Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter June 16, 2017

Preliminary analysis of amplicon high-throughput sequencing as a method for the assessment of fungal diversity in discolored wood

  • Xingxia Ma EMAIL logo , Mingliang Jiang , Junliang Liu , Hao Deng and Shuangyong Wang
From the journal Holzforschung


The diversity of stain fungi is important if wood is inhabited with various fungi, and the discoloration mechanism will be better understood. MiSeq amplicon high-throughput sequencing (Illumina®) is able to detect species richness (the number of species within a community) and species evenness (the sizes of species populations within a community). This study detected fungal diversity in discolored Mongolian pine for the first time by the MiSeq approach, focusing on the nuclear ribosomal internal transcribed spacer-1 (ITS1). The results show that the discolored wood was inhabited by a combination of microorganisms, more than 90% of which belong to Ascomycota fungi at the phylum level. The MiSeq method revealed not only all the inhabited fungal species but also their quantitative relation to each other. The dominant fungal species in sample A are Helotiales (34.1%) and Hypocreales (20.7%). The dominant fungal species in sample B is Nectriaceae (67.9%), while Hypocrea (34.7%) and Sporothrix (27.6%) are the dominant fungal species in sample C. It was confirmed via core microbiome analysis that the following fungi were common stain fungi in the three discolored samples: Fusarium spp., Aspergillus spp., Sporothrix spp., Penicillium spp., Trichoderma spp., Alternaria spp. and Cladophialophora spp.


This research was supported financially by the Central-level Public Welfare Foundation of the Research Institute of Forest New Technology, Chinese Academy of Forestry (CAFYBB2016SY024) and the China National Natural Science Foundation, Ministry of Human Resources and Social Security of the People’s Republic of China (2016).


Clubbe, C.P. (1980) The colonisation and succession of fungi in wood. Document No.: IRG/WP 1107. International Research Group on Wood Preservation, Stockholm.Search in Google Scholar

Fang, Z.D. Methods in Plant Pathology. 3rd ed., China Agricultural Press, Beijing, 1998. pp. 3–427Search in Google Scholar

Gardes, M., Bruns. T.D. (1993) ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Mol. Ecol. 2:113–118.10.1111/j.1365-294X.1993.tb00005.xSearch in Google Scholar PubMed

Handelsman, J., Rondon, M.R., Brady, S.F., Clardy, J., Goodman, R.M. (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol. 5:R245–R249.10.1016/S1074-5521(98)90108-9Search in Google Scholar

Hawksworth, D.L. (1991) The fungal dimension of biodiversity: magnitude, significance, and conservation. Fungal Biol. 95: 641–655.10.1016/S0953-7562(09)80810-1Search in Google Scholar

Huh, N., Jang, Y., Lee, J., Kim, G.H., Kim, J.J. (2011) Phylogenetic analysis of major molds inhabiting woods and their discoloration characteristics. Part 1. Genus Trichoderma. Holzforschung 65:257–263.10.1515/hf.2011.018Search in Google Scholar

Jang, Y., Huh, N., Lee, J., Lee, J.S., Kim, G.H., Kim, J.J. (2011) Phylogenetic analysis of major molds inhabiting woods and their discoloration characteristics. Part 2. Genus Penicillium. Holzforschung 65:265–270.10.1515/hf.2011.019Search in Google Scholar

Kaeberlein, T., Lewis, K., Epstein, S.S. (2002) Isolating “Uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296:1127–1129.10.1126/science.1070633Search in Google Scholar PubMed

Kirker, G.T., Prewitt, M.L., Schultz, T.P., Diehl, S.V. (2012a) Community analysis of preservative-treated southern pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Part 1: Fungal field study. Holzforschung 66:521–527.10.1515/hf.2011.171Search in Google Scholar

Kirker, G.T., Prewitt, M.L., Diehl, W.J., Diehl, S.V. (2012b) Community analysis of preservative-treated southern yellow pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Part 2. Bacteria field study. Holzforschung 66:529–535.10.1515/hf.2011.172Search in Google Scholar

Lee, S., Yamamoto, N. (2015) Accuracy of the high-throughput amplicon sequencing to identify species within the genus Aspergillus. Fungal Biol. 119:1311–1321.10.1016/j.funbio.2015.10.006Search in Google Scholar PubMed

Lee, Y.M., Jang, Y., Kim, G.H., Kim, J.J. (2012) Phylogenetic analysis and discoloration characteristics of major molds inhabiting woods. Part 3. Genus Cladosporium. Holzforschung 66:537–541.10.1515/hf.2011.184Search in Google Scholar

Lee, Y.M., Hong, J.H., Lee, H., Ahn, B.J., Kim, G.H., Kim, J.J. (2013) Phylogenetic analysis of the genus Fusarium and their antifungal activity against wood-decay and sapstain fungi. Holzforschung 67:473–478.10.1515/hf-2012-0124Search in Google Scholar

Lee, Y. M., Lee, H., Jang, Y., Cho, Y., Kim, G. H., Kim, J. (2014) Phylogenetic analysis of major molds inhabiting woods. Part 4. Genus Alternaria. Holzforschung 68:247–251.10.1515/hf-2013-0089Search in Google Scholar

Mikluscak, M.R., Dawson-Andoh, B.E. (2004) Microbial colonizers of freshly sawn yellow-poplar (Liriodendron tulipifera L.) lumber in two seasons: Part 1. Fungi. Holzforschung 58:173–181.10.1515/HF.2004.026Search in Google Scholar

Moreth, U., Schmidt, O. (2000) Identification of indoor rot fungi by taxon-specific priming polymerase chain reaction. Holzforschung 54:1–8.10.1515/HF.2000.001Search in Google Scholar

Morrell, J.J., Dawson-Andoh, B.E. (1998) Biological control: panacea or boondogle. In: Biology and Prevention of Sapstain. A conference sponsored by the Department of Forest Products, Oregon State University. British Columbia, Canada. pp. 39–44.Search in Google Scholar

Morse, A.C., Blanchette, R.A. (2002) Etiology of red stain in Boxelde. Plant Health Prog. doi: 10.1094/PHP-2002-0917-01-RS (Online).101094/PHP-2002-0917-01-RSSearch in Google Scholar

Nelson, P.E., Toussoun, T.A., Marasas, W.F.O. Fusarium species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, 1983.Search in Google Scholar

Pace, N.R.A. (1997) Molecular view of microbial diversity and the biosphere. Science 276:734–740.10.1126/science.276.5313.734Search in Google Scholar PubMed

Pfeffer, A., Hoegger, P.J., Kues, U., Militz, H. (2010) Fungal colonisation of outside weathered modified wood. Wood Sci. Techn. 46:63–72.10.1007/s00226-010-0386-7Search in Google Scholar

Ray, M.J., Dickinson, D.J. (2006) Direct analysis from the wood of the blue staining fungi Aureobasidium pullulans and Hormonema dematioides by denaturing gradient gel electrophoresis. Document No. IRG/WP 06-10595. International Research Group on Wood Protection. Stockholm, Sweden.Search in Google Scholar

Raberg, U., Brischke, C., Rapp, A.O., Hogberg, N., Land, C.J. (2007) External and internal fungal flora of pine sapwood (Pinus sylvestris L.) specimens in above-ground field tests at six different sites in south-west Germany. Holzforschung 61:104–111.10.1515/HF.2007.017Search in Google Scholar

Samuels, G.J. (2006) Trichoderma: systematics, the sexual state, and ecology. Phytopathology 96:95–206.10.1094/PHYTO-96-0195Search in Google Scholar PubMed

Schmidt, O. Wood and Tree Fungi – Biology, Damage, Protection, and Use. Springer Verlag Berlin Heidelberg, Germany, 2006.Search in Google Scholar

Schmidt, E.L., Dietz, M.G. (1985) Arthrographis cuboidea causing pink stain of sodium pentachlorophenoxide-treated red oak. Mycologia 77:316–318.10.2307/3793084Search in Google Scholar

White, T.J., Bruns, T., Lee, S., Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications. Eds. Innis, M.A., Gelfand, D.H., Shinsky, J.J., White, T.J. Academic Press, San Diego, CA. pp. 315–322.10.1016/B978-0-12-372180-8.50042-1Search in Google Scholar

Wilcox, W.W., Dietz, M. (1997) Fungi causing above-ground wood decay in structures in California. Wood Fiber Sci. 29: 291–298.Search in Google Scholar

Zhang, X., Wang, G., Xu, X., Nong, X., Wang, J., Amin, M., Qi, S. (2016) Exploring fungal diversity in deep-sea sediments from Okinawa trough using high-throughput Illumina sequencing. Deep-SEA Res. Part I 116: 99–105.10.1016/j.dsr.2016.08.004Search in Google Scholar

Zink, P., Fengel, D. (1988) Studies on the colouring matter of blue-stain fungi. Part 1. General characterization and the associated compounds. Holzforschung 42:217–220.10.1515/hfsg.1988.42.4.217Search in Google Scholar

Zink, P., Fengel, D. (1989) Studies on the colouring matter of blue-stain fungi Part 2. Electron microscopic observations of the hyphae wall. Holzforschung 43:371–374.10.1515/hfsg.1989.43.6.371Search in Google Scholar

Zink, P., Fengel, D. (1990) Studies on the colouring matter of blue-stain fungi. Part 3. Spectroscopic studies on fungal and synthetic melanins. Holzforschung 44:163–168.10.1515/hfsg.1990.44.3.163Search in Google Scholar

Received: 2017-1-24
Accepted: 2017-5-11
Published Online: 2017-6-16
Published in Print: 2017-9-26

©2017 Walter de Gruyter GmbH, Berlin/Boston

Downloaded on 29.11.2023 from
Scroll to top button