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PROJECT NUMBER:

PROJECT TITLE: Soil Microbial Taxonomic and Functional Diversity as Affected by Land Use and Management

PROJECT DURATION: October 1, 2000 to September 30, 2005

STATEMENT OF THE PROBLEM:

In this multistate project, we consider three aspects of land use and management and how they affect microbial taxonomic and functional diversity. In the first aspect, we determine how land-applied animal wastes affect biogeography, one important component of microbial diversity. In the second aspect, we determine relationships among microbial taxonomic and functional diversity, contaminant bioavailability, and remediation rates for different organic-contaminated soils. In the third aspect, we characterize taxonomic (organismal) and functional (physiological) diversity of bacteria and mycorrhizae in disturbed lands and urban landscapes. A crucial point of the second and third aspects is that they both determine how the rhizosphere, the zone of soil under the influence of plant roots, affects microbial taxonomic and functional diversity.

JUSTIFICATION:

Relation to agriculture, natural resources, rural life, and consumer concerns

Microbial taxonomic and functional diversity is crucial to major ecosystem processes, including the maintenance of fertile soils and the control of nutrient cycles. There is general scientific agreement that human activity is altering ecosystem processes. The importance of these processes is simple: without them, life itself is not possible. However, it is unclear how altering ecosystem processes are reflected in changes in microbial taxonomic and functional diversity, particularly under different land use and management practices.

Biogeographic aspects of land-applied wastes

One important aspect of microbial diversity is biogeography, the study of the global distribution of microorganisms. On the one hand, microorganisms may have a low migration rate and a high rate of speciation and local extinction (Finlay et al., 1999). Under these circumstances, microbial species would become restricted to specific geographical areas and the number of global species would be large. On the other hand, microorganisms may have a high migration rate and a low rate of speciation and local extinction (Finlay et al., 1999). Under these circumstances, microbial species would be pandemic and the number of global species would be small. Research to support either view—or some intermediate view—is surprisingly scanty. However, new molecular methods may permit these positions to be clarified.

One of the most important cases for biogeography involves bacterial pathogens, because biogeography can establish where pathogens come from and this may lead to new approaches to preventing disease (Staley, 1999). A excellent example of this is animal manures. Animal manures are good sources of plant nutrients, especially nitrogen and phosphorus, and land application of these manures is considered good agricultural practice. However, there is the potential for this land application to contribute bacterial pathogens to streams and other water sources through surface runoff, particularly where land area is limited and farmers apply more than the recommended rates. Because of this, agriculture is often implicated when bacterial pathogens are found in surface waters following runoff events from manure-amended lands. It is now possible to use a new technique, ribotyping, to determine the host origin of pathogens.

To understand how ribotyping can solve some fundamental issues of biogeography, it is first necessary to know about fecal coliforms. Fecal coliforms consist of several bacterial genera from the family Enterobacteriaceae that can grow on a selective medium at 44.5˚C for 24 hours. Fecal coliforms normally inhabit the intestinal tract of warm-blooded animals and their presence in soil or water is a good indicator that the soil or water was contaminated by bacterial pathogens. For example, when numbers of fecal coliforms exceed 2,000 per 100 mL of water, the likelihood of bacterial pathogens in the water is 98.1% (Geldreich, 1970). Fecal coliform counts are typically used to monitor U. S. recreational waters. Unfortunately, these counts simply quantify the fecal coliforms and do not identify their host origin. Knowing the host origin of fecal coliforms is important because it may then be possible to direct resources to minimize these bacteria wherever control is possible. For example, if the source of fecal coliforms in a water supply is from humans (e.g., a leaking septic tank), then resources can be directed at finding and eliminating this source.

In the past, the only way to identify the host origin of a bacterium was to observe the bacterium's various phenotypic markers (i.e., observable characteristics expressed by the bacterium, like multiple antibiotic resistance). The main problems with using phenotypic markers are their lack of reproducibility and lack of discriminatory power (ability to distinguish between two closely related strains). However, in recent years, it has become possible to discriminate among subspecies of a bacterium based on its DNA. This process, called genotyping, offers not only increased discriminatory power, but also increased reproducibility. The most common of these genotypic methods include chromosomal DNA restriction analysis, plasmid typing, pulsed field gel electrophoresis, various polymerase chain reaction (PCR) methods, and ribotyping (Farber, 1996).

Each genotypic method has advantages and disadvantages with respect to reproducibility, discriminatory power, ease of interpretation, ease of performance, and subspecies that can be typed. Some of these methods are able to genotype a species to such an extent that a large number of subspecies can be identified—these are the genotypic methods that may allow a subspecies of a bacterium to be associated with a certain host. Ribotyping is one such genotypic method.

Ribotyping is based on ribosomal RNA (rRNA). Ribosomal RNA is present in all bacteria, and is composed of three species, 5S, 16S, and 23S. The DNA in the bacterium that codes for these three species of rRNA is usually present in 2 to 11 copies and is highly conserved (or similar; Grimont and Grimont, 1986). In ribotyping, the DNA is isolated from the bacterium and cut with a special enzyme that only recognizes certain DNA sequences (i.e., a restriction enzyme). The DNA is electrophoresed in a gel and is transferred to a nylon membrane (this is called Southern blotting). The membrane is probed with a chemiluminescent copy of the 5S, 16S, and 23S portions of the DNA and, when properly treated, the membrane gives a pattern that can be scanned with an imager. As a method for distinguishing a subspecies of a bacterium, ribotyping is considered to have excellent reproducibility, good discriminatory power, excellent ease of interpretation, and good ease of performance (Farber, 1996).

In this proposal, the fecal coliform we selected for ribotyping is Escherichia coli. This bacterium was selected for five reasons. First, as a fecal coliform, E. coli is accepted by the American Public Health Association as a good indicator of pathogenic bacteria (Clesceri et al., 1998). Second, most environmental ribotyping has been done with this bacterium. As a result, the methodology for ribotyping this bacterium is established. Third, there is good scientific evidence that specific strains of E. coli are associated with different host species (Faith et al., 1996). Fourth, E. coli does not exist as a stable population in the environment unless the source of contamination is persistent (Savageau, 1983). Fifth, E. coli is easy to isolate and easy to manipulate genetically.

At present, we have ribotyped over 200 E. coli isolates from Georgia and Idaho. These include E. coli isolates from beef cattle, poultry (broilers), sheep, and swine. In addition, we have obtained 70 E. coli isolates from the study of Buchan et al. (1997) to add to our collection. These isolates are from beef cattle (23 isolates), Canada goose (19 isolates), and poultry (28 isolates) from northern Georgia. We have recently obtained the necessary software (GelCompar II) to analyze the gels of these E. coli isolates. Although the comparisons are not yet complete, the gels show clear differences among the ribotypes of the different animal species. Thus, ribotyping shows considerable promise in being able to associate specific E. coli ribotypes with specific animal hosts.

Rhizosphere-enhanced bioremediation of organic contaminants

Improper waste disposal and accidental spills of petroleum, heavy metals, explosives, and other toxic organic materials have contaminated many sites in the United States (Miller and Poindexter, 1994; Skipper and Turco, 1995). One way to reduce or eliminate this contamination is to use bioremediation. Bioremediation is the use of living organisms to reduce or eliminate toxic contaminants. An excellent example of this is the 1989 oil spill from the Exxon Valdez in Prince William Sound, Alaska. Wherever crude oil washed ashore, an oleophilic fertilizer was sprayed on the oil, and the narrowed C:N ratio enhanced growth of the indigenous hydrocarbon-degrading bacteria in the beach sand to degrade the crude oil (Skipper, 1998). This was but one of many early efforts to use microorganisms to remediate organic-contaminated sites. More recently, bioremediation has been used to treat soils contaminated with other organic compounds, including polyaromatic hydrocarbons (PAHs), halogenated compounds (e.g., trichloroethylene), pesticides, polychlorinated biphenyls (PCBs), and explosives (e.g., TNT).

One area of particular interest in bioremediation is the use of plants to enhance bioremediation in the rhizosphere. Rather than spraying on a fertilizer or otherwise amending the soil, here the plant roots exude organic materials into the rhizosphere, and the indigenous soil microorganisms degrade not only the organic materials that the plant provides, but also the contaminant (e.g., Guthrie and Pfaender, 1998; Zhang et al., 1997). The unique partnership of soil microorganisms and plant roots provides a low cost, in situ biological degradation of chemical contaminants (Anderson et al., 1993). There is also some evidence that this rhizosphere-enhanced bioremediation may result in lower contaminant endpoints than can be obtained without plants (Fletcher et al., 1995). This is because rhizosphere-enhanced bioremediation may improve contaminant bioavailability as a result of root exploration and the release of biosurfactants. However, wider application of this technology is hampered by the scarcity of information for predicting success across a broad range of soil–contaminant–climate settings and the lack of a suitable means to monitor remediation progress.

Disturbed lands and urban landscapes

When lands were converted from forest to agriculture decades ago, these lands experienced substantial losses of organic matter and nutrients. The effect on microbial taxonomic diversity was difficult to measure. This is because of fundamental problem in microbiology: the only way to measure taxonomic diversity in the soil was to perform serial dilutions of the soil onto defined agar media. Unfortunately, this method was only capable of isolating a few percent (typically, 1 to 10%) of the total microbial population. For example, it is estimated that currently only 10 and 5% of an estimated 30,000 species of bacteria and 1,500,000 species of fungi are known, respectively (Coleman et al., 1994). However, with the advent of a variety of biochemical and molecular methods, it is now easy to measure not only the taxonomic diversity, but also the functional diversity within soil microbial communities. These methods have the potential to offer much information about the effects of land use and management on soil microbial taxonomic and functional diversity.

Two types of land are of special interest. The first type of land is disturbed land as a result of road construction, industrial and residential development, and surface mining for energy and minerals. The second type of land is urban landscapes that are intensively managed (e.g., golf courses). These lands are of special interest because relatively little research has been conducted on them despite their importance.

Extent of the problem

Biogeographic aspects of land-applied wastes

To date, the use of molecular genetic methods to study biogeography looks promising because some of the methods can discriminate among subspecies of a bacterium. For reasons already identified, we selected E. coli as the test bacterium and ribotyping as the best molecular method to study biogeography of a soil microorganism.

The first part of our objective is to compare E. coli ribotypes from various locations in the United States with the same animal species and determine their geographic variability (e.g., are E. coli ribotypes from cattle different between Georgia and Idaho?). The second part of our objective is to determine their temporal variability (i.e., are the ribotypes of E. coli that were in cattle on June 2000 and June 2001 the same or different?). Although specific strains of E. coli are indeed associated with different host species (Faith et al., 1996), the degree to which some strains are resident and some strains are transient is unknown. A regional research project among several states is ideally suited to test this geographic and temporal variability. Because this work has important public heath implications, we will also create a publicly accessible host source origin library of E. coli ribotypes.

Rhizosphere-enhanced bioremediation of organic contaminants

Under ideal circumstances, sampling and chemical analysis of organic contaminants in the soil is done before and during the treatment process. In this way, contaminant concentrations are monitored and the treatment process continued until remediation goals are met. However, at some contaminated soil sites, this is difficult because the site is in a remote location or the distribution of contaminants is too heterogeneous. Cost may also be an issue. Under these conditions, rhizosphere-enhanced bioremediation may be an attractive option because it requires less material handling, less equipment, fewer on-site operators, and less power consumption than alternatives such as bioreactors, incineration, landfarming, and excavation and removal. Under these conditions, the more remote the site, the greater the cost savings.

Nevertheless, it is important to note that rhizosphere-based treatments often take longer than traditional remediation treatments, and achievable endpoints are often uncertain. How to minimize the time and achieve endpoints with certainty is the focus of this multistate proposal. In the case of minimizing time, more knowledge is needed on the influence of organic contaminants on soil microbial populations and activity, and plant root morphology and development. In the case of endpoints, one way to evaluate this may be to measure soil microbial taxonomic diversity. Under these conditions, these measurements may be a practical monitoring tool.

Disturbed lands and urban landscapes

The objective here is to characterize taxonomic and functional diversity of bacteria and mycorrhizae in disturbed lands and urban landscapes. In the case of disturbed lands, it is important to develop an understanding of how these lands recover. On the one hand, if disturbances have lasting negative effects on the ability of these lands to function ecologically, then it is important to know exactly what is being adversely affected. For example, considerable evidence shows that endomycorrhizal associations in disturbed lands are important because, as beneficial obligate symbionts of roots, mycorrhizal fungi stimulate plant growth and yield through increased nutrient uptake (e.g., P), increase plant resistance to drought and salinity, and increased plant tolerance to root pathogens (Sylvia, 1998). Therefore, if mycorrhizae are being adversely affected, then new strategies must be developed to minimize these effects. On the other hand, if these disturbances have minimal effects, then perhaps current costly regulatory policies can be ameliorated.

A similar case can be made for urban landscapes. It is important to know how these landscapes contribute to soil microbial taxonomic and functional diversity. For example, it is important to know what happens to mycorrhizae under conditions of high fertility, a condition commonly found in homeowner lawns.

Need for and advantages in a cooperative approach

Because of the complexity of studying microbial taxonomic and functional diversity in the soil and rhizosphere, collaboration among scientists with expertise in a variety of aspects of soil microbiology is required. In one sorting, some scientists in the multistate proposal are experts in mycorrhizae, while others specialize in bacteria. In another sorting, some scientists are experts in ribotyping, while others are experts in individual and whole soil FAME analyses. Thus, collaboration among many scientists ensures a diverse array of skills necessary for the complex experiments posed in this multistate proposal.

Similarly, a cooperative approach is also required for equipment. Not all investigators working on a particular objective have all the highly specialized and expensive laboratory equipment needed to conduct the proposed research. Therefore, certain parts of the research are best done at specific locations. For example, isolation of E. coli from the feces of various animals can be done at all the individual state locations, but the ribotyping of these isolates will be done at the University of Georgia which has the necessary gel electrophoresis apparatus, membrane blotting equipment, UV crosslinker, hybridization oven, and imaging system. By working cooperatively, it will possible to conduct research that could only be done with difficulty if the scientists were working individually.

Researchers on the proposed multistate project have had many years of experience in soil microbiology and are highly qualified to conduct the proposed research. As noted in the Critical Review, many of the researchers in this project have collaborated on Regional Projects S-112, S‑170, S‑226, and S-262, and their record of productivity and collaboration is excellent. Of 20 cooperators identified in this multistate proposal, 16 are members of Regional Research Project S-262. Between October 1995 and the present day, researchers in S-262 have generated 3 books, 36 book chapters, 155 journal articles (142 published or in press; 13 submitted), 147 published abstracts, 1 experiment station bulletin, 1 patent (pending), 11 theses or dissertations, and 12 non-refereed publications. Two of the three books and 16 of the journal articles involve collaboration between two or more states. Such productivity and collaboration is critical to the success of the proposed multistate project.

Another need for cooperative work is the need to develop the second edition of our soil microbiology textbook as well as an accompanying lab manual. Although publications cannot be listed objectives, such a publication is important and tangible evidence of the expertise, willingness, and history of cooperation among the participating scientists.

Expected benefit to result when the problem is solved

Biogeographic aspects of land-applied wastes

The studies will assess the geographic and temporal variability of the E. coli ribotypes. Such studies would be useful for understanding the biogeography of bacteria. The proposed studies will also provide the first national database of E. coli ribotypes. By making this database publicly accessible on the world-wide web, these data would be useful to state and federal agencies trying to determine the host origin of nonpoint pollution sources. Such information would be useful in allocating resources to reduce this pollution. For example, if it was determined that fecal coliform contamination was coming from humans and not agricultural animals in a specific instance (a river with high fecal coliform counts), then state and federal agencies could direct resources to look for possible leaking septic tanks and not agricultural runoff.

Rhizosphere-enhanced biotreatment of organic contaminants

The proposed studies will further characterize the microbial taxonomic diversity of rhizosphere microorganisms involved in the bioremediation of contaminated soils. This characterization will improve our ability to use plant–microbial systems to give optimal in situ decomposition at contaminated sites with minimum cost and management. An improved understanding of the time-dependent changes between contaminant concentration and microbial community may be useful for monitoring rhizosphere-enhanced remediation, and provide a biological approach to indicate when bioavailable contaminants are diminished. If this approach is applicable, it should greatly reduce expenses for cleanup of contaminated soils at a number of sites.

Disturbed lands and urban landscapes

With regards to disturbed lands, the research will increase our understanding of the effects of land disturbance on the taxonomic and functional diversity of bacteria and mycorrhizae. This may affect the ability of microbial communities to sustain ecosystem processes (e.g., nutrient cycling). Research will provide a basis for achieving better plant–microbial systems to stabilize disturbed soils and thus reduce erosion and subsequent pollution of surface water. With regards to urban landscapes, the proposed research will provide not only basic information on soil microbial taxonomic and functional diversity, but also how management inputs affect belowground ecological processes and offsite environments such as streams and groundwater.

RELATED CURRENT AND PREVIOUS RESEARCH

Current regional/national priorities

Internationally, microbial diversity is a focus of International Convention on Biological Diversity. All signatory nations have formed special programs to address various aspects of microbial diversity. For example, Canada will conduct a program entitled "Biodiversity in Canadian Agricultural Soils." This is important to the multistate proposal because the Regional Research Project S-262 has links with Canada on this topic and this link will continue in the future. With regards to the United States, soil biodiversity is a component of the USDA NRICGP programs in Ecosystem Science, Soils, and Soil Biology, and a Special Research Program in Global Change, Biodiversity, and Ecosystem Research (PL 89-106). Microbial diversity research is part of Goal 4 of the CSREES "to develop, transfer, and promote....practices that ensure ecosystems achieve a sustainable balance of agricultural activities and biodiversity." With regards to Southern Region, the 1996 SAAESD Strategic Plan established priorities in "Sustainable agriculture" (Priority #3), "Biodiversity" (Priority #10), and "Mitigating environmental problems with intensive animal agriculture operations" (Priority #1). Our proposal will respond directly to Priority #3 and #10, and indirectly to Priority #1.

USDA–CRIS search

A search of the USDA CRIS database shows only one match among the words "microbial taxonomy," "microbial diversity," "taxonomic diversity," and "functional diversity" and any regional research project. The sole match is for the words "taxonomic diversity" and Regional Research Project W-187. This project is analyzing the taxonomic diversity of fungal pathogens and symbiotic fungi associated with insects on North American conifers. This is unrelated to the research in this proposal. There are there no matches for any regional project and the more general terms of "biodiversity" or any combination of the words "microbial community structure," however there are six matches for the word "diversity." Of the six matches, one relates to Regional Research Project S-262 (which is the predecessor to this proposal), one relates to Regional Research Project W-187 (see above), and the remaining four relate to plant germplasm research.

Objectives #2 and #3 in this proposal have a strong connection to the keyword "rhizosphere," but there are no matches to this keyword other than Regional Research Project S-262. (There was a Southern Region Information Exchange Group 25, entitled "The Plant Root Environment," but this SRIEG disbanded in 1998.) There is an indirect match to "rhizosphere" research with Regional Research Project W-147 (Managing Plant–Microbe Interactions in Soil to Promote Sustainable Agriculture), but this regional research project focuses on plant pathogens. On an even more general basis, there are 31 matches to the keyword "soil," but only Regional Research Project S-269 (Biological Control and Management of Soilborne Plant Pathogens for Sustainable Crop Production) bears some semblance to the proposed multistate project. However, its focus is on plant pathogens, not the entire spectrum of soil microorganisms.

With regards to Objective #1 (ribotyping), there were no matches for the keywords "Escherichia coli," "DNA fingerprinting," "fecal coliform," or "ribotyping" for any regional research project. With regards to Objective #2, there are five matches for the word "organic," of which only Regional Research Project W-82 (Pesticides and Other Toxic Organics in Soil and Their Potential for Ground and Surface Water Contamination) applies, but this regional project does not involve plants, a major focus of the new multistate project. With regards to Objective #3, there were two and one match(es) for the keywords "turfgrass" and "disturbed lands," respectively. However, the matches for "turfgrass" were not applicable to the research in this proposal, and the match to "disturbed lands" is for an information exchange group (SRIEG-25) and does not involve plants. Therefore, the proposal has a good relationship to international, national, and regional priorities, and involves research that is not being done by a multistate project elsewhere.

Impact on Science

The proposed research will integrate field and laboratory studies to develop a better understanding of the basic processes affecting microbial taxonomic and functional diversity in soil and rhizosphere. The results will have significant implications for the biogeography of bacteria, the use of plants to reduce or eliminate toxic organic compounds, and the effects of land use on microbial diversity.

Other impacts

A healthy ecosystem is one that is capable of constraining its own fluctuations within certain bounds and maintaining constancy in its species composition and productivity (Tilman, 1996). Ecosystems experiencing stress are characterized by changes in species diversity, nutrient cycling, and productivity (Naeem et al., 1994). Because of the universal presence of microorganisms in all ecosystems and because microorganisms have a much shorter lifespan than most other organisms, we may be able to use microbial taxonomic and functional diversity as an early warning of ecosystem stress.

Literature review

Biogeographic aspects of land-applied wastes

Ribotyping is in its infancy, and there are only eight published papers, only two of which are published in the refereed scientific literature.

The first reports of ribotyping were by Samadpour and Chechowitz (1995) and Simmons et al. (1995). In the case of Samadpour and Chechowitz (1995), 421 of 589 E. coli isolates (71%) from Little Soos Creek (in Washington State) were matched to cow, deer, dog, duck, fish, horse, humans, llama, swine, and poultry. The primary contributors of E. coli to the creek were cows and dogs. In the case of Simmons et al. (1995), they attempted to determine the source of fecal coliforms that were forcing closure of oyster beds in the Chesapeake Bay. Fecal samples were collected from raccoon, waterfowl, otter, muskrat, deer, and humans in the area. The host origin of the E. coli isolates in the oyster beds was matched to raccoons and deer. When these animals were removed by either hunting or trapping, numbers of E. coli declined by up to two orders of magnitude, and this permitted the oyster beds to reopen.

Three subsequent reports were published by the Samadpour team and one report by the Simmons team. Farag and Goldstein (1998) isolated fecal coliforms in Grand Teton National Park (Wyoming) and sent the E. coli isolates to Samadpour for ribotyping. The isolates from Cascade Creek in the park matched source ribotyping patterns of humans, birds, deer, dogs, and elk. This study wasfollowed by a more extensive report the next year involving other locations in the same park (Tippets, 1999). Of 104 E. coli isolates, ribotyping was able to identify the host origin of 69 (66%). A third report by Berghoff (1998) involved the Glen Canyon National Recreational Area, Utah. Again, the E. coli isolates were sent to Samadpour for ribotyping. Ribotyping was able to identify 47 of 248 E. coli isolates (19%). In the fourth report, Simmons and Herbein (1998) initiated studies to determine the source of fecal coliform contamination at a beach in San Diego, CA. Of 83 E. coli isolates, 72 (87%) matched E. coli isolates from harbor seals with a similarity of 80% or better.

In the meantime, two new teams, one at the University of Florida and the University of Georgia began ribotyping. This has resulted in two refereed publications. Parveen et al. (1999) ribotyped a total of 238 E. coli isolates from human and nonhuman sources. The isolates were collected from the Apalachicola National Estuarine Research Reserve (Florida), nearby sewage treatment plants, and directly from animals. The human isolates had 41 different ribotype profiles; the nonhuman isolates had 64 different profiles. When the ribotype profiles were clustered, discriminant analysis showed that 100% of the human profiles and 97% of the nonhuman profiles were correctly classified.

Hartel et al. (1999) used a Riboprinter, an instrument that automates ribotyping, to ribotype E. coli isolates from two small streams in Georgia. Although the RiboPrinter uses only the 16S portion of rRNA as a probe and one restriction enzyme at a time (thereby yielding fewer bands), it automates ribotyping to such an extent that as many as 32 isolates can be processed in one day. The RiboPrinter was able to discriminate among ribotypes of E. coli from a pasture stream, a wooded stream, and cow manure, but the discrimination was insufficient within a site. For this reason, we went to the more discriminatory (but more time-consuming) method involving 5S, 16S, and 23S portions of E. coli rRNA and two restriction enzymes, EcoRI and PvuII. This is the procedure in this proposal.

Rhizosphere-enhanced biotreatment of organic contaminants

The size and activity of microbial populations in the rhizosphere are influenced by the plant species, physical and chemical soil characteristics, and the microbial community (Curl and Truelove, 1985). However, qualitative changes in the rhizosphere microbial population may be more important than the quantitative changes (Macnaughton et al., 1999). Depending upon the plant species, stage of growth, root morphology, and environmental conditions, enhanced biochemical activity of specific soil microorganisms can be favored. By understanding the basic processes that come from the interaction of the plant and the contaminant-degrader microbial populations, enhanced bioremediation strategies may be developed. Such information is particularly important in working with microbial consortia.

There is convincing evidence from both laboratory and field studies that phytoremediation is effective in treating contaminated soils. Although the majority of these studies were conducted in temperate climates (Anderson et al., 1993; Aprill and Sims, 1990; Cunningham and Ow, 1996; Cunningham et al., 1996; Reilley et al., 1996, Schwab et al., 1995; Wiltse et al., 1998), some were conducted in a subarctic climate (e.g., Reynolds et al., 1999). From these studies the main mechanisms for phytoremediation appear to be largely contaminant dependent. For many organic contaminants, especially petroleum compounds, the generally accepted phytoremediation mechanism is enhanced microbial activity in the rhizosphere, which in turn accelerates the rate of degradation of contaminants. Plant-produced compounds may serve as cometabolites for more recalcitrant compounds, and this may result in lower contaminant concentration endpoints than can be obtained without plants (Fletcher et al., 1995).

Understanding the interactions and sequence of processes that govern phytoremediation is complex, but has important practical significance. For example, in ongoing field studies in the Republic of Korea, data show significant reduction of total petroleum hydrocarbons (an integrated measure of petroleum contamination) for vegetated, fertilized, vegetated and fertilized, and control treatments (Reynolds and Koenen, 2000). If total petroleum hydrocarbons is the criterion, then there are no significant differences among treatments. However, if PAHs are the criterion, then a different pattern was observed. In this case, the fertilized and seeded treatment was clearly more effective in reducing concentrations of higher molecular weight PAHs than the other treatments. Moreover, relative to the three other treatments, the fertilized treatment inhibited reduction of higher molecular weight PAHs. These data show that fertilizing and seeding provided more complete degradation of the PAHs fraction of TPH than did fertilizer alone, seeding alone, or the control. Therefore, as cleanup requirements become better defined, rhizosphere-enhanced bioremediation has a greater likelihood of meeting future cleanup guidelines for individual PAH compounds.

Disturbed lands and urban landscapes

In terms of disturbed lands, considerable research has already been conducted, chiefly on lands disturbed by surface mining for mineral and energy resources (e.g., bituminous and lignite coals; Mott and Zuberer, 1991; Tate and Klein, 1985; Cundell, 1977). Such studies show that soil microbial populations reestablish themselves following mining and reclamation activities. The chief variable affecting this reestablishment is revegetation. Thus, the plant provides the carbon inputs necessary for the growth of heterotrophic microbial populations like bacteria, actinomycetes, fungi, and protozoa. These populations form the foundation of the microbial foodwebs needed for ecological restoration of disturbed lands to functional levels exceeding or meeting levels of unmined soils (Kennedy and Smith, 1995; Kennedy, 1999).

Another variable affecting the soil microbial community is the separation and storage of topsoil during mining operations. Some states in the United States require the topsoil be replaced and revegetated with locally adapted plant species, whereas other states permit mining operations to use mixed overburden. There is some evidence that these mixed materials display superior physical and chemical properties compared to unmined, native soil (Dixon et al., 1980). When a spoil area was revegetated with coastal bermudagrass, numbers of bacteria, actinomycetes, fungi, algae, cyanobacteria, and free-living N2-fixing bacteria reached numbers associated with an unmined reference area within 1.5 to 2 years (Mott and Zuberer, 1991). Levels of root infection with arbuscular mycorrhizal fungi also returned to values approaching those of roots of bermudagrass from unmined sites within one to two years (Mott and Zuberer, 1987). In further studies, populations of clover rhizobia easily reestablished themselves in mined soils and they persisted several years following inoculation (Harris and Zuberer, 1993). Similar results were observed with nitrifying bacteria when ammonium sulfate or urea was applied to revegetated, mined lands (Waggoner and Zuberer, 1996). Urease activity was also restored quickly, which is consistent with the rapid restoration of the general heterotrophic population. While all of these and other studies suggest that microbial populations recover fairly quickly, even in mixed overburden materials, they do not address the issues of taxonomic and functional microbial diversity in the reclamation process. This work remains to be done.

In terms of urban landscapes, our understanding of the microbial ecology of intensively managed landscapes is poor. These landscapes constitute the major vegetation in urban and suburban areas where they no doubt have important ecological roles (e.g., water filtration). For example, golf course putting greens were assumed to be relatively "sterile" environments, but substantial numbers of bacteria, actinomycetes and fungi are found in sand-based putting greens (Elliott and Des Jardin, 1999). Kim et al. (1999) found similar results for bacteria in South Carolina putting greens. These studies suggest a rich microbial population associated with the roots even in such artificial sand-based environments.

Of particular interest in the multistate project is the effect of high P fertility on mycorrhizae in urban landscapes. This is because arbuscular mycorrhizae are a major determinant of plant growth response in grasslands. Several studies report the presence of arbuscular mycorrhizae in selected turfgrass species (Koske et al., 1997a, Koske et al., 1997b), and arbuscular mycorrhizae enhance establishment (Gemma et al., 1997b) and drought tolerance (Gemma et al., 1997a) of bentgrass. However, little is known about what, if any, contribution arbuscular mycorrhizae make to turfgrasses in highly fertilized systems. Effective arbuscular mycorrhizae are those which produce the greatest benefit (expressed in terms of increased P acquisition) for the least cost (expressed as C expenditure on mycorrhizas; Graham and Eissenstat, 1994). Although controversial, some researchers have suggested that certain decline diseases of plants may be related to a shift from beneficial to non-beneficial or even parasitic fungi in intensively managed grasslands because of high fertility conditions (Johnson et al., 1992; Johnson, 1993; Schenck and Siqueira, 1987). Glasshouse studies predict that arbuscular mycorrhizae which aggressively colonize roots and stimulate plant growth at low P supply (Abbott and Robson, 1981) will also aggressively colonize at high P supply but may provide no additional P benefit and reduce growth (Graham et al., 1996). Given the pervasive over-application of P to turfgrasses, decreasing input of P fertilizer is most often cited as the approach to increase symbiotic effectiveness. However, such changes in fertilizer management must be based on knowledge of the functional attributes of arbuscular mycorrhizal species and populations. These attributes are: a) the rate and development of colonization of roots (Abbott and Robson, 1981), b) the proliferation of external hyphae in relation to P acquisition (Jakobsen et al., 1992), and c) the carbon cost to support growth and maintenance of the mycorrhizal root (Peng et al., 1993).

OBJECTIVES:

1) to determine the geographic and temporal variability of E. coli ribotypes in the United States;

2) to determine relationships among microbial taxonomic and functional diversity, contaminant bioavailability, and remediation rates for different organic-contaminated soils.

3) to characterize taxonomic and functional diversity of bacteria and mycorrhizae in disturbed lands and urban landscapes.



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