Supplementary information: Phylogenetic composition and properties of bacteria coexisting with the fungus Hypholoma fasciculare in decaying wood

Vendula Valášková, Wietse de Boer, Paulien J.A. Klein Gunnewiek,Martin Pospíšek, Petr Baldrian

Sampling

Samples were collected in the autumn of 2007 in a mixed forest site (forestry Wolfheze managed by Natuurmonumenten) close to Doorwerth village, the Netherlands (N 51° 58' 60”, E 15° 47' 60”, altitude 49 m). The forest has developed on glacial sandy deposits with oak (Quercus robur), beech (Fagus sylvatica), birch (Betula sp.) and pine (Pinus sylvestris) as major species. Age of the trees is variable. Wood sampled from seven tree stumps and trunks (beech 2 samples; birch 2 samples;oak 1 sample;pine 2 samples) with high degrees of decomposition, adjacent to fruiting bodies of Hypholoma fasciculare (Huds. ex Fr. Kummer), was selected for future analysis. The upper 3 cm layer of wood was removed to avoid contamination of bacteria that are present on the surface. The newly exposed wood was surface-sterilized with ethanol and samples were taken using a sterile wood drill. The sampled wood was immediately used for the extraction of bacteria and isolation of DNA. In addition, dry mass, pH, ergosterol content and activity of lignocellulose-degrading enzymes were measured.

Wood analysis

Wood moisture content was determined after drying at 70°C until constant mass was obtained. pH was measured in a suspension of 1 g fresh wood in 10 ml demineralized water after 2h of shaking.

Activity of lignocellulose-degrading enzymes and ergosterol content were determined as indicators of wood decay and fungal biomass, respectively. For enzyme extraction, 1 g of fresh wood was soaked with 4 ml of water, shaken for 1 hour at room temperature and pressed over an iron mesh with 2 mm pore size as described previously (Van der Wal et al., 2007). The resulting liquid was centrifuged (10 min at 10000 x g) and stored at -20°C until analysis.

Laccase (EC 1.10.3.2) activity was measured by monitoring the oxidation of ABTS (2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) (Bourbonnais and Paice, 1990) at 25 °C in citrate–phosphate (100 mM citrate, 200 mM phosphate) buffer (pH 5.0) at 420 nm. Activity of manganese peroxidase (MnP, EC 1.11.1.13) was assayed according to Ngo and Lenhoff (1980)at 25 °Cin succinate-lactate buffer (100 mM, pH 4.5). MBTH (3-methyl-2-benzothiazolinone hydrazone) and DMAB (3,3-dimethylaminobenzoic acid) were oxidatively coupled by MnP, and the resulting purple indamine dye was detected spectrophotometrically at 595 nm. The results were corrected by the activities of samples where manganese was substituted by an equimolar amount of ethylenediaminetetraacetate (EDTA). One unit of enzyme activity was defined as the amount of enzyme forming 1 μmol of reaction product per min.

Activities of endo-1,4-β-glucanase (EC 3.2.1.4) and endo-1,4-β-xylanase (EC 3.2.1.8) were measured with soluble polysaccharide substrates dyed with Remazol Brilliant Blue(carboxymethyl cellulose and birchwood xylan, respectively) using the protocol of the supplier (Megazyme, Ireland). The reaction mixture contained 0.2 ml of 2% dyed substrate in 200 mM sodium acetate buffer (pH 5.0), and 0.2 ml sample. The reaction mixture was incubated at 40°C for 60 min and the reaction was stopped by adding 1 ml of ethanol followed by 10 s vortexing and 10 min centrifugation (Baldrian et al., 2005). The amount of released dye was measured at 595 nm and the enzyme activity was calculated according to standard curves correlating the dye release with the release of reducing sugars. One unit of enzyme activity was defined as the amount of enzyme releasing 1 μmol of reducing sugars per min. All spectrophotometric measurements were performed in a microplate reader (SynergyTM HT, BIO-TEK).

Ergosterol was extracted from 0.25 g wood according to Niemenmaa et al. (2008) and measured using HPLC as described by Van der Wal et al. (2007).DGGE was used to confirm the dominance of H. fasciculare in the fungal community in decomposing wood. The primer pair ITS1f-gc-ITS2 was used for the amplification of the ITS region of fungal 18S rDNA (Bougourne and Cairney, 2005).

Enumeration, isolation and maintenance of bacterial strains

Samples of fresh milled wood were extracted with MES buffer (2-[N-morpholino]ethanesulphonic acid, 1.95g l-1, pH 5.0, 10 ml per 1 g dry mass of wood). The wood suspensions were shaken for 90 min at laboratory temperature on a vortex, sonicated for 2 × 30 s and subsequently shaken for another 30 min using a vortex as described previously (Folman et al., 2008). Dilutions were prepared in sterile MES buffer and 50 μl aliquots of the dilutions were plated in duplicate.

Three different media [1/10 strengthtrypticase soy broth agar, pH 6.5 (TSB6.5) and pH 5.0 (TSB5) as well as water yeast agar, pH 5.0 (WYA)] were used for plating. TSB media contained 1 g l-1 NaCl, 3 g l-1 trypticase soy broth (Oxoid), 20 g l-1 agar and either 1 g l-1 KH2PO4 (TSB6) or 1.95 g l-1 MES (TSB5) as a buffering compound and0.1 g l-1 Delvocid (effective compound Natamycine, DSM Food Specialities) to prevent the growth of fungi. WYA contained 1 g l-1 NaCl, 0.1 g l-1 yeast extract (Difco Technical grade), 1.95 g l-1 MES, 20 g l-1 agar, and 0.1 g l-1 Delvocid. Before autoclaving, the pH was adjusted using HCl and KOH. For preparation of media of pH 5.0, a double-strength agar suspension was autoclaved separately and mixed with media containing the other components after cooling to 55°C, in order to prevent liquefaction of the agar.

Inoculated Petri dishes were incubated at 20°C for 2 weeks and the number of colony forming units (CFUs) was determined.. Colonies were grouped into 10 different types based on morphology and the frequency of these types was estimated. Fiftystrains representing all observed colony morphologies were randomly selected,isolated by sub-culturing and stored at – 80 ˚C (Protect storage system, Technical Service Consultants LTD) until further analysis.

DNA isolation and sequence analysis

Total genomic DNA of bacterial isolates was extracted using the MoBio Ultraclean Soil DNA Isolation Kit (MoBio Laboratories Inc.) following the instructions of the manufacturer. Isolated DNA was used as the template for PCR reactions with 16S rDNA primers 27f (5’-AGAGTTTGATCCTGGCTCAG) and 1492r (5’-GRTACCTTGTTACGACTT) (Edwards et al., 1989). PCR reactions (25 μl) consisted of 1x PCR buffer with MgCl2 (Roche), 200 μM dNTPs (Amersham Biosciences), 0.6 μM 27f, 0.6 μM 1492r, 1.4 U of Fast Start High Fidelity DNA Polymerase (Roche) and 1.2 μl DNA. Cycling conditions were: 94°C for 2 min, 35 cycles (94°C for 30 s, 55°C for 1 min, 72°C for 90 s + 1 s / cycle), and 72°C for 10 min. The PCR products were analyzed on 1.5% (w/v) agarose gels, purified with QIAquick PCR purification kit (Qiagen) and sequenced as a single extension with primer 1492r by Macrogen Inc. (Korea) using an ABI 3730 XL DNA Analyzer (Applied Biosystems).

Total genomic DNA was isolated from wood samples (0.3 g, fresh weight) using the modified method of Miller (Sagova-Mareckova et al., 2008),purified with Geneclean Turbo Kit (Biogenic) and stored at -20°C prior to further analysis.DNA fragments amplified by Dynazyme II polymerase (Finnzymes) using the 27f and 1492r primers were cloned using a CloneJET PCR cloning kit (Fermentas) following the manufacturer’s instructions. Ligated plasmids were transformed into E. coli XL1-Blue cells by electroporation. Transformants were screened for insertion by colony PCR using vector primers pJET1.2 F/ pJET1.2 R as recommended by the manufacturer. PCR products obtained from 20 independent colonies per sample were used for single extension sequencing with primer 1492r carried by Macrogen Inc. (Korea).

Six out of the 140 successfully sequenced clones were identified as most likely chimeras using the previous Chimera Check Software included in the Ribosomal Database Project II (Michigan State University, USA). Remaining 134 sequences were manually edited and corrected prior to BLAST (blastn) search against the nucleotide database at NCBI ( Identification was based on the best blastn match. Clones identified to genera level had similarities of 97% and higher. The distance tree option and Naïve Bayesian rRNA Classifier version 2.0 from Ribosomal Database project II software with confidence threshold set to 80% were also used for identification. When in doubt, clones were identified to higher taxa.

Multiple alignment of obtained sequences was performed using CLUSTALW (Thompson et al., 1994). Phylogenetic trees were computed and drawn using MEGA version 4 (Tamura et al., 2007). The evolutionary history was inferred using the Neighbor-Joining method; evolutionary distances were computed using the LogDet (Tamura-Kumar) method. Rarefaction curves for 16S rDNA sequences were computed using S. Holland’s Analytical Rarefaction software version 1.3 ( The abundance-based richness estimators SCHAO1 and SACE were computed using software implemented at and Aller, 2004a). Phylotypes were identified as identical with similarity cutoff values of 97% and higher, as described previously (Kemp and Aller, 2004b).

Physiological characterization of isolates

The ability of isolates to grow low pHwas tested in semi-solid medium containing 3.0 g l-1 TSB, 1.0 g l-1 MES, 0.25 g l-1 oxalic acid, and 20 g l-1 agar. Before autoclaving, the pH of the medium was adjusted to 4. The low pH agar was prepared as described above. Inoculated plates were incubated at 20ºC.

In vitro antagonism of bacteria against H. fasciculare was tested on agar as described by De Boer et al. (2008).The test media were TSB5 and 2% malt extract agar. Inhibition of fungal growth due to presence of bacteria was evaluated after 2 weeks of incubation at 20ºC.

A metabolic screening of bacterial isolates was performed using Basic Medium (BM) and Basic Medium supplemented with yeast extract (BMY). BM contained 1.95 g l-1 MES, 0.1 g l-1 KH2PO4 , 0.1 g l-1 (NH4)2SO4 , 0.04 g l-1 MgSO4.7 H2O and 0.02 g l-1 CaCl2.2 H2O.BMYadditionally contained0.1 g l-1 of Yeast Extract. Both media were adjusted to pH 6.0 before autoclaving. Lower pH values could not be maintained stable unless organic acids, citric acid or oxalic acid, were added as buffering compounds. However, these organic acids where either metabolized(citric acid) by bacteria or resulted in precipitation (oxalic acid). The following substrates were added to both media at a 5 mM final concentration: D-glucose, D-mannose, D-galactose, L-rhamnose, L-fucose, D-xylose, D-arabinose, N-acetyl-D-glucosamine, D-mannitol, D-cellobiose, D-trehalose, Na-acetate, Na-citrate, oxalic acid, Na-benzoate, ferulic acid, p-coumaric acid, sinapic acid, vannilic acid, syringic acid p-hydroxybenzaldehyde or demineralized water (control). Bacteria were pre-grown on TSB6.5or TSB5 agar plates and resuspended in the basic medium to OD 0.05 (600 nm) as the inoculum. Each well of a microplate contained 200 μl media and was inoculated with 20 μl of bacterial suspension. The plates were incubated at 20ºC while gently shaken and the optical density (600 nm) was measured on days 1, 3, and 10.The OD of control wells was subtracted. Positive growth on a particular substrate was defined as >0.050 increase in absorbance between any two of the sampling times.

Growth of bacterial isolates on colloidal cellulose or chitin was tested on water-yeast-agar (pH 6.5) supplemented with 2 g l-1 of colloidal chitin or cellulose. Halo-formation around colonies was scored as chitinolytic or cellulolytic activity.

Accession numbers

Nucleotide sequences determined in this study were submitted to the Genbank database under accession numbers EU780169-EU780216 for bacterial isolates and FJ198073- FJ198087, FJ198089- FJ198105, FJ198107, FJ198108, FJ198110- FJ198161, FJ198163- FJ198165, FJ198167- FJ198211 for sequences of the clone library derived from wood samples.

References

Baldrian, P., Valášková, V., Merhautová, V., and Gabriel, J. (2005) Degradation of lignocellulose by Pleurotus ostreatus in the presence of copper, manganese, lead and zinc. Research in Microbiology 156: 670-676.

Bougoure, D.S., and Cairney, J.W.G. (2005) Fungi associated with hair roots of Rhododendron lochiae (Ericaceae) in an Australian tropical cloud forest revealed by culturing and culture-independent molecular methods. Environmental Microbiology 7: 1743-1754.

Bourbonnais, R., and Paice, M.G. (1990) Oxidation of nonphenolic substrates - an expanded role for laccase in lignin biodegradation. FEBS Letters 267: 99-102.

De Boer, W., de Ridder-Duine, A.S., Gunnewiek, P., Smant, W., and Van Veen, J.A. (2008) Rhizosphere bacteria from sites with higher fungal densities exhibit greater levels of potential antifungal properties. Soil Biology & Biochemistry 40: 1542-1544.

Edwards, U., Rogall, T., Blocker, H., Emde, M., and Bottger, E.C. (1989) Isolation and direct complete nucleotide determination of entire genes - characterization of a gene coding for 16S-ribosomal RNA. Nucleic Acids Research 17: 7843-7853.

Folman, L.B., Gunnewiek, P., Boddy, L., and de Boer, W. (2008) Impact of white-rot fungi on numbers and community composition of bacteria colonizing beech wood from forest soil. FEMS Microbiology Ecology 63: 181-191.

Kemp, P.F., and Aller, J.Y. (2004a) Bacterial diversity in aquatic and other environments: What 16S rDNA libraries can tell us. FEMS Microbiology Ecology 47: 161-177.

Kemp, P.F., and Aller, J.Y. (2004b) Estimating prokaryotic diversity: When are 16S rDNA libraries large enough? Limnology and Oceanography-Methods 2: 114-125.

Ngo, T.T., and Lenhoff, H.M. (1980) A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions. Analytical Biochemistry 105: 389-397.

Niemenmaa, O., Galkin, S., and Hatakka, A. (2008) Ergosterol contents of some wood-rotting basidiomycete fungi grown in liquid and solid culture conditions. International Biodeterioration & Biodegradation 62: 125-134.

Przybyl, K. (2001) Fungi and bacteria associated with the wet and brown wood in trunk of Betula pendula trees. Acta Societatis Botanicorum Poloniae 70: 113-117.

Sagova-Mareckova, M., Cermak, L., Novotna, J., Plhackova, K., Forstova, J., and Kopecky, J. (2008) Innovative methods for soil DNA purification tested in soils with widely differing characteristics. Applied and Environmental Microbiology 74: 2902-2907.

Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599.

Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994) Clustal-W - improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680.

Van der Wal, A., de Boer, W., Smant, W., and van Veen, J.A. (2007) Initial decay of woody fragments in soil is influenced by size, vertical position, nitrogen availability and soil origin. Plant and Soil 301: 189-201.

Supplementary table 1: Taxonomical identification of bacterial isolates and cloned 16S rDNA fragments from wood decayed by Hypholoma fasciculare.

isolate / Identification / accession number / isolate / identification / accession number
WH1 / Xanthomonadaceae bacterium / EU780169 / WH26 / Burkholderia sp. / EU780193
WH2 / Xanthomonadaceae bacterium / EU780170 / WH27 / Burkholderia glathei / EU780194
WH3 / Dyella sp. / EU780171 / WH28 / Rahnella sp. / EU780195
WH4 / Pedobacter sp. / EU780172 / WH29 / Sphingomonas sp. / EU780196
WH5 / Sphingomonas sp. / EU780173 / WH30 / Xanthomonadaceae bacterium / EU780197
WH6 / Sphingomonas sp. / EU780174 / WH32 / Dyella sp. / EU780198
WH7 / Xanthomonadaceae bacterium / EU780175 / WH33 / Dyella sp. / EU780199
WH8 / Burkholderia sp. / EU780176 / WH34 / Dyella sp. / EU780200
WH9 / Rahnella sp. / EU780177 / WH35 / Dyella sp. / EU780201
WH10 / Burkholderia sp. / EU780178 / WH37 / Xanthomonadaceae bacterium / EU780202
WH11 / Burkholderia sp. / EU780179 / WH38 / Xanthomonadaceae bacterium / EU780203
WH12 / Burkholderia sp. / EU780180 / WH120 / Acidobacteriaceae bacterium / EU780204
WH13 / Acidobacteriaceae bacterium / EU780181 / WH121 / Burkholderia glathei / EU780205
WH14 / Burkholderia sp. / EU780182 / WH123 / Acidobacteriaceae bacterium / EU780206
WH15 / Acidobacteriaceae bacterium / EU780183 / WH124 / Acidobacteriaceae bacterium / EU780207
WH16 / Acidobacteriaceae bacterium / EU780184 / WH141 / Acetobacteraceae bacterium / EU780208
WH17 / Acidobacteriaceae bacterium / EU780185 / WH143 / Caulobacteraceae bacterium / EU780209
WH18 / Acidobacteriaceae bacterium / EU780186 / WH144 / Acetobacteraceae bacterium / EU780210
WH19 / Acidobacteriaceae bacterium / EU780187 / WH145 / Acetobacteraceae bacterium / EU780211
WH20 / Burkholderia sp. / EU780188 / WH146 / Acidobacteriaceae bacterium / EU780212
WH21 / Acidobacteriaceae bacterium / EU780189 / WH147 / Caulobacteraceae bacterium / EU780213
WH22 / Burkholderia sp. / EU780190 / WH148 / Burkholderia sp. / EU780214
WH24 / Burkholderia sp. / EU780191 / WH149 / Rhodospirillaceae bacterium / EU780215
WH25 / Burkholderia sp. / EU780192 / WH150 / Acetobacteraceae bacterium / EU780216
clone / Identification / accession number / clone / identification / accession number
clone102 / Xanthomonadaceae bacterium / FJ198155 / clone411 / Sporosarcina psychrophila / FJ198085
clone104 / Burkholderia glathei / FJ198156 / clone413 / TM7 bacteria / FJ198086
clone108 / Acidocella sp. / FJ198157 / clone414 / Burkholderia sp. / FJ198087
clone111 / Acidobacteriaceae bacterium / FJ198158 / clone416 / Acidobacteriaceae bacterium / FJ198089
clone112 / Bradyrhizobium sp. / FJ198159 / clone417 / Verrumicrobiales bacterium / FJ198090
clone113 / Acidobacteriaceae bacterium / FJ198160 / clone418 / Acidobacteriaceae bacterium / FJ198091
clone114 / Bradyrhizobium sp. / FJ198161 / clone419 / Sporosarcina sp. / FJ198092
clone120 / Labrys monachus / FJ198163 / clone421 / Proteobacterium / FJ198093
clone121 / Acetobacteraceae bacterium / FJ198164 / clone422 / Verrumicrobiales bacterium / FJ198094
clone122 / Planctomycetaceae bacterium / FJ198165 / clone423 / Burkholderia glathei / FJ198095
clone125 / Burkholderia glathei / FJ198167 / clone424 / Actinobacteria bacterium / FJ198096
clone126 / Proteobacterium / FJ198168 / clone429 / Acidobacteriaceae bacterium / FJ198097
clone129 / Methylocapsa sp. / FJ198169 / clone430 / Rhizobiales bacterium / FJ198098
clone131 / Xanthomonadaceae bacterium / FJ198170 / clone434 / Acidobacteriaceae bacterium / FJ198099
clone132 / Burkholderia sp. / FJ198171 / clone435 / Acetobacteraceae bacterium / FJ198100
clone133 / Bdellovibrio like bacterium / FJ198172 / clone436 / Rhizobiales bacterium / FJ198101
clone134 / Beijerinckiaceae bacterium / FJ198173 / clone440 / Acidobacteriaceae bacterium / FJ198102
clone135 / Burkholderia glathei / FJ198174 / clone501 / Bradyrhizobium sp. / FJ198103
clone136 / Bradyrhizobium sp. / FJ198175 / clone502 / Bradyrhizobium sp. / FJ198104
clone137 / Beijerinckiaceae bacterium / FJ198176 / clone503 / Gammaproteobacterium / FJ198105
clone138 / Caulobacteraceae bacterium / FJ198177 / clone505 / Isosphaera sp. / FJ198107
clone139 / Burkholderia sordidicola / FJ198178 / clone506 / Xanthomonadaceae bacterium / FJ198108
clone140 / Acidobacteriaceae bacterium / FJ198179 / clone508 / Paenibacillus sp. / FJ198110
clone141 / Acidobacteriaceae bacterium / FJ198180 / clone509 / Acidobacteriaceae bacterium / FJ198111
clone142 / Sporosarcina psychrophila / FJ198181 / clone510 / Rhizobiales bacterium / FJ198112
clone143 / Verrumicrobiales bacterium / FJ198182 / clone513 / Paenibacillus sp. / FJ198113
clone144 / Bradyrhizobium sp. / FJ198183 / clone517 / Isosphaera sp. / FJ198114
clone145 / Gammaproteobacterium / FJ198184 / clone518 / Bradyrhizobium sp. / FJ198115
clone146 / Burkholderia glathei / FJ198185 / clone602 / Actinobacteria bacterium / FJ198116
clone147 / Flexibacteraceae bacterium / FJ198186 / clone604 / Acidobacteriaceae bacterium / FJ198117
clone148 / Acidobacteriaceae bacterium / FJ198187 / clone605 / Acidisoma sp. / FJ198118
clone149 / Xanthomonadaceae bacterium / FJ198188 / clone606 / Burkholderia sp. / FJ198119
clone150 / Planctomycetaceae bacterium / FJ198189 / clone608 / Acidobacteriaceae bacterium / FJ198120
clone202 / Acidobacteriaceae bacterium / FJ198190 / clone609 / Acidobacteriaceae bacterium / FJ198121
clone203 / Methylocella sp. / FJ198191 / clone611 / Acetobacteraceae bacterium / FJ198122
clone204 / Proteobacterium / FJ198192 / clone612 / Acidobacteriaceae bacterium / FJ198123
clone205 / Acidobacteriaceae bacterium / FJ198193 / clone613 / Sphingobacteriales bacterium / FJ198124
clone206 / Burkholderia glathei / FJ198194 / clone614 / Xanthomonadaceae bacterium / FJ198125
clone207 / Acetobacteraceae bacterium / FJ198195 / clone615 / Burkholderia sp. / FJ198126
clone208 / Paenibacillus sp. / FJ198196 / clone616 / Burkholderia sp. / FJ198127
clone209 / Acidobacteriaceae bacterium / FJ198197 / clone618 / Acidobacteriaceae bacterium / FJ198128
clone210 / TM7 bacteria / FJ198198 / clone619 / Acetobacteraceae bacterium / FJ198129
clone211 / Caulobacteraceae bacterium / FJ198199 / clone620 / Acidobacteriaceae bacterium / FJ198130
clone212 / Acidobacteriaceae bacterium / FJ198200 / clone621 / Acidocella sp. / FJ198131
clone213 / Paenibacillus sp. / FJ198201 / clone623 / Acidobacteriaceae bacterium / FJ198132
clone214 / Beijerinckiaceae bacterium / FJ198202 / clone625 / Methylocystaceae bacterium / FJ198133
clone215 / Paenibacillus sp. / FJ198203 / clone626 / Rhodospirillales bacterium / FJ198134
clone216 / Acetobacteraceae bacterium / FJ198204 / clone627 / Sphingobacteriales bacterium / FJ198135
clone217 / Paenibacillus sp. / FJ198205 / clone629 / Acidobacteriaceae bacterium / FJ198136
clone218 / Acidobacteriaceae bacterium / FJ198206 / clone631 / Burkholderia sp. / FJ198137
clone220 / Gammaproteobacterium / FJ198207 / clone632 / Beijerinckiaceae bacterium / FJ198138
clone221 / Sphingomonas sp. / FJ198208 / clone801 / Acetobacteraceae bacterium / FJ198139
clone222 / Bradyrhizobium sp. / FJ198209 / clone802 / Aquicella sp. / FJ198140
clone224 / Sphingomonadaceae bacterium / FJ198210 / clone803 / Burkholderia phenazinium / FJ198141
clone225 / Verrumicrobiales bacterium / FJ198211 / clone804 / Acetobacteraceae bacterium / FJ198142
clone301 / Acetobacteraceae bacterium / FJ198073 / clone805 / Burkholderia phenazinium / FJ198143
clone318 / Xanthomonadaceae bacterium / FJ198074 / clone806 / Gammaproteobacterium / FJ198144
clone401 / Acidobacteriaceae bacterium / FJ198075 / clone807 / Acidobacteriaceae bacterium / FJ198145
clone402 / Gammaproteobacterium / FJ198076 / clone808 / Caulobacteraceae bacterium / FJ198146
clone403 / Acidobacteriaceae bacterium / FJ198077 / clone809 / Acidobacteriaceae bacterium / FJ198147
clone404 / Acidobacteriaceae bacterium / FJ198078 / clone810 / Acidobacteriaceae bacterium / FJ198148
clone405 / Burkholderia sp. / FJ198079 / clone811 / Acidisoma sp. / FJ198149
clone406 / Gammaproteobacterium / FJ198080 / clone816 / Burkholderia phenazinium / FJ198150
clone407 / Acetobacteraceae bacterium / FJ198081 / clone819 / Massilia sp. / FJ198151
clone408 / Burkholderia glathei / FJ198082 / clone820 / Methylocystaceae bacterium / FJ198152
clone409 / Acetobacteraceae bacterium / FJ198083 / clone821 / Methylocystaceae bacterium / FJ198153
clone410 / Gammaproteobacterium / FJ198084 / clone822 / Acidobacteriaceae bacterium / FJ198154

Supplementary Figure 1. Unrooted Neighbor-joining tree showing the relationships among the 16S rDNA sequences of bacterial clones (circles) and isolates (triangles) of wood decayed byHypholoma fasciculare, and representative sequences of related bacteria (source: Ribosomal Database Project). All bootstrap values >60% are shown (1000 replicates). (A) Phylogenetic relationship of bacteria excluding Proteobacteria. (B) Phylogenetic relationship of bacteria related to Proteobacteria, branch to other bacterial phyla is indicated.

1