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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.

PROCEDURES:

With the exception of Objective #1 (Biogeographic aspects of land-applied wastes), we have extensive experience with all the soil and rhizosphere methods proposed in the Methods section for Objectives #2 and #3 (e.g., BIOLOG: Oka et al., 2000; glomalin production: Wright and Upadhyaya, 1997; mycorrhizae: Douds and Millner, 1999; organic carbon: Franzluebbers et al., 1999; phytoremediation: Olexa et al., 2000; whole soil FAME: Cattelan et al., 1999). For this reason, the procedures for Objective #1 are given in detail, whereas the procedures for Objectives #2 and #3 are given only generally. Similarly, the experiments outlined for Objective #1 are given for the entire 5-year lifespan of the multistate proposal, whereas the experiments for Objectives #2 and #3 are given in groups.

Biogeographic aspects of land-applied wastes

A. Specific experiments and collaborations

Two experiments are proposed. In the first experiment, 500 isolates of E. coli will be obtained from humans and a variety of agricultural and wild animals common to the participating states (AL, DE, GA, PR, TN, TX, WI, USDA-GA, USDA-ID). The number of isolates obtained will be: humans (100), cattle (50), deer (50), dog (50), duck or goose (50), poultry (50), sheep (50), and swine (50). The remaining 50 isolates will be allocated to an animal species that each investigator believes to be important in that location (e.g., elk in Idaho). To maximize ribotype diversity, each human or animal will contribute no more than 5 isolates. For example, 12 different cattle/dairy operations will be visited in order to obtain the necessary 60 cattle E. coli isolates. This experiment will not only form the basis of a national library of E. coli ribotypes, but also will provide information on the biogeographic distribution of the ribotypes. Because of the cost, isolates will initially be ribotyped at one location (GA). As extramural funding becomes available, isolates will also be ribotyped at two other locations (USDA-GA, USDA-ID).

In the second experiment, the temporal variability of E. coli ribotypes in three different agricultural animal species, cattle, swine, and poultry will be tracked. Specific animals of each species will be identified and at least 150 isolates of E. coli will be isolated from at least 5 animals (30 isolates per animal) periodically, depending on the normal agricultural lifespan of on the animal. In the case of cattle, steers will be sampled every two months for one year, after which the cattle will be replaced with another batch of yearling calves. In the case of swine, animals will sampled biweekly for 12 weeks, and in the case of poultry, broilers will be sampled biweekly for 6 weeks. In this experiment, only the locations actively ribotyping isolates will participate in the experiment (DE, GA, USDA-GA, USDA-ID).

B. Methods

Fresh fecal samples will be obtained as aseptically as possible. With the exception of humans, fresh fecal samples will be collected with an ethanol flame-sterilized spatula and will be transferred to sterile Whirl-Pak bags. Collection information will consist of the animal species, person collecting, and the date and place collected. The bags will be kept on ice until the samples are processed. Because of the restrictions on isolating bacteria directly from humans, human isolates of E. coli will be collected from septic tanks. These septic tanks will be from homes without any outside sources of E. coli (e.g., dogs). Septic tanks will be sampled with sterile dilution bottles and will be treated in the same manner as the fecal samples.

To isolate E. coli from fecal samples, a 20-mL sample of 0.1% peptone will be added to the Whirl-Pak bag containing the feces, and the feces will be blended for 30 seconds with a Stomacher blender. The septic tanks samples will be shaken by hand for 1 minute. A 10-µL sample from the Whirlpak bag or dilution bottle will be streaked onto mTEC agar plates. The plates will be wrapped in quadruple Ziploc bags and will be incubated submerged in a water bath at 44.5± 0.2°C for 24 h. All streakings will be done in duplicate. Yellow colonies growing on mTEC agar (Difco) after a 24-h incubation will be considered as presumptive E. coli.

Presumptive isolates of E. coli will be transferred onto tryptic soy agar (TSA) and will be incubated overnight at 35°C. This step will be repeated to ensure pure cultures. After two streakings, each isolate will be streaked a third time onto TSA as well as on urea agar and Simmons citrate agar. In addition to the E. coli isolates, type cultures from the American Type Culture Collection will be used as appropriate controls. These organisms represent almost all the bacteria that can be found on mTEC agar plates. The type cultures include Escherichia coli #11775 (urease, citrate), Klebsiella pneumoniae subspecies pneumoniae #13883 (urease+, citrate+), Citrobacter freundii #8090 (urease+, citrate+), and Enterobacter aerogenes #13048 (urease, citrate+). After overnight incubation at 35°C, presumptive E. coli isolates that can grow on TSA but are urease and citrate will be subjected to an oxidase test. If the isolate is also oxidase, then the isolate will be considered as confirmed E. coli and will be frozen. All other isolates will be autoclaved and discarded. To freeze each E. coli isolate, a loopful of each isolate will be transferred from the third streaking of the TSA plate into two labeled cryovials, each containing a 3.5:1 mixture of saline/phosphate buffer and cryoprotectant. The two cryovials, one representing the working stock and the other the reserve stock, will be kept in separate -80°C freezers.

Confirmed isolates of E. coli will be streaked from the -80°C frozen stocks onto TSA and incubated overnight at 35°C. A single clone will be inoculated into 10 mL of Luria-Bertani broth (Maniatis et al., 1982) and incubated on a rotary shaker at 100 rpm overnight at 35°C. The DNA from a turbid, 1-mL sample will be obtained with a commercial kit (Qiagen DNeasy tissue kit). The DNA will be quantified with a UV spectrophotometer at 260 nm (DNA) and 280 nm (protein). Samples with an acceptable 260:280 ratio (i.e., >1.75) will be used for ribotyping.

A digoxigenin (DIG)-labeled probe will be prepared to yield a reverse transcribed, DIG-labeled probe of E. coli 5S, 16S, and 23S rRNA. The DIG label will be quantified against a kit standard (Roche). To perform a restriction digest of the genomic DNA, a sample of DNA will be added to each of two microfuge tubes and each brought to a specific volume with distilled water. A sample of EcoRI or PvuII with the appropriate restriction enzyme buffer will be added and the mixture will be incubated at 37˚C.

After overnight incubation, loading dye will be added to each tube of restricted DNA. A portion of the DNA will be added to each well of a 0.7% agarose gel. Additional wells will be set aside for DNA ladders of -DNA cleaved with EcoRI and HindIII (molecular weight marker), no DNA control, and DNA from type culture E. coli ATCC #11775. The gel will be submerged in 1X Tris acetate EDTA buffer and electrophoresed at 55 volts for approximately 3 h.

Once the DNA separation is complete, the gel will be placed on a Nytran nylon membrane contained in a vacuum blotting assembly (VacuGene blotting assembly, ). The gel will be sequentially washed with denaturing solution, neutralizing buffer, and 20X transfer buffer. After transfer, the gel will be discarded and the membrane washed in 2X transfer buffer before the DNA on the membrane is fixed with UVirradiation.

The membrane will be hybridized with preheated DIG-labeled probe overnight at 42°C. The membrane will be washed in a series of stringency washes before equilibrating in washing buffer for 1 min. The membrane will be incubated in blocking solution for 60 minutes at room temperature. The blocking solution will be discarded and a new batch of blocking solution containing anti-DIG-alkaline phosphatase will be added. After 30 minutes incubation at room temperature, the membrane will be washed twice for 15 min, and detection buffer will be added for 2 min. The membrane will be removed from detection buffer and will be treated with a chemiluminescent substrate. The chemiluminescence will be quantified with an Alpha Innotech FluorChem 8000 imager and the image saved as a TIFF file.

All banding patterns contained in the image files will be sent to one location (GA) to be quantified with gel analysis software (GelCompar II). The host origin, temporal, and geographic relationships among the isolates will be examined by cluster analysis, and cluster dendrograms will be plotted with the same gel analysis software. Ribotype patterns with respect to geography, time, and host origin will be made publicly available on the multistate project website, <http://eclass.ifas.ufl.edu/dmsa/msp>.

Rhizosphere-enhanced biotreatment of organic contaminants

A. Specific experiments and collaborations

Three groups of experiments are proposed. In the first group of experiments, collaborative lightroom studies with comparable protocols will determine the effect of interactions associated with different plant species, soils, and nutrients on remediation of several unlabelled organic compounds (e.g., PAHs) in soil and rhizosphere. The experiments will incorporate a) a model system of specific organic contaminants, b) whole soil FAME or PLFA (phospholipid fatty acid) and DNA-based techniques to describe changes in microbial community structure, c) BIOLOG plates to assess functional diversity, d) staining techniques to evaluate mycorrhizal colonization, and e) gas chromatography (GC) to determine the concentrations of the remaining parent compound. In this manner, it may be possible to predict the general conditions under which the rhizosphere will stimulate biodegradation of an organic contaminant (AL, AR, DE, FL, NC, SC, WI, Other Coop.-NH, Other Coop.-FL, Other-Coop.-Canada).

In the second group of laboratory experiments, collaborative lightroom studies with comparable protocols will be conducted with labeled organic compounds. These compounds will be selected on the basis of their different degradative patterns from the set of organic compounds in the first group of experiments. The second group of experiments will determine the effect of rhizosphere processes on contaminant bioavailability, with a specific emphasis on plant- or microbially produced biosurfactants. By following the fate of 14C-labelled organic compounds, the degree to which toxic organics may be degraded to intermediates covalently bound to the humic soil fraction (i.e., not to CO2 and H2O) will be studied. The experiments will be conducted with and without plants to assess the role of plants in bioavailability (AL, AR, DE, FL, NC, SC, WI, Other Coop.-NH, Other Coop.-FL, Other-Coop.-Canada).

In the third group of experiments, the results of the second group of experiments will be conducted under field conditions with unlabelled compounds to determine the rhizosphere effects on remediation and bioavailability of organic contaminants in aged and newly contaminated soils (AL, AR, DE, FL, NC, SC, WI, Other Coop.-NH, Other Coop.-FL, Other-Coop.-Canada).

B. Methods

Soils will be amended with toxic organic compounds. Plant species, including grasses and legumes, will be evaluated for germination percentage, growth rate, and root development in these soils under greenhouse, growth chamber, or lightroom conditions. Following plant selection, cooperative greenhouse studies will determine bioremediation in the rhizosphere. The soil will be amended with appropriate amounts of inorganic N, P, and K as indicated by soil test analysis. At each sampling time, pots will be harvested and the quantity of contaminant and degradation products in the plant top and roots and remaining in the rhizosphere and non-rhizosphere soil will be determined. Plant dry matter production and rooting characteristics will be quantified (Bohm, 1979). The microbial population (i.e., bacteria, fungi, degraders) in the rhizosphere and non-rhizosphere (i.e., R/S ratio), will be evaluated at selected sample times. At each sampling time, isolates will be cultured or microbial community structure will be characterized or both.

The soils will be characterized for physical, chemical, and biological properties by standard methods. The physical properties will be texture and water content at -0.03 MPa, the chemical properties will be pH, NH4-N, NO3-N, total N, % organic C, extractable P, K, Ca, Mg, Na, electrical conductivity, and chloride, and the biological properties will be total viable bacteria and fungi.

Other biological properties will be individual and whole soil FAME analyses. Phospholipid fatty acid (PLFA) analyses will be used in instances when the organic contaminant interferes with the whole soil FAME analyses. Ecotypic variations among arbuscular mycorrhizae will be evaluated with a nested PCR approach (Kjøller and Rosendahl, 2000).

Contaminant concentrations will be determined with GC and gas chromatography–mass spectroscopy (GC–MS). Soils will be initially extracted with an appropriate organic solvent using modified EPA methods.

Disturbed lands and urban landscapes

A. Specific experiments and collaborations

Two groups of experiments will be conducted. In the first group of experiments, collaborative studies with comparable protocols will characterize taxonomic and functional diversity of bacteria and mycorrhizae in disturbed soils (e.g., road construction). Lands will be selected with a range of ages and varying amounts of disturbance and revegetation (e.g., coastal bermudagrass to trees) and will be compared to undisturbed sites (DE, MD, PR, TN, TX, WV, USDA-GA, USDA-ID).

In the second group of experiments, collaborative studies with comparable protocols will characterize taxonomic and functional diversity of bacteria and mycorrhizae in urban landscapes (e.g., intensively managed turfgrass systems like golf courses). Experiments will include different grass species. For example, in the case of putting greens, samples from the sand-based root zones will be analyzed for bacterial numbers, BIOLOG carbon-source utilization profiles, whole-soil FAME profiles, water-extractable organic carbon, mycorrhizal infection and possibly quantification of glomalin. If sufficient arbuscular mycorrhizal spores are collected, then they will be submitted to the University of West Virginia for classification. Soils will be sampled periodically during the year to capture seasonal effects on community structure (AL, DE, FL, NC, NE, SC, TX, WV).

B. Methods

Soils from the collaborators will be analyzed for functional diversity (i.e., community level physiological profiles or CLPP) using the commercially available BIOLOG system. This system has been widely adapted for these studies. Soil samples will be collected fresh from the field and refrigerated until analyzed by the BIOLOG system. Ideally, soils will be analyzed immediately upon return to the lab. Appropriate dilutions (i.e., 10-3) will be inoculated into BIOLOG GN plates. The plates will be incubated for 24 to 72 h during which time they will be analyzed with a plate reader to record well-color development. Data from the BIOLOG plates will be analyzed by principal component analysis and other methods to determine the CLPP of the microbial populations.

Taxonomic diversity of the soil microbial communities will be assessed with whole soil FAME profiles. Selected fatty acids will be used as biomarkers for specific groups of microorganisms. The FAME profiles will be analyzed with the software program CANOCO to determine the similarities in microbial communities.

Chemical and physical analysis of the soils will be conducted at Texas A&M University. Microbiological analyses will include enumeration by standard plate count methods, analysis of soil microbial biomass with chloroform-fumigation extraction methods (Brookes et al., 1985), and analysis of microbial communities using the BIOLOG and FAME analyses described above.

Soil samples will be collected, dried in air, and stored at –80oC until they can be extracted for FAME analyses. Extraction will be done under standard protocols developed in our previous regional project research (Franzluebbers et al., 1999) with the exception that prior to the last step of resuspending the extracts in the ether solvent, the GC vials will be sealed and capped under a stream of nitrogen. This eliminates the hazards of mailing flammable solvents. We have shown in earlier research that this method does not adversely affect our ability to determine FAME profiles (Peach et al., 1999). The extracts will be sent to University of Delaware for analysis with the MIDI system (MIDI, Newark, DE).

With regards to mycorrhizae, standard methods (e.g., Sylvia, 1994) will be used to quantify propagules, estimate root colonization, and detect active hyphal lengths of hyphae in soil. Root samples will be collected from vegetation (primarily coastal bermudagrass) at the sampling sites and will be sent to the University of Florida for analysis. The effect of arbuscular mycorrhizae on interplant competition and plant aggressivity will be determined with replacement and additive designs within mesocosm field plots (Snaydon, 1991). Glomalin assays will be conducted to separate the activity of mycorrhizal hyphae from other root-inhabiting fungi. As a specific and persistent glycoprotein (Wright and Upadhyaya, 1997), glomalin will be quantified with an immunofluorescence assay. Detailed methods are available online at <da.gov/nri/smsl/glomalin.htm>.

EXPECTED OUTCOMES:

Biogeographic aspects of land-applied wastes

The proposed research will:

  • identify the degree of geographic variability of E. coli ribotypes among humans, cattle, deer, dog, Canada goose, poultry (broilers), domestic sheep, and swine.

  • identify the degree of temporal variability of E. coli ribotypes in cattle, swine, and poultry (broilers).

  • generate a publicly accessible database of ribotypes of E. coli on the worldwide web for use by resource managers.

Rhizosphere-enhanced biotreatment of organic contaminants

The proposed research will:

  • provide a database of biodegradative strains consisting of organic contaminant degraders isolated from contaminated soils from a diversity of sites across the United States. Such a database will be publicly available on our website and will be similar to the Canadian database at <ak.ca.departments/scsr/department/research/index/html>.

  • create a culture collection of organic contaminant degraders available to researchers upon request.

  • provide guidelines for calculating nutrient amendments for phytoremediating hydrocarbon-contaminated soils.

  • provide an improved knowledge of pollutant bioavailability.

Disturbed lands and urban landscapes

The proposed research will:

  • provide knowledge regarding the effects of disturbed lands and urban landscapes on soil microbial taxonomic and functional diversity.

  • enable land managers to adopt practices that conserve or protect the taxonomic and functional diversity of soil microbes.

  • produce knowledge needed by managers of intensively maintained agroecosystems to make science-based decisions on efficacy of alternative management inputs (e.g., microbial formulations).

ORGANIZATION

Hierarchy

A project chairperson and secretary will be elected at the first meeting of the technical committee. The secretary is automatically the project chairperson-elect for the next year. Therefore, only a new secretary is elected annually after the first year. All voting members of the technical committee are eligible for office, regardless of their affiliation or sponsoring agency. Each of the three multistate objectives will have a chairperson. In addition, there will be two chairpersons, one for the next edition of our textbook and one for the publication of an accompanying laboratory manual. (Because publications may not be listed as objectives, they are not listed in the body of this proposal.) These chairpersons have already been chosen for the multistate project and will be the chairperson for that objective for the entire 5-year-life of the multistate project. However, chairpersons for the objective may be replaced on a vote of the technical committee at the annual meeting. Finally, the technical committee will elect a host chairperson for the annual meeting the next year and designate a general site for the annual meeting.

Duties

The project chairperson, in consultation with the administrative advisor, will: a) notify all technical committee members of the time and place for the formal and informal annual meetings (see below), b) prepare a meeting agenda, c) preside at the meetings, and d) prepare an annual and other reports for the multistate project. The last chairperson of the 5-year project will also prepare the final termination report. The chairperson will distribute the annual report to the technical committee members. The chairperson will also consult with the administrative advisor in the conduct of his or her duties.

The chairpersons for each objective will: a) coordinate research within each objective, b) report on objective progress at the annual meeting, and c) prepare publication of results. Therefore, each chairperson is responsible for achieving the overall objective goal.

The secretary will: a) record the minutes of the annual meeting, and b) prepare and distribute copies of the minutes to all members. In addition, the secretary will perform any other duties as needed by the project chairperson.

The host chairperson will make all arrangements for the technical committee at the formal designated annual meeting site (e.g., lodging). The host chairperson is under the direction of the project chairperson.

Meetings

There will be two meetings per year, one formal meeting at a designated site and another informal meeting at the Soil Science Society of America annual meeting. The formal meeting will be primarily to discuss the three multistate objectives, whereas the Soil Science Society of America annual meeting will be primarily to discuss the two special educational objectives.

Website

The technical committee will maintain a website of the multistate project. Such a website will contain: a) the multistate project with all attachments (if approved), b) minutes of all meetings, c) annual reports, d) progress on the two objectives, and e) hyperlinks to all the technical committee members for further information. The URL for the website is <http://eclass.ifas.ufl.edu/dmsa/msp> and will be maintained by D. Sylvia at the University of Florida. The website is now open.

Organizational chart

ATTACHMENT A

PROJECT LEADERS

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

Principal Leader

State

Agency/Institution

Area of Specialization

Y. Feng

AL

Auburn University

Soil Microbiology

D. C. Wolf

AR

University of Arkansas

Soil Microbiology

J. J. Fuhrmann

DE

University of Delaware

Soil Microbiology

D. M. Sylvia

FL

University of Florida

Mycorrhizal Ecology

J. Graham

FL

University of Florida

Mycorrhizal Agroecology

K. Jayachandran

FL

Florida International University

Soil Microbiology

A. Franzluebbers

GA

USDA–ARS (Watkinsville)

Soil Microbiology

P. G. Hartel

GA

University of Georgia

Soil Microbiology

M. Jenkins

GA

USDA–ARS (Watkinsville)

Soil Microbiology

J. Entry

ID

USDA–ARS (Kimberly)

Soil Microbiology

J. S. Angle

MD

University of Maryland

Soil Microbiology

R. Klucas

NB

University of Nebraska

Plant Pathology

A. G. Wollum

NC

North Carolina State University

Soil Microbiology

C. M. Reynolds

NH

U. S. Army/CRREL

Soil Microbiology

E. C. Schröder

PR

University of Puerto Rico

Soil Microbiology

H. D. Skipper

SC

Clemson University

Soil Microbiology

M. D. Mullen

TN

Univ. of Tennessee

Soil Microbiology

D. A. Zuberer

TX

Texas A&M University

Soil Microbiology

K. Hatzios

VA

Virginia Polytechnic Institute & State University

Administrative Advisor

J. B. Morton

WV

West Virginia University

Environmental Microbiology

W. J. Hickey

WI

University of Wisconsin

Soil Microbiology

J. J. Germida

Canada

University of Saskatchewan

Soil Microbiology

ATTACHMENT B

RESOURCE COMMITMENTS

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

SAES

Project Leaders

SY

PY

TY

Obj. 1

Obj. 2

Obj. 3

Alabama

Y. Feng*

0.20

0.30

X

X

X

Arkansas

D. C. Wolf*

0.10

X

Delaware

J. J. Fuhrmann*

0.15

X

X

X

Florida

D. M. Sylvia*

0.20

0.20

X

X

J. Graham

0.15

0.25

X

Georgia

P. G. Hartel*

0.25

0.25

X

Maryland

J. S. Angle*

0.20

0.10

X

Nebraska

B. Klucas*

0.10

0.10

X

North Carolina

A. G. Wollum*

0.10

0.10

0.10

X

X

Puerto Rico

E. C. Schröder*

0.25

0.50

0.25

X

X

South Carolina

H. D. Skipper*

0.40

0.30

0.20

X

X

Tennessee

M. D. Mullen*

0.10

0.10

X

X

Texas

D. A. Zuberer*

0.25

0.25

X

X

West Virginia

J. B. Morton*

0.25

0.25

0.20

X

Wisconsin

W. J. Hickey*

0.15

0.15

X

X

TOTAL SAES

2.85

1.80

1.80

USDA–ARS

Georgia (Watkinsville)

A. Franzluebbers*

0.10

X

Georgia (Watkinsville)

M. Jenkins*

0.15

X

Idaho (Kimberly)

J. Entry*

0.15

X

X

TOTAL USDA–ARS

0.40

OTHER COOPERATORS

Univ. of Saskatchewan

J. Germida

0.10

X

Florida Internat'l Univ.

K. Jayachandran

0.10

0.10

X

U. S. Army (CRREL)

M. Reynolds*

0.25

X

TOTAL OTHER COOPERATORS

0.45

0.10

GRAND TOTAL FOR PROJECT

3.70

1.90

1.80

*Voting member; SY = Scientist year, PY = Professional years, TY = Technical support years

ATTACHMENT C

CRITICAL REVIEW FOR REGIONAL RESEARCH PROJECT S-262

Introduction

The regional research project had two objectives, one to define diversity of selected beneficial rhizosphere bacteria and fungi in defined soil–plant systems, and the other to elucidate processes controlling colonization and competitiveness of beneficial rhizosphere organisms. In a general sense, these objectives were not new to the project, and really represented a continuation of research by scientists in the Southern Region first begun in Regional Project S-112 (Enhancing Biological Dinitrogen Fixation in Soybeans and Other Legumes; 1976-1981), and continued through Regional Project S-170 (Overcoming Factors Limiting Biological Nitrogen Fixation by Leguminous Plants; 1982-1988), S-226 (Enhancing Beneficial Organisms in the Rhizosphere; 1988-1995), and finally, S-262 (Diversity and Interactions of Beneficial Bacteria and Fungi in the Rhizosphere; 1995-2000). Thus, this regional project has been in existence for almost 25 years under various titles with a continual broad emphasis on the beneficial aspects of bacteria and fungi in the rhizosphere. The reason for this long and involved focus is that the rhizosphere is most complex of all microbial environments and that understanding the interdependency of the biotic and abiotic factors in this environment is an extremely difficult goal. Nevertheless, this understanding is a worthy goal because understanding soil–plant–microbial relationships is one key to understanding ecological processes.

Work accomplished under the original project

This regional project, with its long history, would not be in existence unless its members had not demonstrated a continued excellence in productivity and collaboration. Regional Project S-262 is no exception to this history, and the members of Regional Project S-262 are proud of their accomplishments. In terms of productivity, the regional research project has 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), 12 theses or dissertations, and 12 non-refereed publications. In terms of collaboration, the members of regional research project have had the following collaborations:

a) 12 members from 10 states and Canada collaborated on a textbook, Principles and Applications of Soil Microbiology (completed in 1998). The state affiliation with the various chapters include: AR (Chapter 11, Transformations of carbon and soil organic matter formation), DE (Chapter 10, Microbial metabolism), FL (Chapter 18, Mycorrhizal symbioses; and Chapter 19, Biological control of plant pathogens and nematodes), GA (Chapter 2, The soil habitat), MD (Chapter 7, Viruses), NC (Chapter 1; Introduction and Historical Perspective), SC (Chapter 21, Bioremediation of contaminated soils), TN (Chapter 16, Transformations of other elements), TX (Chapter 12, Biological dinitrogen fixation: Introduction and nonsymbiotic), WV (Chapter 4, Fungi), and Canada (Chapter 15, Transformations of sulfur). To date, over 2200 copies of the textbook have been sold.

b) 4 members from four different states (DE, FL, GA, and TX) collaborated on an accompanying 68-page instructor's manual to the soil microbiology textbook (completed in 1998).

c) 12 different investigators, 9 from the regional project representing nine different states (AL, AR, DE, GA, MD, NC, SC, TX, and WV) and three investigators from outside the region (UT, WA, and Canada) determined differences in bacterial diversity among 14 bulk and rhizosphere soils (completed in 1996).

d) 7 members from 7 states (AR, DE, GA, MD, PR, SC, and TX) determined changes in microbial community structure of the rhizosphere of soybeans during a growing season (completed in 1997).

e) 5 members from 5 states (DE, GA, MD, SC, and TX) determined the misidentification of soil bacteria by fatty acid methyl ester (FAME) and BIOLOG analyses (completed in 1998).

f) 5 members from 5 states (AR, DE, FL, GA, and NH) determined changes in microbial community structure during phytoremediation of pyrene-contaminated soil (completed in 1999).

g) 3 members from three states (AR, DE, and NH) determined the influence of pyrene on individual microbial populations in soil and rhizosphere (completed in 1999).

h) 14 manuscripts (12 accepted, in press, or published, and 2 submitted) are the result of the collaboration between two regional members. Collaborations include DE and GA (5 manuscripts), AR and NH (4 manuscripts), DE and ID (2 manuscripts), USDA and WV (2 manuscripts), and MD and Canada (1 manuscript). These manuscripts, as well as the others identified above, are identified with an asterisk in the section forPublications Issued or Manuscripts Approved for S-262.

Degree to which objectives have been accomplished

Introduction

Although individual investigators claimed time for themselves and various supporting personnel under the aegis of the Regional Research Project S-262, and some support was available to these investigators to travel to the annual meeting, funds allotted to do actual research were extremely limited. Furthermore, rhizosphere research is not a heavily funded research area and individual investigators had only modest success in securing extramural funds to supplement the regional project. To offset these negative aspects, investigators in the regional project relied on low-cost alternatives to conduct regional work or relied on individual investigators or experiment stations willing to subsidize the more expensive research. This accounts for most of our cooperative work.

Under low-cost alternatives, members of the multistate project wrote a soil microbiology textbook, Principles and Applications of Soil Microbiology. This textbook, which was regarded as a special educational objective, was not listed under the multistate objectives because regional research guidelines prohibit publications as objectives. Nevertheless, work on the textbook and its accompanying instructor's manual occupied a considerable portion of our time.

Under the subsidized work, the members of the regional project are grateful to the University of Delaware Experiment Station which allowed us to analyze all our fatty acid methyl ester (FAME) samples without cost, and to the U.S. Army Cold Regions Engineering Laboratory (CRREL, Hanover, NH) for analyzing all our soil samples for organic contaminants without cost. Research was redirected within the objectives to take advantage of these subsidies. This is the reason that Delaware and New Hampshire were involved in 16 of the 19 journal article manuscripts of research collaboration between two or more states. All of this cooperative work, as well as a some other work, is highlighted on the next page.

Objective 1

The objective was redefined in terms using a new methodology, FAME analysis, to identify not only individual bacteria and fungi species in the rhizosphere, but also to determine changes in microbial community structure. Objective #1 was 80% completed. FAME profiles were used to assess the influence of a) different soils, b) ecological succession (time), c) different plant genotypes, d) temperature and matric water potential, and continuous/rotational plots and associated weeds. With regards to different soils, individual FAME profiles of organisms from bulk and rhizosphere soil of bahiagrass grown in 14 different U.S. soils were obtained. The principal result of this research shows that some soils have distinct taxa of bacteria. With regards to ecological succession, whole soil FAME profiles of bulk and rhizosphere soil were obtained from soybeans over a growing season. These results show that individual soils dominate soil microbial community structure. With regards to different plant genotypes, individual FAME profiles were obtained of bacteria from bulk and rhizosphere soil of nodulating and non‑nodulating soybean and various Arabidopsis thaliana mutants, in addition to whole soil FAME profiles of endophyte and non‑endophyte‑infested tall fescue. With regards to temperature and moisture, whole soil FAME profiles were obtained at two different matric water potentials and temperatures. Our collaboration suggests that soil, ecological succession, and matric water potential are important to microbial diversity in the rhizosphere, whereas temperature and plant genotype are not.

The FAME work was then integrated with soils amended with various organic compounds. In one study, a fungicide (Benlate) was added to soil containing juniper or papaya plants. Three studies were conducted soil amended with pyrene and whole soil FAME profiles from bulk and rhizosphere soil of ryegrass were obtained under lightroom and field conditions. Generally, phytoremediation was site-specific.

In addition, scientists collaborated on two other regional manuscripts, one to assess misidentification of soil bacteria by the MIDI system and one to assess plant growth‑promoting rhizobacteria in bulk and rhizosphere soil.

Objective 2

Because most of the cooperative research effort was redirected to Objective #1 and the soil microbiology textbook, this objective was only about 20% completed. Most of the work under this objective was done with mycorrhizae. Specifically, a model system was developed to study early stages of colonization of plant roots by arbuscular mycorrhizae. Several studies investigated the importance of various factors (i.e., iron, phosphite) affecting mycorrhizal colonization and glomalin production, as well as the influence of fumigation, solarization, and fertilization on arbuscular mycorrhizae colonization.

In preparation for the new multistate proposal, several studies assessed community structure of arbuscular mycorrhizae in a) reclaimed strip-mined soils planted with apple, grapes, and other selected plants, b) conventional and low-input (animal manure or manure co-composted with leaves) sustainable farming systems, c) wheat roots grown in moderate to high soil P levels, d) ryegrass grown in hydrocarbon-contaminated soil, and, e) in a combination with one of the bacterial experiments above, soil contaminated with pyrene. As an overall generalization, conventional farming practices tended to decrease arbuscular mycorrhizae activity, while sustainable, organic, low-input practices tended to increase activity.

Incomplete work or areas needing further investigation:

The special education objective identified in Regional Research Project S-262 will continue in the proposed multistate project. Again, this is not listed as an objective because publications may not be listed as objectives. Scientists in the multistate project will write a second edition to the textbook and its accompanying instructor's manual as well as a new laboratory manual. As noted under Hierarchy in the Organization section, the chairpersons for the textbook and laboratory manual have already been selected.

It is the intent in the proposed multistate project to complete Objective #1 of Regional Research Project S-262. With one exception, most of the basic work is done, so FAME analysis will continue in Objective #3 of the multistate proposal as applied work to cover other soil conditions (i.e., disturbed lands and urban landscapes). The one exception is the lack of an interpretive database relating specific fatty acids (biomarkers) to particular microbial taxa. As part of the ongoing research, whole soil FAMEs associated with the various experimental treatments will be analyzed on a routine basis to obtain a more complete perspective of fatty acid dynamics and variability in soils. The University of Delaware is willing to continue a partial subsidy of FAME analysis (now $3 per sample), so it is still possible to continue both this basic and applied work.

Objective #1 in Regional Research Project S-262 also continues to some degree in Objective #2 of the multistate project, but with some modifications. Specifically, as noted in the Procedures of the multistate proposal, chemical interferences were observed with FAME and some organic compounds in soil. To avoid this, scientists working on Objective #2 may switch to phospholipid fatty acid (PLFA) analyses, which may avoid most of these chemical interferences. Again, CRREL is willing to subsidize analysis of toxic organics in soil and this will account for most of the continued emphasis on toxic organics in Objective #2.

Emphasis on processes controlling colonization and competitiveness of beneficial rhizosphere organisms, which was Objective #2 in Regional Research Project S-262, will not continue in the proposed multistate project. Instead, Objective #2 under Regional Research Project S-262 will be replaced with a new objective, Objective #1 (ribotyping) in the multistate project. Therefore, Objective #1 is a new research direction for investigators on the multistate project. Although ribotyping of E. coli is only tangential to rhizosphere work, it has three advantages for the future multistate work. First, biogeographic work is an important part of understanding microbial diversity. Second, this work is mostly molecular genetics and given that most soil microbiological research is moving in this direction, it is important for the multistate project to go in this direction as well. Third, the possibilities of obtaining extramural funding for expanded ribotyping research are excellent.

PUBLICATIONS ISSUED OR MANUSCRIPTS

APPROVED FOR S-262 (1 OCTOBER 1995-PRESENT)

(Note: * = regional collaboration between two or more S-262 scientists)

Books:

Complete (3):

Skipper, H. D., and R. F. Turco. 1995. Bioremediation: Science and applications. Soil Science Society of America Special Publication No. 43, Madison, WI.

*Sylvia, D. M., J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer. 1998. Principles and applications of soil microbiology. Prentice-Hall, Upper Saddle River, NJ.

*Sylvia, D. M., J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer. 1998. Instructor’s manual for principles and applications of soil microbiology. Prentice-Hall, Upper Saddle River, NJ.

Book chapters (36):

*Angle, J. S., and J. V. Gagliardi. 1998. Viruses. p. 132-148. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Angle, J. S., J. V. Gagliardi, M. S. McIntosh, and M. A. Levin. 1996. Enumeration and expression of bacterial counts in the rhizosphere. p. 233-251. In G. Stotzky and J.–M. Bollag (ed.) Soil biochemistry, Vol. 9. Marcel Dekker, NY.

Benny, G. L., R. A. Humber, and J. B. Morton. 2000. Zygomycota: Zygomycetes.In D. J. McLaughlin, P. A. Lemke, and E. McLaughlin (ed.) The mycota, Vol. 7a, Systematics and Evolution. Springer-Verlag, NY (in press).

Donnelly, P. K., and J. A. Entry. 1998. Bioremediation of soils with mycorrhizal fungi. p. 417-432. In D. C. Adriano, J. M. Bollag, W. Frankenberger, and R. Sims (ed.) Bioremediation of contaminated soils. American Society of Agronomy Monograph 37.

Douds, D. D., and G. Nagahashi. 2000. Signaling and recognition events prior to colonization of roots by AM fungi. p. 11-18. In G.K. Podila and D. D. Douds, Jr. (ed.) Current advances in mycorrhiza research. APS Press, St. Paul, MN.

Douds, D. D., Jr., V. Gadkar, and A. Adholeya. 2000. Mass production of VAM fungus biofertilizer. In K. G. Mukerji, B. P. Chamola, and J. Singh (ed.) Mycorrhizal biology. John Wiley & Sons, NY. (in press)

Entry, J. A., and W. H. Emmingham. 1996. Accumulation of lead and zinc form contaminated soil by tree seedlings. p. 229-306. In E.L. Kruger, T. A. Anderson, and J. R. Coats (ed.) Phytoremediation of soil and water contaminants. American Chemical Society Press, Washington, DC.

Entry, J. A., P. A. Rygiewicz, L. S. Watrud, and P. K Donnelly. 1999. The influence of adverse soil conditions on formation and function of arbuscular mycorrhizae.In P. P. Singh and A. K. Sharma (ed.) VA mycorrhizas—interactions in plants, rhizosphere and soils. Oxford Publishing Company, NY (in press).

Entry, J. A., N. C. Vance, M. A. Hamilton, and D. Zabowski. 1995. In situ remediation of soil contaminated with low concentrations of radionuclides. p. 1055-1066. In G. W. Gee and N. R. Wing (ed.) In situ remediation: Scientific basis for current and future technologies. Batelle Press, Richland, WA.

Entry, J. A., L. S. Watrud, R. S. Mannasse, and N. C. Vance. 1996. Phytoremediation of soils contaminated with radionuclides. p. 299-306. In E.L. Kruger, T. A. Anderson and J. R. Coats (ed.) Phytoremediation of soil and water contaminants. American Chemical Society Press, Washington, DC.

*Fuhrmann, J. J. 1998. Microbial metabolism. p. 189-217. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

*Germida, J. J. 1998. Transformations of sulfur. p. 346-368. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Grady, C. P. L., Jr., H. D. Skipper, and W. T. Frankenberger. 1995. Educational needs in bioremediation. p. 305-317. In H. D. Skipper and R. F. Turco (ed.) Bioremediation: Science and applications. Soil Science Society of America Special Publication No. 43, Madison, WI.

Graham, J. H. 2000. Assessing costs of arbuscular mycorrhizal symbiosis in agroecosystems. p. 127-140. In G.K. Podila and D. D. Douds, Jr. (ed.) Current advances in mycorrhiza research. APS Press, St. Paul, MN.

*Graham, J. H., and D. J. Mitchell. 1998. Biological control of soilborne plant pathogens and nematodes. p. 427-446. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Hahn, A., S. F. Wright, and B. Hock. 2000. Immunochemical characterization of arbuscular mycorrhizal fungi. The Mycota, Vol. IX (in press).

*Hartel, P. G. 1998. The soil habitat. p. 21-43. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Jarstfer, A. G., and D. M. Sylvia. 1996. Isolation, culture and detection of arbuscular mycorrhizal fungi. pp. 406-412. In C. J. Hurst et al. (ed.) Manual of environmental microbiology. American Society of Microbiology, Washington, DC.

Jarstfer, A. G., and D. M. Sylvia. 2000. Aeroponic culture of VAM fungi. In A. K. Varma and B. Hock (ed.) Mycorrhiza: Structure, function, molecular biology and biotechnology, 2nd ed. Springer‑Verlag, Berlin (in press).

Jarstfer, A. G., and D. M. Sylvia. 2000. Isolation, culture and detection of arbuscular mycorrhizal fungi. p. 406-412. In C. J. Hurst et al. (ed.) Manual of environmental microbiology. American Society of Microbiology, Washington, DC (in press).

Morton, J. B. 1996. Mycorrhizae. p. 324-327. In Anonymous (ed.) McGraw–Hill 1997 yearbook of science and technology. McGraw–Hill, NY.

Morton, J. B. 1999. Evolution of endophytism in arbuscular mycorrhizal fungi of the Glomales. p. 121-140. In C. W. Bacon and J. H. White (ed.) Microbial endophytes. Marcel Dekker, NY.

*Morton, J. B. 1998. Fungi. p. 72-93. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Morton, J. B. 1999. Evolution of endophytism in arbuscular mycorrhizal fungi of Glomales. p. 121-140. In C. W. Bacon, and J. H. White. (ed.) Microbial endophytes. Marcel Dekker, NY.

Morton, J. B., R. E. Koske, S. L. Stürmer, and S. P. Bentivenga. 2000. Protocols for measurement of diversity among arbuscular fungi. In G. Mueller, A. Rossman, and G. Bills (ed.) Biological diversity handbook series: Standard methods for fungi. Smithsonian Institute Press, Washington, DC (in press).

*Mullen, M. D. 1998. Transformations of other elements. p. 369-386. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Pedersen, C. T. and D. M. Sylvia. 1996. Mycorrhiza: Ecological implications for plant interactions. p. 195-222. In K. G. Mukerji (ed.) Concepts in mycorrhizal research. Kluwer Academic Publishers, The Netherlands.

*Skipper, H. D. 1998. Bioremediation of contaminated soils. p. 469-481. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Stockel, D. L., E. C. Mudd, and J. A. Entry. 1996. Herbicide mobility and degradation in riparian wetlands. p. 114-132. In E. L. Kruger, T. A. Anderson, and J. R. Coats (ed.) Phytoremediation of soil and water contaminants. American Chemical Society, Washington, DC.

Sylvia, D. M. 1999. Fundamentals and applications of arbuscular mycorrhizae: A "biofertilizer" perspective. p. 705-723.  In J. O. Siqueira et al. (ed.) Soil fertility, biology, and plant nutrition interrelationships. Viçosa: SBCS, Lavras: UFLA/DCS.

*Sylvia, D. M. 1998. Mycorrhizal symbioses. p. 408-426. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Sylvia, D. M., and D. O. Chellemi. 2000. Interactions among root-inhabiting fungi and their implications for biological control of root pathogens. Adv. Agron. (In press).

*Wagner, G. H., and D. C. Wolf. 1998. Carbon transformations and soil organic matter formation. p. 218-258. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice-Hall, Upper Saddle River, NJ.

Wolf, D. C., and L. C. Purcell. 1999. Ineffective nitrogen fixation. p. 9-10. In G. L. Hartman, J. B. Sinclair, and J. C. Rupe (ed.) Compendium of soybean diseases, 4th ed. APS Press, St. Paul, MN.

*Wollum, A. G., II. Introduction and historical perspective. p. 3-20. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

*Zuberer, D.A. 1998. Biological dinitrogen fixation: Introduction and nonsymbiotic. p. 295-321. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Refereed Journals (published, in press, or accepted; 142)

Angle, J. S., M. A. Levin, J. V. Gagliardi, and M.S. McIntosh. 1995. Validation of microcosms for examining the survival of Pseudomonas aureofaciens (lacZY) in soil. Appl. Environ. Microbiol. 61:2835-2839.

Arredondo–Peter, R., M. S. Hargrove, G. Sarath, J. Moran, J. Logrman, J. S. Olson, and R. V. Klucas. 1997. Rice hemoglobins: Gene coding, analysis, and oxygen‑binding kinetics of a recombinant protein synthesized in Escherichia coli. Plant Physiol. 115:1259‑1266.

Aziz, T., D. M. Sylvia, and R. F. Doren. 1995. Activity and species composition of arbuscular mycorrhizal fungi following soil removal. Ecol. Appl. 5:776-784.

Bago, B., P. E. Pfeffer, D. D. Douds, Jr., J. Brouillette, G. Bécard, and Y. Shachar-Hill. 1999. Carbon metabolism in spores of the arbuscular mycorrhizal fungus Glomus intraradices as revealed by NMR spectroscopy. Plant Physiol. 121:263- 271.

Bentivenga, S. P., and J. B. Morton. 1995. A monograph of the genus Gigaspora incorporating developmental patterns of morphological characters. Mycologia 87:720-732.

Bentivenga, S. P., and J. B. Morton. 1996. Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae. Proc. Nat. Acad. Sci. 93:559-5662.

Bentivenga, S. P., J. D. Bever, and J. B. Morton. 1997. Genetic variation of morphological characters within a single isolate on the endomycorrhizal fungus, Glomus clarum. Am. J. Bot. 84:1211‑1216.

Bever, J. D., and J. B. Morton. 1999. Heritable variation of spore shape in a population of arbuscular mycorrhizal fungi: Suggestions of a novel mechanism of inheritance. Am. J. Bot. 86:1209-1216.

Bever, J. D., J. B. Morton, J. Antonovics, and P. A. Schultz. 1996. Host-dependent sporulation and species diversity of arbuscular fungi in a mown grassland. J. Ecol. 84:71-82.

Bhgwat, A. A., K. C. Gross, R. E. Tully, and D. L. Keister. 1996. Beta glucan synthesis in Bradyrhizobium japonicum: Characterization of a new locus (ndvC). J. Bacteriol. 178:4635-4642.

*Cattelan, A. J., P. G. Hartel, and J. J. Fuhrmann. 1998. Bacterial composition in the rhizosphere of nodulating and non-nodulating soybean. Soil Sci. Soc. Am. J. 62:1549-1555.

*Cattelan, A. J., P. G. Hartel, and J. J. Fuhrmann. 1999. Screening plant growth-promoting rhizobacteria (PGPR) to promote early soybean growth. Soil Sci. Soc. Am. J. 63:1670-1680.

Cha, D. K., J. J. Fuhrmann, D. W. Kim, and C. M. Golt. 1999. Fatty acid methyl ester (FAME) technology for monitoring Nocardia levels in activated sludge. Water Res. 33:1964-1969.

Chellemi, D. O., F. M. Rhoads, S. M. Olson, J. R. Rich, D. Murray, G. Murray, and D. M. Sylvia. 1999. An alternative, low-input production system for fresh market tomato. J. Altern. Agric. 14:59-68.

Douds, D. D. 1997. A procedure for the establishment of Glomus mosseae in dual culture with Ri T‑DNA transformed carrot roots. Mycorrhiza 7:57‑61.

Douds, D. D., Jr., and P. D. Millner. 1999. Biodiversity of arbuscular mycorrhizal fungi in agroecosystems. Agric. Ecosyst. Environ. 74:77-93.

Douds, D. D., G. Nagahashi, and G. D. Abney. 1996. The differential effects of cell wall-associated phenolics, cell walls, and cytosolic phenolics of host and non-host roots on the growth of two species of AM fungi. New Phytol. 133:289-294.

Douds, D. D., P. E. Pfeffer, and Y. Shachar-Hill. 2000. Application of in vitro methods to study carbon uptake and transport by AM fungi. Plant Soil (in press).

Douds, D. D., Jr., L. Galvez, G. Bécard, and Y. Kapulnik. 1998. Regulation of mycorrhizal development by plant host and AM fungus species in alfalfa. New Phytol. 138:27-35.

Douds, D. D., L. Galvez, M. Franke–Snyder, M. Reider, and L. E. Drinkwater. 1997. Effect of compost addition and crop rotation upon VAM fungi. Agric. Ecosyst. Environ. 65:257‑266.

El–Kenawy, Z., J. S. Angle, P. van Berkum, and R. L. Chaney. 1997. Zinc and Cd effects on the early stages of nodulation of white clover. Agron. J. 89:875‑880.

Entry, J. A. 1999. Influence of nitrogen on atrazine and 2,4-dichlorophenoxyacetic acid degradation in blackwater and redwater forested wetland soils. Biol. Fertil. Soils 29:348-353.

Entry, J. A. 1999. Influence of nitrogen on cellulose and lignin degradation in blackwater and redwater forested wetland soils. Biol. Fertil. Soils (in press).

Entry, J. A., and W. H. Emmingham. 1995. The influence of dairy manure on atrazine and 2,4-dichlorophenoxyacetic acid mineralization in pasture soils. Can. J. Soil Sci. 75:379-383.

Entry, J. A., and W. H. Emmingham. 1995. Sequestration of 137Cs and 90Sr from soil by seedlings of Eucalyptus tereicornis. Can. J. For. Res. 25:1044-1047.

Entry, J. A., and W. H. Emmingham. 1996. Nutrient content and extractability in riparian soils supporting forests and grasslands Appl. Soil Ecol. 4:119-124.

Entry, J. A., and W. H. Emmingham. 1996. The influence of vegetation on microbial degradation of atrazine and 2,4-dichlorophenoxyacetic acid in riparian soils. Can. J. Soil Sci. 76:101-106.

Entry, J. A., and W. H. Emmingham. 1998. Influence of forest age on forms of carbon in Douglas-fir soils in the Oregon Coast Range. Can. J. For. Res. 28:390- 395.

Entry, J. A., and C. C. Mitchell. 1998. Soil C, N, and crop yields in Alabama’s long-term "Old Rotation" cotton experiment. Soil Till. Res. 47:331-338.

Entry, J. A., and R. E. Sojka. 2000. Influence of polyacrylamide on movement of microorganisms in irrigation water. Environ. Pollut. (in press).

Entry, J. A., and L. S. Watrud. 1998. Accumulation of 137Cs and 90Sr in Alamo switchgrass. Water Air Soil Pollut. 104:339-352.

Entry, J. A., and L. S. Watrud. 1999. Accumulation of 137Cs and 90Sr from contaminated soil by three grass species inoculated with mycorrhizal fungi. Environ. Pollut. 104:449-457.

Entry, J. A., and L. S. Watrud. 2000. Influence of organic amendments on accumulation of 137Cs and 90Sr from contaminated soil by three grass species. Water Air Soil Pollut. (in press).

Entry, J. A., P. K. Donnelly, and W. H. Emmingham. 1995. Atrazine and 2,4-D mineralization in relation to microbial biomass in soils of young, second, and old-growth forests. Appl. Soil Ecol. 2:77-84.

Entry, J. A., P. K. Donnelly, and W. H. Emmingham. 1995. Mineralization of atrazine and 2,4-D in soils inoculated with Phanerochaete crysosporium and Trappea darkeri. Appl. Soil Ecol. 3:85-90.

Entry, J. A., C. C. Mitchell, and C. B. Backman. 1997. Influence of management practices on soil organic matter, microbial biomass and cotton yield in Alabama’s "Old Rotation." Biol. Fertil. Soils 24:353-358.

Entry, J. A., C. A. Strausbaugh, and R. E. Sojka. 2000. Wood chip–polyacrylamide cores as a carrier for biocontrol bacteria suppresses Verticllium wilt on potato. Biocontrol Sci. Technol. (accepted)

Entry, J. A., D. W. Reeves, C. B. Backman, and R. L. Raper. 1996. Influence of tillage and wheel traffic on microbial biomass, residue decomposition and extractable nutrients in a coastal plain soil. Plant Soil 180:129-137.

Entry, J. A., D. W. Reeves, C. B. Backman, and R. L. Raper. 1996. Influence of compaction from wheel traffic and tillage on arbuscular mycorrhizae infection and nutrient uptake by Zea mays. Plant Soil 180:139-146.

Entry, J. A., B. H. Wood, J. E. Edwards, and C. W. Wood. 1997. Influence of organic by-products and nitrogen source on chemical and microbiological status of an agricultural soil. Biol. Fertil. Soils 24:196-204.

*Entry, J. A., R. K. Hubbard, J. E. Thies, and J. J. Fuhrmann. 2000. Influence of vegetation in riparian filterstrips on coliform bacteria. I. Movement and survival in surface and groundwater. J. Environ. Qual. (in press).

*Entry, J. A., R. K. Hubbard, J. E. Thies, and J. J. Fuhrmann. 2000. Influence of vegetation in riparian filterstrips on coliform bacteria. II. Survival in soil. J. Environ. Qual. (in press).

Entry, J. A., G. B. Runion, S. A. Prior, R. J. Mitchell, and H. H. Rogers. 1998. Influence of CO2 enrichment and nitrogen fertilization on tissue chemistry and carbon allocation in longleaf pine seedlings. Plant Soil 200:3-11.

Entry, J. A., D. W. Reeves, E. C. Mudd, W. J. Lee, E. A. Guertal, and R. L. Raper. 1996. Influence of wheel traffic and tillage on microbial biomass, residue decomposition and extractable nutrients in a Coastal Plain soil. Plant Soil 180:129-137.

Entry, J. A., N. C. Vance, M. A. Hamilton, D. Zabowski, L. S. Watrud, and D. C. Adriano. 1996. Phytoremediation of soils contaminated with low concentrations of radionuclides. Water Air Soil Pollut. 88:167-176.

Espeleta, J. F., D. M. Eissenstat, and J. H. Graham. 1999. Citrus root responses to localized dry soil: A new approach to study of mycorrhizal effects on the roots of mature trees. Plant Soil 206:1-10.

Fang, C. W., M. Radosevich, and J. J. Fuhrmann. 2000. Atrazine and phenanthrene degradation in grass rhizosphere soil as related to microbial community structure. Soil Biol. Biochem. (accepted)

Farmer, D. J., and D. M. Sylvia. 1998. Variation in the ribosomal DNA internal transcribed spacer of a diverse collection of ectomycorrhizal fungi. Mycol. Res. 102:859-865.

Feng, Y., R. D. Minard, and J.–M. Bollag. 1998. Photolytic and microbial degradation of 3,5,6-trichloro-2-pyridinol. Environ. Toxicol. Chem. 17:814-819.

Feng, Y., J.–H. Park, T. C. Voice, and S. A. Boyd. 2000. Bioavailability of soil-sorbed biphenyl to bacteria. Environ. Sci. Technol. (in press)

Franzluebbers, A. J., F. M. Hons, and D. A. Zuberer. 1995. Soil organic carbon, microbial biomass, and mineralizable carbon in sorghum manage­ment systems. Soil Sci. Soc. Am. J. 59:460-466.

Franzluebbers, A. J., F. M. Hons, and D. A. Zuberer. 1995. Tillage and crop effects on seasonal soil carbon and nitrogen dynamics. Soil Sci. Soc. Am. J. 59:1618-1624.

Franzluebbers, A. J., F. M. Hons, and D. A. Zuberer. 1995. Tillage-induced seasonal changes in soil physical properties affecting soil CO2 evolution under intensive cropping. Soil Till. Res. 34:41-60.

Franzluebbers, A. J., F. M. Hons, and D. A. Zuberer. 1996. Seasonal dynamics of active soil carbon and nitrogen pools under intensive cropping in conventional and no tillage. J. Plant Nut. Soil Sci. 159:343-349.

Franzluebbers, A. J., F. M. Hons, and D. A. Zuberer. 1998. In situ and potential CO2 evolution from a Fluventic Ustochrept in south central Texas as affected by tillage and cropping intensity. Soil Till. Res. 47:303‑308.

Franzluebbers, A. J., R. L. Haney, F. M. Hons, and D. A. Zuberer. 1996. Active fractions of organic matter in soils with different texture. Soil Biol. Biochem. 28:1367-1372.

Franzluebbers, A. J., R. L. Haney, F. M. Hons, and D. A. Zuberer. 1999. Assessing biological soil quality with chloroform fumigation-incubation: Why subtract a control? Can. J. Soil Sci. 79:521-528.

Franzluebbers, A. J., R. L. Haney, F. M. Hons, and D. A. Zuberer. 1996. Determination of microbial biomass and nitrogen mineralization following rewetting of dried soil. Soil Sci. Soc. Am. J. 60:1133-1139.

Franzluebbers, A. J., S. F. Wright, J. A. Stuedemann, and H. H. Schomberg. 2000. Soil aggregate distribution and glomalin in pastures of the southern Piedmont USA. Soil Sci. Soc. Am. J. (in press)

*Franzluebbers, A. J., N. Nazih, J. A. Stuedemann, J. J. Fuhrmann, H. H. Schomberg, and P. G. Hartel. 1999. Soil carbon and nitrogen pools under low- and high-endophyte-infected tall fescue. Soil Sci. Soc. Am. J. 63:1687-1694.

Gagliardi, J. V., and J. S. Karns. 2000. Leaching of E. coli O157:H7 in diverse soils under various agricultural management practices. Appl. Environ. Microbiol. 66:877-883.

Gagliardi, J. V., J. S. Buyer, J. S. Angle, and E. Russek–Cohen. Comparison of soil microbial community structural and functional analyses following inoculation of genetically engineered and non-engineered pseudomonads to wheat roots in diverse soils. Soil Biol. Biochem. (accepted)

Glucksman, A. M., H. D. Skipper, J. S. Domingo, and R. L. Brigmon. 2000. Use of fatty acid methyl esters to detect microorganisms from water. J. Appl. Bacteriol. (accepted).

Graham, J. H., and L. K. Abbott. 2000. Wheat responses to aggressive and nonaggressive arbuscular mycorrhizal fungi. Plant Soil (in press).

Graham, J. H., and D. M. Eissenstat. 1998. Field evidence for carbon costs of citrus mycorrhizas. New Phytol. 140:103-110.

Graham, J. H., D. L. Drouillard, and N. C. Hodge. 1996. Carbon economy of sour orange in response to different Glomus spp. Tree Physiol. 16:1023-1029.

Graham, J. H., L. W. Duncan, and D. M. Eissenstat. 1997. Carbohydrate allocation patterns in citrus genotypes as affected by phosphorus nutrition, mycorrhizal colonization and mycorrhizal dependency. New Phytol. 135:335‑343.

Graham, J. H., N. C. Hodge, and J. B. Morton. 1995. Fatty acid methyl ester profiles for characterization of glomalean fungi and their endomycorrhizae. Appl. Environ. Microbiol. 61:58-64.

Griffiths, R. P., J. A. Entry, E. R. Ingham, and W. H. Emmingham. 1997. Chemistry and microbiological activity of forest and pasture riparian zone soils along three Pacific Northwest streams. Plant Soil 190:169-178.

Haney, R. L., A. J. Franzluebbers, F. M. Hons, and D. A. Zuberer. 1999. Soil C extracted with water or K2SO4:pH effect on determination of microbial biomass. Can. J. Soil Sci. 79:529-533.

Haney, R. L., S. A. Senseman, F. M. Hons, and D. A. Zuberer. 1999. Effect of glyphosate on soil microbial activity and biomass. Weed Sci. 48:89-93.

Hartel, P. G., W. I. Segars, N. Stern, J. Steiner, and A. Buchan. 1999. Ribotyping to determine the host origin of Escherichia coli isolates in different water samples. p. 377-382. In D. S. Olsen and J. P. Potyondy (ed.) Wildland hydrology. American Water Resources Association Technical Publications Series TPS-99-3, Herndon, VA.

Ibekwe, A. W., J. S. Angle, R. L. Chaney, and P. van Berkum. 1997. Zinc and Cd toxicity to alfalfa and its microsymbiont. J. Environ. Qual. 25:1032‑1040.

Ibekwe, M., J. S. Angle, R. L. Chaney, and P. van Berkum. 1997. Zinc and Cd effects on Rhizobium leguminosarum bv. trifolii and while clover using chelate buffered solution. Soil Sci. Soc. Am. J. 62:204‑211.

Ibekwe, M. A., P. van Berkum, R. L. Chaney, and J. S. Angle. 1997. Differentiation of clover rhizobia isolated from metal contaminated and control soils with varying pH. Soil Sci. Soc. Am. J. 61:1679‑1685.

Jarstfer, A. G., P. Farmer–Koppenol, and D. M. Sylvia. 1998. Tissue magnesium and calcium affect development and reproduction of an arbuscular mycorrhiza. Mycorrhiza 7:237-242.

Johnson, N. C., Graham, J. H., and F. A. Smith. 1997. Functioning of mycorrhizal associations along the mutualism‑parasitism continuum. New Phytol. 135:575‑585.

Kennedy, J. L., J. C. Stutz, and J. B. Morton. 1999. Glomus eburneum and G. luteum, two previously undescribed species of arbuscular mycorrhizal fungi, with emendation of G. spurcum. Mycologia91:1083-1093.

Lee, W. J. , C. W. Wood, D. W. Reeves, J. A. Entry, and R. L. Raper. 1996. Interactive effects of wheel-traffic and tillage on soil carbon and nitrogen. Comm. Soil Sci. Plant Anal. 27:3027-3043.

Marshall, S. B., M. L. Cabrera, C. W. Wood, L. C. Braun, M. D. Mullen, and E. A. Guertal. 1999. Denitrification from fescue pastures fertilized with broiler litter. J. Environ. Qual. 28:1978-83.

McCulley, R., T. Boutton, S. Archer, D. Zuberer, F. Hons, and A. Hubbard. 1997. Spatial–temporal variation in soil respiration and microbial biomass in a subtropical savanna parkland. Bull. Ecol. Soc. Amer. 78:283.

Melhorn, C. G., M. D. Mullen, D. D. Tyler, and B.N. Duck. 1998. Biological and biochemical soil properties in no‑till corn with different cover crops. J. Soil Water Conserv. 53:219-224.

Miller, J., A. G. Wollum, II, and J. B. Weber. 1997. Degradation of 14C primusulfuron in soil from four depths under sterile and nonsterile conditions. J. Environ. Qual. 26:440‑445.

Miller, J., A. G. Wollum, II, and J. B. Weber. 1997. Degradation of 14 C atrazine and 14C metolachlor in soil from four depths under sterile and nonsterile conditions. J. Environ. Qual. 26:633‑638.

Mitchell, C. C., and J. A. Entry. 1998. Soil C, N and crop yields in Alabama’s long-term "Old Rotation" cotton experiment. Soil Till. Res. 47:331-338.

Morton, J. B. 1995. Taxonomic and phylogenetic divergence among five Scutellospora species (Glomales, Zygomycetes) based on comparative developmental sequences. Mycologia 87:127-137.

Morton, J. B. 1996. Redescription of Glomus caledonium based on correspondence of spore morphological characters in type specimens and a living reference culture. Mycorrhiza 6:161-166.

Morton, J. B., S. P. Bentivenga, and J. D. Bever. 1995. Discovery, measurement, and interpretation of diversity in symbiotic endomycorrhizal fungi (Glomales, Zygomycetes). Can. J. Bot. 73 (Suppl. 1):S25-S32.

Morton, J. B., J. D. Bever, and F. L. Pfleger. 1997. Taxonomy of Acaulospora gerdemannii and Glomus leptotichum, synanamorphs of one anamorphic fungus in Glomales. Mycol. Res. 101:625‑631.

Mpepereki, S., A. G. Wollum, and F. Makonese. 1996. Growth temperature characteristics of indigenous Rhizobium and Bradyrhizobium isolates from Zimbabwean soils. Soil Biol. Biochem. 28:1537‑1539.

Mullen, M. D., C. G. Melhorn, D. D. Tyler, and B. N. Duck. 1998. Biological and biochemical soil properties in no-till corn with different cover crops. Soil Water Conserv. 53:219-224.

Nagahashi, G., and D. D. Douds. 1997. Appressorium formation by AM fungi on isolated cell walls of carrot roots. New Phytol. 136:299‑304.

Nagahashi, G. and D. Douds, Jr. 1999. A rapid and sensitive bioassay with practical application for studies on interactions between root exudates and arbuscular mycorrhizal fungi. Biotechnol. Tech. 13:893-897.

Nagahashi, G., and D. D. Douds, Jr. 2000. Partial separation of root exudate components and their effects upon the growth of germinated spores of AM fungi. Mycol. Res. (in press).

Nagahashi, G., D. D. Douds, and G. D. Abney. 1996. Phosphorus amendment inhibits hyphal branching of the VAM fungus Gigaspora margarita directly and indirectly through its effect on root exudation. Mycorrhiza 6:403-408.

Nagahashi, G., D. D. Douds, Jr., and G. Bécard. 1999. Recognition and communication events between arbuscular mycorrhizal fungi and host roots. Curr. Top. Plant Biol. 1:63-75.

Nagahashi, G., D. Douds, Jr., and M. Buee. 2000. Light-induced hyphal branching of germinated AM fungus spores. Plant Soil (in press).

*Nichols, T. D., D. C. Wolf, H. B. Rogers, C. A. Beyrouty, and C. M. Reynolds. 1997. Rhizosphere microbial populations in contaminated soils. Water Air Soil Pollut. 95:165‑178.

*Oka, N., P. G. Hartel, and J. J. Fuhrmann. 1997. Effect of plant genotype on rhizobacterial composition of Arabidopsis thaliana. p. 437-440. In A. Ogoshi, K. Kobayashi, Y. Homma, F. Kodama, N. Kondo, and S. Akino (ed.) Plant growth-promoting rhizobacteria—present status and future prospects. OECD, Paris.

*Oka, N., P. G. Hartel, O. Finlay–Moore, J. Gagliardi, D. Zuberer, J. J. Fuhrmann, J. S. Angle, and H. D. Skipper. 2000. Misidentification of soil bacteria by fatty acid methyl ester (FAME) and BIOLOG analyses. Biol. Fertil. Soils (in press).

*Olexa, T. J., T. J. Gentry, P. G. Hartel, D. C. Wolf, J. J. Fuhrmann, D. M. Sylvia, and C. M. Reynolds. 2000. Mycorrhizal colonization and microbial community structure in the rhizosphere of annual ryegrass grown in pyrene-amended soils. Int. J. Phytoremed. (accepted).

Pawlowska, T.E., D. D. Douds, and I. Charvat. 1999. In vitro propagation and life cycle of an arbuscular mycorrhizal fungus Glomus etunicatum. Mycological Res. 103:1549-1556.

Pedersen, C. T., and D. M. Sylvia. 1997. Limitations in the use of benomyl in evaluating mycorrhizal functioning. Biol. Fertil. Soils 25:163-168.

Pedersen, C. T., D. M. Sylvia, and D. G. Shilling. 1999. Pisolithus arhizus ectomycorrhiza affects plant competition for phosphorus between Pinus elliottii and Panicum chamaelonche. Mycorrhiza 9:199-204.

Pfeffer, P. E., D. D. Douds, G. Bécard, and Y. Shachar–Hill. 1999. Carbon uptake and the metabolism and transport of lipids in an arbuscular mycorrhiza. Plant Physiol. 120:587-598.

Ramirez, M. E., D. W. Israel, and A. G. Wollum, II. 1997. Phenotypic and genotypic diversity of similar serotypes of soybean bradyrhizobia from two soil populations. Soil Biol. Biochem. 29:1539‑1545.

Ramirez, M. E., D. W. Israel, and A. G. Wollum, II. 1997. Phenotypic characterization of soybean bradyrhizobia in two soils of North Carolina. Soil Biol. Biochem. 29:1547‑1555.

Redecker, D., J. B. Morton, and T. D. Bruns. 2000. Ancestral lineages of arbuscular mycorrhizal fungi (Glomales). Mol. Phylogen. Evol. 14:276-284.

Redecker, D., J. B. Morton, and T. D. Bruns. 2000. Molecular phylogeny of Glomus sinuosum and Sclerocystis coremioides places both taxa firmly in Glomus. Mycologia 92:282-285.

*Reynolds, C. M., D. C. Wolf, T. J. Gentry, L. B. Perry, C. S. Pidgeon, B. A. Koenen, H. B. Rogers, and C. A. Beyrouty. 1999. Plant enhancement of indigenous soil micro-organisms: A low-cost treatment of contaminated soils. Polar Rec. 35:33-40.

Rillig, M. C., S. F. Wright , M. F. Allen, and C. B. Field. 1999. Rise in carbon dioxide changes soil structure. Nature 400:628.

*Rogers, H. B., C. A. Beyrouty, T. D. Nichols, Jr., D. C. Wolf, and C. M. Reynolds. 1996. Selection of cold-tolerant plants for growth in soils contaminated with organics. J. Contam. Soil 5:171-186.

Runion, G. B., J. A. Entry, S. A. Prior, R. J. Mitchell, and H. H. Rogers. 1998. Effects of elevated atmospheric CO2 and water stress on tissue chemistry and carbon allocation in Pinus palustris seedlings. Tree Physiol. 19:329-335.

Salinas–Garcia, J. R., F. M. Hons, J. E. Matocha, and D. A. Zuberer. 1997. Soil carbon and nitrogen dynamics as affected by long-term tillage and nitrogen fertilization. Biol. Fertil. Soils 25:182-188.

Schultz, P. A., J. D. Bever, and J. B. Morton. 1999. Acaulospora colossica sp. nov. from an old field in North Carolina and morphological comparisons with similar species, A. laevis and A. koskei. Mycologia 91:676-683.

Schutter, M. E., and J. J. Fuhrmann. 1999. Microbial responses to coal fly ash under field conditions. J. Environ. Qual. 28:648-652.

Séjalon-Delmas, N., A. Magnier, D. D. Douds, and G. Bécard. 1998. Cytoplasmic autofluorescence of an arbuscular mycorrhizal fungus Gigaspora gigantea and non destructive fungal observations in planta. Mycologia 90:921-926.

Skipper, H. D., A. G. Ogg, and A. C. Kennedy. 1996. Root biology of grasses and ecology of rhizobacteria for biological control. Weed Technol. 10:610-620.

Stürmer, S. L., and J. B. Morton. 1997. Developmental patterns defining morphological characters in spores of species in Glomus (Glomales, Zygomycetes). Mycologia 89:72‑81.

Stürmer, S. L., and J. B. Morton. 1999. Scutellospora rubra, a new arbuscular mycorrhizal species from Brazil. Mycol. Res. 103:949-954.

Stutz, J. C., and J. B. Morton. 1996. Successive pot cultures reveal high species richness of arbuscular endomycorrhizal fungi in arid ecosystems. Can. J. Bot. 74:1883-1889.

Stutz, J. C., R. Copeman, C. A. Martin, and J. B. Morton. 2000. Patterns of species composition and distribution of arbuscular mycorrhizal fungi in arid regions of southwestern North America and Namibia, Africa. Can. J. Bot. (in press)

Surange, S., A. G. Wollum, II, K. Kumar, and C. S. Nautiyal. 1997. Characterization of Rhizobium from root nodules of leguminous trees growing in alkaline soils. Can. J. Microbiol. 45:1500‑1515.

Sylvia, D. M. 1998. Taskforce on technology-assisted graduate instruction at the University of Florida. J. Nat. Resour. Life Sci. Educ. 27:159-161.

Sylvia, D. M. 2000. Short root densities, ectomycorrhizal morphotypes, and associated phosphatase activity in a slash pine plantation. J. Sustainable For. (in press).

Sylvia, D. M., and D. O. Chellemi. 2000. Interactions among root-inhabiting fungi and their implications for biological control of root pathogens. Adv. Agron. (in press).

Sylvia, D. M., and A. G. Jarstfer. 1997. Distribution of mycorrhiza on competing pines and weeds in a southern pine plantation. Soil Sci. Soc. Am. J. 61:139-144.

Sylvia, D. M., A. Alagely, D. Kent, and R. Mecklenburg. 1998. Mycorrhiza of landscape trees produced in raised beds and containers. J. Arboric. 24:308-315.

Syvertsen, J. P. and J. H. Graham. 1999. Phosphorus supply and arbuscular mycorrhizas increase growth and net gas exchange responses of two Citrus spp. grown at elevated CO2. Plant Soil 208:209-219.

Tavaria, F. K., and D. A. Zuberer. 1998. Effect of low pO2 on colonization of maize roots by a genetically altered Pseudomonas putida [PH6(L1019)]. Biol. Fertil. Soils 26:43-49.

Vance, N. C., and J. A. Entry. 2000. Soil processes important to restoration of a headwaters catchment in the Siskiyou Mountains. For. Ecol. Management (in press).

Waggoner, P. J., and D.A. Zuberer. 1996. Response of nitrification and nitrifying bacteria in mine spoil to urea or ammonium sulfate. Soil Sci. Soc. Am. J. 60:477-486.

Walworth, J. M., C. R. Woolard, J. F. Braddock, and C. M. Reynolds. 1997. Enhancement and inhibition of soil petroleum biodegradation through the use of fertilizer nitrogen: An approach to determining optimum levels. J. Contam. Soils. 6:465‑480.

Wright, S. F. A fluorescent antibody assay for hyphae and glomalin from arbuscular mycorrhizal fungi. Plant Soil (in press)

Wright, S. F., and R. L. Anderson. Aggregate stability and glomalin in alternative crop rotations for the central Great Plains. Biol. Fertil. Soils. (in press)

Wright, S. F., and A. Upadhyaya. 1998. A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant Soil 198:97-107.

Wright, S. F., and A. Upadhyaya. 1998. Comparison of N-linked oligosaccharides of glomalin from arbuscular mycorrhizal fungi and soils by capillary electrophoresis. Soil Biol. Biochem. 13:1853-185.

Wright, S. F., and A. Upadhyaya. 1999. Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps. Mycorrhiza 8:283-285.

Wright, S. F., J. L. Starr, and I. C. Paltineanu. 1999. Changes in aggregate stability and concentration of glomalin, a glycoprotein produced by arbuscular mycorrhizal fungi, during transition from plow- to no-till management. Soil Sci. Soc Am. J. 63:1825-1829.

*Wright, S. F., M. Franke–Snyder, J. B. Morton, and A. Upadhyaya. 1996. Time-course study and partial characterization of a protein on hyphae of arbuscular mycorrhizal fungi during active colonization of roots. Plant Soil 181:193-203.

Xiong, K., and J. J. Fuhrmann. 1996. Comparison of rhizobitoxine-induced inhibition of b-cystathionase from different bradyrhizobia and soybean genotypes. Plant Soil 186:53‑61.

Xiong, K., and J. J. Fuhrmann. 1996. Soybean response to nodulation by wild‑type and an isogenic Bradyrhizobium elkanii mutant lacking rhizobitoxine production. Crop Sci. 36:1267‑1271.

Refereed Journals (submitted; 13):

Bago, B., P. E. Pfeffer, D. D. Douds, J. Brouillette, G. Bécard, and Y. Shachar–Hill. Carbon metabolism in spores of the arbuscular mycorrhizal fungus Glomus intraradices as revealed by NMR spectroscopy. Plant Physiol.

Bever, J. D., P. A. Schultz, A. Pringle, and J. B. Morton. 2000. Arbuscular mycorrhizal fungi: More diverse than meets the eye and the ecological tale of why. BioScience

*Franke–Snyder, M, D. D. Douds, L. E. Drinkwater, P. Wagoner, and J. B. Morton. Diversity of communities of arbuscular (AM) fungi present in conventional versus low-input agricultural sites in eastern Pennsylvania, USA. Applied Soil Ecol.

Gagliardi, J.V., and J. S. Karns. Survival of E. coli O157:H7 from manure and irrigation water in soil and on cover crops. Soil Sci. Soc. Am. J.

*Gagliardi, J. V., R. S. Angle, J. J. Germida, R. C. Wyndham, C. P. Chanway, R. J. Watson, C. Greer, H. H. Yu, T. McIntyre, M. A. Levin, E. Russek–Cohen, S. Rosolen, J. Nairn, A. Seib, T. Martin–Heller, and G. Wisse. Intact soil-core microcosms for pre-release testing of introduced microbes: Comparison with multi-site field releases in diverse soils and climates. Can. J. Microbiol.

*Gentry, T. J., D. C. Wolf, C. M. Reynolds, and J. J. Fuhrmann. Pyrene influence on soil microbial populations. Bioremed. J.

Nagahashi, G., D. D. Douds, M. Buee, and G. Bécard. Light-induced hyphal branching of germinated AM fungus spores. New Phytol.

Nagahashi, G., D. D. Douds, M. Buee, and G. Bécard. Morphological effects and ecological implications of the effect of root exudate signals upon growth of germinated AM fungus spores. New Phytol.

*Nazih, N., O. Finlay–Moore, P. G. Hartel, and J. J. Fuhrmann. 2000. Whole soil fatty acid methyl ester (FAME) profiles of early soybean rhizosphere as affected by temperature and matric water potential. Soil Biol. Biochem.

Pidgeon, C. S., and C. M. Reynolds. Rhizosphere effect on treatment rates, sequence, and endpoints in a petroleum-contaminated Alaskan soil. Int. J. Phytoremed.

Scantling, M. K., D. C. Wolf, A. L. Waldroup, and C. M. Reynolds. Influence of poultry waste on bioremediation of motor oil-contaminated soil. J. Environ. Qual.

van Berkum, P., and J. J. Fuhrmann. 2000. Evolutionary relationships among the soybean bradyrhizobia reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence. Int. J. Syst. Bacteriol.

Vasilas, L. M., B. L. Vasilas, J. J. Fuhrmann, J. T. Sims, C. M. Hamilton, R. W. Taylor, and W. F. Ritter. Agronomic and environmental implications of different rates of broiler litter amendments to soybeans. Agron. J.

Experiment Station and Extension Bulletins: 1

Patents: 1

Theses: 12 (4 Ph.D. and 8 M.S.)

Abstracts and Proceedings: 147

Non-Refereed Publications: 12

Newspaper, Magazines, etc.: 0

SIGNATURES

Project Title: Soil Microbial Taxonomic and Functional Diversity as Affected by Land

Use and Management

_______________________________________ ________________________

Administrative Advisor Date

_______________________________________ ________________________

Chair, Regional Association of Directors Date

_______________________________________ ________________________

Chair, Multistate Review Committee Date

_______________________________________ ________________________

Administrator, CSREES Date

REFERENCES

Abbott, L. K., and A. D. Robson. 1981. Infectivity and effectiveness of vesicular-arbuscular mycorrhizal fungi: Effect of inoculum source. Aust. J. Agric. Res. 1:631-639.

Anderson, T. A., E. A. Guthrie, and B.T. Walton. 1993. Bioremediation in the rhizosphere. Environ. Sci. Technol. 27:2630-2636.

Aprill, W., and R. Sims. 1993. Evaluation of the use of prairie grasses for stimulating polycyclic aromatic hydrocarbon treatment in soil. Chemosphere 20:253-265.

Berghoff, K. 1998. Beach sediment bacterial contamination and microbial source tracking study. Report from the Resource Management and Science Division, Glen Canyon National Recreation Area, National Park Service, Utah.

Bohm, W. 1979. Methods of studying root systems. Ecological studies. Vol. 33. Springer Verlag, NY.

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

Buchan, A., M. Alber, M. A. Moran, and R. E. Hodson. 1997. An evaluation of subtyping methods for the identification of fecal pollution sources. p. 365-368. In K. J. Hatcher (ed.) Proceedings of the 1997 Georgia Water Resources Conference, March 20-22, Athens, Georgia.

Cattelan, A. J., P. G. Hartel, and J. J. Fuhrmann. 1999. Screening plant growth-promoting rhizobacteria (PGPR) to promote early soybean growth. Soil Sci. Soc. Am. J. 63:1670-1680.

Clesceri, L. S., A. E. Greenberg, and A. D. Eaton. 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, American Water Works Association, and Water Environment Federation, Washington, DC.

Coleman, D. C, J. Dighton, K. Ritz, and K. E. Giller. 1994. Perspectives on the compositional and functional analysis of soil communities. p. 261-271. In K. Ritz, J. Dighton, and K. E. Giller (ed.) Beyond the biomass: Compositional and functional analysis of soil microbial communities. John Wiley & Sons, NY.

Cundell, A. M. 1977. The role of microorganisms in the revegetation of strip-mined land in the Western United States. J. Range Management 30:299-305.

Cunningham, S. D., and D. W. Ow. 1996. Promises and prospects of phytoremediation. Plant Physiol. 110:715-719.

Cunningham, S. D., T. A. Anderson, A. P. Schwab, and F. C. Hsu. 1996. Phytoremediation of soils contaminated with organic pollutants. Adv. Agron. 56:55-114.

Curl, E. A., and B. Truelove. 1985. The rhizosphere. Adv. Ser. Agric. Sci. 15. Springer–Verlag, NY.

Dixon, J. B., H. S. Arora, F. M. Hons, P. E. Askenasy, and L. P. Hossner. 1980. Chemical, physical, and mineralogical properties of soils, mine soil, and overburden associated with lignite mining. p. 12-21. In L. R. Hossner (ed.) Reclamation of surface-mined lignite spoil in Texas. The Texas Agricultural Experiment Station and The Center for Energy and Mineral Resources, College Station, TX.

Douds, D. D., Jr., and P. D. Millner. 1999. Biodiversity of arbuscular mycorrhizal fungi in agroecosystems. Agric. Ecosyst. Environ. 74:77-93.

Elliott, M. L., and E. A. Des Jardin. 1999. Effect of organic nitrogen fertilizers on microbial populations associated with bermudagrass putting greens. Biol. Fertil. Soils 28:431-435

Faith, N. G., J. A. Shere, R. Brosch, K. W. Arnold, S. E. Ansay, M. S. Lee, J. B. Luchansky, and C. W. Kasper. 1996. Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin. Appl. Environ. Microbiol. 62:1519-1525.

Farag, A., and J. Goldstein. 1998. Water quality in the backcountry of Grand Teton National Park. Report from the Environmental and Contaminants Research Center, USGS Biological Resources Division, Jackson, WY.

Farber, J. M. 1996. An introduction to the hows and whys of molecular typing. J. Food Protect. 59:1091-1101.

Finlay, B. J., G. F. Esteban, J. L. Olmo, and P. A. Tyler. 1999. Global distribution of free-living microbial species. Ecography 22:138-144.

Fletcher, J. S., P. K. Donnelly, and R. S. Hegde. 1995. Biostimulation of PCB-degrading bacteria by compounds released from plant roots. p. 131-136. In R. E. Hinchee, D. B. Anderson, and R. E. Hoeppel (ed.) Bioremediation of recalcitrant organics. Battelle Press, Columbus, OH.

Franzluebbers, A. J., N. Nazih, J. A. Stuedemann, J. J. Fuhrmann, H. H. Schomberg, and P. G. Hartel. 1999. Soil carbon and nitrogen pools under low- and high-endophyte-infected tall fescue. Soil Sci. Soc. Am. J. 63:1687-1694.

Geldreich, E. E. 1970. Applying bacteriological parameters to recreational water quality. J. Am. Water Works Assoc. 62:113-120.

Gemma, J. N., R. E. Koske, N. Jackson, and K. M. De Anthonis. 1997a. Mycorrhizal fungi improve drought resistance in creeping bentgrass. J. Turfgrass Sci. 73:15-29.

Gemma, J. N., R. E. Koske, E M. Roberts, and N. Jackson. 1997b. Enhanced establishment of bentgrasses by arbuscular mycorrhizal fungi. J. Turfgrass Sci. 73:9-14.

Graham, J. H., and D. M. Eissenstat. 1994. Host genotype and the formation and function of VA mycorrhizae. Plant Soil 159:179-185.

Graham, J. H., D. L. Drouillard, and N. C. Hodge. 1996. Carbon economy of sour orange in response to different Glomus spp. Tree Physiol. 16:1023-1029.

Grimont, F., and P. A. D. Grimont. 1986. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur/Microbiol. (Paris) 137B:165-175.

Guthrie, E. A., and F. K. Pfaender. 1998. Reduced pyrene bioavailability in microbially active soils. Environ. Sci. Technol. 32:501-508.

Harris, P.A., and D.A. Zuberer. 1993. Subterranean clover enhances production of ‘Coastal’ bermudagrass in the revegetation of lignite mine spoil. Agron. J. 85:236-241.

Hartel, P. G., W. I. Segars, N. Stern, J. Steiner, and A. Buchan. 1999. Ribotyping to determine the host origin of Escherichia coli isolates in different water samples. p. 377-382. In D. S. Olsen and J. P. Potyondy (ed.) Wildland hydrology. American Water Resources Association Technical Publications Series TPS-99-3, Herndon, VA.

Jakobsen, I., L. K. Abbott, and A. D. Robson. 1992. External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. I. Spread of hyphae and phosphorus inflow into roots. New Phytol. 120:371-380.

Johnson, N. C. 1993. Can fertilization of soil select less mutualistic mycorrhizae? Ecol. Applic. 3:749-757.

Johnson, N. C., P. J. Copeland, R. K. Crookston, and F. L. Pfleger. 1992. Mycorrhizae: Possible explanation for yield decline with continuous corn and soybean. Agron. J. 84:387‑390.

Kennedy, A. C. 1999. Bacterial diversity in agroecosystems. Agric. Ecosyst. Environ. 74:65-76.

Kennedy, A. C. and K. L. Smith. 1995. Soil microbial diversity and the sustainability of agricultural soils. p. 75-86. In H. P. Collins, G. P. Robertson and M. J. Klug (ed.) The significance and regulation of soil biodiversity. Kluwer Academic Publishers, The Netherlands.

Kim, J. H., H. D. Skipper, L. C. Miller, A. R. Mazur, and M. L. Elliott. 1999. Seasonal shifts of rhizobacteria in new USGA greens. Agron. Abstr. p. 135.

Kjøller, R., and S. Rosendahl. 2000. Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR and SSCP (single stranded conformation polymorphisms). Plant Soil (in press)

Koske, R. E., J. N. Gemma, and N. Jackson. 1997a. Mycorrhizal fungi associated with three species of turfgrass. Can. J. Bot. 75:320-332.

Koske, R. E., J. N. Gemma, and N. Jackson. 1997b. A preliminary survey of mycorrhizal fungi in putting greens. J. Turfgrass Sci. 73:2-8.

Macnaughton. S. J., J. R. Stephen, A. D. Venosa, G. A. Davis, U. J. Chang, and D. C. White. 1999. Microbial population changes during bioremediation of an experimental oil spill. Appl. Environ. Microbiol. 65:3566–3574.

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning. A laboratory manual. Cold Spring Harbor, NY.

Miller, R. V., and J. S. Poindexter. 1994. Strategies and mechanisms for field research in environmental bioremediation. American Academy Microbiol., Washington, DC.

Mott, J. B., and D. A. Zuberer. 1987. Occurrence of vesicular- arbus­cular mycorrhizae in mixed overburden mine spoils of Texas. Reclama­tion Reveget. Res. 6:145-156.

Mott, J. B., and D. A. Zuberer. 1991. Natural recovery of microbial populations in mixed overburden surface-mine spoils of Texas. Arid Soil Res. and Rehab. 5:21-34.

Naeem, S., L. J. Thompson, S. P. Lawer, J. H. Lawton, and R. M. Woodfin. 1994. Declining biodiversity can alter the performance of ecosystems. Nature 368:734-737.

Oka, N., P. G. Hartel, O. Finlay–Moore, J. Gagliardi, D. Zuberer, J. J. Fuhrmann, J. S. Angle, and H. D. Skipper. 2000. Misidentification of soil bacteria by fatty acid methyl ester (FAME) and BIOLOG analyses. Biol. Fertil. Soils (in press).

Olexa, T. J., T. J. Gentry, P. G. Hartel, D. C. Wolf, J. J. Fuhrmann, D. M. Sylvia, and C. M. Reynolds. 2000. Mycorrhizal colonization and microbial community structure in the rhizosphere of annual ryegrass grown in pyrene-amended soils. Int. J. Phytoremed. (accepted).

Parveen, S., K. M. Portier, K. Robinson, L. Edmiston, and M. L. Tamplin. 1999. Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution. Appl. Environ. Microbiol. 65:3142-3147.

Peach, A., J. J. Fuhrmann, and D. A. Zuberer. 1999. Diversity of microbial communities in east Texas lignite mine soils. Agron. Abstr. p. 227.

Peng, S., D. M. Eissenstat, J. H. Graham, and K. Williams. 1993. Growth depression in mycorrhizal citrus at high phosphorus supply: Analysis of carbon costs. Plant Physiol. 101:1063‑1071.

Reilley, K. A., M. K. Banks, and A. P. Schwab. 1996. Organic chemicals in the environment. J. Environ. Qual. 25:212-219.

Reynolds, C. M., and B. A. Koenen. 2000. Rhizosphere-enhanced remediation project
at Osan Air Base, South Korea, April 2000. CRREL Contract Report, Hanover, NH (in press).

Samadpour, M., and N. Chechowitz. 1995. Little Soos Creek microbial source tracking. Report to Surface Water Management Division, King County Department of Public Works, Seattle, WA.

Savageau, M. A. 1983. Escherichia coli habitats, cell types, and molecular mechanisms of gene control. Am. Naturalist 122:732-743.

Schenck, N. C., and J. O. Siquiera. 1987. Ecology of VA mycorrhizal fungi in temperate agroecosystems. p. 2-4. In D. M. Sylvia, L. L. Hung, and J. H. Graham (ed.) Mycorrhizae in the next decade—Practical solutions and research priorities. Univ. of Florida, Gainesville.

Schwab, A. P., M. K. Banks, and M. Arunachalam. 1995. Biodegradation of polycyclic aromatic hydrocarbons in rhizosphere soil. p. 23-29. In D. B. Hinchee, T. A. Anderson, and R .E. Hoeppel (ed.) Bioremediation of recalcitrant organics. Battelle Press, Columbus, OH.

Simmons, G. M., Jr., and S. A. Herbein. 1998. Potential sources of Escherichia coli (E. coli) to Children's Pool in La Jolla, California. Final Report for the City of San Diego and the County of San Diego Department of Environmental Health.

Simmons, G. M., Jr., S. A. Herbein, and C. M. James. 1995. Managing nonpoint fecal coliform sources to tidal inlets. Universities Council on Water Resources. Water Resources Update, Issue 100:64-74.

Skipper, H. D. 1998. Bioremediation of contaminated soils. p. 469-481. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Skipper, H. D., and R. F. Turco. 1995. Bioremediation: Science and applications. Soil Science Society of America Special Publication No. 43, Madison, WI.

Snaydon, R. W. 1991. Replacement or additive designs for competitive studies. J. Appl. Ecol. 28:930-946.

Staley, J. T. 1999. Bacterial biodiversity: A time and place. ASM News 65:681-687.

Sylvia, D. M. 1994. Vesicular-arbuscular mycorrhizal (VAM) fungi. p. 351-378. In R.W. Weaver et al. (ed.) Methods of soil analysis, Part 2. Microbiological and biochemical properties. Soil Science Society of America, Madison, WI.

Sylvia, D. M. 1998. Mycorrhizal symbioses. p. 408-426. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ.

Tate, R. L., and D. A. Klein, D.A. 1985. Soil reclamation practices: Microbiological analysis and applications. Marcel Dekker Inc., NY.

Tilman, D. 1996. Biodiversity: Population versus ecosystem stability. Ecology 77:350-363.

Tippets, N. 1999. Backcountry water quality testing in Grand Teton National Park—1998 Summer season. Report from the Environmental and Contaminants Research Center, USGS Biological Resources Division, Jackson, Wyoming.

Waggoner, P. J., and D. A. Zuberer. 1996. Response of nitrification and nitrifying bacteria in mine spoil to urea or ammonium sulfate. Soil Sci. Soc. Am. J. 60:477-486.

Wiltse, C. C., W. L. Rooney, Z. Chen, A. P. Schwab, and M. K. Banks. 1998. Greenhouse evaluation of agronomic and crude oil-phytoremediation potential among alfalfa genotypes. J. Environ. Qual. 27:169-173.

Wright, S. F., and A. Upadhyaya. 1997. Extraction of an abundant and unusual protein from soil and comparison with hyphal protein of arbuscular mycorrhizal fungi. Soil Sci. 161:575-586.

Zhang, Y. M., W. J. Maier, and R. M. Miller. 1997. Effect of rhamnolipids on the dissolution, bioavailability and biodegradation of phrenanthrene. Environ. Sci. Technol. 31:2211-2217.

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