The Krean Jurnal f Micrbilgy, Vl. 42, N. 2, June 2006, p. 116-124 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: 042-829-7593, Fax: 042-829-7590 E-mail: 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
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
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 (500-580 bp)» w (19). ditrect cunt; TDC) d w» w DNA ey EtBr w m w z w w, ù m 1.7 10 10 cells/g sil, ù m 4.46 10 11 cells/g sil. w s q (plate cunt; PC) w ³ d w, ù m 4.9 10 7 CFU/g sil, ù m 3.71 10 7 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 1.5 10 10 cells/g sil ³ 88.2%, ù m 1.91 10 11 cells/g sil ³ 43%ƒ ³ y.» sq w 300-5000 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.
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.
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.
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 95 24 CPA-37 Afipia brmeae 99 14 CPA-46 Uncultured alpha prtebacterium 98 12 CPA-50 Uncultured sil bacterium clne 95 4 CPA-73 Uncultured sil bacterium clne 95 11 CPA-97 Uncultured bacterium 92 2 CPA-99 Uncultured bacterium clne 96 13 CPA-117 Uncultured bacterium clne 99 8 CPA-120 Uncultured bacterium clne 98 21 CPA-125 Uncultured alpha prtebacterium clne 98 20 CPA-137 Bradyrhizbium sp. 97 30 γ-prtebacteria CPA-20 Uncultured bacterium clne 98 15 CPA-70 Uncultured silbacterium clne 98 29 CPA-75 Bacterium Ellin405 99 19 Pine δ-prtebacteria CPA-21 Uncultured delta prte bacterim clne 95 18 frest sil Acidbacteria CPA-44 Uncultured Acidbacteriaceae bacterium 97 25 CPA-59 Uncultured eubacterium WD261 99 27 CPA-68 Uncultured silbacterium clne 98 16 CPA-133 Uncultured Acidbacteriales bacterium 99 23 Actinbacteria CPA-52 Uncultured silbacterim clne 99 22 CPA-67 Uncultured eubacterium 98 26 CPA-92 Uncultured eubacterium WD294 90 1 CPA-122 Uncultured actinbacterium clne 98 10 planctmycete CPA-1 Uncultured plan ctmycete clne 91 7 CPA-24 Uncultured frest sil bacterium clne 97 17 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 DSSD32 95 28 α-prtebacteria CBA-2 Afipia sp. 93 20 CBA-26 Uncultured sil bacterium 93 24 CBA-57 Uncultured bacterium 92 13 CBA-107 Bacterium Ellin5003 94 26 γ-prtebacteria CBA-25 Uncultured bacterium 90 25 CBA-44 Uncultured gamma prtebacterium 98 6 Acidbacteria CBA-1 Uncultured bacterium 95 21 CBA-37 Uncultured sil bacterium clne 92 19 CBA-58 Uncultured Acidbacteria bacterium 94 18 CBA-67 Uncultured bacterium clne 97 2 CBA-77 Uncultured sil bacterium 92 14 CBA-88 Uncultured sil bacterium 92 23 CBA-93 Uncultured eubacterium WD205 92 12 Quercus CBA-106 Uncultured bacterium clne 90 4 frest sil Actinbacteria CBA-11 Uncultured actinbacterium 94 15 CBA-36 Curtbacterium sp. 95 5 CBA-43 Curtbacterium flaccumfaciens 97 7 CBA-45 Curtbacterium flaccumfaciens 98 10 CBA-47 Curtbacterium sp. 91 11 CBA-49 Curtbacterium sp. 90 8 CBA-63 Curtbacterium sp. 97 1 CBA-76 Curtbacterium sp. 92 16 CBA-92 Curtbacterium sp. 97 9 CBA-79 Curtbacterium sp. 94 22 planctmycete CBA-97 Uncultured bacterium 91 17 Verrucmicrbia CBA-70 Uncultured Verrucmicrbiabacterium 93 3
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.
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». 2006 Bigreen21 y ( y: 20050301-034-384-006-01-00) w,. š x 1., y,. 2005. Á x.. 2. w, ûª,, ½,, k. 2000. Ÿ x ü ù w w y. w wz. 89, 41-48. 3. y,, Takashi Smeya. 2003. x DVC w ù m ³ sƒ. w wz. 39, 181-186. 4. y, x. 1995.» m ³ m r ³. w wz 21, 319-324. 5. Á l Œ(1) ž Œ G. (ž, ). 129-154. 6. Alexander, M. 1985. Intrductin t sil micrbilgy. Jhn Wiley & Sns, New yrk. 7. Berg, B. and G. Agren. 1984. 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, 2880-2888. 8. Blmfield, S., G. Stewart., C. Ddd., R. Bth., and E. Pwer. 1998. The viable but nnculturable phenmenn explained. J. Micrbil. 144, 1-3. 9. Chandler, D. P., R. W. Schreckhise, J. L. Smith, and H. Bltn Jr. 1997. Electrelutin t remve humic acids frm sil DNA and RNA extracts. J. Micrbil. 61, 273-278. 10. Clwell, R. R., P. R. Braytn, D. J. Grimes, D. B. Rzak, S. A. Huq, and L. M. Plamer. 1985. Viable but nn-culturable Vibri chlera and related pathgens n the envirnment: Implicatins fr release f genetically engineered micrrganisms. Bitechnl. 3, 817-820. 11. Curtis, T. P., W. T. Slan, and J. C. Scannell. 2002. Estimatin prkarytic diversity and its limis. Prc. Natl. Acad. Sci. USA 99, 10494-10499. 12. Hlms, E. and V. Jensen. 1972. Aerbic chemrgantrphic bacteria f a Dnaish beech frest. Oiks, 23, 248-260. 13. Insam, H. and K. Haselwandter. 1989. Metablic qutient f the sil micrflra in relatin t plant successin. Oeclgia 79, 174-178. 14. Jhnsn, J. L. 1994. 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. 683-700. 15. Kim, J. G. and N. K. Chang. 1989. Litter prductin and decmpsitin in the pinus rigida plantatin in Mt. Kwan-ak. Krean J. Ecl. 12, 9-20. 16. Kim, J. H. and H. W. Lee. 1989. Grwth f sil micrrganism fr the leachates frm leaf litter. Krean J. Ecl. 12, 67-74. 17. Kgure, K., U. Simidu, and N. Taga. 1984. An imprved direct
124 Sng-Ih Han et al. Kr. J. Micrbil viable cunt methd fr aquatic bacteria. Arch. Hydrbil. 102, 117-122. 18. Kgure, K., U. Simidu, N. Taga, and R. R. Clwll. 1987. Crrelatin f directin f direct viable cunts with hetertrphic activity fr marine bacteria. Appl. Envirn. Micrbial. 53, 2332-2337. 19. Lane, D. J. 1991. 16S/23S rrna sequencing, In Stackebrandt, E., M. Gd fellw (ed.), Nucleic acid techniques in bacterial systematics, Jhn Wiley and Sns, Chichester. pp. 115-175. 20. Mun, H. T. and H. T. J. 1994. Litter prductin and decmpsitin in the Quercus acutissima and pinus rigida frest sil. Krean J. Ecl. 17, 345-353. 21. Mun, H. T. and J. H. Kim. 1992. 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, 147-155. 22. Park, B. K. and M. R. Kim. 1985. The decmpsitin rate f litter and sil micrrganisms in slpe directins. Krean J. Ecl. 8, 31-37. 23. Saitu, N. and M. Nei. 1987. The neighbr-jining methd: a new methd fr recnstructing phylgenetic trees. Ml. Bil. Evl. 4, 406-425. 24. Thmsn, J. D., D. G. Higgins, and T. J. Gibsn. 1994. 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, 4673-4680. 25. Trsvik, V., J. Gsksyr, and F. L. Daae. 1990 High diversity in DNA f sil bacteria. Appl. Envirn. Micrbil. 56, 782-787. 26. Tsai, Y. L. and B. H. Olsn. 1991. Rapid methd fr direct extractin f dna frm sil and sediments. Appl. Envirn. Mcrbil. 57, 1070-1074. 27. Vaneechutte M., R. Rssau, P. De Vs, M. Gillis, D. Janssens, N. Paepe, A. De Ruck, T. Fiers, G. Claeys, and K. Kersters. 1992. Rapid identificatin f bacteria f the Cmamamnadaceae with amplified ribsmal DNA restrictin analysis(ardra). FEMS Micrbil. Lett. 93, 227-234. (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 302-729, 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.