A protocol specialized for microbial DNA extraction from living poplar wood


  • Xiao Li YU Huazhong Agricultural University, College of Horticulture and Forestry Sciences/ Hubei Engineering Technology Research Center for Forestry Information, Wuhan 430070 (CN)
  • Xing Yi HU Hubei Academy of Forestry, Wuhan 430075 (CN)
  • Xiu Xiu WANG Huazhong Agricultural University, College of Horticulture and Forestry Sciences/ Hubei Engineering Technology Research Center for Forestry Information, Wuhan 430070 (CN)
  • Xin Ye ZHANG Hubei Academy of Forestry, Wuhan 430075 (CN)
  • Ke Bing DU Huazhong Agricultural University, College of Horticulture and Forestry Sciences/ Hubei Engineering Technology Research Center for Forestry Information, Wuhan 430070 (CN)




bacterial DNA, DNA quality, fungal DNA, Populus, wet heartwood


Microbial DNA extraction is a critical step in metagenomic research. High contents of chemical substances in wood tissues always cause low microbial DNA yield and quality. Up to date, almost no specialized methods involved in microbial DNA extraction from living wood were reported. In this study, an improved protocol (M1) concerning microbial DNA extraction from living poplar wood was developed. We compared microbial DNA yield and quality by M1 with those by other seven methods, including PowerSoil DNA isolation kit (M2), two soil microbial DNA extraction methods (M3 and M4), poplar genomic DNA extraction method from wood (M5), and microbial DNA extraction method from herb stems (M6), isolating bacteria (M7) and isolating fungus (M8). Results showed that M1 yielded much better quality and concentration of microbial DNA than the other methods (M2-M8) from both poplar wetwood and sapwood tissues. Following M1 protocol, 1 g of wetwood sample could yield 272.27 ng/ul (vol=50 ul) pure microbial DNA with the absorption ratios of 1.87 (A260/A230) and 1.66 (A260/A280). For 1 g of sapwood sample, these values were 361.83 ng/ul, 1.85 and 2.24, respectively. These DNA could be stably visualized by agarose gel electrophoresis and amplified by primer sets of bacteria (16S V3-V4, 16S-V4, 16S V4-V5) and fungus (ITS1, ITS2). While, the other seven methods only obtained less or contaminated microbial DNA, which could not be amplified stably by aforementioned primer sets. Our protocol provided an approach for microbial community study in living poplar wood in a more accurate way by molecular biology techniques.


Amann RI, Ludwig W, Schleifer KH (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Review 59(1):143-169. https://doi.org/10.1128/mr.59.1.143-169.1995

Cenis JL (1992) Rapid extraction of fungal DNA for PCR amplification. Nucleic Acids Research 20(9):2380. https://doi.org/10.1093/nar/20.9.2380

Corapcioglu MO, Huang CP (1987). The adsorption of heavy metals onto hydrous activated carbon. Water Research 21(9):1031-1044. https://doi.org/10.1016/0043-1354(87)90024-8

Daniel R (2004). The soil metagenome- a rich resource for the discovery of novel natural products. Current Opinion Biotechnology 15(3):199-204. https://doi.org/10.1016/j.copbio.2004.04.005

Demeke T, Jenkins GR (2010). Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Analytical and Bioanalytical Chemistry 396(6):1977-1990. https://doi.org/10.1007/s00216-009-3150-9

Desai C, Madamwar D (2007). Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments. Bioresource Technology 98(4):761-768. https://doi.org/10.1016/j.biortech.2006.04.004

Grîu T, Lunguleasa A (2016). Salix viminalis vs. Fagus sylvatica-fight for renewable energy from woody biomass in Romania. Environmental Engineering and Management Journal 15(2):413-420. http://omicron.ch.tuiasi.ro/EEMJ/

Jeremic D, Cooper P, Srinivasan U (2004). Comparative analysis of balsam fir wetwood, heartwood, and sapwood properties. Canadian Journal of Forest Research 34(6):1241-1250. https://doi.org/10.1139/X03-287

Johansson T, Hjelm B (2013). Frequency of false heartwood of stems of poplar growing on farmland in Sweden. Forests 4(1):28-42. https://doi.org/10.3390/f4010028

Karakousis A, Tan L, Ellis D, Alexiou H, Wormald PJ (2006). An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR. Journal of Microbiological Methods 65(1):38-48. https://doi.org/10.1016/j.mimet.2005.06.008

Logan EM, Pulford ID, Cook GT, MacKenzie AB (1997). Complexation of Cu2+ and Pb2+ by peat and humic acid. European Journal of Soil Science 48(4):685-696. https://doi.org/10.1046/j.1365-2389.1997.00123.x

Magnani GS, Cruz LM, Weber H, Bespalhok JC, Daros E, Baura V, Yates MG, Monteiro RA, Faoro H, Pedrosa FO, … Souza EM (2013). Culture-independent analysis of endophytic bacterial communities associated with Brazilian sugarcane. Genetics and Molecular Research 12(4):4549-4558. https://doi.org/10.4238/2013.October.15.3

Maropola MKA, Ramond JB, Trindade M (2015). Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench). Journal of Microbiological Methods 112:104-117. https://doi.org/10.1016/j.mimet.2015.03.012

Martin L, Cochard H, Mayr S, Badel E (2021). Using electrical resistivity tomography to detect wetwood and estimate moisture content in silver fir (Abies alba Mill.). Annals of Forest Science 78:65. https://doi.org/10.1007/s13595-021-01078-9

Martin-Laurent F, Philippot L, Hallet S, Chaussod R, Germon JC, Soulas G, Catroux G (2001). DNA extraction from soils: old bias for new microbial diversity analysis methods. Applied and Environmental Microbiology 67(5):2354-2359. https://doi.org/10.1128/AEM.67.5.2354-2359.2001

Moré MI, Herrick JB, Silva MC, Ghiorse WC, Madsen EL (1994). Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment. Applied and Environmental Microbiology 60(5):1572-1580. https://doi.org/10.1128/aem.60.5.1572-1580.1994

Motková P, Vytrasová J (2011). Comparison of methods for isolating fungal DNA. Czech Journal of Food Sciences 29:S76-S85. https://doi.org/10.17221/266/2011-CJFS

Moya R, Muñoz F, Jeremic D, Berrocal A (2009). Visual identification, physical properties, ash composition, and water diffusion of wetwood in Gmelina arborea. Canadian Journal of Forest Research 39(3):537-545. https://doi.org/10.1139/X08-193

Nakada R, Okada N, Nakai T, Kuroda K, Nagai S (2019). Water potential gradient between sapwood and heartwood as a driving force in water accumulation in wetwood in conifers. Wood Science and Technology 53:407-424. https://doi.org/10.1007/s00226-019-01081-4

Pereira P, Ibáñez F, Rosenblueth M, Etcheverry M, Martínez-Romero E (2011). Analysis of the bacterial diversity associated with the roots of maize (Zea mays L.) through culture-dependent and culture-independent methods. ISRN Ecology 2011:1-10. https://doi.org/10.5402/2011/938546

Pindi PK, Srinath RR, Shanker AS (2013). Novel approaches of genomic DNA isolation for identification of cultivable bacteria. Jundishapur Journal of Microbiology 6(10):e8339. https://doi.org/10.5812/jjm.8339

Rondon MR, August PR, Bettermann AD, Brady SF, Grossman TH, Liles MR, ... Goodman RM (2000). Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Applied Environmental Microbology 66(6):2541-2547. https://doi.org/10.1128/AEM.66.6.2541-2547.2000

Sakamoto Y, Kato A (2002). Some properties of the bacterial wetwood (watermark) in Salix sachalinensis caused by Erwinia salicis. IAWA Journal 23(2):179-190. https://doi.org/10.1163/22941932-90000296

Schink B, Ward JC (1984). Microaerobic and anaerobic bacterial activities involved in formation of wetwood and discoloured wood. IAWA Bulletin 5(2):105-109. https://doi.org/10.1163/22941932-90000872

Seo GT, Ohgaki S (2001). Evaluation of refractory organic removal in combined biological powdered activated carbon-microfiltration for advanced wastewater treatment. Water Science and Technology 43(11):67-74. https://doi.org/10.2166/wst.2001.0668

Sessitsch A, Hardoim P, Döring J, Weilharter A, Krause A, Woyke T, ... Reinhold-Hurek B (2012). Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis. Molecular Plant-Microbe Interactions 25(1):28-36. https://doi.org/10.1094/MPMI-08-11-0204

Terrat S, Christen R, Dequiedt S, Leliévre M, Nowak V, Regnier T, … Ranjard L (2012). Molecular biomass and meta taxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure. Microbial Biotechnology 5(1):135-141. https://doi.org/10.1111/j.1751-7915.2011.00307.x

Thomas RJ (1975). The effect of polyphenol extraction on enzyme degradation of bordered pit tori. Wood and Fiber Science 7(3):207-215. http://ovidsp.ovid.com/ovidweb.cgi

Torsvik V, Ovreas L (2002). Microbial diversity and function in soil: from genes to ecosystems. Current Opinion Microbiology 5(3):240-245. https://doi.org/10.1016/S1369-5274(02)00324-7

Tsai YL, Olson BH (1991). Rapid method for direct extraction of DNA from soil and sediments. Applied Environmental Microbiology 57(4):1070-1074. https://doi.org/10.1128/AEM.57.4.1070-1074.1991

Verbylaite R, Beišys P, Rimas V, Kuusiene S (2010). Comparison of ten DNA extraction protocols from wood of European aspen (Populus tremula L.). Baltic Forestry 16(1):35-42. https://doi.org/10.1051/forest/2009090

Verma D, Satyanarayana T (2011). An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries. Applied Biochemistry and Biotechnology 165(2):454-464. https://doi.org/10.1007/s12010-011-9264-5

Větrovský T, Baldrian P (2013). The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses. PLoS One 8(2):e57923. https://doi.org/10.1371/journal.pone.0057923

Wang XQ, Jiang ZH, Ren HQ (2008) Distribution of wet heartwood in stems of Populus xiaohei from a spacing trial. Scandinavian Journal of Forest Research 23(1):38-45. https://doi.org/10.1080/02827580701763706

Wang ZP, Gu Q, Deng FD, Huang JH, Megonigal JP, Yu Q, ... Han XG (2016). Methane emissions from the trunks of living trees on upland soils. New Phytol 211(2):429-439. https://doi.org/10.1111/nph.13909

White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds). PCR protocols: a guide to methods and applications. San Diego: Academic Press, pp 315-322.

Yeates C, Gillings MR (1998). Rapid purification of DNA from soil for molecular biodiversity analysis. Letters in Applied Microbiology 27(1):49-53. https://doi.org/10.1046/j.1472-765X.1998.00383.x

Yeates C, Gillings MR, Davison AD, Altavilla N, Veal DA (1997). PCR amplification of crude microbial DNA extracted from soil. Letters in Applied Microbiology 25(4):303-307. https://doi.org/10.1046/j.1472-765X.1997.00232.x

Zhou J, Bruns MA, Tiedje JM (1996). DNA recovery from soils of diverse composition. Applied and Environmental Microbiology 62(2):316-322. https://doi.org/10.1128/AEM.62.2.316-322.1996



How to Cite

YU, X. L., HU, X. Y., WANG, X. X., ZHANG, X. Y., & DU, K. B. (2022). A protocol specialized for microbial DNA extraction from living poplar wood. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 50(4), 12822. https://doi.org/10.15835/nbha50312822



Research Articles
DOI: 10.15835/nbha50312822