Source: UNIV OF MASSACHUSETTS submitted to
AMAZON RAINFOREST MICROBIAL OBSERVATORY: FUNCTIONAL DIVERSITY, TAXONOMIC DIVERSITY, AND RESPONSE TO ECOSYSTEM CONVERSION
 
PROJECT DIRECTOR: Nusslein, K. R. Tiedje, J. M. Bohannan, B. J. Rodrigues, J. L.
 
PERFORMING ORGANIZATION
MICROBIOLOGY
UNIV OF MASSACHUSETTS
AMHERST,MA 01003
 
NON TECHNICAL SUMMARY: Understanding the causes and consequences of biological diversity is a central goal of biology. Studies of the diversity of plants and animals have shown that biodiversity is heterogeneously distributed across the Earth, with a few regions that contain the majority of plant and animal diversity. One of the best studied is the rainforest of Amazonia, Brazil. This region is under great threat; conversion to agriculture has resulted in the loss of over 17% of the original rainforest. At this rate, the Amazon rainforest could cease to exist by the end of the century. Despite its great plant and animal diversity, and its long history of diversity studies, the Amazon rainforest is among the least understood ecosystems regarding bacterial diversity. There is reason to believe that microbial diversity could be very high in this region. First, the high diversity of macroorganism species in the region reflects the great environmental variation present, variation that could also result in very high microbial diversity. Second, the existence of such high macroorganism diversity could provide a wide variety of substrates for microbial metabolism. However, there currently exist only three very limited studies of bacterial diversity in the Amazon rainforest, and these studies present very conflicting views regarding the diversity of bacterial life. In addition, nothing is known how agriculutural land use change influences soil bacterial diversity patterns in this region. We propose to establish a Microbial Observatory (MO) in the Amazon rainforest. The proposed Observatory will have three overarching goals: to search for biological novelty, to describe the bacterial communities of Amazon rainforest soil, and to determine the effect of agricultural conversion on these communities. We will use intensive and extensive culture-independent methods, combined with novel culture-dependent methods, to achieve these goals. This proposal describes a four-year, multi-PI effort to investigate the bacterial diversity of the native Amazonian rainforest, and how agriculturally driven ecosystem conversion can alter this diversity. The research plan will be completed through a strong interdisciplinary and collaborative effort between the University of Massachusetts (PI Nnsslein), Michigan State University (Co-PI Tiedje), the University of Texas at Arlington (Co-PI Rodrigues), and the University of Oregon (Co-PI Bohannan). This proposal offers the following advantages: It determines rainforest soil microbial diversity. It is focused on the largest planet ecosystem. It combines different taxonomic and functional levels of diversity analysis. It utilizes three measures of diversity. It considers spatial diversity analysis. Discovery of novelty through isolation of Acidobacteria. Consideration of two microbial groups with different ecophysiological strategies. Archive for a large soil databank available to other researchers.
 
OBJECTIVES: An increasing demand for grain, fibers, and biofuel has accelerated the relentless conversion of the Amazon ecosystem to agriculture, creating a new frontier of deforestation of 1 million sq. km. The consequences of this shift are expected to be significant to global emissions of greenhouse gases, global warming, soil degradation, and water resources. Macro-biological studies of the diversity of plant and animals have revealed the rainforest as one of the biodiversity hotspots. However, the Amazon rainforest, credited as the largest biological reservoir of animal and plant species on Earth, is among the least understood ecosystems regarding bacterial diversity. The forest soil microbial diversity is poorly characterized and the only three studies currently present conflicting views about its microbiological diversity. Understanding the causes, patterns, and consequences of soil microbiological diversity and its importance to the rainforest soil ecosystem function is a central goal of this Microbial Observatory. We propose to establish baseline diversity at broad coverage in the Amazon rainforest, and determine how microbial diversity changes in response to land use by ecosystem conversion from rainforest to agriculture. Our conceptual model for this research is guided by three questions: 1. Do magnitude and spatial scaling of microbial diversity mirror that of tropical plant and animal diversity 2. Are spatial patterns in soil microbial diversity less pronounced at higher levels of taxonomic resolution[unclear, remove altogether] 3. Is the bacterial diversity in Amazon Forest reduced by agricultural development The range of Expected Outputs and Impacts of the proposed research will be consolidated through enhancing international collaboration with a team of Brazilian biogeochemists and microbiologists. It will allow postdoctoral research associates and undergraduate students to interact with their Brazilian peers to exchange scientific information, giving them a chance to think globally and to learn from different perspectives, and will provide educational opportunities through training on state of the art genome sequencing techniques, metagenomics annotation, spatial patterns of functional genes, isolation, and taxonomic classification. By disseminating our results widely, we will improve understanding of microbial diversity at different levels of resolution: spatial distribution patterns, and gene patterns linked to habitat. We will not only establish previously unavailable base line data on tropical soil diversity, but investigate also the impact ecosystem conversion has on that diversity. This will allow us to better estimate consequences of common agricultural conversions. In addition, we will define the level of resolution at which soil bacterial diversity is established.
 
APPROACH: Soil is often avoided in Microbial Ecology studies in favor of environmental extremes to encounter a more manageable level of bacterial diversity. In this MO we directly target soil microbial diversity in the largest plant ecosystem in the world. We will assess the genomic variability of members of the genus Burkholderia as one measure of diversity, and follow the shift in Burkholderia diversity from native rainforest via the impact of deforestation to agricultural monoculture. Measurements of diversity of N2 fixation characteristics of Burkholderia strains corroborate this study at a functional level. This work will contribute better estimates soil microbial diversity in Amazonia, one of largest biological reservoir of world, and how and at what level of resolution that diversity follows a pattern. Our strategy to discover biological novelty and how it is influenced by agriculturally driven land use change is as follows. We will determine tropical bacterial diversity at several levels of resolution using culture dependent vs. culture independent approaches, and investigate how diversity is influenced by landscape changes. Our initial attempt to answer "Who is there" will be discovery driven to establish a baseline of bacterial diversity in the Amazon. Dominant and rare species composition will be described at high coverage using a variant of the 454 sequencing technology, called Ultra-Deep Sequencing. Species composition will be paralleled by a characterization of the diversity of known functional genes based on the extensive gene microarray termed GeoChip and round up the core datasets for diversity. To address the question whether agricultural development alters this diversity we will engage a hypothesis driven research approach in which we employ both culture independent as well as culture dependent techniques across a chronosequence of four land uses. The above listed DNA based core diversity analyses will be applied to all treatments. To deepen our insight into diversity shifts we will then focus on the common soil bacterial genera Burkholderia and Acidobacteria as a proxy of influences on diversity. In an extension of the massively parallel 454 sequencing technique we termed Multi Locus Community Profiling we will characterize and compare the functional and taxonomic diversity of defined target genes of both genera within the soil community. For select soil samples traditional clone library analysis of the gene for nitrogen fixation, nifH, and recA will confirm MLCP. Burkholderia diversity can then be efficiently speciated using a Burkholderia Phyloarray. The array will help with grouping the isolates. Culture independent analyses will be paralleled by strategic attempts to isolate a large variety of Burkholderia and Acidobacteria. Hypothesizing that different ecological strategies for survival are imprinted in the isolates genomes, we will apply Multi Locus Sequencing Typing to investigate genomic patterns of isolates relative to soil treatments.
 
CRIS NUMBER: 0216773 SUBFILE: CRIS
PROJECT NUMBER: MAS0200804556 SPONSOR AGENCY: NIFA
PROJECT TYPE: NRI COMPETITIVE GRANT PROJECT STATUS: NEW MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Jan 1, 2009 TERMINATION DATE: Dec 31, 2012

GRANT PROGRAM: BIOLOGY OF PLANT-MICROBE ASSOC.
GRANT PROGRAM AREA: Plant Systems

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
131019911006.110%
131064011006.110%
131401011006.130%
136019911006.310%
136064011006.310%
136401011006.330%

CLASSIFICATION HEADINGS
KA131 - Alternative Uses of Land
KA136 - Conservation of Biological Diversity
S0199 - Soil and land, general
S4010 - Bacteria
S0640 - Tropical forests
F1100 - Bacteriology
G6.1 - Ensure Clean Water and Air
G6.3 - Protect and Manage Forests and Rangelands


RESEARCH EFFORT CATEGORIES
BASIC 70%
APPLIED 10%
DEVELOPMENTAL 20%

KEYWORDS: amazon rainforest~soil microbiology~soil microbial diversity~land use change~microbial observatory~454 sequencing~biological novelty~spatial diversity~acidobacteria~burkholderia~multi locus community profiling~soybean

PROGRESS: Jan 1, 2010 TO Dec 31, 2010
OUTPUTS: Land use change is one of the greatest threats to biodiversity worldwide, especially in tropical forests. The Amazon Rainforest is the Earth's largest reservoir of plant and animal diversity and it has been subject to especially high rates of land use change, primarily to cattle pasture. This conversion is predicted to result in a significant loss of biodiversity if it continues at current rates. These estimates are based solely on studies of plants and animals, and it is unclear how microbial diversity responds to such land use change. Past studies suggest that soil microbial diversity may actually increase in response to ecosystem conversion in the Amazon. However, these studies have been limited in sampling depth and extent, and have focused exclusively on local diversity. Sampling and Processing. We sampled again and more extensively in the spring of 2010. Unfortunately, after some of the sampling sites had been outsourced to a company for selective logging we found them completely destroyed and no longer useful for additional sampling. New sampling sites were established and sampled after their future use was confirmed with land owners. Additional secondary forest sites were added. We collected and archived over 165 new soil cores from the sampling expeditions in 2010. These samples have been divided for chemical and physical analyses, cultivation of the target groups Acidobacteria and Burkholderia, and isolation of total soil DNA for 454 pyrosequencing of the 16S rRNA gene. In addition, we have under cultivation over 800 nitrogen fixers from these samples. This year we also established a vegetation profile. The site selected for ARMO is the Fazenda Nova Vida, a research station that is part of the Large Scale Biosphere-Atmosphere Experiment in Amazonia (www.lbaeco.org/lbaeco/). The general plant community composition at Fazenda Nova Vida has been described previously by one of our collaborators in Brazil, and we have now confirmed this data set. In brief, the pastures are dominated by the grasses Urochloa brizantha, Urochloa decumbens (formerly the genus Brachiaria), or Panicum maximum (or a mix of two of the three). The primary forest is a typical wet Terra Firme forest of the "Open Forest with Palms" variety. Identified species in the primary forest include Orbignya phalerata, Tabebuia spp., Erisma uncinatum, and Vismia guianensis, although the majority of species are unidentified. Group Meetings. During our travels to the sampling site we had a planned stop over at the University of Sao Paulo to meet with all Brazilian collaborators over the period of 1.5 days. During this meeting we shared all current datasets, discussed ways to improve on ways to get import permits from the USDA, defined a new DNA extraction strategy to be used by all project members, improved our sampling strategy, and established a list of planned manuscripts and rules for authorship. We also defined overlapping interests with our Brazilian collaborators and potentials for future funding to extend this project in the study of land use change from forest to soybean plantations. PD Nusslein presented the most recent finding at the USDA PD meeting in January 2011. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: 2010-01-01 TO 2010-12-31 During this cycle we have obtained high quality genomic DNA from weathered soils in past projects (PD Klaus Nusslein has extensive experience with Hawaiian soils). Our extraction protocols have been tested with the Amazon Rainforest soils on a total of 165 soil samples to extract DNA for ultra deep sequencing of the 16S rRNA gene via 454 pyrosequencing. Due to problems with the 454 reaction cocktail delivered by the company we are still delayed in the complete sequencing of this sample set (currently 92% completed). Functional genes for both ammonia oxidation and methane oxidation shift in their phylogenetic diversity with land use, and land use treatment specific groups establish over time. We have submitted a proposal to the DOE-JGI Community Sequencing Program to establish a metagenomics proposal after the white paper was accepted. Although the importance of our research goals was confirmed by the reviewers our project was not chosen for sequencing. After intense discussions with the program officers at a DOE-JGI meeting this year we will resubmit this proposal for next year's funding cycle. After the analyses of the year one sampling data set we show that conversion of the Amazon Rainforest into cattle pasture significantly increases local taxonomic richness and phylogenetic diversity of soil bacteria, but decreases community turnover and average similarity, resulting in biotic homogenization of bacterial biodiversity. This homogenization is driven in part by the loss of endemic forest soil bacteria, and seems to be of sufficient magnitude to result in a net loss of bacterial diversity. In summary: (i) Land use change "randomizes" microbial communities, but reduces community similarity. (ii) Recovery (primary to secondary rainforest) does seem to happen on the scale of community composition, and the effect of ecosystem conversion is consistent across years (same in pastures; p <0.001). (iii) The Impact of land use is long lasting. (iv) Microbial biogeography responds to land use change in a way similar to plants, but microbial diversity (alpha, beta and gamma) responds in a way different from plants. (v) Land use change alters the structures of aerobic ammonia oxidizing and of methane oxidizing bacteria; (vi) Species abundance of culturable nitrogen fixing isolates changes with land use. Given that soil bacteria represent the majority of biodiversity in terrestrial ecosystems and are intimately involved in ecosystem functions (including carbon sequestration) this loss should be of concern, and microbial biodiversity should be taken into account when assessing the impact of land use change in tropical forests.

PUBLICATION INFORMATION: 2010-01-01 TO 2010-12-31
No publications reported this period

PROJECT CONTACT INFORMATION
NAME: Cromack, P.
PHONE: 413-545-5913
FAX: 413-545-1977