Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Phylogenetic Characteristics of viable but Nonculturable Bacterial Populations in a Pine Mushroom (Tricholoma matsutake) Forest Soil

송이 자생군락 토양 내 난배양성 세균군집의 계통학적 특성

  • Kim, Yun-Ji (Institute of Microbial Ecology & Resources, Mokwon University) ;
  • Whang, Kyung-Sook (Institute of Microbial Ecology & Resources, Mokwon University)
  • 김윤지 (목원대학교 미생물생태자원연구소) ;
  • 황경숙 (목원대학교 미생물생태자원연구소)
  • Published : 2007.09.30
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Abstract

The CFDA (6-carboxyfluorescein diacetate) direct viable count method and plate count (PC) method using conventional nutrient broth (NB) medium and $10^{-2}$ diluted NB (DNB) medium were applied to samples collected from Mt. Yongdoo In Andong, in an effect to determine the number of living bacteria pine mushroom forest soil. The number of living bacteria determined via plate count in NB medium comprised $5{\sim}8%$ of the CFDA direct viable count, and the bacteria in the DNB medium comprised $40{\sim}47%$. This result indicated that viable but nonculturable (VBNC) bacteria existed in the pine mushroom forest soil at a high percentage. The phylogenetic characteristics of the VBNC bacterial populations in the samples of pine mushroom (Tricholoma matsutake) forest soil were analyzed via the direct extraction of DNA and 16S rDNA-ARDRA. The 115 clones from pine mushroom forest soil were clustered into 31 different RFLP phylotypes by ARDRA. Based on the 16S rDNA sequences, the 31 ARDRA clusters were classified into 6 phylogenetic groups: ${\alpha}-,\;{\beta}-,\;{\gamma}-Proteobacteria$, Acidobacteria, Actinobacteria and Firmicutes. Among these bacterial populations, approximately 85% were classified as members of phylum Acidobacteria. The Acidobacteria phylum was shown to exist abundantly in the pine mushroom forest soil.

송이 자생군락 토양 내 세균군집의 정량적 평가를 수행한 결과 CFDA 형광염색법을 이용해 직접 계수된 생균수는 $7.4{\pm}1.19{\times}10^8{\sim}1.07{\pm}0.17{\times}10^9cells/g$ soil로 육즙영양배지(nutrient broth, NB)에서 배양된 생균수는 CFDA 계수치의 $5{\sim}8%$로 계수되었으며, $10^{-2}$으로 희석한 NB(DNB)배지에서는 $40{\sim}47%$의 계수치를 나타내었다. 이상의 결과로부터 송이 자생군락 토양내에는 배양이 곤란한 난배양성(viable but non-culturable; VBNC)세균이 다수 존재해 있는 것으로 추정되었다. 송이 자생군락 토양내 세균군집의 계통학적 특성을 검토하기 위해 토양으로부터 직접 DNA를 추출하고 16S rDNA-ARDRA cluster 분석을 통하여 대표 clone의 16S rDNA 염기서열 분석을 수행하였다. 송이 자생군락 토양으로부터 구축된 총 115 clone은 31 ARDRA cluster로 분류되었으며, ${\alpha}-,\;{\beta}-,\;{\gamma}-$ Proteobacteria, Acidobacteria, Actinobacteria 그리고 Firmicutes의 6개 계통군이 확인되었다. 이들 계통군 중 약 85%가 Acidobacteria 계통군에 속하여 압도적인 우점군임이 확인되어 매우 독특한 계통학적 특성을 나타내었다.

Keywords

References

  1. 강안석, 김양섭, 차동열, 박용환. 1988. 송이버섯 발생과 관련되는 기후 특성 분석 연구. 한국균학회지 16, 256-256
  2. 김인기, 염곡효, 황경숙. 2002. 형광현미경을 이용한 음용지하수내 배양 불능 세균의 관찰 및 정량적 평가. 한국미생물학회지 38, 180-185
  3. 문형태. 1994. 삼림생태계의 낙엽생산량, 분해량 및 분해 과정에 따른 화학적 구성원의 변화. 한국과학재단
  4. 박진숙, 황경숙, 천종식. 2005. 미생물의 분류동정 실험법, 월드사이언스
  5. 송인근, 김유영, 조홍범, 최영길. 1999 . 토양세균 군집의 대사 다양성과 16S rDNA의 제한 효소 지문분석에 의한 유전적 다양성 비교.한국미생물학회지 35, 72-77
  6. 이재곤, 이창국, 장희진, 곽재진. 2004. Purge and Trap Headspace 법에 의한 솔잎 (Pinus densiflora s.)휘발성 성분. 한국식품영양학회 17,260-265
  7. 차동열. 1981. 송이버섯에 대한 연구현황. 한국균학회지 9, 164-165
  8. 황경숙, 유승헌. 1995. 유기영양분 농도에 따른 토양 세균의 증식양상과 통상 및 편성 저영양세균의 분리. 한국미생물학회지 21, 319-324
  9. Barns, S.M., S.L. Takala, and C.R. Kuske. 1999. Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment. Appl. Environ. Microbiol. 65, 1731-1737
  10. Berg, B. and G. Agren. 1984. Decomposition of needle litter and its organic chemical component: theory and field experiments. Longterm decomposition in a Scots pine forest III. Can. J. Bot. 62, 2880-2888 https://doi.org/10.1139/b84-384
  11. Bloomfield, S.F., GS.A.B. Stewart, C.E.R. Dodd, I.R. Booth, and E.G.M. Power. 1998. The viable but nonculturable phenomenon explained. Microbiology 144, 1-3
  12. Borneman, J. and E.W. Triplett. 1997. Molecular microbial diversity in soils from Eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl. Environ. Microbiol. 63, 2647-2653
  13. Chandler, D.P., R.W. Schreckhise, J.L. Smith, and H. Bolton, Jr. 1997. Electroelution to remove humic acids from soil DNA and RNA extracts. J. Microbiol. Methods 28, 11-19 https://doi.org/10.1016/S0167-7012(96)00957-8
  14. Hugenholtz, P., B.M. Goebel, and N.R. Pace. 1998. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180,4765-4774
  15. Ito, J. 1994. The present status of Matsutake production technique in Japan. Korean J Mycol. 9, 219-222
  16. Johnson, J.L. 1994. Similarity analysis ofrRNAs, pp. 683-700. In P. Gerhardt, R.G.E. Murray, W.A. Wood, and N.R. Krirg (ed.), Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, USA
  17. Kim, J.G. and N.K. Chang. 1989. Litter production and decomposition in the pinus rigida plantation in Mt. K wan-ak. Korean J. Ecol. 12, 9-20
  18. Kishimoto, N., Y. Kosako, and T. Tano. 1991. Acidobacterium capsulatum gen. nov., sp. nov.: an acidophilic chemoorganotrophic bacterium containing menaquinone from acidic mineral environment. Curr. Microbiol. 22, 1-7 https://doi.org/10.1007/BF02106205
  19. Lagac, L., M. Pitre, M. Jacques, and D. Roy. 2004. Identification of the bacterial community of maple sap by using amplified ribosomal DNA (rDNA) restriction analysis and rDNA sequencing. Appl. Environ. Microbiol. 70, 2052-2060 https://doi.org/10.1128/AEM.70.4.2052-2060.2004
  20. Lane, D.J. 1991. 16S/23S rRNA sequencing, pp. 115-175. In E. Stackebrandt and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics, John Wiley and Sons, Chichester, UK
  21. Lee, C.-Y., O.-P. Hong, M.-J. Jung, and Y.-H. Han. 1997. Effect of carbon sources and vitamins on myclial growth of Tricholoma matsutake DGUM 26001. Korean J. Mycol. 25, 226-232
  22. Massol-Deya, A.A., D.A. Odelson, R.F. Hickey, and J.M. Tiedje. 1995. Bacterial community fingerprinting of amplified 16S and 16S-23S ribosomal DNA gene sequences and restriction endonuclease analysis (ARDRA), pp. 3.3.2/1.3.3.2/8. In A.D.L. Akkermans, J.D. van Elsas, and F.J. de Bruijn (ed.) Molecular Microbial Ecology Manual, Kluwer, Netherlands
  23. Mark, G.W., J.V. McArthur, and L.J. Shimkets. 1997. Bacterial diversity of a carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa. Appl. Environ. Microbiol. 63, 1505-1514
  24. Mun, H.T. and J.H. Kim. 1992. Litter fall decomposition, and nutrient dynamics of litter in red pine (pinus densiflora) and Chinese thuja (Thuja orientalis) stands in the lime stone area. Korean J. Ecol. 15, 147-155
  25. Ogawa, M. 1981. Mycorrhiza in the pine forest-the ecological study ofmatsutake as a microorganism. Korean J. Mycol. 9, 225-227
  26. Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425
  27. Stackebrandt, E., W. Liesack, and B.M. Goebel. 1993. Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis. FASEB J. 7, 232-236 https://doi.org/10.1096/fasebj.7.1.8422969
  28. Thomson, J.D., D.G. Higgins, and T.J. Gibson. 1994. CLUSTAL W; improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680 https://doi.org/10.1093/nar/22.22.4673
  29. Torsvik, V., J. Gosksoyr, and F.L. Daae. 1990. High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56, 782-787
  30. Tsai, Y.L. and B.H. Olson. 1991. Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57, 1070-1074
  31. Vaneechoutee, M., R. Rossau, P. De Vos, M. Gillis, D. Janssens, N. Paepe, A. De Rouck, T. Fiers, G. Claeys, and K. Kersters. 1992. Rapid identification of bacteria of the Comamamonadaceae with amplified ribosomal DNA restriction analysis (ARDRA). FEMS Microbiol. Lett. 93,227-234 https://doi.org/10.1111/j.1574-6968.1992.tb05102.x