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Bacterial diversity of terra preta and pristine forest soil from the Western Amazon

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Abstract

The survey presented here describes the bacterial diversity and community structures of a pristine forest soil and an anthropogenic terra preta from the Western Amazon forest using molecular methods to identify the predominant phylogenetic groups. Bacterial community similarities and species diversity in the two soils were compared using oligonucleotide fingerprint grouping of 16S rRNA gene sequences for 1500 clones (OFRG) and by DNA sequencing. The results showed that both soils had similar bacterial community compositions over a range of phylogenetic distances, among which Acidobacteria were predominant, but that terra preta supported approximately 25% greater species richness. The survey provides the first detailed analysis of the composition and structure of bacterial communities from terra preta anthrosols using noncultured-based molecular methods.

Introduction

Terra preta anthrosols in the Amazon basin are nutrient rich soils that contain thick, dark colored, surface horizons with a high organic matter content (Lima et al., 2002) and are noted for their exceptional fertility and ability to accumulate stable organic carbon. Prior studies suggest these soils were formed by pre-Columbian, Amerindians, who practiced a “slash and char” agriculture (Mann, 2002). The ability of terra preta to accumulate stable organic matter has attracted attention as a possible means for increasing fertility and carbon storage in tropical soils (Sombroek, 1966; Lehmann et al., 2003), and has provoked questions regarding the role of microorganisms in the formation and plant growth promotion characteristics of these soils (Thies and Suzuki, 2003). The objective of this study was to carry out a survey of the bacterial diversity in terra preta and pristine forest soil as the basis for studies on the ecology of these forest soils.

To characterize bacterial diversity in the terra preta and pristine forest soil, samples were collected from two adjacent locations in the Jamari National Forest, of Rondonia, Brazil (latitude: 8° 45′ 0 S, longitude: 63° 27′ 0 W). The soil from the pristine forest was a clay loam ultisol having a 1 cm A horizon under a layer of moist duff. The B horizon consisted of a highly oxidized ultisol with an acid pH of 3.1 and had negligible organic matter. The terra preta samples were collected at a site two hundred meters distant where the soil contained a deep, organic matter rich, sandy loam in the A horizon that was approximately 1 m thick before transitioning into the underlying clay ultisol. Intact soil cores measuring 10 cm in depth and 8 cm in width were collected from the surface horizon below the organic litter layer using sterile, stainless steel soil coring sleeves. The cores were shipped immediately to the University of California, Riverside, USA, where they were frozen at −80 °C. At the time of analysis, three cores from each site were combined into one composite sample and sieved to pass a 1.0 mm screen. The composite samples were analyzed for their chemical and physical characteristics and metal contents (Supplemental Table 1, online supplementary material) and were subjected to molecular analyses to identify the predominant bacteria and to describe the bacterial community structures at different phylogenetic distances based on comparisons of 16S rRNA gene sequences recovered from the samples.

The analysis of bacterial diversity employed a two step procedure in which the first step was to sort 16S rRNA clone libraries with a method referred to as oligonucleotide fingerprinting of ribosomal genes (OFRG) (Valinsky et al., 2002). This was followed by sequencing of random clones from the taxonomic clusters generated by OFRG to identify the predominant bacteria. The gene sequences were also used to construct phylogenetic trees based on actual sequence data. To produce the 16S rRNA gene clone libraries, near full-length (1465 bp) small-subunit rRNA gene sequences were PCR amplified from soil DNA. The purified DNA was ligated into pGEM-T (Promega) and transformed into competent Escherichia coli JM109 (Promega). Libraries of the rRNA genes were represented by 768 clones for each sample, which were arrayed on replicate nylon membranes for hybridization with 33P DNA end-labeled oligonucleotide probes as described by Valinsky et al., (2002). Signal intensities with background correction were obtained using ImaGene 4.0 software (Biodiscovery) and were transformed for every probe into three values: 0, 1, and N, where 0 and 1 indicate negative and positive hybridization events, respectively, and N indicates an uncertain assignment. This process creates a hybridization fingerprint for each clone, which is a vector of values resulting from hybridizations with all probes. Estimates of diversity using 16S rRNA gene sequences were examined based at 95%, and 90% similarities using the PHYLIP-formatted distance matrices generated by the computer program PAUP 4.0 (Sinauer Associates, Inc.).

To identify the taxonomic groups separated by OFRG, nucleotide sequences were determined for 76 and 49 rRNA gene clones that were randomly selected from within each of the clusters generated by OFRG. The 16S rRNA gene sequences were submitted to the BLAST server (Basic Local Alignment Search Tool), National Center for Biotechnology Information (NCBI) to determine the closest matching sequences in the GenBank and to infer phylogenetic affiliations. Novel sequences were deposited at GenBank and were assigned accession numbers AY326512–AY326636genbank:AY326512genbank:AY326513genbank:AY326514genbank:AY326515genbank:AY326516genbank:AY326517genbank:AY326518genbank:AY326519genbank:AY326520genbank:AY326521genbank:AY326522genbank:AY326523genbank:AY326524genbank:AY326525genbank:AY326526genbank:AY326527genbank:AY326528genbank:AY326529genbank:AY326530genbank:AY326531genbank:AY326532genbank:AY326533genbank:AY326534genbank:AY326535genbank:AY326536genbank:AY326537genbank:AY326538genbank:AY326539genbank:AY326540genbank:AY326541genbank:AY326542genbank:AY326543genbank:AY326544genbank:AY326545genbank:AY326546genbank:AY326547genbank:AY326548genbank:AY326549genbank:AY326550genbank:AY326551genbank:AY326552genbank:AY326553genbank:AY326554genbank:AY326555genbank:AY326556genbank:AY326557genbank:AY326558genbank:AY326559genbank:AY326560genbank:AY326561genbank:AY326562genbank:AY326563genbank:AY32564genbank:AY326565genbank:AY326566genbank:AY326567genbank:AY326568genbank:AY326569genbank:AY326570genbank:AY326571genbank:AY326572genbank:AY326573genbank:AY326574genbank:AY326575genbank:AY326576genbank:AY326577genbank:AY326578genbank:AY326579genbank:AY326580genbank:AY326581genbank:AY326582genbank:AY326583genbank:AY326584genbank:AY326585genbank:AY326586genbank:AY326587genbank:AY326588genbank:AY326589genbank:AY326590genbank:AY326591genbank:AY326592genbank:AY326593genbank:AY326594genbank:AY326595genbank:AY326596genbank:AY326597genbank:AY326598genbank:AY326599genbank:AY326600genbank:AY326601genbank:AY326602genbank:AY326603genbank:AY326604genbank:AY326605genbank:AY326606genbank:AY326607genbank:AY326608genbank:AY326609genbank:AY32610genbank:AY326611genbank:AY326612genbank:AY326613genbank:AY326614genbank:AY326615genbank:AY326616genbank:AY326617genbank:AY326618genbank:AY326619genbank:AY326620genbank:AY326621genbank:AY326622genbank:AY326623genbank:AY326624genbank:AY326625genbank:AY326626genbank:AY326627genbank:AY326628genbank:AY326629genbank:AY326630genbank:AY326631genbank:AY326632genbank:AY326633genbank:AY326634genbank:AY326635genbank:AY326636.

Phylogenetic trees based on the sequenced clones were constructed using the 16S rRNA gene sequence data. Nucleotide sequences were aligned using Clustal X, after which an evolutionary distance matrix was generated using the program MEGA3 (Kumar et al., 2004) using the N-J method. Bootstrap analyses of the neighbor-joining data were conducted based on 1000 samplings to assess the stability of the phylogenetic relationships. Rarefaction curves and estimates of species diversity at different phylogenetic distances were determined using the computer program DOTUR (Schloss et al., 2004). Comparisons of the species coverage and overall community similarities at different phylogenetic distances were determined using the program LIBSHUFF (Singleton et al., 2001).

Section snippets

Results and discussion

The Amazon contains diverse soils of which only a few have been characterized with respect to their microbiology. To date there has been only one prior survey of Amazon soils that used molecular methods to analyze soil microbial diversity in which 100 clones were analyzed (Borneman and Triplett, 1997). The survey data presented here thus provide a more comprehensive survey and the first analysis of terra preta using molecular methods. Inspection of the taxonomic trees generated by OFRG showed

Acknowledgments

The authors gratefully acknowledge Lea Valinsky and James Borneman for assistance with oligonucleotide fingerprinting methods, and Steven Qi for technical assistance.

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