Soil Biology & Biochemistry 39 (2007) 684–690

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Bacterial diversity of terra preta and pristine forest soil from the Western Amazon
Jong-Shik Kima, Gerd Sparovekb, Regina M. Longoc, Wanderley Jose De Meloc, David Crowleya,Ã
b a Department of Environmental Sciences, University of California, Riverside, CA, USA Department of Soil Science, ESALQ, University of Sao Paulo, Piracicaba CP 9, CEP 13418-900, Brazil c Department of Technology, Universidade Estadual Paulista, Jaboticabal, SP, CEP 14884-900, Brazil

Received 25 May 2006; received in revised form 1 August 2006; accepted 11 August 2006 Available online 18 September 2006

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. r 2006 Elsevier Ltd. All rights reserved.
Keywords: Bacterial diversity; Forest soils; Microbial ecology; Terra preta

1. 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
ÃCorresponding author. Tel.: +1 951 827 3785; fax: +1 951 827 3993.

E-mail address: (D. Crowley). 0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2006.08.010

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: 81 450 0 S, longitude: 631 270 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 À80 1C. At the time of analysis, three cores from

Libraries of the rRNA genes were represented by 768 clones for each sample. Comparisons of the species coverage and overall community similarities at different phylogenetic distances were determined using the program LIBSHUFF (Singleton et al. and N indicates an uncertain assignment. with terra preta having overall greater diversity (Fig. and 90% similarities using the PHYLIP-formatted distance matrices generated by the computer program PAUP 4. respectively. respectively). 2001). This process creates a hybridization fingerprint for each clone. The purified DNA was ligated into pGEM-T (Promega) and transformed into competent Escherichia coli JM109 (Promega). nucleotide sequences were determined for 76 and 49 rRNA gene clones that were randomly selected from within each of the clusters generated by OFRG. Other commonly used measures of diversity including Simpsons Index of Diversity and Chao I. Calculation of Shannon index values using DOTUR yielded significantly different (P40:05) values for terra preta and forest soil (5. Rarefaction curves and estimates of species diversity at different phylogenetic distances were determined using the computer program DOTUR (Schloss et al. As shown using rarefaction. after which an evolutionary distance matrix was generated using the program MEGA3 (Kumar et al. 3). 1. near full-length (1465 bp) small-subunit rRNA gene sequences were PCR amplified from soil DNA. Novel sequences were deposited at GenBank and were assigned accession numbers AY326512–AY326636. 2). Comparison of the forest soil sample set against terra preta revealed that all of the OTU diversity in the forest soil was represented in terra preta and that the forest sample was not significantly different from the terra preta (P ¼ 0:64. Using the program LIBSHUFF to compare the similarities of the communities from each soil at different phylogenetic distances.ARTICLE IN PRESS J.2 and 4. In contrast.. 2002). (2002). 2004). Bootstrap analyses of the neighbor- joining data were conducted based on 1000 samplings to assess the stability of the phylogenetic relationships. The 16S rRNA gene sequences were submitted to the BLAST server (Basic Local Alignment Search Tool). 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.37.). and the number of branches that represent different taxonomic groups.0 mm screen..-S. Nucleotide sequences were aligned using Clustal X. in that it contained additional sequences that did not occur in the forest soil. The survey data presented here thus provide a more comprehensive survey and the first analysis of terra preta using molecular methods. Kim et al. with values of 60% and 68% for the forest soil and terra preta samples sets. 2004) using the N-J method. comparison of the terra preta versus the forest soil showed that terra preta was significantly different (P ¼ 0:05). there was significant separation of OTU richness (95% similarity level) as sample size increased above 50 for each sample set (Fig. This was followed by sequencing of random clones from the taxonomic clusters generated by OFRG to identify the predominant bacteria. it was determined that overall coverage of sequence diversity was high. which is a vector of values resulting from hybridizations with all probes. / Soil Biology & Biochemistry 39 (2007) 684–690 685 each site were combined into one composite sample and sieved to pass a 1. The composite samples were analyzed for their chemical and physical characteristics and metal contents (Supplemental Table 1.. 2. Altogether. Inspection of the taxonomic trees generated by OFRG showed that the two soils differed with respect to the number of operational taxonomic units (OTUs).. data not shown). 1). respectively (Fig. Signal intensities with background correction were obtained using ImaGene 4. which were arrayed on replicate nylon membranes for hybridization with 33P DNA end-labeled oligonucleotide probes as described by Valinsky et al. Estimates of diversity using 16S rRNA gene sequences were examined based at 95%. 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. these analyses suggest that bacterial species richness was approximately 25% greater for terra preta than that in the forest soil. To produce the 16S rRNA gene clone libraries. Results and discussion The Amazon contains diverse soils of which only a few have been characterized with respect to their microbiology. 1997). Enumeration of the unique 16S rRNA gene sequences yielded 396 OTUs from terra preta as compared to 291 OTUs in the forest soil (Table 1). which employ independent mathematical approaches to measuring diversity revealed the same phenomenon. Inc. . National Center for Biotechnology Information (NCBI) to determine the closest matching sequences in the GenBank and to infer phylogenetic affiliations. where 0 and 1 indicate negative and positive hybridization events. The greatest difference in 16S rRNA gene sequences between terra preta and the forest soil occurred for OTUs having up to 5% evolutionary distance (95% 16S rRNA similarity). To identify the taxonomic groups separated by OFRG. Phylogenetic trees based on the sequenced clones were constructed using the 16S rRNA gene sequence data. The gene sequences were also used to construct phylogenetic trees based on actual sequence data. 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.0 (Sinauer Associates.0 software (Biodiscovery) and were transformed for every probe into three values: 0.. and N.

Representatives of the a. whereas the Y and O subgroups are largely absent from acid soils. Complete coverage of the two communities was obtained at greater than 90% similarity. The differences in community composition revealed by the LIBSHUFF analysis were confirmed by the phylogenetic analyses based on DNA sequencing. most of the cloned bacterial sequences were in the A and G subgroups. Kim et al. Taxonomic cluster analysis of 16S rRNA gene sequences from terra preta and adjacent pristine forest soil based on oligonucleotide fingerprinting of 16S rRNA gene sequences.ARTICLE IN PRESS 686 J. and Verrucomicrobia. The A subgroup is thought to be the most phylogenetically diverse. Terra preta contained sequences from the Y subgroup. 700 clones per sample. 2004). Our results are largely in agreement with these suppositions. corresponding to species and genera. Here. 1.. with ca. This earlier survey suggested that the A and G subgroups are ubiquitous. comprising from 12% of the noncultured species surveyed in Austrian forest soils under pine to 35% in spruce-fir-beech soils in Europe (Hackl et al. b. The 16S rRNA clone libraries were generated for single samples consisting of three composited replicate soil cores (litter layer removed) taken from the top 10 cm of the soil profile at each location. Acidobacterium was predominant. whereas mostly a Proteobacteria were found in the forest soil. suggesting that the large data set that was analyzed here.. and g Proteobacteria were similarly abundant in terra preta. In the phylogenetic tree generated by DNA sequencing. Among the major groups represented. represented all of the major bacterial phyla that were present in the soils. which purportedly favors non-acid soils. Other bacterial groups that were common to both two soils included the Proteobacteria. with deep branches in the phylogenetic tree that may represent distinct lineages. Actinobacteria. The coverage curves converged at 95% evolutionary distance and then separated again at distances from 96% to 90%.-S. 1999). Planctomy- cetes. as compared to 9 from the forest soil (Fig. The phylogenetic trees included two possible new clades of Acidobacterium that were distinct from the previously described subgroups for this phylum. Only the A subgroup was found in the acid soil from the pristine forest. comprising approximately 30% of the bacteria in terra preta and 50% of the forest soil. Identities of the major bacterial groups by direct sequencing of clones representing different clusters revealed 14 phylogenetic groups in terra preta. 4). At least four subgroups of these bacteria have been recognized previously using specific 16S rRNA gene primers in a survey of 43 soils and sediments representing a range of environments (Barns et al. Acidobacterium sp are common in many forest soils around the world.05 Actinobacteria Fig. the BLAST analysis revealed three possible . / Soil Biology & Biochemistry 39 (2007) 684–690 Terra Preta Nitrospira Proteobacteria Chloroflexi Acidobacterium Bacillus Proteobacteria Preserved Forest Acidobacterium Acidobacterium alpha-Pro Actinobacteria CFB gamma-Pro beta-Pro Verrucomicrobia Actinobacteria alpha-Pro Acidobacterium Verrucomicrobia Actinobacteria Delta-Pro Chloroflexi gamma-Pro beta-Pro Planctomycetes Proteobacteria Acidobacterium Verrucomicrobia 0. indicating that there were also significant differences between the communities at deeper phylogenetic levels.

rhizosphere effects. Based on prior knowledge.37 0.1 0.05 evolutionary distance 0.0030 0.20 0.0 0. . D 0.60 0. Table 1 Measures of bacterial species diversity based on OFRG analysis of microbial communities from terra preta and Amazon forest soil 1.2 0.015 413 291/768 196 142 4. Kim et al. Here.80 0. 2. Vertical bars indicate 95% confidence intervals. Gene sequences were differentiated by oligonucleotide fingerprint grouping at 0. The relationship between organic matter inputs from different overstory trees. composition of forest soils is still not understood. It may then be possible to unravel the ecology of these bacterial communities and study the role of specific bacterial groups that contribute to the many interesting properties of these soils. both soils supported abundant plant growth and carried similar above ground vegetation consisting of a diverse community of Amazon forest tree species and understory plants.10 evolutionary distance Shannon index Simpsons index Estimated richness Chao1 (0. and the bacterial community Fig. Rarefaction analysis of 16S rRNA gene sequences from terra preta and forest soil. and a second welldefined cluster of five clones that were most closely related to the Y-subgroup.34 277 new subgroups of Acidobacterium.40 0.0020 0. There was also increased earthworm activity and soil aggregation in terra preta that may provide a wide range of niches and thereby contribute to increased bacterial species diversity. soil pH is likely to be one of the most important selection factors affecting the microbial species composition in different soils (Fierer and Jackson.ARTICLE IN PRESS J. C 0.00 0.2 0.-S. 2006). LIBSHUFF analysis of 16S rRNA gene sequences from terra preta (closed circles) compared to adjacent forest soil (open circles) showing differences in composition of the bacterial communities (y-axis) over a range of evolutionary distances (D) (x-axis).5 0.00 Coverage. Two new clades of Acidobacterium were found in the terra preta.05 evolutionary distance) Terra preta Forest soil 396/742 287 210 5.0005 (Cx-Cxy)2 Index OTU richness Unique OFRG groups / clones 0. Solid lines indicate the value of (CX-CXY)2 for samples at each value of D.4 Evolutionary distance. one that was most closely related to the G subgroup. The new clade of Acidobacterium in the forest soil was comprised by four clones that occurred within the A-subgroup and were most similar to previous accessions designated as Holophaga. / Soil Biology & Biochemistry 39 (2007) 684–690 687 350 300 Terra Preta Forest Soil Number of OTUs observed 250 200 150 100 50 0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 Number of sequences sampled Fig.05 dissimilarity level.0015 0.3 0.0025 0. 3. but likely contributes to differences in diversity and community composition along the forest floor. Broken lines indicate the P ¼ 0:05 value of (CX-CXY)2 for the randomized samples.0010 0. Additional surveys and comparisons at different locations will be needed to characterize other locations with terra preta.

-S. (90%) 54 776-1 Uncultured gamma proteobacterium (92%) 47-1 Nitrosococcus halophilus (90%) 82 99 1083-1 Uncultured beta proteobacterium (95%) 966-1 Uncultured beta proteobacterium (89%) 99 1251-1 Burkholderia sp. (98%) 2-1 46-1 Uncultured Planctomycete (93%) 99 426-1 Anaeromyxobacter dehalogenans (90%) 293-1 Uncultured delta proteobacterium (92%) 89 685-1 Geobacter sulfurreducens (87%) 99 1074-1 Geobacter sulfurreducens (87%) 24-1 Syntrophus gentianae (88%) 900-1 Uncultured delta proteobacterium (92%) 221-1 Uncultured delta proteobacterium (95%) 94 772-1 Uncultured delta proteobacterium (95%) 99 1154-1 Uncultured delta proteobacterium (96%) 1001-1 Uncultured delta proteobacterium (96%) 98 0. / Soil Biology & Biochemistry 39 (2007) 684–690 869-1 Uncultured Acidobacteria (95%) 22-1 Uncultured Acidobacteria ( 95%) G 91 26-1 Uncultured Acidobacteria (94%) 99-1 Uncultured Acidobacteria (96%) 73 210-1 Uncultured Acidobacteria (92%) 79 99 1192-1 Uncultured Acidobacteria (95%) 997-1 Uncultured Acidobacteria (92%) New 99 483-1 Uncultured Acidobacteria (87%) 99 894-1 Uncultured Acidobacteria (93%) 291-1 Uncultured Acidobacteria (95%) 1086-1 Uncultured Acidobacteria (92%) 18-1 Uncultured Acidobacteria (92%) 991-1 Uncultured Acidobacteria (97%) 83 A 99 1271-1 Uncultured Acidobacteria (94%) 97 1272-1 Uncultured Acidobacteria (94%) 9999 1005-1 Uncultured Acidobacteria (93%) 99 1091-1 Uncultured Acidobacteria (97%) Y 395-1 Uncultured Acidobacteria (93%) 70 1093-1 Uncultured Acidobacteria (91%) 99 1267-1 Uncultured Acidobacteria (92%) 99 1363-1 Uncultured Acidobacteria (93%) New 99 7-1 Uncultured Acidobacteria (93%) 87 501-1 Uncultured Acidobacteria (91%) 81 31-1 Uncultured Acidobacteria (93%) 40-1 Streptomyces kasugaensis (94%) 99 37-1 Streptomyces kasugaensis (94%) 34-1 Streptomyces kasugaensis (94%) 42-1 Streptomyces kasugaensis (94%) 99 41-1 Streptomyces kasugaensis (94%) 911-1 Uncultured Rubrobacteridae (93%) 75 15-1 Uncultured Rubrobacteridae (95%) 99 43-1 Paenibacillus macerans (95%) 99 1078-1 Bacillus cereus (98%) 1077-1 Bacillus cereus (99%) 99 993-1 Bacillus bataviensis (96%) 99 307-1 Bacillus bataviensis (97%) 98 1180-1 Uncultured Chloroflexi (87%) 1067-1 Uncultured Chloroflexi (92%) 99 12-1 Uncultured Chloroflexi (94%) 98 99 4-1 Parachlamydia acanthamoebae (93%) 3-1 Parachlamydia acanthamoebae (94%) 99 27-1 Uncultured Verrucomicrobia (95%) 28-1 Uncultured Verrucomicrobia (95%) 30-1 Uncultured Verrucomicrobia (95%) 1164-1 Nitrospira moscoviensis (96%) 1163-1 Nitrospira moscoviensis (95%) 99 6-1 Uncultured Bacteroidetes (92%) 5-1 Uncultured Bacteroidetes (92%) 99 140-1 candidate division TM7 (89%) 227-1 Afipia genosp (93%) 99 128-1 Afipia genosp (93%) 99 594-1 Uncultured alpha proteobacterium (97%) 98 711-1 Bradyrhizobium japonicum (98%) 99 1100-1 Bradyrhizobium japonicum (98%) 597-1 Mesorhizobium sp. (A) Terra preta soil (B) Forest soil. (98%) 99 592-1 Uncultured beta proteobacterium (94%) 99 591-1 Uncultured beta proteobacterium (94%) 78 142-1 Burkholderia sp. Phylogenetic trees for unique 16S rRNA gene sequences based on rRNA gene sequencing.05 91 99 Acidobacterium Actinobacteria Bacillus Chloroflexi Chlamidiae Verrucomicrobia Nitrospira Bacteroidetes TM7 Alpha Proteobacteria Gamma Proteobacteria Beta Proteobacteria Unknown Planctomycetes Delta Proteobacteria (A) Fig. (98%) 141-1 Burkholderia sp. . Kim et al.ARTICLE IN PRESS 688 J. 4.

Kluwer Academic Publishers. Management. 2002. (96%) 670-2 Afipia felis (89%) 100 178-2 Uncultured alpha proteobacterium (95%) 100 288-2 Azospirillum amazonense (92%) 337-2 Acidosphaera rubrifacien (95%) 87 1503-2 Acidosphaera rubrifacien (91%) 100 100 1529-2 Acidosphaera rubrifacien (93%) 1202-2 Uncultured actinobacterium (92%) 1309-2 Acidothermus cellulolyticus (92%) 100 1532-2 Frankia sp. Kumar.R. N. Applied and Environmental Microbiology 70. Netherlands. Quantitative comparisons of 16S rRNA gene sequence libraries from environmental samples. Geoderma 110. Bodrossy.2006. (92%) 100 271-2 100 1126-2 Uncultured planctomycete (90%) 345-2 Nostocoida limicola (96%) 530-2 Parachlamydia acanthamoebae (93%) 546-2 Uncultured Verrucomicrobia (96%) 241-2 Uncultured Verrucomicrobia (89%) 100 941-2 Uncultured Verrucomicrobia (88%) 100 0. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Amazon Soils: A Reconnaissance of the Soils of the Brazilian Amazon Region. Mello.. 1997. Handelsman. S. / Soil Biology & Biochemistry 39 (2007) 684–690 98 340-2 Uncultured Acidobacteria (92%) 355-2 Uncultured Acidobacteria (91%) 100 339-2 Uncultured Acidobacteria (92%) 753-2 Uncultured Acidobacteria (92%) 100 169-2 Uncultured Acidobacteria (92%) 1209-2 Uncultured Acidobacteria (92%) 1496-2 Uncultured Acidobacteria (92%) 466-2 Uncultured Acidobacteria (93%) 1151-2 Uncultured Acidobacteria (93%) 1215-2 Uncultured Acidobacteria (92%) 1216-2 Uncultured Acidobacteria (92%) 100 81-2 Uncultured Acidobacteria (94%) 92-2 Uncultured Acidobacteria (94%) 100 100 1031-2 Uncultured Acidobacteria (95%) 1219-2 Uncultured Acidobacteria (88%) 244-2 Uncultured Acidobacteria (96%) 821-2 Uncultured Acidobacteria (94%) 83 760-2 Uncultured Acidobacteria (95%) 100 99 958-2 Uncultured Acidobacteria (94%) 99 829-2 Uncultured Acidobacteria (93%) 648-2 Uncultured Acidobacteria (94%) 462-2 Uncultured Acidobacteria (92%) 100 55-2 Uncultured Acidobacteria (96%) 100 157-2 Uncultured Acidobacteria (96%) 100 90-2 Frateuria aurantia (96%) 100 845-2 Uncultured gamma proteobacteria (96%) 96 1052-2 Frateuria aurantia (96%) 1048-2 Burkholderia unamae (97%) 99 1131-2 Burkholderia tropica (98%) 100 1236-2 Uncultured gamma proteobacterium (94%) 100 52-2 Uncultured alpha proteobacterium (97%) 71 557-2 Bradyrhizobium genosp (99%) 99 98 722-2 Sphingomonas sp.G..R. Applied and Environmental Microbiology 70.M.. Larget. C. (Continued) Acknowledgments The authors gratefully acknowledge Lea Valinsky and James Borneman for assistance with oligonucleotide fingerprinting methods. S.. Sombroek. 300pp. Rathbun. H.R. Science 297.B.. The real dirt on rain forest fertility. J.1016/j.. Ker... 5485–5492. J. Sessitsch. Triplett. The diversity and biogeography of soil bacterial communities..C. Kim et al. B. Properties. D.. B. C. 2004. M.. R. 2004. W. . 2647–2653. 4. S. Brief Bioinformation 5. S. 1966. 2004.R. Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment. Lima.. C. Borneman. Lehmann. Comparison of diversities and compositions of bacterial populations inhabiting natural forest soils. Centre for Agricultural Publications and Documentation.W.. Nei.. Schloss. Gilkes..V. 2006. 1731–1737. 2001.-S... 5057–5065. Furlong.D. and Steven Qi for technical assistance. J. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation.. 010 References Barns. J.. The Netherlands.ARTICLE IN PRESS J..C... S. Fierer. J. R. Pedogenesis and pre-Colombian land use of ‘‘Terra Preta Anthrosols’’ (‘‘Indian black earth’’) of Western Amazonia. Singleton. Amazonian Dark Earths: Origin. A. 1–17. Applied and Environmental Microbiology 65.. Mann.05 689 A Acidobacteria New A Gamma Proteobacteria Beta Proteobacteria Alpha Proteobacteria Actinobacteria Unknown Planctomycetes Chlamydiae Verrucomicrobia (B) Fig. Proceedings of the National Academy of Sciences 103. 626–631.. Zechmeister-Boltenstern.N... 920–923.L. 523pp. Hackl. Kern. Takala. Tamura. M.B. E. D. 150–163. Applied and Environmental Microbiology 63.08. 2002. P. L. W. Supplementary data Supplementary data associated with this article can be found in the online version at doi:10. Glaser. K. Kuske.A. Appendix A..E.. 2003.W. Jackson. Woods. Whitman. Applied and Environmental Microbiology 67..J.soilbio.. 1999.. W. 4374–4376.L. E. Schaefer. Wageningen.. Integration of microbial ecology and statistics: a test to compare gene libraries.

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