP Version 4.04 (SAS Institute, 2001). For the leachate analyses, a separate two-factor analysis was performed on the leachate volume and RS2G, total coliforms, and fecal streptococci concentrations. In these analyses manure application method and date of the rain event were subplot treatment factors. For the RS2G survival in soil analyses, a separate two-factor analysis was performed on each set of cores that were rained on at different times after manure application. In these analyses manure application method and soil depth were subplot treatment factors. For each analysis, a Student's t test (P = 0.05) was calculated by JMP software for comparisons among treatment means. For comparisons between RS2G and total coliform concentrations within a sample we performed a paired t test analysis. Unless stated otherwise, values reflect the mean ± SE of three replications.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of Time Before First Rain on RS2G Leaching
RS2G was mixed with the manure before it was applied to the cores. We varied the length of time before applying 1650 mL of water (equivalent to 50.8 mm of rain) to each core and collecting the leachate to evaluate the mobility of bacteria through the soil cores following a rain event. The volume of liquid manure (167 mL) applied to each core was insufficient to produce any leachate. The volume of leachate collected was not affected by the length of time before the first rainfall event, but it was affected by the manure application procedure (Table 1). Less leachate was collected from cores in which the manure was incorporated into soil than from cores in which the surface was disturbed before manure application (tilled) or undisturbed (no-till). The manure solids created a visible manure layer over the surface of both the tilled and no-till treated columns but not over the columns in which the manure was incorporated into the soil surface.
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After two equivalent rain events, if the length of time before the first rainfall event increased from 4 to 8 d there was a substantial (8- to 100-fold) decrease in the RS2G concentration in the leachate (compare Experiments 1 and 3 in Table 3). However, there was only a slight, if any, decrease in total coliform and only a 4- to 16-fold decrease in leachate fecal streptococci concentrations if the length of time before the first rain event increased from 4 to 8 d. There were more RS2G, total coliforms, and fecal streptococci leached from cores following the second rainfall event on Day 16 than following the first rainfall event on Day 8 (compare Day 8 in Table 1 and Experiment 3 in Table 3). This is in contrast to what was observed when the first rainfall event occurred 4 d after manure application and the second rainfall occurred 8 d after manure application (compare the results from Day 4 in Table 2 and Experiment 2 in Table 3). Taken together, these results suggest there is a dynamic interplay between the length of time between manure application and first rainfall event and the leachability of manure-derived bacteria following a second rainfall event.
Predicting Leachability of RS2G
A regression analysis (Fig. 1)
was performed with leachate concentrations of RS2G measured from cores that experienced a single rainfall event on the 4th, 8th, or 16th day after manure application and from cores that experienced three rainfall events after manure application. Linear regression parameters for E. coli RS2G concentrations in the leachate from cores that received one rainfall event at various days were r2 = 0.81; P < 0.0001; slope = -0.218; y intercept = 7.4 log CFU 100 mL-1; x intercept = 34 d; x intercept, 95% lower confidence limit = 30 d; and x intercept, 95% upper confidence limit = 40 d. Linear regression parameters for E. coli RS2G concentrations in leachate over the 16-d period for cores that received three rainfalls were r2 = 0.57; P value < 0.0001; slope = -0.207; y intercept = 7.13 log CFU 100 mL-1; x intercept = 56 d; x intercept, 95% lower confidence limit = 43 d; and x intercept, 95% upper confidence limit = 84 d. Although other models may be better than the linear regression model for predicting the leachability of RS2G the linear regression models suggest that RS2G would continue to leach through the soils for about 34 to 56 d after manure application, depending on the timing before and frequency of rainfall events.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Following a rain event, percolating water transports RS2G cells through the core, which redistributes RS2G in the soil profile, potentially to become residents of these newly colonized habitats. Most of the RS2G cells were found near the soil surface and fewer were found throughout the remaining portions of the core. Our observation that RS2G cells can survive for extended periods (16 d) before the first rain event clearly indicates that either the nutrients provided by the manure facilitate this long-term survival, that E. coli RS2G is able to compete with both the indigenous soil microbiota and the manure microbiota for nutrients and microhabitats in the soil, or that E. coli RS2G or other manure bacteria produce toxic compounds that decrease the competitive ability of indigenous soil bacteria and consequently increase the survival capabilities of RS2G. We only examined the culturable RS2G cells and it is possible that we underestimated the total number of RS2G cells in the leachate that were injured yet viable, but not culturable (Rompre et al., 2002; Roszak and Colwell, 1987). Leachates collected from the no-manure treated cores also had relatively high concentrations of the indicator organisms. We evaluated the concentrations of total coliforms and fecal streptococci in our leachate samples to have an alternative assessment of the mobility of bacteria through the soil cores. It is reasonable to assume that the fecal streptococci concentrations in the leachate obtained from manure-treated cores should equal or exceed their concentrations in the leachate from the control (no-manure) cores. Yet, this was not the case. In general, there were fewer fecal streptococci in the leachate from manure-treated cores than from the no-manure treated cores (Tables 2 and 3), suggesting that manure treatments either killed or prevented the leaching of soil fecal streptococci. This decrease could be due to death of soil fecal streptococci because they are susceptible and/or sensitive to toxic compounds present in the manure or they are unable to compete for nutrients or microsites with manure bacteria introduced into the soil. The dynamic nature of changes in the population structure of fecal streptococci has also been reported in runoff water containing fresh cattle feces (Doran and Linn, 1979).
There was no difference among the manure application procedures and the control cores in the total coliform concentrations in leachate collected from those cores, although there were discrepancies between the RS2G and total coliform concentrations in the leachates from the manure-treated cores. Similarly, leachates collected from cores in which the manure was spiked with RS2G should have total coliform counts that equal or exceed those for RS2G. Yet, this was clearly not the case when the time before the first rain event following manure application increased from 4 to 16 d (Table 1). These results could be explained by a poor ability of RS2G to grow on the indicator medium when they are stressed or that over time there is a decrease in RS2G concentrations that occurs concurrently with an increase in either indigenous soil total coliforms or preferential survival of non-RS2G total coliforms present in the manure. These results suggest that RS2G enumerations based on the use of the total coliform indicator medium would underestimate the true RS2G concentrations. Similar discrepancies between indicator media and selective media for enumeration of specific E. coli populations have been reported previously (Gagliardi and Karns, 2002).
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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symbols indicate samples obtained from cores that received a rainfall event on the 4th, 8th, or 16th day after manure application. (B) The 
symbols indicate samples obtained from cores that received three rainfall events on the 4th, 8th, and 16th day after manure application. Since there was no statistically significant effect of manure application on RS2G concentrations in the leachate, the three replicate cores for each manure application procedure were pooled to generate a total of nine samples per rainfall event.