Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter January 13, 2020

Direct bioautography for the screening of selected tropical wood extracts against basidiomycetes

  • Regina Wanschura EMAIL logo , Matthias Baumgartner , Claudia U. Linder , Elisabeth Windeisen , J. Philipp Benz and Klaus Richter
From the journal Holzforschung


To understand the reasons for the high durability of tropical wood species, the chemistry of the extractives needs to be elucidated. As these extractives consist of a great variety of components differing in quantity and composition, the analysis is often time-consuming. To focus on the key bioactive substances, bioassay-guided fractionation is helpful, but the established bioassay methods cannot be readily adapted to basidiomycete fungi that are commonly used for the respective durability tests, because they do not sporulate easily in laboratory settings. The research therefore aims at developing a direct bioautography using homogenized hyphae from basidiomycetes, to overcome this restriction. Extracts from four tropical wood species were analyzed regarding their potential bioactivity on two selected basidiomycete fungi. To this end, the chemically complex mixtures and extract constituents were resolved by a two-dimensional planar chromatography and the metabolites were located by characteristic zones of fungal growth inhibition, which was accentuated by a color reaction. The bioactive fractions were analyzed by gas chromatography/mass spectrometry (GC/MS). Potentially responsible compounds could be identified, such as the alkaloid bicuculline from Mezilaurus itauba, which has not been described in this species yet. The presented bioassay method can be used as a rapid screening method for bioactive components from wood.


The authors would like to thank Valerija Erceg, Selina Götz and Martina Schwarzmüller for their valuable contribution by performing parts of the analysis.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.


AbuMweis, S.S., Barake, R., Jones, P.J.H. (2008) Plant sterols/stanols as cholesterol lowering agents: a meta-analysis of randomized controlled trials. Food. Nutr. Res. 52. Doi: 10.3402/fnr.v52i0.1811.10.3402/fnr.v52i0.1811Search in Google Scholar

Berridge, M.V., Tan, A.S. (1993) Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch. Biochem. Biophys. 303:474–482.10.1006/abbi.1993.1311Search in Google Scholar

Berridge, M.V., Herst, P.M., and Tan, A.S. (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 11:127–152.10.1016/S1387-2656(05)11004-7Search in Google Scholar

Bisset, N.G., Wolff, R.E., Chavanel, V., Lantz, J.P. (1971) Constitutants Sesquiterpenique et Triterpeniques des resins du genre Shorea. Phytochemistry 10:2451–2463.10.1016/S0031-9422(00)89892-1Search in Google Scholar

Brito Pereira Bezerra Martins, A.O., Albuquerque Siebra, A.L., de Morais Oliveira, C.D., Leite, G.O., Melo Coutinho, H.D., Athayde, M.L., Bolignon, A.A., Andrade, J.C., Oliveira, L.R., Kerntopf, M.R., Barbose, R., Alves Santana Cesário, F.R., Coutinho, T.S., Quintans-Júnior, L.J., Alencar de Menezes, I.R. (2018) Antimicrobial gastroprotective and healing effect of the hydroalcoholic extract of Astronium fraxinifolium. Lett. Drug Des. Discov. 15:325–334.10.2174/1570180814666170213160114Search in Google Scholar

Choi, S.M., Ruddick, J.N.R., Morris, P.I. (2001) The effect of storage and subculturing on in vitro fruit body formation and spore production in Gloeophyllum sepiarium and Oligoporus placentus. Int Res Group Wood Preservation, Stockholm, Sweden, Document No IRG/WP/01-20232.Search in Google Scholar

Choi, S.M., Ruddick, J.N.R., Morris, P.I. (2002) The copper tolerance of mycelium vs Spores for two brown rot fungi. Int Res Group Wood Preservation, Stockholm, Sweden, Document No IRG/WP/02–10422.Search in Google Scholar

Clausen, C.A., Coleman, R.D., Yang, V.W. (2010) Fatty acid–based formulations for wood protection against mold and sapstain. Forest Prod. J. 60:301–304.10.13073/0015-7473-60.3.301Search in Google Scholar

Croan, S.C., Highley, T.L. (1991) Conditions for carpogenesis and basidiosporogenesis by brown rot fungi basidiomycete Gloeophyllum trabeum. Mater. Org. 27:1–9.Search in Google Scholar

Custódio, D.L., da Veiga Junior, V.F. (2014) Lauraceae alkaloids. RSC Adv. 4:21864–21890.10.1039/C4RA01904KSearch in Google Scholar

Da Silva, V.C., Napolitano, A., Eletto, D., Rodrigues, C.M., Pizza, C., Vilegas, W. (2011) Characterization of gallotannins from Astronium species by flow injection analysis-electrospray ionization-ion trap-tandem mass spectrometry and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Eur. J Mass Spectrom. 17:365–375.10.1255/ejms.1141Search in Google Scholar PubMed

Deklerck, V., De Windt, I., Defoirdt, N., Van den Bulcke, J., Beeckman, H., Espinoza, E., Van Acker, J. (2017) Assessing the natural durability for different tropical timber species using the mini-block test. Proceedings IRG Annual Meeting. Ghent, Belgium. Document No. IRG/WP 17-10886.Search in Google Scholar

Deutsches Institut für Normung e.V. (1994) EN 350-1: Natürliche Dauerhaftigkeit von Vollholz. Teil 1: Grundsätze für die Prüfung und Klassifikation der natürlichen Dauerhaftigkeit von Holz. Berlin, Beuth 1994–10.Search in Google Scholar

Deutsches Institut für Normung e.V. (1996) EN 113: Prüfverfahren zur Bestimmung der vorbeugenden Wirksamkeit gegen holzzerstörende Basidiomyceten. Bestimmung der Grenze der Wirksamkeit. Berlin, Beuth 1996–11.Search in Google Scholar

Dewanjee, S., Gangopadhyay, M., Bhattacharya, N., Khanra, R., Dua, T.K. (2015) Bioautography and its scope in the field of natural product chemistry. Pharm. Biomed. Anal. 5:75–84.10.1016/j.jpha.2014.06.002Search in Google Scholar PubMed PubMed Central

Etxeberria, A., Mendarte, S., Larregla, S. (2011) Determination of viability of Phytophthora capsici oospores with the tetrazolium bromide staining test versus a plasmolysis method. Rev. Iberoam. Micol. 28:43–49.10.1016/j.riam.2010.11.005Search in Google Scholar PubMed

Favre-Godal, Q, Queiroz, E.F., Wolfender, J.L. (2013) Latest developments in assessing antifungal activity using TLC-bioautography: a review. J. AOAC Int. 96:1175–1188.10.5740/jaoacint.SGEFavre-GodalSearch in Google Scholar

Freimoser, F.M., Jakob, C.A., Aebi, M., Tuor, U. (1999) The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities. Appl. Environ. Microbiol. 65:3727–3729.10.1128/AEM.65.8.3727-3729.1999Search in Google Scholar

Gabel, L. (2008) Zur Effizienz der Zerschäumungsanalyse im halbtechnischen Maßstab am Beispiel von Humulus lupulus L. und Rosmarinus officinalis L. Dissertation. Technische Universität München.Search in Google Scholar

Gérard, J., Guibal, D., Paradis, S., Vernay, M., Beauchêne, J., Brancheriau, L., Châlon, I., Daigremont, C., Détienne, P., Fouquet, D., Langbour, P., Lotte, S., Thévenon, M.F., Méjean, C., Thibaut, A. (2011) Tropix 7. CIRAD in Google Scholar

Gottlieb, O.R. (1972) Review article: Chemosystenmatics of the Lauraceae. Phytochemistry 11:1537–1570.10.1016/0031-9422(72)85001-5Search in Google Scholar

Hadi, S. (2009) The isolation of hopeaphenol, a tetramer stilbene, from Shorea ovalis Blume. Adv. Nat. Appl. Sci. 3:107–113.Search in Google Scholar

Helfenstein, A., Vahermo, M., Nawrot, D., Demirci, F., Iscan, G., Krogerus, S., Yli-Kauhaluoma, J., Moreira, V.M., Tammela, P. (2017) Antibacterial profiling of abietane-type diterpenoids. Bioorg. Med. Chem. 25:132–137.10.1016/j.bmc.2016.10.019Search in Google Scholar

Hidore, M.R., Nabavi, N., Sonleitner, F., Murphy, J.W. (1991) Murine natural killer cells are fungicidal to Cryptococcus neoformans. Infect. Immun. 59:1747–1754.10.1128/iai.59.5.1747-1754.1991Search in Google Scholar

Hillis, W.E., Yazaki, Y. (1973) Polyphenols of Intsia heartwoods. Phytochemistry 12:2491–2495.10.1016/0031-9422(73)80461-3Search in Google Scholar

Hirano, Y., Kondo, R., Sakai, K. (2003) Novel stilbenoids isolated from the heartwood of Shorea laeviforia. J. Wood. Sci. 49:339–343.10.1007/s10086-002-0481-ySearch in Google Scholar

Hostettmann, K. (1991) Assays for bioactivity. In: Methods in Plant Biochemistry. Eds. Dey, PM., Harborne, JB. Vol. 6. Academic Press, San Diego. p. 360.Search in Google Scholar

Imai, T., Inoue, S., Matsushita, N.O.Y., Suzuki, R., Sakurai, M., de Jesus, J.M.H., Finger, S.K.O.Z., Fukushima, K. (2008) Heartwood extractives from the Amazonian trees Dipteryx odorata, Hymenaea courbaril, and Astronium lecointei and their antioxidant activities. J. Wood Sci. 54:470–475.10.1007/s10086-008-0975-3Search in Google Scholar

Kawamura, F., Ohara, S., Nishida, A. (2004) Antifungal activity of constituents from the heartwood of Gmelina arborea: part 1. Sensitive antifungal assay against Basidiomycetes. Holzforschung 58:189–192.10.1515/HF.2004.028Search in Google Scholar

Kilic, A., Niemz, P. (2012) Extractives in some tropical woods. Eur. J. Wood Wood Prod. 70:79–83.10.1007/s00107-010-0489-8Search in Google Scholar

Kirker, G.T., Blodgett, A.B., Arango, R.A., Lebow, P.K., Clausen, C.A. (2013) The role of extractives in naturally durable wood species. Int. Biodeterior. Biodegradation. 82:53–58.10.1016/j.ibiod.2013.03.007Search in Google Scholar

Liu, X., Han, R., Wang, Y., Li, X., Zang, M., Yan, Y. (2014) Fungicidal activity of a medium-chain fatty acids mixture comprising caprylic, pelargonic and capric acids. Plant Pathol. J. 13:65–70.10.3923/ppj.2014.65.70Search in Google Scholar

Mäkinen, K.K. (2014) 3 – Authorised EU health claims for xylitol and sugar-free chewing gum (SFCG). Foods, Nutrients and Food Ingredients with Authorised EU Health Claims. 46–72.10.1533/9780857098481.2.46Search in Google Scholar

Meshulam, T., Levitz, S.M., Christin, L., Diamond, R.D. (1995) A simplified new assay for assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-2H)-tetrazolium-5-carboxanilide (XTT). J. Infect. Dis. 172:1153–1156.10.1093/infdis/172.4.1153Search in Google Scholar

Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55–63.10.1016/0022-1759(83)90303-4Search in Google Scholar

Osmonova, N. (2011) Screening of antimicrobial effects of selected fungi and studies on antibiotic constituents of Bulgaria inquinans (Pers.) Fr. (Bulgariaceae) and Meripilus giganteus (Pers.:Fr.) P.Karst. (Meripilaceae). Dissertation. University of Hamburg.Search in Google Scholar

Patel, N., Oudemans, P.V., Hillman, B., Kobayashi, D.Y. (2013) Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica. Antonie van Leeuwenhoek 103:1271–1280.10.1007/s10482-013-9907-3Search in Google Scholar

Pohl, C.H., Kock, J.L.F., Thibane, V.S. (2011) Antifungal free fatty acids: a review. In: Science Against Microbial Pathogens: Communicating Current Research and Technological Advances. Ed. Méndez-Vilas, A. Formatex Research Center, Badajoz, Spain. pp. 61–71.Search in Google Scholar

Råberg, U., Edlund, M.L., Terziev, N., Land, C.J. (2005) Testing and evaluation of natural durability of wood in above ground conditions in Europe – an overview. J. Wood Sci. 51:429–440.10.1007/s10086-005-0717-8Search in Google Scholar

Rahalison, L.M., Hamburger, M., Hostettmann, K. (1991) A bioautographic agar overlay method for the detection of antifungal compounds from higher plants. Phytochem. Anal. 2:199–203.10.1002/pca.2800020503Search in Google Scholar

Rennerfelt, E. (1948) Thujaplicin, a fungicidal substance in the heartwood of Thujja plicata. Physiol. Plant. 1:245–254.10.1111/j.1399-3054.1948.tb07128.xSearch in Google Scholar

Rutiaga-Quinones, J.G. (2001) Chemische und biologische Untersuchungen zum Verhalten dauerhafter Holzarten und ihrer Extrakte gegenüber holzabbauenden Pilzen. Dissertation. Technische Universität München.Search in Google Scholar

Schultz, T.P., Nicholas, D.D. (2000) Naturally durable heartwood: evidence for a proposed dual defensive function of the extractives. Phytochemistry 54:47–52.10.1016/S0031-9422(99)00622-6Search in Google Scholar

Schwager und Lange (1998) Biologischer Holzschutz. Literaturstudie über akzessorische Bestandteile dauerhafter Holzarten mit resistenzwirksamer Aktivität. Münster. Landwirschaftsverlag. Schriftenreihe Nachwachsende Rohstoffe.Search in Google Scholar

Silva, R., Nagheim, T.J., Mesquita, A.A.L., Gottlieb, O. (1983) y-Lactones from Mezillaurus synandra. Phytochemistry 22:772–773.10.1016/S0031-9422(00)86983-6Search in Google Scholar

Singh, T., Sing, A.P. (2012) A review on natural products as wood protectant. Wood Sci. Technol. 46:851–870.10.1007/s00226-011-0448-5Search in Google Scholar

Smeds, A.I., Eklund, P.C., Monogioudi, E., Willför, S.M. (2012) Chemical characterization of polymerized products formed in the reactions of matairesinol and pinoresinol with the stable radical 2,2,diphenyl-1-picrylhydrazyl. Holzforschung 66:283–294.10.1515/hf.2011.151Search in Google Scholar

Snow, A.D., Castillo, G.M., Nguyen, B.P., Choi, P.Y., Cummings, J.A., Cam, J., Hu, Q., Lake, T., Pan, W., Kastin, A.J., Kirschner, D.A., Wood, S.G., Rockenstein, E., Masliah, E., Lorimer, S., Tanzi, R.E., Larsen, L. (2019) The Amazon rain forest plant Uncaria tomentosa (cat’s claw) and its specific proanthocyanidin constituents are potent inhibitors and reducers of both brain plaques and tangles. Sci. Rep. 9:561.10.1038/s41598-019-38645-0Search in Google Scholar

Starke, K. (2013) Grundlagen der Pharmakologie des Nervensystems. In: Allgemeine und spezielle Pharmakologie und Toxikologie. 11. Auflage. Hrsg. Aktories, K., Förstermann, U., Hofmann F und Starke, K. München, Elsevier GmbH. pp. 115–116.10.1016/B978-3-437-42523-3.00002-6Search in Google Scholar

Stirling, R., Kus, S., Uzunovic, A. (2016) Inhibition of basidiospore germination by western red cedar heartwood extractives. Int. Biodeterior. Biodegradation. 114:145–149.10.1016/j.ibiod.2016.06.008Search in Google Scholar

Stockert, J.C., Horobin, R.W., Colombo, L.L., and Blázquez-Castro, A. (2018) Tetrazolium salts and formazan products in cell biology: viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochem. 120:159–167.10.1016/j.acthis.2018.02.005Search in Google Scholar

Wagenführ, R., Scheiber, C. (1974) Holzatlas, 3.Auflage. VEB Fachbuchverlag Leibzig, Germany 1989:311–315.Search in Google Scholar

Wanschura, R., Windeisen, E., Richter, K. (2016) Screening of bioactive extracts of a tropical hardwood species and identification of key substances. Proceedings of the 14th EWLP and IUFRO scientific workshop on Biorefinery, Autrans.Search in Google Scholar

Wanschura, R., Windeisen, E., Richter, K. (2017) Establishing a screening method for bioactive extractives of wood species against basidiomycetes. IUFRO Division 5 Conference and 60th SWST Convention Proceedings, Vancouver.Search in Google Scholar

Wanschura, R., Windeisen, E., Richter, K. (2018) Application of foam fractionation to wood. Wood Sci. Technol. 53:349–371.10.1007/s00226-019-01087-ySearch in Google Scholar

Watanabe, M., Maemura, K., Kanbara, K., Tamayama, T., Hayasaki, H. (2002) GABA and GABA receptors in the central nervous system and other organs. Int. Rev. Cytol. 213:1–47.10.1016/S0074-7696(02)13011-7Search in Google Scholar

Wedge, D., Nagle, D.G. (2000) A New 2D-TLC Bioautography method for the discovery of novel antifungal agents to control plant patogens. J. Nat. Prod. 63:1050–1054.10.1021/np990628rSearch in Google Scholar PubMed

Willför, S., Hemming, J., Reunanen, M., Holmbom, B. (2003) Phenolic and lipophilic extractives in Scots pine knots and stemwood. Holzforschung 57:359–372.10.1515/HF.2003.054Search in Google Scholar

Willför, S., Eklund, P., Sjöholm, R., Reunanaen, M., Sillanpää, R., von Schoultz, S., Hemming, J., Nisula, L., Holmbom, B. (2005) Bioactive phenolic substances in industrially important tree species. Part 4: identification of two new 7-hydroxy divanillyl butyrolactol lignans in some spruce, fir, and pine species. Holzforschung 59: 413–417.10.1515/HF.2005.067Search in Google Scholar

Yanez, Y., Aura, M.P., Pedro, P., Duz, D. (1988) Neolignans from Mezillaurus itaúba. Phytochemistry 25:1953–1956.10.1016/S0031-9422(00)81182-6Search in Google Scholar

Yang, D.Q. (2009) Potential utilization of plant and fungal extracts for wood protection. Forest Prod. J. 59:97–103.Search in Google Scholar

Supplementary Material

The online version of this article offers supplementary material (

Received: 2019-05-22
Accepted: 2019-11-05
Published Online: 2020-01-13
Published in Print: 2020-08-27

©2020 Walter de Gruyter GmbH, Berlin/Boston

Downloaded on 30.11.2023 from
Scroll to top button