Brief Summary of Minutes of Annual Meeting

Objective 1: to determine the geographic variability of E. coli ribotypes in the United States. We conducted collaborative experiments to determine the geographic variability of E. coli ribotypes in the United States. With the help of researchers from AL, DE, GA, IN, NY, PR, TN, TX, and USDA over the 5-year project, the objective was accomplished. We determined that there was good ribotype separation among host animal species at one location, and that the ability to match environmental isolates to a host origin database depended on a large number of environmental and host origin isolates that were not geographically separated. We extended the changes of ribotypes of E. coli with geography to time. Our results suggested that the majority of ribotypes were transient and unique to each sampling time; therefore, a large number of E. coli isolates per host were necessary to establish a host origin database that was independent of changes with time. We also extended this work to flow conditions, and established that ribotype changed with flow conditions. Finally, we established that ribotypes changed with an animals diet. All these results were discouraging because if ribotypes of E. coli changed with geography, time, flow rate, and diet, then a permanent host origin database large to encompass these patterns would be time-consuming and expensive to construct. Rather than continue with E. coli, we decided to test another species of fecal bacteria, Enterococcus faecalis, for changes in ribotype patterns with geography. We completed another collaborative manuscript this bacterium and the results were similar to that of E. coli. Again, these results are discouraging because a permanent host origin database large to encompass these patterns would be time-consuming and expensive to construct. Therefore, with two exceptions, we decided to move from library-dependent bacterial source tracking (BST) methods to library-independent methods. The first exception was a multi-year project to investigate at-risk or impaired watersheds in Virginia with two library-dependent BST methods, antibiotic resistance analysis and pulsed-field gel electrophoresis. The watersheds ranged from completely urban or suburban basins (all homes in the watershed served by sewers) to mixed-use basins dominated by agricultural activities (most homes served by onsite systems) to forested basins within the boundaries of a state park (no homes). Water and sediment samples were collected and analyzed for E. coli and enterococci. The goal of the project was to use the two library-dependent methods to identify some fecal sources that were apparent (should be present) and some sources that were not (should not be present, e.g., livestock sources should not be present in the completely suburban watershed). Based on the locations of the sample sites and their localized sources of contamination, two libraries, comprised of known source isolates collected from the county and the greater northern Virginia area, were used to analyze the patterns of isolates recovered from membrane-filtered stream water samples. The average rate of correct classification for the 1,490-isolate, four-way (human, domestic pets, wildlife, birds) urban library was 80.5%. The average rate of correct classification for the 2,012-isolate, four-way (human, livestock, wildlife, birds) rural library was 84.1%. To date, the major sources in the rural watersheds have been livestock and wildlife (with a minor human signature at certain locations), and the major sources in the urban areas have been birds and wildlife. After storm events, the livestock signatures were elevated in rural areas and wildlife signatures were elevated in urban areas. The second exception was to compare various library-dependent, genotypic BST methods for their strengths and weaknesses. All the methods correctly identified the dominant host source in the majority of samples, but false positive rates were as high as 57%. One S-297 researcher continued this research with another research team examining both genotypic and phenotypic methods. Their results were similar. Four projects with library-independent methods were conducted. The first project was to make BST quicker, more effective, and less expensive, and led to the development of targeted sampling as a prelude to BST. Targeted sampling is like the childrens game of hot and cold. The sampling is first divided into baseflow and stormflow conditions because runoff during stormflow typically increases fecal bacteria counts 10- to 100-fold. By combining local knowledge, sampling, and resampling of the water during one of these two flow conditions, it was possible to identify hotspots of fecal contamination quickly and easily. Targeted sampling also reduced the effects of bacterial changes with geography and time, and this reduction made existing BST methods better and less expensive by reducing the environmental complexity. At this point, targeted sampling has been conducted during both base- and storm-flow conditions in marine waters along the Georgia Coast, but has not yet been conducted in freshwater or in other states. The second project was to determine the limitations of fluorometry as a BST method. Fluorometry works by detecting optical brighteners from laundry detergents in environmental waters because these compounds fluoresce when exposed to UV light. Therefore, fluorometry is not a bacterial source tracking method per se, but a chemical source tracking method. Furthermore, because these fluorescent compounds are associated with human sewage, the method discriminates only between human and nonhuman sources of fecal contamination. Preliminary tests by researchers from GA and VA concluded that fluorometry was an acceptable, inexpensive method to detect human sewage in fresh and marine waters. The third project was to pursue enterococcal speciation as a simple method to discriminate between human and non-human fecal contamination. We determined that Ent. faecalis had a host range essentially restricted to humans and wild birds. Our results suggest that unless the fecal loading rate from migratory or resident wild birds is high, water samples collected during baseflow conditions with high numbers of Ent. faecalis may indicate human fecal contamination. The fourth project was a proof-in-concept project for a species-specific ribosomal DNA gene biosensor to detect Enterococcus faecalis rapidly in environmental samples (water and human feces). The biosensor used sulforhodamine B (SRB) dye-encapsulated liposome technology in a competitive assay to detect the presence of a synthetic 16S rRNA gene sequence specific to Ent. faecalis. Two different target analyte sequences were hybridized with liposomes that had reporter probes (ssDNA oligonucleotides) attached to them. The bioassay strip was sensitive when used with synthetic target sequences and could be run in under 20 minutes, but was not able to detect the target sequence amplified from environmental isolates or ATCC strains of the Ent. faecalis 16S rRNA gene. The positive signal generated with the control synthetic sequence suggests that perhaps secondary structures (e.g., hairpins) may inhibit the hybridization of the target analyte with the capture and reporter probes. Objective 2: To determine relationships among microbial taxonomic and functional diversity, contaminant bioavailability, and remediation rates for different organic-contaminated soils. A 5-year collaborative project was conducted to evaluate rhizosphere-enhanced bioremediation of organic contaminants. The participants included researchers from AL, AR, DE, FL, IN, NH, NC, OK, SC, WI, and Canada. We first evaluated the interactions of different plant species, soils, and nutrients to remediate soils contaminated with such organic contaminants as crude oil, nitroaromatic explosives, hexadecane, and pyrene. We began our evaluations by testing 21 warm- and cool-season grasses and legumes for their ability to germinate, survive, and grow in crude oil-contaminated soil. The most appropriate warm-season grasses were pearl millet (Pennisetum glaucum) and sudangrass (Sorghum sudanense), and the most appropriate cool-season grasses were ryegrass (Lolium multiflorum) and fescue (Festuca arundinacea). In soil amended with pyrene and sprigged with bermudagrass (Cynodon dactylon), the number of pyrene-degrading microbes was significantly higher than in soil not amended with pyrene. These results suggest that the presence of the contaminant and the plant increases the potential of the rhizosphere microbial community to degrade pyrene. In soil amended with pyrene containing no plants, whole soil fatty acid methyl ester (FAME) analysis indicated a shift in the composition of the soil microbial community in the pyrene-amended soil compared to the unamended soil. We then evaluated the influence of rhizosphere on soil remediation of spilled crude oil at a petroleum storage tank facility. The treatments were: 1) non-vegetated non-fertilized control, 2) fescue-ryegrass mixture plus fertilizer, and 3) bermudagrass-fescue mixture plus fertilizer. Vegetation was successfully established at the site, even though the soil had an initial total petroleum hydrocarbon concentration of 9,175 mg/kg. While alkylated two-ring naphthalenes degraded equally in all treatments, the larger three-ring alkylated phenanthrenes-anthracenes and dibenzothiophenes degraded more in the vegetated, fertilized plots than in the non-vegetated, non-fertilized plots. In this field study, the increased biodegradation of the more recalcitrant alkylated polycyclic aromatic hydrocarbon compounds in the crude oil-contaminated soil most likely occurred because of the increased rhizosphere soil volume (associated with increased root length) and because the added nutrients increased total bacterial, fungal, and polycyclic aromatic hydrocarbon (PAH) degrader numbers. In a separate study, pre-established bins of soil (0.9-m diameter) were amended with pyrene in a 10-month experiment under field conditions. After a 175-d lag period, the rate of pyrene loss followed first-order kinetics, with a rate constant significantly higher in nonvegetated than vegetated treatments. The delay of pyrene dissipation in the rhizosphere may be because of the presence of easily degradable organic material from the plant roots. The successful bioremediation of sites contaminated with aromatic compounds typically relies on the presence and stimulation of aromatic hydrocarbon-degrading bacteria from the indigenous microbial population. Although aerobic bioremediation is the common treatment, there is no direct biological assay to document the bioremediation during treatment. We developed a technique based on real time PCR amplification of aromatic oxygenase genes to detect and quantify a number of aromatic catabolic genes in environmental samples. Each primer set was specific for a family of oxygenase genes (e.g., toluene dioxygenase). Based on available published sequences, PCR primer sets were identified which targeted biphenyl dioxygenase, naphthalene dioxygenase, toluene dioxygenase, toluene/xylene monooxygenase, phenol monooxygenase, and ring hydroxylating-toluene monooxygenase genes. The primer sets and real-time PCR methods were then used to demonstrate the effectiveness of the approach, first in laboratory enrichment microcosms, and then at contaminated field sites. At the field sites, aromatic oxygenase genes were detected in groundwater monitoring wells with current or recent petroleum contamination, but not in wells with no history of contamination. Genes were no longer detected after contaminant concentrations reached zero. We determined that certain bacteria were uniquely adapted to interact with humic acids in a manner that allowed these organisms to access and degrade PAHs sorbed by humic acids. Although we isolated a wide variety of bacteria that degraded non-sorbed phenanthrene, only three strains of Burkholderia sp. and one strain of Delftia acidovorans degraded humic acid-sorbed phenanthene. We termed this phenotype characteristic competence. Competence was not a graded characteristic; bacteria either degraded humic acid-sorbed phenanthene or they did not. The characteristics of competent cells that support this phenotype, and the nature of the interaction occurring between humics and bacterial cells, are unknown. However, competence determinants were physically associated with the cells; diffusible agents (e.g., biosurfactants) did not have a significant role. As yet, the known physical differences between competent and closely related non-competent strains are phospholipid fatty acid content and extracellular matrix production. To the best of our knowledge, this is the first report of specific groups of bacteria adapting to interact with humic acids. We evaluated the microbial activity and community structure in soils contaminated with the nitroaromatic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydrol-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). Our results suggest that natural attenuation of these compounds in highly contaminated soils may not be feasible. Because nonionic surfactants had little effect on bacterial growth, these surfactants may help overcome this natural attenuation. We studied the characteristics of isolates from two bacterial species, Pseudomonas putida and Rhodococcus erythropolis, to degrade soil-sorbed biphenyls. Both isolates showed strong tendencies to attach to soils. The pseudomonad was more motile and had a higher chemotactic response to biphenyl than the Rhodococcus isolate. It appears that attachment to soil, motility, and chemotaxis are important characteristics that influence the bacterial access to soil-sorbed biphenyl. Bacteria with higher chemotactic response and moderate cell surface hydrophobicity may access soil-sorbed biphenyl more efficiently than bacteria with a lower chemotactic response and less cell surface hydrophobicity. We determined the influence of soil properties on the degradation of 14C- and 13C-labelled glucose or hexadecane. Even though the parent compound was rapidly degraded (half-life <2 d), only 34% of the C was evolved as 14CO2 after 275 d, with the remaining C incorporated into microbial biomass and soil organic matter. Studies with 13C-labelled glucose show that the initial microbes that metabolized the C underwent degradation themselves, the C of which was then incorporated into phospholipid fatty acids of other members of the microbial community. There was sufficient 13C incorporated into phospholipid fatty acids that it was possible to assess what members were active in C degradation. Objective 3. To characterize taxonomic and functional diversity of bacteria and mycorrhizae in disturbed lands and urban landscapes. We conducted collaborative experiments to evaluate the effects of various disturbances or land-management systems on the microbial abundance and community structure and function of soil communities. Over the 5-year project, participants included researchers from AL, GA, IN, NC, NY, OR, SC, and TX. We divided land use into four management systems: pasture, cropping, turfgrass, and mycorrhizae. In pasture systems, we conducted both short-term growth studies and long-term field experiments with scientists from GA, TX, and USDA to determine changes in soil organic C and N during growth of tall fescue uninfected or infected with the fungus, Neotyphodium. We thought that plant metabolites produced in association with the endophytic fungus could alter soil microbial activity and how it processed C and N in soil. Tall fescue forage accumulation was greater with than without the fungus, confirming previous results that fungus infection also confers greater fitness to plants. Microbial communities under endophyte-infested and non-infested fescue were different in terms of C source utilization (BIOLOG); this difference suggested that the endophyte affected the metabolic capacities of the microbial populations in the soil. In cropping systems, we conducted three studies on the effects of continuous cotton management systems on soil biological properties. In the first study, phospholipid profiles suggested that microbial communities associated with conventional tillage and no tillage continuous cotton systems were dissimilar, and that the tillage effect varied with soil depth and time. The second study compared a standard cotton production system with an intensive cotton cropping system that maximized the production of crop residues and legume N inputs. Again, the phospholipid profiles showed that soil microbial communities under conventional tillage were different from those under the conservation tillage treatment, and that soil microbial communities shifted with soil depth. In the third study, we investigated several soil microbiological parameters in a cotton crop-management system: soil organic C, microbial biomass C, aggregate size classes, and water-extractable carbon (80°C for 16 h), and total glomalin in soils from plots under different tillage, fertility (0 and 90 kg N ha-1), and rotation schemes (conventional till continuous cotton, reduced-till cotton-corn rotation, and reduced-till continuous cotton). Soil from the reduced-till and cotton-corn rotation treatments contained the more organic matter compared with continuous cotton plots. No major differences were found in aggregate size classes among the treatments, except that the >2000-micrometer fraction increased in the cotton-corn rotation at the expense of the 53- to 250-micrometer fraction in the plots receiving 90 kg N ha-1. Soil microbial biomass carbon ranged from approximately 270 to 550 mg kg-1 across all treatments, including N application and depth. Hot-water extractable C ranged from approximately 261 to 445 mg kg-1 in all treatments including N and depth. Total glomalin ranged from approximately 2300 to 2800 mg kg-1 soil, and was well correlated with soil organic C (r2=0.87). Total glomalin C accounted for roughly 7 to 9% of the soil organic C. In a 3-year cropping system with plants other than cotton, we determined the effects of different agricultural management practices on soil and rhizosphere microbial communities of corn (Zea mays) and soybean (Glycine max). Corn and soybean plants grown under different tillage (no-till or plow) and rotation (monoculture or rotation) treatments were collected over different growth stages (V1, V2, V3, V6, V9, and maturity) during three growing seasons. When rhizospheres of corn and soybean plants were compared using PCR amplification of the small subunit rRNA gene and separated by denaturing gradient gel electrophoresis, the banding patterns of the microbial rhizosphere community grouped mainly according to agronomic treatment. This effect was more evident in the bacterial versus the eukaryotic communities, and in corn versus soybean rhizospheres. We identified the most significant bands in the corn rhizosphere that were consistently associated with improved plant growth for all three seasons. Nucleotide sequencing identified one of the four bands as Burkholderia species. We chose this species of bacteria for subsequent laboratory plant growth experiments and found significant differences in plant growth when the plants were inoculated with these isolates. We used a polyphasic approach (combining traditional and molecular methods) to assess changing soil and crop management strategies (or naturally occurring soil stresses) on soil microbial community abundance, activity, and diversity in the rhizosphere of transgenic (Bt) corn and rice. After 3 years of field trials, the transgenic plants had no adverse effects on the abundance, activity, or diversity of microbial communities compared with their non-transgenic isolines. Residues of two transgenic Bt corn varieties decomposed at the same rate as their non-transgenic counterparts and were colonized by similar microbial communities. Residues placed on the soil surface decomposed significantly slower than those buried at 5-cm depth, and were colonized by distinctly different bacterial and fungal communities. In the case of corn plant parts, cobs, stalks and leaves were colonized by distinctly different bacterial and fungal communities and decomposed at different rates, ranging from low to high, respectively. However, transgenic versus non-transgenic plants were not a significant factor. We conducted several long-term experiments with wheat to determine changes in bacterial populations with different tillage systems. Bacterial populations varied during the year, exhibiting a general decrease in the late fall and an increase in the early spring. Bacterial numbers increased in a no-till, summer-fallow system when compared to conventional tillage--contrary to some reports that suggest that microbial populations will decrease in these management systems. With respect to carbon dioxide flux, the flux in conventionally tilled fields was almost five times greater than no-till fields. After determining C and N mineralization rates of crop residue components, we developed a C sequestration model, CQESTR, to estimate residue loss and C sequestration, but the model was not sufficiently user friendly. Therefore, we developed a more user-friendly model by rewriting the user manual, adding a program tutorial, and inserting a Help function in the Microsoft Windows interface. In turfgrass systems, scientists from AL, NC, SC, and TX investigated various aspects of the microbiology of intensively managed turfgrass systems including golf courses, sports fields, and home lawns. Turfgrass systems differ from agricultural systems because, except for aeration, they are essentially zero-tillage systems maintained with plenty of water and nutrients. Therefore, they really represent highly productive, managed grasslands. We showed that sports turfs contained abundant microbial populations (bacteria and fungi) throughout the growing seasons. Further there was relatively little seasonal decline, and populations were relatively stable over periods of several years when turf maintenance practices were adequate. Furthermore, in terms of species composition, the populations were similar to those found in other agricultural systems. In mycorrhizal systems, four major studies were conducted. In the first study, a 5-year experiment was initiated to measure how alternative land management practices for fresh market tomato production affect soil health. The prevailing system uses raised beds and fumigation with methyl bromide for control of soil-borne nematodes, weeds, and root fungal pathogens. We tried five different management programs in replicated 1-acre plots over a 3-year period prior to a 2-year resumption of the prevailing system: 1) conventional tomato production using nematicide/fungicide and herbicides; 2) continuously maintained disk fallow, 3) weed fallow in which endemic plants colonized the plots; 4) bahiagrass conservation tillage where strips were tilled in the grass sod before tomato plantation, and 5) organic management consisting of cover crops of millet and Sunn hemp (a tropical legume) rotations and annual broadcast of broiler chicken litter mixed with urban plant debris. We monitored changes in soil fungal communities with length heterogeneity PCR and non-metric multivariate analysis. After three years, fungal communities in soil under continuous tomato production and continuous disk-fallow cultivation were similar. Communities in soil that were left undisturbed in a weed fallow system were similar to communities in a perennial pasture grass rotation. Communities within an organically managed system were unique. Following traditional tomato production in the fourth year, communities in the organic and pasture grass systems remained unique, whereas communities in the weed fallow were similar to communities under continuous tomato production or continuous cultivation. Fungal communities were dominated by a 341-bp rDNA amplicon fragment. Cloning and sequencing indicated the dominant fragment belong to Fusarium spp., which was further confirmed by the ITS 1 sequence from single spore isolates of F. oxysporum f. sp. lycopersici. The relative abundance of the 341-bp fragment was greatly decreased in organic and pasture grass systems that which also had a lower incidence of Fusarium wilt in the tomato crop. At the end of each crop season, tomato rhizosphere soil and fibrous roots of tomato and cover crops were collected. Roots were cleared, stained and examined for arbuscular mycorrhizae (AM) and other root endophyte colonization. Rhizosphere soil was assayed for mycorrhizal infection potential (MIP) based on maize seedling colonization after 35 days. Bahiagrass and weed fallow systems promoted the highest AM colonization in the tomato crop and produced moderate MIP levels and spore numbers in rhizosphere soils. Cover crops had moderate levels of AM colonization. The disk fallow, conventional and organic systems supported lower AM colonization of tomato roots, but systems that created the most disturbance (conventional and disk fallow), promoted the highest MIP and spore counts from trap cultures. Thus, managements potentially most disruptive to AM fungal communities produced the highest inoculum density, but the least infective inoculum with respect to the tomato crop grown under nutrient supply. Organic management with the highest P supply was least conducive for development of MIP or AM colonization of tomato. In contrast, the organic system supported one non-AM endophyte at high incidence (Microdochium bolleyi). In the other systems, a diversity of non-AM endophytes was observed, including a dark septate endophyte (Phialophora spp.). Considering the companion analysis of the bacterial and fungal diversity, communities were more similar and diverse in the systems that produced the least disturbance (bahiagrass sod and weed fallow). Thus, soil microbial activity and diversity in the agro-ecosystems studied were most affected by soil nutrient supply and disturbance. In the second major mycorrhizal study, we determined if sugarcane yield decline was linked with early and rapid colonization by AM or not. Fallow management (up to 1 year) of sugarcane soils followed by repeated tillage (to break up the root crown and to reduce weed cover before replanting) produced up to a 30% increase in biomass at the first cutting, but only a slight increase at the second cutting, and no increase at the third cutting. Treatments with soil biocides duplicated the fallowing effect. In most instances, no soil microorganisms deleterious to sugarcane roots were identified. Arbuscular mycorrhizae were trapped from successively planted sugarcane fields, and three Glomus isolates were selected to reconstitute a steamed local muck in glasshouse experiments. Muck treated or not treated with steam received a combined extract of the non-steamed field soil and pot cultures of each isolate to check for the deleterious effect of microorganisms that pass through 20-micrometer sieve openings. Roots in non-steamed soil were rapidly colonized in advance of shoot development. In reconstituted soils, rate of mycorrhizal development varied with the Glomus isolate. Shoot biomass gain was lowest in non-steamed soil, and highest in steamed soil, with no effect of the extract from the non-sterile field/pot culture soils. Total biomass gain was inversely related to colonization rate among soil and Glomus isolates. Reduction of biomass gain compared to the steamed soil treatment was best predicted by early root colonization for the three Glomus spp. and soil treatments at 2 weeks after shoot emergence, and to a lesser extent by later colonization at 4 and 6 weeks. In the third study, we evaluated arsenic uptake in the hyperaccumulating fern, Pteris vittata (Chinese brake fern), a fern that grows naturally in soils of the southern United States. A greenhouse experiment was conducted with and without P. vittata, where arbuscular mycorrhizae from an As-contaminated soil colonized the plants. Three levels of arsenic (0, 50, and 100 mg kg-1) and three levels of P (0, 25, and 50 mg kg-1) were used. Arbuscular mycorrhizae not only tolerated As amendment, but also their presence increased frond dry mass at the highest As application rate. The AM fungi increased As uptake across the range of P levels, while P uptake generally increased only when there was no As amendment. In the fourth study, we studied fungal and bacterial dynamics in relatively nutrient-rich and nutrient-poor wetland plant communities. The dominant wetland plant communities (Panicum, Cladium, Typha, Salix, mixed herbaceous, and slough area plants) were sampled seasonally from nutrient-rich and nutrient-poor areas. We measured ergosterol concentration, percentage root colonization by mycorrhizal fungi, and total bacterial counts in detrital and soil samples. Mycorrhizal fungi were not influenced by water level, and both mycorrhizal and total fungi had seasonal patterns that were influenced by the plant community. We continued these studies in greenhouse experiments. No arbuscular mycorrhizal fungal community had a consistent impact on plant growth and nutrition. For one wetland plant, flooding eliminated mycorrhizal fungal colonization, and in free-drained treatments, P amendment suppressed colonization. For the same wetland plant, some mycorrhizal communities affected shoot and root P concentrations, but there were no significant plant growth responses. For another wetland plant, the mycorrhizal association was suppressed, but not eliminated, by flooding and P amendment. Mycorrhizal colonization improved plant growth and P nutrition at lower P levels, but conferred no benefit or was detrimental at higher P levels. Objective #1 We established that the ribotypes of bacteria most widely used for bacterial source tracking, E. coli and the fecal enterococci, changed considerably with geography, time, and flow conditions. In addition, we showed that the ribotypes of E. coli changed with diet. These results were discouraging because they suggest that library-dependent BST methods will require a large host origin database (thousands of isolates) in order to encompass this genetic variability. Such a library will be time-consuming and expensive to construct. In the case were we did have libraries of sufficient size, our studies showed that library-dependent BST methods did work, and the information should provide city and county officials with information to improve impaired waters. We developed targeted sampling as a prelude to BST as a way to reduce the effects of bacterial changes with geography and time. Targeted sampling is like the childrens game of hot and cold, and by combining local knowledge, sampling, and resampling of the water during one of these two flow conditions, it was possible to identify hotspots of fecal contamination quickly and easily. By reducing the environmental complexity, we made existing BST methods quicker, more effective, and less expensive. We also extended BST research to fluorometry. This chemical BST method works by detecting optical brighteners from laundry detergents in environmental waters from malfunctioning septic drain fields and leaking sewer pipes. Because these fluorescent compounds are only associated with human sewage, the method discriminates between human and nonhuman sources of fecal contamination. Regulatory and municipal authorities in several states are currently evaluating fluorometry. The fluorescent signals appeared to be stable over seasons in different water conditions. Whenever fluorescent plumes were found, BST tests demonstrated a human signature in every case where it was performed. Therefore, our tests suggest that fluorometry was an acceptable, inexpensive method to detect human sewage in fresh and marine waters. We also tried enterococcal speciation as a simple method to discriminate between human and non-human fecal contamination, and confirmed that Ent. faecalis had a host range essentially restricted to humans and wild birds. Our results suggest that unless the fecal loading rate from migratory or resident wild birds is high, water samples collected during baseflow conditions with high numbers of Ent. faecalis indicate human fecal contamination. We started development of a species-specific primer that works in a biosensor to detect synthetic sequences of Ent. faecalis. This development is a positive first step in the development of an inexpensive new technique to detect pathogens from the environment. Objective #2 Although phytoremediation is a common and inexpensive form of environmental remediation, its efficacy is highly variable. This variability reduces our confidence in phytoremediation to remove toxic organic contaminants from the environment. Our goal was to define how biogeochemical factors and their interactions influence the phytoremediation of crude oil. We showed that adding fertilizer and establishing vegetation increased microbial populations, which, in turn, consistently reduced contaminant concentrations of crude oil. Our results suggest that agronomic practices are important to consider when developing systems to phytoremediate crude oil-contaminated sites. By assessing the degradation potential of the indigenous microbial community, determining degrader numbers, and detailing their molecular makeup, we identified important microbial information necessary to assess remediation technologies. In this manner, we enhanced studies of contaminated site ecology, and ultimately established a coherent management strategy for cleanup of crude oil-contaminated soils. For bioremediation as humic acid-sorbed PAHs, our studies show that some organisms may be uniquely suited to overcome many of the bioavailability limitations that hinder biodegradation of PAHs in soil. More broadly, because humic acids are the most pervasive organic molecules to which bacteria are exposed, niches occupied by bacteria adapted to interact with humic acids could have far-reaching implications. Objective #3 In pasture systems, agricultural management practices (e.g., crop rotations, reduced tillage, organic matter amendments, or introduction of toxins) may alter the nature of the soil environment and influence the composition and activity of the microbial community. Our studies suggest that it may be possible to manage naturally occurring plant metabolites to increase agronomic productivity or to sustain environmental quality. In the short term, our investigations with toxic alkaloids in tall fescue indicated soil responses to these alkaloids were relatively minor, but statistically significant. These compounds also increased tall fescue fitness. In the long term, soil biochemical responses to toxic alkaloids led to greater soil organic C sequestration, thereby mitigating greenhouse gas emissions. In cropping systems, our cotton management studies indicated that there is a potential for increased C storage in soils managed with reduced tillage. Less N was needed for cotton production in rotation and reduced till systems than in conventionally tilled systems. Changes in tillage practices not only increased microbial biomass C, but also shifted microbial community structure. However, how these changes affect the long-term sustainability of cotton cropping systems is still unclear. Continued use of conventional farming systems (i.e., plowing, rod-weeding, and summer fallow) were detrimental to soil quality and sustainable crop production because these systems reduce organic matter. Our data should encourage producers to convert from conventional tillage to conservation tillage (e.g., direct seed), thus reducing erosion and improving soil health by increasing the soil organic matter.

PUBLICATIONS (* denotes collaboration between two or more S-297 members) Books *Edge, T., J. Griffith, J. Hansel, V. J. Harwood, M. Jenkins, A. Layton, M. Molina, C. Nakatsu, R. Oshiro, M. Sadowsky, J. Santo Domingo, O. Shanks, G. Stelma, J. Stewart, D. Stoeckel, B. Wiggins, and J. Wilbur. 2005. Microbial source tracking guide document. U. S. Environmental Protection Agency, Office of Research and Development, EPA/600-R-05-064. *Sylvia, D. M., J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer. 2005. Principles and applications of soil microbiology, 2nd edition. Prentice-Hall, Upper Saddle River, NJ. *Sylvia, D.M., P.G. Hartel, J.J. Fuhrmann, and D. Zuberer (ed.) 2005. Instructors manual: Principles and applications of soil microbiology, 2nd edition. Prentice Hall, Upper Saddle River, NJ. Book Chapters Chaney, R. L., Y.M. Li, J. S. Angle, A. J. M. Baker, R. D. Reeves, S. L. Brown, F. A. Homer, M. Malik, and M. Chin. 2000. 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