Microbes Environ. Vol. 23, No. 1, 49–56, 2008 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.23.49

Community Structure of Soil in a Tropical Rainforest Several Years After Fire

SHIGETO OTSUKA1*, IMADE SUDIANA2, AIICHIRO KOMORI1, KAZUO ISOBE1, SHIN DEGUCHI1†, MASAYA NISHIYAMA1‡, HIDEYUKI SHIMIZU3, and KEISHI SENOO1 1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan; 2Research Centre for Biology, The Indonesian Institute of Sciences (LIPI), Cibinong Science Centre, Jalan Raya Jakarta-Bogor Km. 46, Cibinong 16911, Indonesia; and 3Asian Environment Research Group, National Institute for Environmental Studies, 16–2 Onogawa, Tsukuba, Ibaraki 305–8506, Japan (Received July 18, 2007—Accepted November 22, 2007)

The bacterial community structure in soil of a tropical rainforest in East Kalimantan, Indonesia, where forest fires occurred in 1997–1998, was analysed by denaturing gradient gel electrophoresis (DGGE) with soil samples collected from the area in 2001 and 2002. The study sites were composed of a control forest area without fire damage, a lightly- burned forest area, and a heavily-burned forest area. DGGE band patterns showed that there were many common bac- terial taxa across the areas although the vegetation is not the same. In addition, it was indicated that a change of vegeta- tion in burned areas brought the change in bacterial community structure during 2001–2002. It was also indicated that, depending on a perspective, community structure of soil bacteria in post-fire non-climax forest several years after fire can be more heterogeneous compared with that in unburned climax forest. The dominant soil bacteria in the field of the present study were Acidobacteria, Actinobaceria, and Alphaproteobacteria based on the DNA sequences of DGGE bands, although they were not dominant among the culturable bacteria from the same soil samples. Key words: soil, bacteria, community structure, tropical rainforest, forest fire

Forest fires often occur in Indonesia mainly due to human community ecology. Mabuhay et al.12,13) examined the effect activities such as clearing for large-scale plantation, and the of forest fire on soil in Hiroshima, Japan, and reported that fires that occurred in 1997–1998 in Kalimantan Island (also microbial biomass and abundance were high in the unburned called Borneo Island in Malaysia) were aggravated by the El area. Acea and Carballas1) pointed out that changes in physi- Niño phenomenon. Wildfire can be a part of the ecosystem ological groups of microorganisms may occur after fire. and a necessary event in some areas, where it maintains the There is, yet, little information on the impact of a vegetation diversity of the ecosystem by decreasing the dominance of change after fire on the microbial community in soil in tropi- competitive species and releasing nutrients23). In such areas, cal rainforests. there are plants whose seeds germinate only after fire, stimu- In this study, the bacterial community diversity in soil sev- lated by heat or smoke. In tropical Asia including Kaliman- eral years after fire in a tropical rainforest in Kalimantan tan Island, however, fire is generally not a part of the ecosys- Island was evaluated by denaturing gradient gel electro- tem and no natural forest is expected to adapt to fire. phoresis (DGGE) analysis targeting the 16S rRNA gene (16S Kalimantan Island is extremely rich in tropical organisms, on rDNA). DGGE analysis can reveal the DNA sequence-based which the harmful impact of fire is expected to be serious, structure of a bacterial community including nonculturable since the loss of “key organisms” in tropical rainforests can populations, and has been used in many environments as a significantly affect the recovery of forest ecosystems14). The method for the evaluation of bacterial community concern for the impact of forest destruction on natural eco- structure3,6). In addition, some of the DGGE bands were systems has so far chiefly been restricted to higher excused, cloned, and sequenced in order to examine taxo- plants11,21). nomic properties. Taxonomic groups of 70 culturable bacte- Microorganisms including bacteria play an important role ria were also revealed, in order to compare them with those in the soil ecosystem, for example, in the decomposition of detected in a denaturing gradient (DG) gel. organic matter, and it is, therefore, imperative to reveal their Materials and Methods * Corresponding author. E-mail: [email protected]; Research field Tel: +81–3–5841–5176; Fax: +81–3–5841–8042. ° † The research field was located in Bukit Bangkirai (0 80'S, Present address: National Agricultural Research Center for Tohoku 116°70'E), East Kalimantan, Indonesia. Soil type in the research Region, National Agriculture and Food Research Organization, 4 field is mostly Ultisols (USDA classification). The average annual Akahira, Shimo-kuriyagawa, Morioka-shi, Iwate 020–0198, Japan rainfall reaches approximately 2,500 mm. From June until October ‡ Present address: Faculty of Environmental Studies, Nagasaki Uni- (the dry season), the rainfall is generally lower than in the other versity, 1–14 Bunkyo-cho, Nagasaki-shi, Nagasaki 852–8521, months of the year. The average annual temperature reaches Japan approximately 26°C. 50 OTSUKA et al.

Three sampling plots were set depending upon the degree of fire fore, the samples had been stored at 4°C for about two weeks after damage in 1997–1998, composed of a control natural forest area the sampling, and were then adapted to test conditions at 30°C with without fire damage (Plot K), a lightly-burned forest area (Plot LD), each soil’s moisture content at 60% of the maximum water holding and a heavily-burned forest area (Plot HD). Before the fires, the capacity for three weeks. areas containing Plots LD and HD were parts of a natural forest of the same type as Plot K. The total areas of Plots K, LD, and HD Soil texture, soil pH, and levels of total carbon, total nitrogen, and were 10,000 m2, 10,100 m2, and 10,000 m2, respectively, and the available nitrogen three plots were approximately 500–1,000 m from one another. It Soil texture was determined by a particle-size analysis using a 17) seems that after the fire, some plants survived in Plot LD and few in pipette method . Soil pH (H2O) was measured potentiometrically Plot HD, and new plants had started growing in both plots. At the with a soil-water ratio of 1:2.5. Total carbon (total C) and total time of 2002, Plots K, LD, and HD had 280, 128, and 94 species of nitrogen (total N) levels were determined in an automatic highly tree, and the Shannon Index of tree diversity of the plots were 2.07, sensitive N-C analyser, a Sumigraph NC-90A (Sumika Chemical 1.86, and 1.33, respectively (Wakiyama, S. and N. Sukigara, Japan Analysis Service, Tokyo, Japan), equipped with a GC-8A gas chro- Wildlife Research Center, Taito-ku, Tokyo, Japan. Unpublished matograph (Shimadzu, Kyoto, Japan). The amount of available data). The three most frequent trees were Shorea laevis, Madhuca nitrogen (available N) was determined by the phosphate buffer solu- kingiana, and Macaranga glaberrimus (all climax plants) in Plot K; tion method18). M. kingiana, Durio acutifolius, and Macaranga gigantea (the former two, climax plants; the latter one, a pioneer plant) in Plot DNA extraction and PCR LD; and M. gigantea, Mallotus paniculatus, and Mallotus tri- DNA was directly extracted from 0.5 g of each soil sample and TM chocarpa (all pioneer plants) in Plot HD (Wakiyama, S. and N. purified using the UltraClean Soil DNA Kit (MO Bio Laborato- Sukigara. Unpublished data). ries, Carlsbad, CA, USA). The final volume of extracted DNA solu- In addition, Plot IC was set for comparison, which had been tion was 20 µl. The DNA solution was diluted twenty-fold with dominated by a grass Imperata cylindrica, and had had no trees sterile distilled water and used as a PCR template. The universal after severe fire damage before 1997 until the present study. Plot IC prokaryotic PCR primers, 357F (5'-CCTACGGGAGGCAGCAG- was 1–1.5 km from the other plots and located between a valley and 3') and 520R (5'-GTATTACCGCGGCTGCTGG-3') corresponding a haul road, which might be the main reason no tree had so far to positions 341–357 and 538–520 of Escherichia coli 16S rDNA grown in this area. The total area of Plot IC was about 1,800 m2. numbering, respectively, were used to obtain fragments targeting The soil type in Plot IC is also Ultisols. the 16S rDNA V3 variable region of about 200 bp (including prim- ers). The GC-clamp of Muyzer et al.16) was attached to the 357F Soil sampling and test conditions primer to enable DGGE. PCR was run in a thermal cycler, a Gene- Plots K, LD, and HD were dissected into 10 m×10 m squares, AmpTM PCR System 2700 (Applied Biosystems, Foster City, CA, and five squares each were selected randomly. Bulk soils of the A USA), in 0.2-ml tubes using 50-µl reaction volumes. The reaction horizon were collected from the centre of each square in September mixture contained 1 µl of the template DNA solution, 200 nM of both the primers, 200 µM each of nucletides, 250 µM of MgCl2, 2 U 2001 and July 2002. At the same time, three points were selected ® randomly in Plot IC, and soils of the A horizon were collected from of AmpliTaq Gold DNA polymerase (Applied Biosystems) and 5 each point. Soil from one point of Plot HD (HD3, cf. Table 1) con- µl of 10×reaction buffer. The template was substituted with sterile tained post-fire fragmented charcoal. Plant roots were removed distilled water in the negative control sample. The initial denatur- carefully from the soil. The research field was far from the laborato- ation step was done at 94°C for 5 min. The amplification was car- ries and the soil samples could not be analysed immediately. There- ried out with 5 cycles including denaturation at 94°C for 30 sec,

Table 1. Thickness, texture, pH (H2O), and levels of total carobon (total C), total nitrogen (total N), and available nitrogen (available N) of the A horizon of soil in 2001

1) 2) −1 −1 Available N Sampling point pH (H2O) −1 1) Thickness (cm) Soil texture Total C (g kg ) Total N (g kg ) (mg kg ) K1 8 S 3.97 13.2 0.71 3.47 K2 7.5 SL 4.11 23.0 1.08 4.95 K3 7 S 4.54 9.60 0.63 2.22 K4 9 SL 4.37 16.4 1.06 2.31 K5 8 SiL 4.47 23.0 1.37 3.66 LD1 5 SL 4.56 14.0 0.95 1.26 LD2 6.5 SL 4.47 23.7 1.41 1.97 LD3 5.5 L 5.06 14.3 1.09 1.24 LD4 5 SL 4.96 13.7 0.81 1.02 LD5 5 SL 4.69 11.1 0.84 1.55 HD1 10 S 4.16 28.4 1.79 1.73 HD2 8 SL 4.42 19.7 1.26 1.06 HD3 16 SL 3.81 44.0 1.28 3.76 HD4 6 S 4.67 29.8 1.55 1.90 HD5 8.5 S 4.39 5.60 0.36 8.18 IC1 7.5 SL 4.34 10.5 0.57 n.m. IC2 n.m. SL 4.70 16.2 0.91 n.m. IC3 n.m. SL 4.56 12.6 0.76 n.m. 1) n.m. represents “not measured”. 2) S, SL, SiL, and L represent Sandy, Sandy Loam, Silt Loam, and Loam, respectively. Soil Bacterial Community in a Tropical Forest 51 annealing at 50°C for 30 sec, and DNA extension at 72°C for 60 was extracted from the gel piece by a 24-hour incubation at 4°C. sec, followed by 25 cycles including denaturation at 94°C for 30 s, The supernatant of the DNA extract was used as template DNA in annealing at 55°C for 30 sec, and DNA extension at 72°C for 60 the PCR re-amplification using the same primers, 357F and 520R, sec. The final extension was done at 72°C for 7 min. Amplification as described above. Amplicons were passed through MicroSpinTM of PCR products of the proper size was confirmed by electrophore- S-400 HR Columns (Amersham Pharmacia Biotech, Uppsala, Swe- sis through a 1.5% agarose gel, followed by staining with ethidium den) to remove remaining dNTPs and primers, and cloned with the ® bromide. pGEM -T Easy System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Plasmids containing the clones ® DGGE analysis were extracted and purified using a Qiagen Plasmid Mini Kit The DGGE was carried out using a DCodeTM Universal Mutation (Qiagen, Valencia, CA, USA). The sequences of five clones each Detection System (Bio-Rad Laboratories, Hercules, CA, USA), from one DNA band were determined with the DTCS Quick Start with an acrylamide concentration in the gel of 8% and a denaturing Kit for Dye Terminator Cycle Sequence (Beckman Coulter, Fuller- gradient of 35% to 65%. The amplicons were electrophoresed with ton, CA, USA) and Genetic Analysis System CEQ 8000 (Beckman two DGGE markers that sandwich most of the DGGE bands. The Coulter). Prior to the sequencing, another PCR amplification was markers were composed of 16S rDNA fragments amplified by PCR done with the extracted plasmid as template DNA, using the same from DNAs of Staphylococcus aureus IAM12544T and Nocardiop- primers (357F-GC and 520R) as described above; the amplicons sis literi IFO13360T, with the same primers and reaction conditions were electrophoresed again on a DG gel to confirm whether they are as mentioned above. The electrophoreses were run for 6 h at 120 V. ® the same as the original bands. Subsequently, the gels were stained with SYBR Green I (Bio- Whittaker Molecular Applications, Rockland, ME, USA) according Sequence-based identification of bacteria in a DG gel. to the manufacturer’s instructions, and a digital image of the gel The sequence of a cloned DNA band was compared with avail- was obtained with an image scanner, the Epi-Light UV FA 500 sys- able sequences in the GenBank database using the Blast tem (Yamato Scientific, Tokyo, Japan). DNA bands were detected programme2). Based on the similarity of the V3 region of 16S with the software Image Gauge version 3.45 (Fuji Photo Film, rDNA, the taxon of the bacterium that has the sequence was esti- Tokyo, Japan). Based on the relative positions of DNA bands mated at the phylum/class level. A neighbour-joining tree including between the two markers (c.f. Fig. 1, pointed by arrows), each band the clone and other members of the estimated taxon was constructed was numbered and its absorbance was measured. The data was used using Clustal W version 1.7, and the validity of the estimation was for a cluster analysis with KyPlot software (K. Yoshioka, 1997 to confirmed. 1999, version 2.0 beta 4). The cluster analysis was carried out with the following conditions: Significance Level, 0.05; Data Type, Raw Identification of culturable bacteria Data; Measure, Standardized Euclidean; Clustering Method, Ward, Equal quantites of soil samples from all sampling points in Plot which was output in Squared Distance. K, HD, or IC were mixed well. Ten grams of soil sample from each plot with 90 ml of sterilized water was placed in a 100-ml cup, then Extraction and sequencing of DGGE bands homogenised at room temperature at 10,000 rpm for 1 min using a Thirty selected bands in the DG gel applied with the samples of Waring blender (Sakuma Seisakusho, Tokyo, Japan). The suspen- 2001 were carefully excised under UV illumination and then placed sion was serially diluted 10-fold and 0.1 mL aliquots were spread in 20 µl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). DNA on 1/20 nutrient broth agar plates. The agar plates were incubated at 30°C for five days. In this study, a more concentrated nutrient broth and longer incubation period caused some bacterial colonies to grow large enough to engulf many other colonies. One agar plate each for Plots K, HD, and IC on which around 20–30 colonies formed was selected, and all colonies on the plate were isolated. A total of 70 bacterial colonies were obtained. The almost full length of 16S rDNA of the bacteria was ampli- fied by PCR with the following primers; 27F, 5'-AGAGTTTGATC- CTGGCTCAG-3' and 1525R, 5'-AAAGGAGGTGATCCAGCC-3'. PCR was run in a thermal cycler, GeneAmpTM PCR System 2700 (Applied Biosystems) in 0.2-ml tubes using 50 µl reaction volumes. The reaction mixture contained 1 µl of cell solution made from each colony, 200 nM of the both primers, 200 µM each of nucletides, 250 ® µM of MgCl2, 1 U of AmpliTaq Gold DNA polymerase (Applied Biosystems) and 5 µl of 10×reaction buffer. Initial denaturation step was done at 94°C for 5 min. The amplification was carried out with 25 cycles including denaturation at 94°C for 30 s, annealing at 55°C for 30 sec, and DNA extension at 72°C for 60 sec. The final exten- sion was done at 72°C for 7 min. Amplification of PCR products of the proper size was confirmed by electrophoresis through a 1.5% agarose gel, followed by staining with ethidium bromide. The amplificons were purified using MicroSpinTM S-400 HR Columns (Amersham Pharmacia Biotech). Cycle sequencing reac- tions were performed using the Thermo Sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP (Amer- sham Pharmacia Biotech), with the primers 27F, 1525R, and the Fig. 1. DGGE profile of the 16S rDNA V3 region amplified by PCR following additional primers; 704F, 5'-GGTGAAATGCGYAGA-3' from DNA directly extracted from soil collected in 2001 (a) and 2002 and 789R, 5'-GGGGTATCTAATCCC-3'. The reaction products (b). Bands between the two DGGE markers pointed to by arrows were were run on a 4% Long Ranger (FMC Bio Products, Rochland, ME, provided for a cluster analysis. K, LD, HD, and IC represent samples from Plot K (unburned forest area), Plot LD (lightly-damaged forest US) using a DSQ-1000L DNA Sequencer (Shimadzu). area), Plot HD (heavily-damaged forest area), and Plot IC (grassland Based on the almost full-length 16S rDNA sequence, the isolates area without trees). were identified phylogenetically by the same method as that used 52 OTSUKA et al. for the identification of bacteria that appeared as DGGE bands described above.

Results and Discussion

First, it should be noted that the results presented here are those from soil samples collected several years after fire. Therefore, the anticipated direct damage of fire such as heat and smoke could not be examined. Thickness, texture, pH (H2O), and the levels of total C, total N, and available N of the A horizon of soil in Plot K (unburned forest area), Plot LD (lightly-damaged forest area), Plot HD (heavily-damaged forest area), and Plot IC (grassland area without trees) in 2001 are shown in Table 1. Small differences were recognised in these properties depending on sampling points, but not on plots. For instance, the thickness and the total C content of the A horizon were greatest on average in Plot HD, followed by Plots K and LD in descending order. The relationship between these values and the degree of fire damage was obscure based on the properties shown in Table 1. The largest value for total C, at sampling point H3, was apparently due to post-fire frag- mented charcoal included in the soil (cf. Materials and Meth- ods). Of the DGGE band patterns derived from the samples of 2001 (Fig. 1a), none were identical. Although the vegetation is not the same among the plots, many bands were common to several samples from all plots, indicating that common bacterial taxa are dominant across the plots. In the band pat- terns derived from the samples of 2002 (Fig. 1b), many bands were again common to several samples from all plots, Fig. 2. Dendrograms constructed by Ward’s method from DGGE but the concentration or the presence/absence of some bands band patterns obtained from the soil samples collected in 2001 (a) and 2002 (b). K, LD, HD, and IC represent samples from Plot K (unburned was not the same as those of 2001. forest area), Plot LD (lightly-damaged forest area), Plot HD (heavily- In the dendrogram derived from the data of 2001 (Fig. 2a), damaged forest area), and Plot IC (grassland area without trees), respec- it seems that the community structure was not affected by the tively. Samples with the same name were collected from the same point. chemical properties of soil examined (Table 1), but affected by the plot difference to some extent. The samples of Plots K and LD were not separated clearly and formed a cluster tion of Plots LD and HD made the community structure of together (Cluster K-LD). Four samples of Plot HD formed a soil bacteria in the two plots similar in 2002. The difference cluster (Cluster HD) but they were more distant from the in the vegetation may cause the difference in soil organic remaining one sample of Plot HD (HD1) than those of Plots matter and soil temperature depending on light intensity, etc., K and LD. In the dendrogram derived from the data of 2002 which would affect the soil bacteria. There is another possi- (Fig. 2b), the formation of clusters differed from that of bility, that the community structure changes depending on 2001. The samples of Plot K and only one sample from Plot the time of year. However, considering that the samples were LD formed a cluster together (Cluster K). The other four collected in September 2001 and July 2002, and that the samples of Plot LD formed a cluster with the samples of Plot study area is in a tropical rainforest climate where the tem- HD (Cluster LD-HD). The formation of this cluster means perature is stable and the dry season lasts from June to Octo- that the community structure of bacteria in Plots LD and HD ber, the difference in environmental factors depending on the was more similar to each other than to that in Plot K in 2002. sampling months does not seem significant. After the fire, some parts of trees had survived in Plot LD, The samples from Plot IC clustered well and were located which were common to those in Plot K, however, pioneer far from the other samples, meaning that the soil bacteria in trees such as M. gigantea and Vernonia arborea increased in this grassland have distinct community structures. Plot HD Plots LD and HD after September 2001 (Wakiyama, S. and was burned as well as Plot IC, but the community structure N. Sukigara. Unpublished data). It has been illustrated that of the soil bacteria in Plot HD was more similar to that in plant community structure influences bacterial community Plots K and LD than that in Plot IC. Many infant trees were composition4,15). It was also suggested that changes in vegeta- growing in Plot HD but there were almost no trees in Plot IC, tive community structure in the years following fire have the which is considered to be one of the chief reasons for the dif- potential to be the more dominant driver and shaper of the ference in the community structure of soil bacteria between soil microflora than the direct impact of the fire disturbance Plots HD and IC. itself 9). Therefore, it is possible that the change of the vegeta- Fierer and Jackson8) performed a continental-scale exami- Soil Bacterial Community in a Tropical Forest 53

Tabel 2. Taxonomic properties and nearest relatives of the 16S rDNA clones extracted from denaturing gradient gels

Sequence Accession 3) 4) 2) 5) Identical to 1) 2) Phylum/Class Nearest match (accession number ) % ID Plot name number (if any) A AB362940 Verrucomicrobia/— Uncultured clone (DQ450782) 98 K L B AB362941 Actinobacteria/Actinobacteria Uncultured clone (AB254795, etc.) 100 K C-1 AB362942 /Alphaproteobacteria Uncultured clone (AM500711, etc.) 100 K C-2 AB362943 — Uncultured clone (EF430159) 95 K D-1 AB362944 — Uncultured clone (AY662019) 97 K D-2 AB362945 Acidobacteria/Acidobacteria Uncultured clone (AM697576, etc.) 99 K D-3 AB362946 Proteobacteria/ Schlegelella thermodepolymerans (AY152824, etc.) 95 K Caenibacterium thermophilum (AF125876) D-4 AB362947 Proteobacteria/— Uncultured clone (AB247833, etc.) 87 K E AB362948 Acidobacteria/Acidobacteria Uncultured clone (EF074764, etc.) 100 K O F AB362949 Proteobacteria/— Uncultured clone (EF516057, etc.) 100 K P G AB362950 Acidobacteria/Acidobacteria Uncultured clone (EF072504, etc.) 100 K H-1 H-1 AB362951 Acidobacteria/Acidobacteria Uncultured clone (EF072504, etc.) 100 K G H-2 AB362952 Actinobacteria/Actinobacteria Uncultured clone (EF429901, etc.) 99 K I-1 AB362953 Proteobacteria/Alphaproteobacteria Uncultured clone (DQ829409) 100 K I-2 AB362954 Acidobacteria/Acidobacteria Uncultured clone (EF075535, etc.) 100 K J AB362955 Actinobacteria/Actinobacteria Uncultured clone (AF257824, etc.) 99 K K AB362956 Actinobacteria/Actinobacteria Uncultured clone (EF429901, etc.) 100 K R, Y L AB362957 Verrucomicrobia/— Uncultured clone (DQ450782) 98 LD A M AB362958 Proteobacteria/Alphaproteobacteria Uncultured clone (AM500711, etc.) 100 LD N AB362959 Acidobacteria/Acidobacteria Uncultured clone (AM697576, etc.) 100 LD O AB362960 Acidobacteria/Acidobacteria Uncultured clone (EF074764, etc.) 100 LD E P AB362961 Proteobacteria/Alphaproteobacteria Uncultured clone (EF516057, etc.) 100 LD F Q AB362962 Proteobacteria/Alphaproteobacteria Uncultured clone (EF520978, etc.) 100 LD W, X R AB362963 Actinobacteria/Actinobacteria Uncultured clone (EF429901, etc.) 100 LD K, Y S AB362964 Bacteroidetes/Sphingobacteria Uncultured clone (AJ575720) 98 HD T AB362965 Acidobacteria/Acidobacteria Uncultured clone (DQ829482, etc.) 99 HD U AB362966 Proteobacteria/— Uncultured clone (EF074274, etc.) 98 HD V-1 AB362967 Acidobacteria/Acidobacteria Uncultured clone (AY963483, etc.) 99 HD V-2 AB362968 Acidobacteria/Acidobacteria Uncultured clone (EF520958, etc.) 100 HD V-3 AB362969 — Uncultured clone (DQ195629) 90 HD W AB362970 Proteobacteria/Alphaproteobacteria Uncultured clone (EF520978, etc.) 100 HD Q, X X AB362971 Proteobacteria/Alphaproteobacteria Uncultured clone (EF520978, etc.) 100 HD Q, W Y AB362972 Actinobacteria/Actinobacteria Uncultured clone (EF429901, etc.) 100 HD K, R Z-1 AB362973 Proteobacteria Uncultured clone (EF074274, etc.) 95 IC Z-2 AB362974 Acidobacteria/Acidobacteria Uncultured clone (EF019667, etc.) 99 IC Z-3 AB362975 Acidobacteria/Acidobacteria Uncultured clone (EF516733, etc.) 100 IC a AB362976 Proteobacteria/— Uncultured clone (EF075729, etc.) 100 IC b AB362977 Acidobacteria/Acidobacteria Uncultured clone (EF516884, etc.) 100 IC c AB362978 Acidobacteria/Acidobacteria Uncultured clone (EF520958, etc.) 100 IC d-1 AB362979 Acidobacteria/Acidobacteria Uncultured clone (EF019667, etc.) 100 IC d-2 AB362980 Actinobacteria/Actinobacteria Rhodococcus coprophilus (AJ306402, etc.) 99 IC Catenulispora sp. (AJ865860, etc.) etc. 1) Sequence name corresponds to the DGGE band name in Fig. 1. DGGE bands C, D, H, V, Z, and d had more than one sequence. All the sequences of each of these bands were confirmed to have the same motility on a DG gel. 2) Accession number is for DDBJ, EMBL, and GenBank. 3) A minus sign, “—”, means “unidentifiable” at the phylum or class level. 4) An uncultured bacterium whose genus name is not given is described as an “uncultured clone” regardless of its title in the DDBJ, EMBL, and GenBank database. 5) The plot is where the sample was collected in which the sequence was detected, and does not mean the sequence is specific to the plot. nation of soil bacterial communities, and reported that bacte- area including Plot HD was intrinsically the tropical rainfor- rial diversity was unrelated to site temperature, latitude, and est. The pH of the soil from the plots was about 4–5 depend- other variables that typically predict plant and animal diver- ing on sampling points but not on plot type. These factors sity, and community composition was largely independent of might result in many common bacterial taxa being dominant geographic distance. Fierer and Jackson8) also reported that in all the plots. the diversity and richness of soil bacterial communities dif- Plot K had the largest number of plant species, and the fered by ecosystem type, and these differences could largely branch distances in the dendrogram within Plot K were be explained by soil pH. The vegetation differed temporally smaller than those within Plots LD and HD. It is possible that among the plots in this study, but the ecosystem type of the a stable, climax tropical rainforest composed of diverse 54 OTSUKA et al.

Table 3. List of islates and their taxonomic properties Isolate Accession % Phylum/Class Genus 3) 2) ID1) number2) Closest type strain (accession number ) ID SBK-1 AB366342 Actinobacteria/Actinobacteria Streptomyces Streptomyces purpureus LMG19368T (AJ781324) 98 SBK-2 AB366343 Actinobacteria/Actinobacteria Streptomyces Streptomyces purpureus LMG19368T (AJ781324) 96 SBK-3 AB366344 Actinobacteria/Actinobacteria Streptomyces Streptomyces glauciniger FXJ14T (AY314782) 98 SBK-4 AB366345 /“” Bacillus cereus ATCC14579T (AF290547), etc. 99 SBK-5 AB366346 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 99 SBK-6 AB366347 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 99 SBK-7 AB366348 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 99 SBK-8 AB366349 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 99 SBK-9 AB366350 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 100 SBK-10 AB366351 Firmicutes/“Bacilli” Bacillus Bacillus sphaericus ATCC14577T (DQ286299) 99 SBK-11 AB366352 Firmicutes/“Bacilli” Bacillus Bacillus odysseyi NBRC100172T (AF526913) 95 SBK-12 AB366353 Firmicutes/“Bacilli” Bacillus Bacillus odysseyi NBRC100172T (AF526913) 98 SBK-13 AB366354 Firmicutes/“Bacilli” Bacillus Bacillus subtilis ssp. subtilis DSM10T (AJ276351) 98 SBK-14 AB366355 Firmicutes/“Bacilli” Bacillus Bacillus subtilis ssp. subtilis DSM10T (AJ276351) 99 SBK-15 AB366356 Firmicutes/“Bacilli” Paenibacillus Paenibacillus chinjuensis WN9T (AF164345) 98 SBK-16 AB366357 Proteobacteria/Alphaproteobacteria Sphingomonas Sphingomonas echinoides ATCC14820T (AB021370) 97 SBK-17 AB366358 Proteobacteria/Alphaproteobacteria Sphingomonas Sphingomonas echinoides ATCC14820T (AB021370) 99 SBK-18 AB366359 Proteobacteria/Betaproteobacteria Burkholderia cepacia ATCC25416T (U96927), etc. 99 SBK-19 AB366360 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia fungorum LMG16225T (AF215705) 100 SBK-20 AB366361 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia fungorum LMG16225T (AF215705) 100 SBK-21 AB366362 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia mimosarum PAS44 T (AY752958) 98 SBK-22 AB366363 Proteobacteria/Gammaproteobacteria Enterobacter Enterobacter asburiae JCM6051T (AB004744) 100 SBK-23 AB366364 Proteobacteria/Gammaproteobacteria Pseudomonas Pseudomonas aeruginosa ATCC10145T (AF094713) 98 SBH-1 AB366323 Actinobacteria/Actinobacteria Streptomyces Streptomyces purpureus LMG19368T (AJ781324) 98 SBH-2 AB366324 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 99 SBH-3 AB366325 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 100 SBH-4 AB366326 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 99 SBH-5 AB366327 Firmicutes/“Bacilli” Bacillus Bacillus luciferensis LMG18422T (AJ419629) 97 SBH-6 AB366328 Firmicutes/“Bacilli” Bacillus Bacillus megaterium DSM32T (D16273) 99 SBH-7 AB366329 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia terricola LMG20594T (AY040362) 97 SBH-8 AB366330 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia terricola LMG20594T (AY040362) 98 SBH-9 AB366331 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia stabilis LMG14294T (AF148554) 99 SBH-10 AB366332 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia cepacia ATCC25416T (U96927), etc. 99 SBH-11 AB366333 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia cepacia ATCC25416T (U96927), etc. 97 SBH-12 AB366334 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia plantarii LMG16020T (U96932) 97 SBH-13 AB366335 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia cepacia ATCC25416T (U96927), etc. 99 SBH-14 AB366336 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia stabilis LMG14294T (AF148554) 99 SBH-15 AB366337 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia fungorum LMG16225T (AF215705) 99 SBH-16 AB366338 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia unamae MTl-641T (AY221956) 97 SBH-17 AB366339 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia sacchari LMG19450T (AF263278) 97 SBH-18 AB366340 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia sacchari LMG19450T (AF263278) 97 SBH-19 AB366341 Proteobacteria/Gammaproteobacteria Enterobacter Enterobacter hormaechei CIP103441T (AJ508302) 99 SBI-1 AB366295 Actinobacteria/Actinobacteria Leifsonia Leifsonia xyli ssp. xyli JCM9733T (AB016985) 97 SBI-2 AB366296 Actinobacteria/Actinobacteria Leifsonia Leifsonia xyli ssp. xyli JCM9733T (AB016985) 97 SBI-3 AB366297 Actinobacteria/Actinobacteria Leifsonia Leifsonia xyli ssp. xyli JCM9733T (AB016985) 96 SBI-4 AB366298 Actinobacteria/Actinobacteria Microbacterium Microbacterium resistens DMMZ1710T (Y14699) 95 SBI-5 AB366299 Actinobacteria/Actinobacteria Streptomyces Streptomyces griseoflavus LMG19344T (AJ781322) 98 SBI-6 AB366308 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 100 SBI-7 AB366301 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 99 SBI-8 AB366302 Firmicutes/“Bacilli” Bacillus Bacillus cereus ATCC14579T (AF290547), etc. 99 SBI-9 AB366303 Firmicutes/“Bacilli” Bacillus Bacillus subtilis ssp. subtilis DSM10T (AJ276351) 99 SBI-10 AB366304 Firmicutes/“Bacilli” Bacillus Bacillus subtilis ssp. subtilis DSM10T (AJ276351) 99 SBI-11 AB366305 Firmicutes/“Bacilli” Bacillus Bacillus subtilis ssp. subtilis DSM10T (AJ276351) 99 SBI-12 AB366306 Firmicutes/“Bacilli” Bacillus Bacillus mycoides ATCC6462T (AF155956) 100 SBI-13 AB366307 Firmicutes/“Bacilli” Bacillus Bacillus thuringiensis ATCC10792T (AF290545) 98 SBI-14 AB366310 Firmicutes/“Bacilli” Bacillus Bacillus megaterium DSM32T (D16273) 99 SBI-15 AB366309 Firmicutes/“Bacilli” Bacillus Bacillus megaterium DSM32T (D16273) 99 SBI-16 AB366300 Firmicutes/“Bacilli” Paenibacillus Paenibacillus elgii NBRC100335T (AY090110) 99 SBI-17 AB366311 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia stabilis LMG14294T (AF148554) 99 SBI-18 AB366312 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia cepacia ATCC25416T (U96927), etc. 99 SBI-19 AB366313 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia cenocepacia LMG16656T (AF148556) 96 SBI-20 AB366314 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia cepacia ATCC25416T (U96927), etc. 99 SBI-21 AB366315 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia mimosarum PAS44 T (AY752958) 98 SBI-22 AB366316 Proteobacteria/Betaproteobacteria Burkholderia Burkholderia mimosarum PAS44 T (AY752958) 99 SBI-23 AB366317 Proteobacteria/Betaproteobacteria Ralstonia syzygii ATCC49543T (U28237) 98 SBI-24 AB366318 Proteobacteria/Betaproteobacteria Ralstonia Ralstonia insidiosa AU2944T (AF488779) 98 SBI-25 AB366319 Proteobacteria/Gammaproteobacteria Dyella Dyella koreensis NBRC100831T (AY884571) 96 SBI-26 AB366320 Proteobacteria/Gammaproteobacteria Pseudomonas Pseudomonas mosselii CIP105259T (AF072688) 100 SBI-27 AB366321 Proteobacteria/Gammaproteobacteria Dyella Dyella japonica JCM21530T (AB110498) 97 SBI-28 AB366322 Proteobacteria/Gammaproteobacteria Dyella Dyella japonica JCM21530T (AB110498) 96 1) Isolates whose ID begins with SBK, SBH, and SBI were isolated from Plots K, HD, and IC, respectively. 2) Accession number is for DDBJ, EMBL, and GenBank. 3) Each isolate does not necessarily belong to the species to which its closest type strain belongs to. Soil Bacterial Community in a Tropical Forest 55 plants provides a somewhat homogeneous environment in lum is often detected in PCR-based analyses. Therefore, soil compared with a non-climax forest. there should be other reasons for Firmicutes being dominant The taxa of the bacteria identified based on the sequences in cultured bacteria but less detected in PCR-based analyses. of DGGE bands are listed in Table 2. Some clones were con- It is possible that Firmicutes is one of the dominant taxa firmed to be different from the original bands by additional among culturable bacteria but not dominant in total bacteria. DGGEs with them used as template DNA, and the data from Culturable bacteria in soil comprise a minor fraction of the such clones were not included in Table 2. As a result, the 30 total bacteria5,7,19,22), and there still are many as yet uncultured bands were composed of 33 different sequences (41 bacteria in soil, which would explain the discrepancy sequences are listed in Table 1 with duplications). It was between the dominant members of cultured bacteria and confirmed that bands showing the same motility in a DG gel those detected in PCR-based analyses. mostly contained at least one same sequence. In conclusion, many bacterial taxa were commonly domi- The sequence d-2 in Table 2 could be of Rhodococcus or nant in soil in which the vegetation was not the same, while a Catenulispora, but it is necessary to compare a longer change of vegetation in burned areas brought a change in sequence with those of the type strains of the type species of bacterial community structure. It can be said that, depending Rhodococcus and Catenulispora to identify its genus. The on the viewpoint, post-fire non-climax forest can have a nearest relatives of a bacterium that has the sequence D-3 are more heterogeneous community structure of soil bacteria Schlegelella thermodepolymerans and Caenibacterium ther- several years after fire, than unburned climax forest. In order mophilum, but the bacterium is possibly an unknown species to discover the dominating factor for such community struc- based on the low sequence similarity (% ID). Although 16S turing, it is inevitable to reveal more about the characteristics rDNA data of numerous known bacteria have already been of soil bacteria, including not-yet-cultured bacteria. registered in the current DNA databases, the nearest relatives of bacteria that have the 31 sequences (without duplication) Acknowledgements other than d-2 and D-3 were uncultured clones (Table 1), indicating that most of the soil bacteria are unknown and We are grateful to Dr. Herwint Simbolon and Mr. Entis Sutisna, Research Centre for Biology, The Indonesian Institute of Sciences uncultured. Actinobacteria, Acidobacteria, and Alphaproteo- (LIPI), for their support in the field survey and soil sampling. We bacteria were shown to be dominant bacteria in the soil used are grateful to Dr. Eizi Suzuki, Faculty of Science, Kagoshima Uni- in this study. Janssen10) reviewed reports on the abundance of versity, for information on tropical trees, and also to Dr. Hiroki Rai, bacterial taxa in soil at the phylum level, and concluded that Graduate School of Agricultural and Life Sciences, The University Actinobacteria, Acidobacteria, and Alphaproteobacteria are of Tokyo for technical support with DGGE. This study was finan- often abundant in soil and Bacteroidetes, Firmicutes, and cially supported by the Global Environment Research Fund (E-2 and E-051) of the Ministry of the Environment, Japan. Planctomycetes are generally less abundant, which is sup- ported by the present results. The results of phylogenetic identification of 70 bacterial References isolates are listed in Table 3. Many of the isolates belonged 1) Acea, M.J., and T. Carballas. 1996. Changes in physiological groups to taxa that could not be identified at the species level only of microorganisms in soil following wildfire. FEMS Microbiol. 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