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1 The Krean Jurnal f Micrbilgy, Vl. 42, N. 2, June 2006, p Cpyright 2006, The Micrbilgical Sciety f Krea 16S rdna-ardra w ù ù m ü VBNC ³ mw p w Á½ Áy * w w k ³ d (DVC) sq (PC) w ù ù m sw ³ sƒ w, DVC w ³ w sq w ³ 1% ùkû. l m ü sq š w ù (viable but nn culturable; VBNC) ³ 99% w q. VBNC ³ w w m l DNA wš 16S rdna-ardra mw mw p mw. ù ù m l ƒƒ 111 clnes, 108 clnes z wš HaeIII r 30 grups 26 grups ARDRA grup w. ƒ ARDRA grup l t clne w 16S rdna» w, ù m α-prtebacteria (12 clnes), γ-prtebacteria (3 clnes), δ-prtebacteria (1 clne), Flexibacter/Cytphaga (1 clne), Actinbacteria (4 clnes), Acidbacteria (4 clnes), š Planctmycetes (5 clnes) 7 m y, ù m α-prtebacteria (4 clnes), γ-prtebacteria (2 clnes), Actinbacteria (10 clnes), Acidbacteria (8 clnes), Planctmycetes (1 clne), š Verrucmicrbia (1 clne) 6 w m y., ù ù m ü w 99% VBNC ³ y ³ mw w y. Key wrds ý 16S rdna-ardra, phylgeny, Pine frest sil, Quercus frest sil, viable but nn-culturable (VBNC) bacteria k l œ ù, ù ƒ, š m» xk» w. ù w k w w (5, 7), m w ƒ w m ù m w ƒ m y w w m k w. m k»z,, x, w ù w m yw r w» (5). ½» w ù w w yù yw y, ù mw ù w w k y (15). ù» mw w ù d w wd Ÿ m d w» d x w, w w,, ƒ yw w ql sw. p w *T whm crrespndence shuld be addressed. Tel: , Fax: kswhang@mkwn.ac.kr yw š, w yw y ù w w w (5, 6, 13, 16, 22). ù ù ü w š ù w w d ù d ü p w (15, 20, 21). m w w wš m kw ƒ» w š w ù ³(VBNC; viable but nn-culturable) w w w š (3, 8, 11). kw mw š y l w DNA» q w» w w wš. p, ARDRA (amplified rdna restrictin analysis) 16S rdna swš wz w y wš w kw Ÿ w š (8, 25, 27). ù (Quercus acutissima) ù (Pinus densiflra) wd Ÿ m ü sw kw p w» w sq w sƒ wš sq w š w ù ³(viable but nn-culturable bacteria; VBNC) s 116

2 Vl. 42, N. 2 ù ù m ü VBNC ³ mw p 117 y w VBNC ³ sww m ü mw p mw. m œ ù (Pinus densiflra) ù (Quercus acutissima) w. ƒ m l dm(0-20 cm) wdm(20-40 cm) wš wd Ÿ m w. m ƒƒ 1g w 100 ml ³ hmgenizer (AceAM-7, Nikn Seiki C.) 15,000 rpm 2 jš ³ w z w (4). ³ d ³ d (TDC) w xÿ DNA ey ethidium brmide (EtBr) w.» m 200 µl 0.2 µm nuclepre filterƒ funnel w z 0.1 M phsphate buffer (ph 7.2) 0.01% EtBr ƒw 3 w xÿx (Leica DMLS, Leica C.)w ³ d w. ³ 20 w s³ w (3, 4). ³ d w ³ d ³ d (direct viable cunt; DVC) Kgure (17, 18) xw w. m xk 15,000 rpm 10 3z w w m ü» w. m xk 500 µl 100 µl DNA w w (nalidixic acid, 100 µg/ml; pipemidic acid, 50 µg/ml; pirmidic acid, 50 µg/ml) 200 µl» (DNB, acetic acid) yww z 200 µl ³ 1000 µl w.» yw 28 C 36 w 6 w EtBr w z xÿ x (Leica DMLS, Leica c.) w Á y s w ³ d w (3). sq w ³ d sq (plate cunt; PC) w ³ d m ³ w z 5 petri dish 1 ml wš NB (nutrient brth; peptne 1%, beef extract 1%, NaCl 0.5%) 10-2 w DNB w w y w w. 28 C 1,200 w sq x clny» ³ d w (4). Ttal DNA w ƒ m l ttal DNA Rapid (26) w. m 5g 120 mm phsphate buffer (ph 8.0) 10 ml xkw m ü humic acid PCR s w w. m slutin I (150 mm NaCl, 100 mm EDTA, D.W 100 ml, lyszyme 1 g, ph 8.0) 8 ml ƒwš 37 C 2 k z, Slutin (100 mm NaCl, 500 mm Tris-HCl, D.W 100 ml, SDS 10 g, ph 8.0) 8 ml ƒw -70 C deep freezer 65 C wš 7,000 rpm 10 w d. z 5 M NaCl 2.7 µl, 10% CTAB 2.1 µl chlrfrm:isamylalchl=24:1(v/v) ƒwš 15,000 rpm 10 w. 1.6 M NaCl sw 13% PEG ƒw DNA pellet. DNA pellet z 750 µl D.W ƒw 37 C š 10 M NH 4 OAc 190 µl 2 ethanl š isprpanl ƒw DNA wš 70% ethanl 1 ml w z œ (Micr Vac MV-100, TOMMY)w. DNA wš» (Mupid-21, Gel dcumentatin system, Bi-Rad) y w (1, 9). 16S rdna PCR s E. cli 16S rdna cnserved sequence» w 27F (5'-AGAGTTTGATCC-TGGCTCAG-3') primer 1492R (5'- AAGGAGGTGATCCAGCCGC-3') primer w. Ttal DNA PCR w yw» w DNA w w. 16S rdna PCR 94 C 5 w 94 C denaturatin 1, 55 C annealing 1, 72 C extentin 1 30z wš, 72 C 10 final extentin Perkin Elmer (GeneAmpR PCR System 9700, Applied Bisystems) w w (1). PCR s» (Mupid-21, Gel dcumentatin system, Bi-Rad)w y w (1). 16S rdna Clning PCR s s 16S rdna pgem T-Easy Vectr (Prmega, USA) 3:1 wš T4-DNA ligase ƒw 4C 12 ligatinw. Cmpetent cell (C.P cell) 200 ml LB (Difc. USA) E.cli DH5 wš O.D 0.4ƒ ¾ 37 C k w ³ z wš 5 M CaCl 2 10 ml ice 10 ew z, 6,000 rpm 10 w cell wš 2 ml CaCl2 ƒw. CP cell 50 µl ligatin DNA 5 µl y ww z ice 1 ew 42 C 45 heat shckwš LB 450 µl ƒw 37 C 1 k w z X-gal (20 mg/ml) 1 ml, ITPG (20 mg/ml) 100 µl š ampicillin (20 mg/ml) 1 ml sw LB w w 37 C 24 w. LB plate clny blue-white clny w x y w clne library z T7 (5'-TAATACGACTCACT ATAGGG-3') primer SP6 (5'-TATTTAGGTGACACTATAG-3') primer

3 118 Sng-Ih Han et al. Kr. J. Micrbil w clny PCR w wš 16S rdna vectr insert y w (24). Clny PCR 95 C, 5 w 94 C denaturatin 30, 60 C annealing 30, 72 C extensin 1 30z wš, 72 C 7 final extensin Perkin Elmer (GeneAmpR PCR System 9700, Applied Bisystems) w w (1). Amplified Ribsmal DNA Restrictin Analysis (ARDRA) clne 16S rdna PCR s w wz w z ƒ clne w. 4 bases w 2.5U HaeIII (5'...GG CC...3', 3'...CC GG...5') w PCR prduct 1 µg, 10X buffer 2 µl, enzyme 1 µl, D. W yww 20 µl tube z 37 C 4 w. wz w 4% agarse gel (1X TAE buffer; 40 mm Tris-acetate, 1 mm EDTA) w 1X TAE buffer 100 V, 300 ma 1 30» w z ethidium brmide (EtBr) 30 w UV (Gel dcumentatin system, Bi-Rad)w y w. y band pattern Gelcmpar II sftware (versin 4.0; Applied Maths, Belgium) w ƒ clne w (27). 16S rdna» w 16S rdna x ABI PRISM BigDye Terminatr Cycle Sequencing Ready Reactin Kit (Applied Bisystems) w» w. Sequencing PCR BigDye 1.3 µl, T7 primer 1 µl, 16S rdna sample 1 µl (100 ng), 2X buffer 3.4 µl ³ 13.3 µl yww z cycle sequencing w. PCR 100% ethanl 50 µl 3 M sdium acetate (ph 5.2) 2 µl ƒw z 15,000 rpm 25 e jš. 250 µl 70% ethanl w k z HiDi Frmamide 20 µl ƒw 95 C 2 denaturatin w z þƒ jš ABI PRISM 310 Genetic Analyser (Applied Bisystems) w 16S rdna ( bp)» w (19). ditrect cunt; TDC) d w» w DNA ey EtBr w m w z w w, ù m cells/g sil, ù m cells/g sil. w s q (plate cunt; PC) w ³ d w, ù m CFU/g sil, ù m CFU/g sil ³ 0.01~0.28% û e ùkü. l m ü sq š w ù ³ w q. ³ d (DVC) y» DNA w w ƒwš w s kw Á y s(» w w ³) xÿ w w m w š ³ w viable w VBNC ³ w w (3, 10). DVC w ù ù m ü w VBNC ³ sƒ ww. Kgure (17, 18) w DVC m w» w DVC (3) w ³ d ww, ù m ü DVC cells/g sil ³ 88.2%, ù m cells/g sil ³ 43%ƒ ³ y.» sq w ùkü, DVC w ³ w sq w ³ (PC) ù m 0.33%, ù m 0.02% ƒ w ³ 1% ùkû (Fig. 1).,» s ¾ w ù x š w VBNC ³ m ü 99% w. p, ù m ù m VBNC ³ ³ kw DNA» m 16S rdna» hmlgy DDBJ/NCBI/ GenBank database BLAST prgram w w. ƒ» alignment Clustal X prgram w w m w w w (23, 24). š ³ ³ d ù ù Ÿ d m ü ³ (ttal Fig. 1. Cmparisn f the number f bacteria btained by ttal direct cunt (TDC), direct viable cunting (DVC) and plate cunting (PC) cllected frm Pine and Quercus frest sil.

4 Vl. 42, N. 2 ù ù m ü VBNC ³ mw p 119 p sƒƒ. z ƒ clne w wz (HaeIII, Prmega) wš r w ³ mw., ù m l 111 clnes 30 16S rdna Clning ARDRA pattern» ùkù m ü sq w š w VBNC ³ 99% w y»» w m ü w w w w. w w» w w š ARDRA, DGGE š RFLP w w» w w š (14, 27). ù ù m ü VBNC ³ mw w w Rapid DNA w. ƒ m l DNA 16S rdna s clningw ù m l 111 clnes, ù m l 108 clnes ƒƒ. grups (100%-level) ARDRA pattern y (Fig. 2) ù m l 108 clnes 26 grups (70%-level) ARDRA pattern y (Fig. 3). 16S rdna» w ù ù m l z ƒ clne 16S rdna-ardra pattern ƒ ARDRA grup l t clnes w 16S rdna» wš, Gene Bank database BLAST search v w» 16S rdna» w. ù m l 30 t clnes α-prtebacteria (12 clnes), γ-prtebacteria (3 clnes), δ-prtebacteria (1 clne), Fig. 2. Analysis f micrbial cmmunity in a frest sil by using ARDRA. UPGMA dendrgram f cluster analysis f ARDRA based n the HaeIII 16S rdna. 16S rdna genes were extracted frm pine frest sil.

5 120 Sng-Ih Han et al. Kr. J. Micrbil Fig. 3. Analysis f micrbial cmmunity in frest sil by using ARDRA. UPGMA dendrgram f cluster analysis f ARDRA based n the HaeIII 16S rdna. 16S rdna genes were extracted frm Quercus frest sil. Flexibacter/Cytphaga (1 clne), Actinbacteria (4 clnes), Acidbacteria (4 clnes), š Planctmycetes (5 clnes) 7 m y. w, ù m α- prtebacteria (4 clnes), γ-prtebacteria (2 clnes), Actinbacteria (10 clnes), Acidbacteria (8 clnes), Planctmycetes (1 clne), š Verrucmicrbia (1 clne) 6 m y (Table 1). clnes 16S rdna»» w y ³ y m ü w VBNC ³ mw w w. ù ù m ü VBNC ³ m w p» Fig. 2 3 ùkü ARDRA pattern w ù m ARDRA grups 6 clnes sww ARDRA grups (grup 6, 8, 9, 12, 14, 15, 16, 20, 25) ³ w (Fig. 4). ù ARDRA grups Rhdplanes sww grup 8 (6 clnes), grup 12 (7 clnes) š Bradzrhiybium Afipia sww grup 9 (10 clnes), grup 20 (10 clnes), grup 14 (12 clnes) 5 grups α-prtebacteria y, grup 15 (6 clnes) γ-prtebacteria w.

6 Vl. 42, N. 2 ù ù m ü VBNC ³ mw p 121 Table 1. The clsest micrrganism f the dminant clnes n ARDRA grups frm Pine and Quercus frest sil Surce TaxnmicGaffiliatin n. f clnes GGTheGclset micrrganisms Similarity (%) ARDRA grup α-prtebacteria CPA-22 Afipia brmeae 99 9 CPA-34 Uncultured bacterium clne CPA-37 Afipia brmeae CPA-46 Uncultured alpha prtebacterium CPA-50 Uncultured sil bacterium clne 95 4 CPA-73 Uncultured sil bacterium clne CPA-97 Uncultured bacterium 92 2 CPA-99 Uncultured bacterium clne CPA-117 Uncultured bacterium clne 99 8 CPA-120 Uncultured bacterium clne CPA-125 Uncultured alpha prtebacterium clne CPA-137 Bradyrhizbium sp γ-prtebacteria CPA-20 Uncultured bacterium clne CPA-70 Uncultured silbacterium clne CPA-75 Bacterium Ellin Pine δ-prtebacteria CPA-21 Uncultured delta prte bacterim clne frest sil Acidbacteria CPA-44 Uncultured Acidbacteriaceae bacterium CPA-59 Uncultured eubacterium WD CPA-68 Uncultured silbacterium clne CPA-133 Uncultured Acidbacteriales bacterium Actinbacteria CPA-52 Uncultured silbacterim clne CPA-67 Uncultured eubacterium CPA-92 Uncultured eubacterium WD CPA-122 Uncultured actinbacterium clne planctmycete CPA-1 Uncultured plan ctmycete clne 91 7 CPA-24 Uncultured frest sil bacterium clne CPA-26 Uncultured bacterium clne 92 5 CPA-33 Uncultured bacterium 93 6 CPA-91 Uncultured sil bacterium clne 91 3 Flexibacter/Cytphapa CPA-79 Uncultured bacterium DSSD α-prtebacteria CBA-2 Afipia sp CBA-26 Uncultured sil bacterium CBA-57 Uncultured bacterium CBA-107 Bacterium Ellin γ-prtebacteria CBA-25 Uncultured bacterium CBA-44 Uncultured gamma prtebacterium 98 6 Acidbacteria CBA-1 Uncultured bacterium CBA-37 Uncultured sil bacterium clne CBA-58 Uncultured Acidbacteria bacterium CBA-67 Uncultured bacterium clne 97 2 CBA-77 Uncultured sil bacterium CBA-88 Uncultured sil bacterium CBA-93 Uncultured eubacterium WD Quercus CBA-106 Uncultured bacterium clne 90 4 frest sil Actinbacteria CBA-11 Uncultured actinbacterium CBA-36 Curtbacterium sp CBA-43 Curtbacterium flaccumfaciens 97 7 CBA-45 Curtbacterium flaccumfaciens CBA-47 Curtbacterium sp CBA-49 Curtbacterium sp CBA-63 Curtbacterium sp CBA-76 Curtbacterium sp CBA-92 Curtbacterium sp CBA-79 Curtbacterium sp planctmycete CBA-97 Uncultured bacterium Verrucmicrbia CBA-70 Uncultured Verrucmicrbiabacterium 93 3

7 122 Sng-Ih Han et al. Kr. J. Micrbil Fig. 4. Distributin f the ARDRA grups btained by restrictin analysis f 16S rdna with the endnuclease HaeIII frm Pine frest sil. Fig. 6. Distributin f the ARDRA grups btained by restrictin analysis f 16S rdna with the endnuclease HaeIII frm Quercus frest sil. š grup 16 (6 clnes) grup 25 (7 clnes) Acidbacteria w š, grup 6 (6 clnes) Planctmycetes m w (Fig. 5). w, ù m ARDRA grups 6 clnes sww 8 ARDRA grups (3, 6, 7, 15, 20, 22, 24, 26) ARDRA grup w (Fig. 6). ù m ARDRA grups Bradzrhiybium, Afipia sww grup 20 (7 clnes), grup 24 (11 clnes) š Azspirillum sww grup 26 (8 clnes) 3 grups α-prtebacteria m w y, grup 6 (7 clnes) γ-prtebacteria w. Grup 3 (6 clnes) Verrucmicrbia w š, grup 7 (12 clnes), grup 15 (6 clnes), grup 22 (7 clnes) Actinbacteria m w (Fig. 7). ù ù m ü 99% w sƒ VBNC ³ mw p mw, ù m ü sw ³ 50% α-prtebacteria m ƒ m y š, Acidbacteria Plantmycetes m ƒƒ 15% w m ùkû. ù m ü ³ 40% Actinbacteria m ƒ m y š, Acidbacteria m 17% w. p Verrucmicrbia m ù m ùkù p m w.» Ÿ x ü y ù e ù w w w w ù w w y ù j ùkü š šw (2). ù» mw w ù d w wd Ÿ m d w. w w,,,» m ü yw w ql sw. p w yw š, w yw y ù w w w (5, 6, 13, 16, 22). e ( ù ) y ( ù ) m m ƒ ƒ ³ w š q w.» w ù m l ³ w w w y Ÿ d m w ³ Bacillus s x ³, ³, Arthrbacter xk ³, ³ w (14). Fig. 5. Phylgenetic trees shwing the relatinships amng the 16S rdna sequences f clnes t dminant ARDRA grups frm Pine frest sil.

8 Vl. 42, N. 2 ù ù m ü VBNC ³ mw p 123 Fig. 7. Phylgenetic trees shwing the relatinships amng the 16S rdna sequences f clnes t dminant ARDRA grups frm Quercus frest sil. wr e m ³ flra ³ Arthrbacter, Bacillus, Micrcccus, Streptmyces 4 90% wš š š (12).» w m ³ w, ù ù m ü 99% w sƒ VBNC ³ mw w w w š m l DNA w 16S rdna-ardra» mw w VBNC ³ y ³ mw w y. m k ü VBNC ³ w» w k w VBNC ³ w w» Bigreen21 y ( y: ) w,. š x 1., y, Á x.. 2. w, ûª,, ½,, k Ÿ x ü ù w w y. w wz. 89, y,, Takashi Smeya x DVC w ù m ³ sƒ. w wz. 39, y, x » m ³ m r ³. w wz 21, Á l Œ(1) ž Œ G. (ž, ) Alexander, M Intrductin t sil micrbilgy. Jhn Wiley & Sns, New yrk. 7. Berg, B. and G. Agren Decmpsitin f needle litter and its rganic chemical cmpnent: thery and field experiments. Lngterm decmpsitin in a Scts pine frest III. Can. J. Bt. 62, Blmfield, S., G. Stewart., C. Ddd., R. Bth., and E. Pwer The viable but nnculturable phenmenn explained. J. Micrbil. 144, Chandler, D. P., R. W. Schreckhise, J. L. Smith, and H. Bltn Jr Electrelutin t remve humic acids frm sil DNA and RNA extracts. J. Micrbil. 61, Clwell, R. R., P. R. Braytn, D. J. Grimes, D. B. Rzak, S. A. Huq, and L. M. Plamer Viable but nn-culturable Vibri chlera and related pathgens n the envirnment: Implicatins fr release f genetically engineered micrrganisms. Bitechnl. 3, Curtis, T. P., W. T. Slan, and J. C. Scannell Estimatin prkarytic diversity and its limis. Prc. Natl. Acad. Sci. USA 99, Hlms, E. and V. Jensen Aerbic chemrgantrphic bacteria f a Dnaish beech frest. Oiks, 23, Insam, H. and K. Haselwandter Metablic qutient f the sil micrflra in relatin t plant successin. Oeclgia 79, Jhnsn, J. L Similarity analysis f rrnas, In Gerhardt, P., R.G.E. Murray, W. A. Wd, N. R. Krirg (ed.), Methds fr general and mlecular bacterilgy. American Sciety fr Micrbilgy, Washingtn, DC Kim, J. G. and N. K. Chang Litter prductin and decmpsitin in the pinus rigida plantatin in Mt. Kwan-ak. Krean J. Ecl. 12, Kim, J. H. and H. W. Lee Grwth f sil micrrganism fr the leachates frm leaf litter. Krean J. Ecl. 12, Kgure, K., U. Simidu, and N. Taga An imprved direct

9 124 Sng-Ih Han et al. Kr. J. Micrbil viable cunt methd fr aquatic bacteria. Arch. Hydrbil. 102, Kgure, K., U. Simidu, N. Taga, and R. R. Clwll Crrelatin f directin f direct viable cunts with hetertrphic activity fr marine bacteria. Appl. Envirn. Micrbial. 53, Lane, D. J S/23S rrna sequencing, In Stackebrandt, E., M. Gd fellw (ed.), Nucleic acid techniques in bacterial systematics, Jhn Wiley and Sns, Chichester. pp Mun, H. T. and H. T. J Litter prductin and decmpsitin in the Quercus acutissima and pinus rigida frest sil. Krean J. Ecl. 17, Mun, H. T. and J. H. Kim Litterfall decmpsitin, and nutrient dynamics f litter in red pine (pinus densiflra) and Chinese thuja (Thuja rientalis) stands in the lime stne area, Krean J. Ecl. 15, Park, B. K. and M. R. Kim The decmpsitin rate f litter and sil micrrganisms in slpe directins. Krean J. Ecl. 8, Saitu, N. and M. Nei The neighbr-jining methd: a new methd fr recnstructing phylgenetic trees. Ml. Bil. Evl. 4, Thmsn, J. D., D. G. Higgins, and T. J. Gibsn CLUSTAL W; imprving the sensitivity f prgressive multiple sequence alignment thrugh sequence weighting, psitins specific gap penalties and weight matrix chice. Nucleic Acids Res. 22, Trsvik, V., J. Gsksyr, and F. L. Daae High diversity in DNA f sil bacteria. Appl. Envirn. Micrbil. 56, Tsai, Y. L. and B. H. Olsn Rapid methd fr direct extractin f dna frm sil and sediments. Appl. Envirn. Mcrbil. 57, Vaneechutte M., R. Rssau, P. De Vs, M. Gillis, D. Janssens, N. Paepe, A. De Ruck, T. Fiers, G. Claeys, and K. Kersters Rapid identificatin f bacteria f the Cmamamnadaceae with amplified ribsmal DNA restrictin analysis(ardra). FEMS Micrbil. Lett. 93, (Received April 25, 2006/Accepted June 1, 2006) ABSTRACT : Cmparisn f Phylgenetic Characteristics f Viable but Nn-Culturable (VBNC) Bacterial Ppulatins in the Pine and Quercus Frest Sil by 16S rdna-ardra Sng-Ih Han*, Yun-Ji Kim, and Kyung-Sk Whang (Department f Bitechnlgy and Institute f Micrbial Eclgy Resurces, Mkwn University, Daejen , Krea) In this study was perfrmed t analyze quantitatively the number f viable but nn-culturable bacteria in the Pine and Quercus frest sil by imprved direct viable cunt (DVC) and plate cunt (PC) methds. The number f living bacteria f Pine and Quercus frest sil by PC methd were less then 1% f DVC methd. This result shwed that viable but nn-culturable (VBNC) bacteria existed in the frest sil with high percentage. Diversity and structure f VBNC bacterial ppulatins in frest sil were analyzed by direct extracting f DNA and 16S rdna-ardra frm Pine and Quercus frest sil. Each f them btained 111 clnes and 108 clnes frm Pine and Quercus frest sil. Thirty different RFLP types were detected frm Pine frest sil and twenty-six different RFLP types were detected frm Quercus frest sil by HeaIII. Frm ARDRA grups, dminant clnes were selected fr determining their phylgenetic characteristics based n 16S rdna sequence. Based n the 16S rdna sequences, dminant clnes frm ARDRA grups f Pine frest sil were classified int 7 majr phylgenetic grups : α-prtebacteria (12 clnes), γ-prtebacteria (3 clnes), δ-prtebacteria (1 clne), Flexibacter/Cytphaga (1 clne), Actinbacteria (4 clnes), Acidbacteria (4 clnes), Planctmycetes (5 clnes). Als, dminant clnes frm ARDRA grups f Quercus frest sil were classified int 6 majr phylgenetic grups : α-prtebacteria (4clnes), γ-prtebacteria (2 clnes), Actinbacteria (10 clnes), Acidbacteria (8 clnes), Planctmycetes (1 clne), and Verrucmicbia (1 clne). Result f phylgeneric analysis f micrbial cmmunity frm Pine and Quercus frest sils were mstly cnfirmed at uncultured r unidentified bacteria, VBNC bacteria f ver 99% existent in frest sil were cnfirmed variable cmpsitin f unknwn micrrganism.