Evaluation of Microbial Ecology in Pasture Ecosystems with Long-term Poultry Litter Additions

Final Report for GS03-030

Project Type: Graduate Student
Funds awarded in 2003: $9,990.00
Projected End Date: 12/31/2005
Region: Southern
State: Arkansas
Graduate Student:
Major Professor:
Dr. Mary Savin
University of Arkansas
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Project Information

Summary:

Microorganisms are the primary decomposers in the environment, and thus facilitate nutrient cycling by producing enzymes, retaining nutrients in biomass on a short-term time scale, and mineralizing and increasing availability of nutrients to other organisms. Microorganisms are numerous and participate in a multitude of ecological interactions that alter their environment, fitness, and the fitness of the organisms interacting with them. Despite their importance, microorganisms are difficult to study because of their microscopic size and short generation times. The potential for rapid population turnover and genetic exchange among microorganisms makes their adaptations to environmental changes another element to consider in studying the effects of microorganisms on soil quality. The understanding of how soil management practices, such as the application of poultry litter to pasture land, influence microbial dynamics and how these changes may affect long-term system sustainability is limited. In this study, soil microbial community dynamics were studied in small grassland plots receiving annual applications of fertilizers. Plots received one of two rates of untreated poultry litter, alum-treated poultry litter, inorganic N fertilizer, or no amendment each spring since 1995. Fertilizer rates were 2.24 Mg/ha and 8.98 Mg/ha for the litters, or 65 kg N/ha and 260 kg N/ha for ammonium nitrate treatments. For this research, data were collected from extraction and incubation techniques traditionally employed in soil microbiology. In addition, microbial community diversity was investigated using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis. Biological activity was measured by quantifying dehydrogenase, acid and alkaline phosphatases, and beta -glucosaminidase enzyme potentials. Available nutrient pools were measured for microbial biomass, inorganic N, soluble reactive phosphorus, and dissolved organic C. Additionally, the amount of antibiotic resistance expressed by bacterial isolates was quantified to evaluate whether resistance to antibiotics was increasing due to land applications of poultry litter. The utilization of several microbial assessment tools was conducted to improve understanding of soil microbial communities in poultry litter-amended grasslands in terms of microbial community size, functions, and diversity, and potential for the indigenous populations to develop antibiotic resistance. The goal of this research was to acquire knowledge to use toward the development of management practices that simultaneously improve nutrient cycling and soil quality.

Introduction

The importance of animal agriculture and the need to promote management practices designed to reduce nutrient leaching and run-off necessitate studies that examine the effect of manure applications on soil microbial communities and nutrient dynamics. Manure is a valuable source of organic matter and nutrients required to support plant growth and maintain soil quality. In the past the application of manure to cropland from animal-based agricultural operations had been considered an efficient and effective use of resources. However, expanding urban development combined with consolidation of animal production operations have resulted in limited acreage receiving excessive amounts of manure, which can and has led to increased loss of nutrients via surface run-off and subsurface leaching.

Parham et al. (2002) demonstrated the positive role of microorganisms in facilitating the decomposition of manure and increasing the plant availability of nutrients in field plots with a 70-yr history of regular cattle manure or inorganic fertilizer applications. Microbial biomass, pH, and microbial activity as indicated by dehydrogenase and alkaline phosphatase enzyme measurements, increased in the manure-amended soils as compared to the soils treated with inorganic fertilizers.

In addition to containing organic and inorganic nutrients, animal manures can contain antibiotic residues or antibiotic resistant microorganisms. The use of antibiotics in preventing and treating human infections and in animal production to increase growth rates and reduce disease has been a prevalent practice in recent history that has come under much scrutiny. The prolific use of antibiotics has resulted in the presence of antibiotic resistance in a variety of microorganisms and in a variety of environments. Bacterial isolates showing multiple antibiotic resistance have been found in poultry litter (Kelley et al., 1998), in fecal samples from cattle feedlots (Dargatz et al., 2003), and in swine manure slurries (Smalla et al., 2000). Compounding the problem of increased antibiotic resistance is the prevalence of genetic elements encoding multiple resistance or associated with resistance to other stresses such as heavy metals (Smalla et al., 2000). However, it remains unclear to what extent the presence of antibiotic residues or antibiotic resistant microbes in land-applied wastes contribute to the spread of antibiotic resistance in the environment. Facilitated spread of antibiotic resistance could have profound implications for human and animal health because of the difficulty in treating infections caused by antibiotic resistant pathogenic organisms.

In this study the goal was to gain a comprehensive understanding of the long-term effects of repeated poultry litter applications to grass plots on microbial community functions, activities, and diversity.

References:

Dargatz, D.A., P.J. Fedorka-Clay, S.R. Ladely, C.A. Kopral, K.E. Ferris and M.L. Headrick. 2003. Prevalence and antimicrobial susceptibility of Salmonella spp. Isolated from US cattle feedlots in 1999 and 2000. J. Appl. Microbiol. 95: 753-761.

Kelley, T.R., O.C. Pancorba, W.C. Merka, and H.M. Barnharts. 1998. Antibiotic resistance of bacterial litter isolates. Poultry Sci. 77:243-247

Parham, J.A., S.P. Deng, W.R. Raun, and G. V. Johnson. 2002. Long-term cattle manure application in soil I. Effect on soil phosphorus levels, microbial biomass C, and dehydrogenase and phosphatase activities. Biol. Fertil. Soils. 35:328-337

Smalla, K., H. Heuer, A. Gotz, D. Niermeyer, E. Krogerrecklenfort and E. Tietze. 2000. Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity in IncQ-like plasmids. Appl. Environ. Microbiol. 67: 3542-3548.

Project Objectives:

Three specific research objectives were addressed:

1. Assess the impact of repeated annual poultry litter additions on soil microbial biomass and enzyme activity.

2. Assess the impact of annual poultry litter additions or inorganic fertilizers on soil microbial diversity.

3. Evaluate the contribution of repeated land application of poultry litter to the levels of antibiotic resistance expressed in indigenous environmental microorganisms.

Research

Materials and methods:

Study Site: Twenty-eight plots (1.52 x 3.01m, with 5% slope) growing tall fescue (Lolium arundinacea Schreb) located at the University of Arkansas Main Agricultural Experiment Station in Fayetteville had been established in 1995 on a Captina silt loam (fine-silty, siliceous, mesic Typic Fragiudult). Plots either received no inputs (control) or single applications in the spring of one of the following fertilizers: alum-treated poultry litter applied at 2.24 Mg/ha; alum-treated poultry litter applied at 8.98 Mg/ha; untreated poultry litter applied at 2.24 Mg/ha; untreated poultry litter applied at 8.98 Mg/ha; ammonium nitrate applied at 65 kg N/ha, or ammonium nitrate applied at 260 kg N/ha. Rates for ammonium nitrate inorganic fertilizer were based on the year one N concentrations measured in the alum-treated litter. Each treatment was replicated in four plots in a completely randomized design. When the study was started in 1995, treatments averaged Mehlich III test P values of 131 ( 1) mg P/kg.

Sampling: Soils were sampled prior to fertilizer applications, which occurred on May 30 in 2003 and April 19 in 2004, and approximately 10 days, 1 month, and 6 months following fertilizer applications. Sampling dates were May 9, June 9, July 6, and Nov 22 in 2003 and April 5, April 29, May 19 and Nov 9 in 2004. Eight surface soil samples (1.7-cm diameter, 5-cm depth) were collected aseptically and bulked per plot using a stratified random sampling scheme. Samples were stored on ice until transported back to the lab. Subsamples were removed within 24 hours of initial collection and frozen at -80oC until DNA was extracted. Remaining soil was refrigerated at 4oC and processed within 1 week of sampling.

Methods Under Objective 1

Microbial biomass C and N were measured using the chloroform-fumigation-extraction method to determine the biomass and nutrient cycling capacity of the microbial community (Vance et al., 1987). Unfumigated soil (15 g moist) was extracted with 0.5 M K2SO4 at a ratio of 1:2 soil:extract (wt:vol) ratio. Samples were shaken for 30 min on a reciprocating shaker and filtered through Whatman 42 filter paper. At the same time, 15 g of soil was fumigated with ethanol-free chloroform for 24 hrs. Chloroform vapors were removed and the soil was extracted in the same manner as the unfumigated soil. Total organic carbon (TOC) in extracts was measured with a Rosemont DC–190 High-Temperature TOC Analyzer (Tekmar-Dohrmann, Cincinnati, OH) in 2003 and a TOC-V PC-Controlled TOC Analyzer (Shimadzu Inc., Columbia, MD) in 2004. A sub-sample of each K2SO4 extract was oxidized and analyzed for microbial biomass N by the alkaline persulfate oxidation method (Cabrera and Beare, 1993). Total persulfate nitrogen (TPN) oxidized to nitrate was analyzed colorimetrically on an auto-nutrient analyzer (Skalar, Norcross, GA). Microbial biomass C and N were calculated from the difference between C or N in the fumigated and unfumigated soil extracts, expressed per g dry soil (determined by oven-drying soil at 105oC for 24 hours) and multiplied by appropriate correction factors (Vance et al., 1987; Brookes et al., 1985). C in the unfumigated extracts was used to calculate dissolved organic C per g dry soil and N in the unfumigated extracts was used to calculate total dissolved N per g dry soil. Microbial biomass P measurements were attempted, but not continued nor reported because the chloroform-fumigation-extraction method did not yield reliable results for microbial biomass P. Soluble reactive phosphorus (SRP) was extracted with milli-Q water at a 1:10 soil:water (wt/vol) ratio and analyzed colorimetrically on a Skalar San plus segmented flow analyzer (Norcross, GA).

Enzyme activities of dehydrogenase, phosphomonoesterase (acid & alkaline phosphatases), and Beta -glucosaminidase were used to assess biological activity. Methods based on production of colored products resulting from the enzymatic cleavage of the original substrate were used (Tabatabai, 1994). Dehydrogenase enzyme activity is based on colorimetric determinations of 2,3,5-triphenyl formazen (TPF) produced during the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by soil microorganisms. Briefly, 10 g air-dried soil mixed with 0.1 g CaCO3 was divided into 3 g sub-samples, mixed with 0.5 ml 3% TTC and 1.25 ml of milli-Q water, and incubated at 37oC for 24 hrs. Following incubation, 10 ml of methanol was added, the soil solution shaken for 1 min and filtered through Whatman 40 filter paper. The filter paper was quantitatively rinsed with methanol until the reddish color was removed from the filter. Filtrate volume was adjusted to 50 ml and absorption measured at 485 nm (Tabatabai, 1994).

Phosphomonoesterase activity is based on the colorimetric determination of p-nitrophenol released by phosphatase activity, when soil is incubated with buffered sodium p-nitrophenyl phosphate solution (Tabatabai, 1994). Briefly, 1 g of sieved, moist soil was incubated for 1 hr at 37oC in the presence of 4 ml of modified universal buffer at pH 6.5 for acid phosphatase and pH 11 for alkaline phosphatase assays, and 1 ml of p-nitrophenyl-phosphate solution. To stop the reaction, 1 ml of 0.5 M CaCl2 and 4 ml of 0.5 M NaOH was added, the solution filtered through Whatman 40 filter paper, and absorption measured at 405 nm (Tabatabai, 1994).

Beta-glucosaminidase activity is based on the colorimetric determination of p-nitrophenol released when soil is incubated with buffered p-nitrophenyl-N-acetyl- beta -D-glucosaminide solution (Parham and Deng, 2000). Briefly, 1 g of sieved, moist soil was incubated for 1 hr at 37oC in the presence of 4 ml of 0.1 M acetate buffer pH 5.5, and 1 ml of 10 mM p-nitrophenyl-N-acetyl- beta -D-glucosaminide solution. To stop the reaction, 1 ml of 0.5 M CaCl2 and 4 ml of 0.5 M NaOH was added, the solution filtered through Whatman 40 filter paper, and absorption measured at 405 nm (Parham and Deng, 2000).

Means and standard errors were calculated for each treatment for nutrient and enzyme analyses, and data were analyzed by performing analysis of variance (ANOVA) procedures.

Methods Under Objective 2

A polymerase chain reaction (PCR) – denaturing gradient gel electrophoresis (DGGE) approach was used to analyze diversity of bacterial communities by amplifying and separating 16S rRNA gene fragments. Soil and litter DNA was extracted using the bead-beating protocol outlined by Bio101 for the FastDNA Spin kit for soil DNA isolation (Qbiogene, Carlsbad, CA). DNA was quantified by fluorimetry using Hoescht 33258 dye and calf thymus DNA as a standard. DNA was amplified in PCR reaction mixtures containing 50 mM KCL, 10 mM Tris-Cl (pH 8.3), 1.5 mM MgCl2, 0.001% (w/v) gelatin, 200 µM dNTP, 0.2 µM each primer (338F-GC and 518R primers specific for Eubacteria), and 400 ng of bovine serum albumin (BSA) per µl in a total volume of 50 µl. To each reaction mixture, 1.25 units of AmpliTaq polymerase (Applied Biosystems, Foster City, CA) and 10 ng template DNA were added. Amplification was performed in an MJ Research (Waltham, MA) thermal cycler as follows: initial denaturation at 94oC for 5 min(s); followed by 30 cycles of denaturation, annealing and extension at 94oC for 30 s, 55oC for 30 s and 72oC for 30 s, respectively; with a final extension at 72oC for 20 min. Products were resolved on 1.2% (wt:vol) agarose gels, stained with ethidium bromide and quantified against a molecular mass standard (BioRad Laboratories, Hercules, CA) using Kodak’s EDAS 290 gel documentation system and Kodak’s ID image analysis software (Eastman Kodak Co., New Haven, CT).

PCR products were analyzed by DGGE, which involves the separation of the PCR products in a polyacrylamide gel (37.5:1 bisacrylamide:acrylamide) containing a linear gradient of denaturant, established chemically with urea and formamide. The melting properties of amplified PCR products vary by base sequences, resulting in the cessation of DNA migration at different locations (distances) in the gel. Bands of unique migration distances are assumed to represent unique phylotypes. The end product of DGGE is the production of a picture of the diversity of the microbial community (Heuer et al., 1999). PCR products were resolved by DGGE with a denaturing gradient of 55-70% on a 8% acrylamide gel in 1x TAE (40 mM Tris-acetate (pH 8.0), 20 mM sodium acetate, 1 mM EDTA). DGGE gels were run for 16 hr at 70 V and then stained with SYBR Green for 20 min. DNA banding patterns were imaged using Kodak’s EDAS 290 gel documentation system and Kodak’s ID image analysis software (Eastman Kodak Co., New Haven, CT) and were analyzed using Quantity One (BioRad Laboratories, Hercules, CA). Bands were identified and migration distances determined. The presence and absence of bands with the same migration distances were used to construct similarity matrices and dendrograms to visualize clustering of samples. Branches and nodes connect communities; therefore, differences in branch distances reflect dissimilarities among communities.

Methods Under Objective 3

The ability of a bacterial species to grow on antibiotic treated agar media was interpreted as antibiotic resistance. Ten-fold serial dilutions of litter and soil were prepared in 0.85% NaCl solutions (Zuberer, 1994). Dilutions were spread onto 0.1X tryptic soy agar (TSA) plates and incubated at 25°C for 7 days to yield plates containing 20 – 300 isolates for viable bacterial plate counts. Forty-eight, representative isolates were selected per treatment, transferred to 2 of 48 wells in 96-well plates, and stamped onto 0.1X TSA agar plates containing antibiotics. Antibiotics tested included bacitracin at 0, 0.25, 2.5, and 12.5 units/ml, monensin at 0, 14, 72, and 316 µM., and tetracycline at 0, 23, 113, 225 µM. Plates were incubated at 25°C for 7 days to allow for colony development of cultivatable antibiotic resistant microorganisms. Plates were then scored for presence or absence of the colonies. The number of isolates growing on plates at a given concentration of antibiotic divided by the number of isolates on plates containing no antibiotics was calculated to give a percent resistance at each concentration of antibiotic for controls and each litter treatment (five treatments in total). The isolates remaining in the 96-well plates were frozen at –80oC for DNA extraction. Means and standard errors were calculated for each treatment and data were analyzed by performing analysis of variance (ANOVA) procedures.

References:

Brookes, P.C., A. Landman, G. Pruden, D.S. Jenkinson. 1985. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. & Biochem. 17: 837-842

Cabrera, M.L., and M.H. Beare. 1993. Alkaline persulfate oxidation for determining total nitrogen in microbial biomass extracts. Soil Sci. Soc. Am. J. 57: 1007-1012

Heuer, H., K. Hartung, G. Wieland, I. Kramer, and K. Smalla. 1999. Polynucleotide probes that target a hypervariable region of 16S rRNA genes to identify bacterial isolates corresponding to bands of community fingerprints. Appl. Environ. Microbiol. 65:1045-1049

Parham, J.A. and S.P. Deng, 2000. Detection, quantification and characterization of β-glucosaminidase activity in soil. Soil Biol. & Biochem. 32:1183-1190

Tabatabai, M.A. 1994. Soil enzymes p. 775-833. In R.W. Weaver et al. (ed.) Methods of Soil Analysis. Part 2. 1st ed. SSSA Book Series 5. SSSA, Madison, WI

Vance, E.D., P.C. Brookes and D.S. Jenkinson. 1987. An extraction method for measuring soil microbial biomass C. Soil Biol. Biochem. 19: 703-707

Zuberer, D.A. 1994. Recovery and enumeration of viable bacteria p. 119-144. In R.W. Weaver et al. (ed.) Methods of Soil Analysis. Part 2. 1st ed. SSSA Book Series 5. SSSA, Madison, WI

Research results and discussion:

Objective 1

We had expected that microbial biomass and enzyme activity would be higher in plots receiving poultry litter. In fact, few significant differences were found among microbial biomass C values across treatments. Microbial biomass N was consistently lowest in the high ammonium nitrate fertilized plots, although not always significantly lower at the P < 0.05 level. Inorganic N availability was highly dependent on year. Inorganic N concentrations suggested that timing of applications may have significant impact on functioning or importance of specific microbial groups, but that effects on microorganisms of fertilizer treatments were not observable at the level of biomass C.

Biological activity was assessed by measuring dehydrogenase, beta-glucosaminidase, and acid and alkaline phosphatase enzyme potentials. Alkaline phosphatase potentials, but not acid phosphatase nor beta-glucosaminidase, were higher immediately following litter applications and were significantly correlated with soluble reactive phosphorus concentrations across treatments before and after litter additions. Data did indicate that repeated litter additions at the high rates have resulted in elevated beta-glucosaminidase and acid and alkaline phosphatase enzyme activities over time as compared to the control or soil receiving the high rate of ammonium nitrate. There was a general trend of decreasing enzyme activity with time across sample dates each year. Ammonium nitrate applications at the high rate resulted in reduced enzyme activity, suggesting that the ecosystem was stressed. Dissolved organic C data suggested the system was not C limited in the high N treatment, and the finding of reduced biological activity requires further study. In contrast, phosphatase activities associated with the high rates of litter applications indicated that the ecosystem was biologically active and capable of mineralizing phosphorus.

Objective 2

We had expected that poultry litter amendments would alter dominant microorganisms and thus diversity of community structure. In fact, treatments did not result in discernible differences by PCR-DGGE with sole exception being that high rate of ammonium nitrate resulted in clustering of those replicates (Figures 1 and 2). Our DGGE results do not indicate that poultry litter additions have affected the total bacterial community, but that high rates of N have. However, ancillary nutrient and enzyme data suggest that use of techniques revealing further refinement in resolution are warranted to reveal changes in specific populations that impact nutrient cycling of N and P.

Fig. 1. May 2003 DGGE gel of amplified 16SrRNA gene fragments from soil samples collected prior to treatment applications. From left to right, lanes 1-4 are unfertilized control, lanes 5-8 and 9-12 are alum-treated poultry litter applied at 2.24 Mg/ha and 8.98 Mg/ha, respectively, lanes 13-16 and 17-20 are untreated poultry litter applied at 2.24 Mg/ha and 8.98 Mg/ha, respectively, and lanes 21-24 and 25-28 are ammonium nitrate applied at 65 kg N/ha and 260 kg N/ha, respectively.

Fig. 2. June 2003 DGGE gel of amplified 16SrRNA gene fragments from soil samples collected 10 days following fertilizer applications. From left to right, lanes 1-4 are unfertilized control, lanes 5-8 and 9-12 are alum-treated poultry litter applied at 2.24 Mg/ha and 8.98 Mg/ha, respectively, lanes 13-16 and 17-20 are untreated poultry litter applied at 2.24 Mg/ha and 8.98 Mg/ha, respectively, and lanes 21-24 and 25-28 are ammonium nitrate applied at 65 kg N/ha and 260 kg N/ha, respectively.

Objective 3

We had hypothesized that poultry litter amendments would result in increased expression of antibiotic resistance in culturable bacteria. Isolates cultivated from untreated and alum-treated poultry litter were screened for resistance to bacitracin and monensin. Isolates from both untreated (PL) and alum-treated (AL) litters showed greater resistance to monensin than bacitracin at the highest concentration tested. Furthermore, isolates from AL expressed greater resistance to monensin than isolates from PL at the two highest concentrations.

Consistent with the findings for the poultry litter itself, isolates from soil one month following amendment application showed greater resistance to monensin than bacitracin. Similarly, six months following amendment application, the same trend continued. Of all three antibiotics, isolates expressed the least resistance to the concentrations of tetracycline tested. Individual soil treatment effects were inconsistent and varied depending on sampling time. Further analysis is required to develop more comprehensive understanding of the impact of animal waste applications to land in terms of antibiotic resistance in the indigenous bacterial communities.

Because DGGE of 16S rRNA gene fragments produced gels with numerous faint bands, and litter treatments and controls did not clearly separate from each other, amplified DNA bands of antibiotic resistant isolates could not be compared to the total soil community. We are however continuing to explore other methods to assess the diversity of the culturable bacterial community expressing antibiotic resistance to selected antibiotics.

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:

The following abstracts and presentations were made between 2003 and 2005. A Master of Science thesis is in preparation to be defended for a December 2005 graduation, and we anticipate three peer-reviewed scientific papers to be published.

ABSTRACTS

Tomlinson, P. J. and M. C. Savin. 2005. Microbial community structure of long-term poultry litter amended soils. In Annual Meetings Abstracts [CD-ROM]. ASA, CSSA, and SSSA, Madison, WI. In press.

Tomlinson, P. J. and M. C. Savin. 2005. Assessment of dehydrogenase, phosphatase and beta-glucosaminidase enzyme potentials in soil receiving long-term poultry litter additions. In Meetings Abstracts Soil Ecology Society 10th Biennial International Conference, Argonne, IL, May 22-25, 2005.

Tomlinson, P. J. and M. C. Savin. 2004. Antibiotic resistance in run-off and soil receiving poultry litter. In Annual Meetings Abstracts [CD-ROM]. ASA, CSSA, and SSSA, Madison, WI.

Tomlinson, P. J. and M. C. Savin. 2004. Poultry litter, an influence on antibiotic resistance in soil? In Invited Papers & Abstracts of Contributed Papers [CD-ROM]. Southern Branch ASA, Madison, WI.

Tomlinson, P. J., K. R. Payne, K. R. Brye, and M. C. Savin. 2003. Microbial dynamics in long-term research plots receiving alum-treated and untreated poultry litter. In Annual Meetings Abstracts [CD-ROM]. ASA, CSSA, and SSSA, Madison, WI.

OTHER ORAL and POSTER PRESENTATIONS

M. C. Savin and P. J. Tomlinson. 2005. Enzymes in soils with long-term poultry litter additions. Cooperative Regional Project S-297 Biodiversity and Microbial Community Structure in Soil and Rhizosphere annual meeting, Fayetteville, AR, June 26 – 28, 2005.

Tomlinson, P. J. 2005. Multi-dimensional assessment of microbial communities in soils receiving long-term applications of poultry litter. Crop, Soil and Environmental Sciences Department Exit Seminar, Fayetteville, AR, Mar. 28, 2005.

Tomlinson, P. J., and M. C. Savin. 2005. Microbial community dynamics assessed in soils receiving poultry litter. Gamma Sigma Delta Student Oral Presentation Contest, Fayetteville, AR.

Tomlinson, P. J., and M. C. Savin. 2004. Microbial dynamics in long-term research plots receiving alum-treated and untreated poultry litter. Cooperative Regional Project S-297 Biodiversity and Microbial Community Structure in Soil and Rhizosphere annual meeting, Pendleton OR, June 6 – 7, 2004.

M. C. Savin and P. J. Tomlinson. 2004. Antibiotic resistance of bacteria isolated from run-off and soils receiving poultry litter. Arkansas Water Resources Center Conference, Fayetteville, AR, Apr. 20-21, 2004.

Tomlinson, P. J., K. R. Payne, K. R. Brye, and M. C. Savin. 2004. Microbial dynamics in long-term research plots receiving alum-treated and untreated poultry litter. Gamma Sigma Delta Student Poster Contest, Fayetteville, AR. (3rd Place Master’s Student Div.)

Tomlinson, P. J., and M. C. Savin. 2003. Influence of poultry litter amendments on soil microbial communities. Cooperative Regional Project S-297 Biodiversity and Microbial Community Structure in Soil and Rhizosphere annual meeting, Blacksburg, WV, May, 24 – 26, 2003.

Project Outcomes

Project outcomes:

1. Applications of untreated and alum-treated poultry litters tended to result in a lower microbial biomass C/N ratio, suggesting bacterial dominance.

2. The application of poultry litter, alum-treated poultry litter, and ammonium nitrate fertilizers at high rates impacted the dynamics of the soil microbial community. High rates of poultry litter and alum-treated litter increased phosphatase activities, while the high application rate of ammonium nitrate resulted in a reduction in the activity of dehydrogenase and alkaline phosphatase, suggesting that the ecosystem was stressed

3. Increased alkaline phosphatase activity ten days following untreated and alum-treated litter applications indicated that the ecosystem was biologically active and capable of mineralizing phosphorus.

4. Acid and alkaline phosphatase potentials were increased in the high litter rate treatments as compared to control, supporting the conclusion that many cycles of microbial growth, death, and decay are required to permanently increase phosphatase activity (Tabatabai and Dick, 2002).

5. Changes within microbial communities resulting from management practices such as the application of poultry litter to grasslands may require the use of techniques that can achieve a greater level of resolution of the community diversity (such as denaturant gradient gel electrophoresis focused on specific functional groups of microorganisms or terminal restriction fragment length polymorphisms (T-RFLP).

6. Greater resistance to the antibiotics monensin and bacitracin was expressed in alum-treated (AL) than untreated litter (PL), but those differences were not maintain in soil isolates.

7. Greater resistance to monensin and bacitracin in soils receiving the high rate of untreated poultry litter (L4) as compared to the control was measured one month after litter application, but not six months after amendments.

8. Resistance to antibiotics in soil isolates tended to follow the pattern of monensin > bacitracin > tetracycline which is what may be expected based on organisms targeted by each antibiotic.

9. Antibiotic resistance in the environment is a topic receiving much attention in recent years. There is still relatively little that is known about how the use of antibiotics and the consequent land application of animal wastes affect the resistance acquired and expressed by environmental communities. The level of antibiotic resistance measured appeared to be a function of the antibiotic tested, management treatment, and time since application. Our interest in this area has led us into multi-institutional collaboration with other soil microbiologists affiliated with the multi-state S-297 soil microbiology research group. We are attempting to obtain funding to study this issue and the multi-state research group is making it one of its two primary objectives in it new regional research project (SDC-316, approve deferred at this time).

References:

Tabatabai, M.A. and W. A. Dick 2002. Enzymes in soil p. 567-596. In R. G. Burns and R. P. Dick (ed.) Enzymes in the Environment. Marcel Dekker, Inc., New York, NY

Recommendations:

Areas needing additional study

Objective 1

The differences in nitrogen concentrations found between the 2003 and 2004 spring samplings suggested that timing of fertilizer application may be very important to microbial functioning and nutrient cycling. Differences at the time of application in abiotic environmental factors, such as moisture and temperature, may have contributed to differences in the activity of microbial functional groups, such as nitrifier populations, and hence forms and amounts of nitrogen measured in the soil.

Additionally, phosphatase enzymes cleaving P will affect the availability of inorganic P. The leaching versus run-off or organism uptake potential of available P will also depend on litter application timing in spring and abiotic constraints on microbial activity. More study is required to determine most appropriate timing of applications.

Objective 2

Because DGGE of PCR products amplified with primers for Eubacterial 16S rRNA gene fragments amplified many bands, distinguishing bands was difficult and did not result in clear separation of litter treatments from each other or from the control. However, due to results obtained for nutrient data, we feel that investigation of more specific bacterial groups warrants further research efforts. We plan to amplify ammonia oxidizers to attempt to gain insight into N cycling in land receiving continued poultry litter inputs. We also observed a substantial number of actinomycetes in the culturable community isolated from soil. Actinomycetes, comprise an ecologically important group of gram-positive bacteria known for both the decomposition of recalcitrant, lignocellulosic compounds and for the inherent production of antibiotics. We will thus attempt to use PCR-DGGE to amplify and interpret how poultry litter applications affect the diversity of the soil actinomycete community.

Alternatively, T-RFLP may be an alternative approach to reveal community diversity because it tends to resolve many more peaks than DGGE and may provide the information about bacterial diversity that was not obtainable by DGGE of 16S rRNA gene fragments.

Objective 3

A proposed approach for assessing diversity of the culturable, antibiotic resistant community is the use of rRNA intergenic spacer analysis (RISA). We have performed several preliminary assessments of this method and it appears to show promise. Our plan, contingent upon securing additional funding, is to sequence a portion of the 16S gene fragment of at least one representative for each uniquely sized RISA amplicon. DNA sequences will allow us to investigate the phylogenetic placement and identity of the culturable, antibiotic resistant isolates and to compare across treatments communities that are resistant to different antibiotics.

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 or SARE.