Source: UNIVERSITY OF FLORIDA submitted to
COLLABORATIVE RESEARCH: HOW ARE ACHAEL DIVERSITY, ABUNDANCE, AND FUNCTIONAL REGULATED IN AGROECOSYSTEMS?
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0214983
Grant No.
2008-35319-04593
Project No.
FLA-MCS-004745
Proposal No.
2008-04347
Multistate No.
(N/A)
Program Code
51.8A
Project Start Date
Aug 1, 2008
Project End Date
Jul 31, 2012
Grant Year
2008
Project Director
Triplett, E.
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
MICROBIOLOGY & CELL SCIENCE
Non Technical Summary
The archaea are one of the three domains of life. Despite their relatively high abundance in cultivated soils, soil archaea have never been cultured and their role is not clear. This work will be determining those environmental factors that are correlated with (and may also drive) microbial diversity, high throughput analysis of microbial diversity, culturing, and the biochemistry and physiology of ammonia oxidation. Recent evidence suggests that at least some of the soil archaea are involved in ammonia oxidation and they may play a dominant role in this function. But the ammonia oxidizing activity of these soil organisms is unknown. There is also some evidence that soil Archaea may be far more abundant in cultivated soils than in uncultivated soils. However, this needs to be tested in a rigorous way in multiple locations. The specific effects of disturbance, succession, N fertilization, and cropping systems on the abundance and diversity of these soil organisms is not known. The effects of the physical and chemical properties of soils on archaea are unknown. This proposal addresses all of these questions. Archaea may be the most abundant ammonia-oxidizing organisms in cultivated soils and this may have important implications for agriculture. We believe this work will lay the ground work for an understanding of Archaea in soil that may one day influence agricultural practices.
Animal Health Component
(N/A)
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110110320%
1252420106015%
1364099106025%
9017310209010%
9037110303010%
2054099110320%
Knowledge Area
901 - Program and Project Design, and Statistics; 903 - Communication, Education, and Information Delivery; 125 - Agroforestry; 102 - Soil, Plant, Water, Nutrient Relationships; 205 - Plant Management Systems; 136 - Conservation of Biological Diversity;

Subject Of Investigation
4099 - Microorganisms, general/other; 7310 - Experimental design and statistical methods; 2420 - Noncrop plant research; 0110 - Soil; 7110 - Research on research management;

Field Of Science
1060 - Biology (whole systems); 3030 - Information and communication; 2090 - Statistics, econometrics, and biometrics; 1103 - Other microbiology;
Goals / Objectives
1. The abundance and diversity of archaea in treatments that vary in disturbance, succession, and N fertilization regimes; 2. Archaeal and bacterial abundance and diversity in response to changes in cropping systems and soil pH; 3. Limit of soil archaeal diversity without statistical extrapolation using pyrosequencing of amplified 16S rRNA genes; 4. Abundance and diversity of archaea in 100 soils that represent a broad array of soil/ecosystem types, and 5. Ammonia oxidizing activity of soil archaea compared to the ammonia oxidizing bacteria.
Project Methods
Sites to be sampled for Objectives 1-3. Three sites were determined to be ideal for Objectives 1-3: Kellogg Biological Station (KBS) LTER (http://lter.kbs.msu.edu/) in Michigan, the Cedar Creek LTER (http://www.cedarcreek.umn.edu/) in Minnesota, and the Rothamsted Research experiments in England (http://www.rothamsted.bbsrc.ac.uk/resources/ExperimentsGuide.html). Sampling protocol. In year one, we will sample each plot of each treatment from each experiment three times each month during the growing season. The frequency of sampling may change in subsequent years based on year one results. For each sampling a 1 cm-wide and 10 cm-long core will be taken of the topsoil. Each sample will be frozen immediately in liquid nitrogen; frozen samples will be stored at -80 degrees centigrade until DNA extraction. Minnesota and Michigan soil samples will be shipped to Colorado and Florida, respectively for DNA extraction. SPECIFIC EXPERIMENTS TO BE SAMPLED AT EACH SITE: KBS-LTER: Cropping systems KBS-LTER: Succession KBS-LTER: Nitrogen Deposition KBS-LTER: Nitrogen Fertilization Cedar Creek LTER: Nitrogen deposition: Experiment 001 Cedar Creek LTER: Nitrogen deposition: Experiment 002 Rothamsted Research long-term liming experiments Rothamsted Broadbalk experiment: Effects of N fertilizer and cultivation DNA extraction, ARISA and subsequent data analysis, quantitative PCR. DNA will be extracted from all soil samples and ARISA will be used to obtain bacterial and archaeal fingerprint patterns from these soils. Automated ribosomal intergenic spacer analysis (ARISA), first described by Fisher and Triplett (31), will be used to screen samples and identify broad patterns in archaeal and bacterial diversity across the collected samples. Relative abundances of archaea will be estimated by normalizing qPCR results with archaeal-specific primers to the results with bacterial-specific primers yielding an approximate archaeal:bacterial ratio. Bacterial abundances will be determined using the bacterial-specific qPCR protocol. PYROSEQUENCING - primer choice and single library construction The two primers for the amplification and subsequent pyrosequencing of archaeal 16S rRNA are 770F (ATTAGATACCCSGGTAGTCC) and UA1204r (TTMGGGGCATRCNKACCT). PYROSEQUENCING - multiple library construction Multiple 16S rRNA libraries in one 454 run will be done.The region downstream of the A adaptor will be sequenced. The 8-base bar code will be matched with a particular sample allowing up to 256 libraries to be sequenced in one 454 run. ANALYSIS OF PYROSEQUENCING DATA - single libraries: About 400,000 reads of 16S rRNA genes will be obtained in one run from the 454 GS FLX. Alignment, tree building, and clustering of these many reads will be done by Rob Knight and coworkers at the University of Colorado (CU) and Luiz Roesch and Alberto Riva at UF. They have assembled a set of tools that allow reasonably rapid analysis of such large datasets. Analysis of 400,000 16S rRNA reads from single DNA sample will be performed. ANALYSIS OF PYROSEQUENCING DATA - multiple libraries: We will analyze up to 100 16S rRNA samples sequenced with 4,000 reads.

Progress 08/01/08 to 07/31/12

Outputs
Target Audience: The target audience was the community of scientists interested in agricultural soils, microbes in agricultural soils, and those interested in nitrogen abundance and utilization in soils. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? For the University of Florida portion of this project, we have completed two PhD degrees, trained two postdoctoral reseachers, and provided significant research experiences for seven undergraduates. How have the results been disseminated to communities of interest? Through publication in refereed journals. What do you plan to do during the next reporting period to accomplish the goals? This is the final report.

Impacts
What was accomplished under these goals? The objective of this work was to determine the drivers of the diversity, abundance, and function of archaea in agroecosytems. We have accomplished this objective. As a result of our work, we now know that agroecosystems around the world possess a higher abundance of archaea that non-agricultural soils. We also know that most of the soil archaea in agricultural soils are probably ammonia oxidizers, but they remain uncultured (at least in pure culture) which prevents the needed biochemical evidence to support this role. However, tools have been developed by our group to distinguish the amount of ammonia oxidation done by archaea rather than bacteria. In addition, as a result of this USDA-funded work, we now know that the primer driver of archaea abundance in agricultural soils is free ammonia concentration, which, in turn, is driven by nitrogen fertilization and soil pH. The high abundance of ammonia oxidizing archaea in agricultural soils is reversible. That is, one land is removed from agricultural production and allowed to be reclaimed by native vegetation, the relative abundance of archaea falls to very low levels. The presence of high numbers of archaea in agricultural soils is so significant and distinctive that the ammonia oxidizing archaea can be considered a biomarker for land use. The highest abundance of these organisms found to date is in the soil of the Everglades Agricultural Area in Florida. Non-agricultural soils consistently have a high relative abundance of Bradyrhizobium, a bacterial genus well known for its ability to form nitrogen-fixing nodules on the roots of many legumes but also capable, in some cases, of free living nitrogen fixation in soils. We have also learned with this USDA support that the predominant genus of archaea in soil is Ca. Nitrososphaera. No members of this genus have yet been cultured but we have obtained an enrichment that is about 50% Ca. Nitrososphaera. A genome sequence for one of these strains is now available and has tentatively been given the species name of Ca. Nitrososphaera evergladensis. This genome sequence suggests that this organism is capable of using a very efficient pathway for CO2 fixation. This may have important consequences for future work to sequester CO2 to mitigate the harmful effects of CO2 pollution. We believe that this collective body of work has contributed significantly to our understanding of archaea in soil and suggests an important application for these organisms in CO2 sequestration. Our work has results in the publication of 19 papers in a variety of important journals across all years with all collaborators.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Zhalnina, K.V., Dias, R., Dorr de Quadros, P., Davis-Richardson, A.G., Camargo, F.A.O., Clark, I.M., McGrath, S.P., Hirsch, P.R., Triplett, E.W. 2014. Soil pH and C:N ratio predict microbial diversity and composition in the Rothamsted Park Grass experiment. Microbial Ecology, in revision.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Zhalnina, K.V., Dias, R., Leonard, M.T., Dorr de Quadros, P., Camargo, F.A.O., Drew, J.C., Farmerie, W.G., Daroub, S.H., Triplett, E.W. 2014. Candidatus Nitrososphaera evergladensis genome shows enhanced adaptive ability of ammonia-oxidizing archaea to survive in soil. PLOS ONE, in press.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhalnina, K., Dorr de Quadros, P., Gano, K.A., Davis-Richardson, A.G., Fagen, J.R., Brown, C.T., Giongo, A., Drew, J.C., Sayavedra-Soto, L.A., Arp, D.J., Camargo, F.A.O., Daroub, S.H., Clark, I.M., McGrath, S.P., Hirsch, P.R., Triplett, E.W. 2013. Ca. Nitrososphaera and Bradyrhizobium are inversely correlated and related to agricultural practices in long-term field experiments. Frontiers in Microbiology 4:104.