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Natural resistance of five woods to Phanerochaete chrysosporium degradation
International Biodeterioration & Biodegradation 64 (2010) 711e715
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Natural resistance of five woods to Phanerochaete chrysosporium degradation Luciana S. Oliveira a, Andréa L.B.D. Santana a, Cláudia A. Maranhão a, Rita de Cássia M. de Miranda b, Vera Lúcia A. Galvão de Lima c, Suzene I. da Silva d, Márcia S. Nascimento b, *, Lothar Bieber a a
Departamento de Química Fundamental e CCEN, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, Pernambuco, Brazil Departamento de Antibióticos e CCB, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, Pernambuco, Brazil c Departamento de Economia Doméstica, Universidade Federal Rural de Pernambuco, Dois Irmãos, 52171-030 Recife, Pernambuco, Brazil d Departamento de Botânica, Universidade Federal Rural de Pernambuco, Dois Irmãos, 52171-030 Recife, Pernambuco, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 May 2010 Received in revised form 5 August 2010 Accepted 6 August 2010 Available online 28 September 2010
This research evaluated the natural resistance of five woods to the white-rot wood-destroying fungus Phanerochaete chrysosporium under laboratory conditions and in nature. The studied species were Hymenaea stigonocarpa, Anadenanthera colubrina, Caesalpinia ferrea, Manilkara huberi and Delonix regia. The natural resistance to decay is one of the most important properties of wood, mainly assigned to lignin and extractives of wood. A. colubrina has the highest content of extractives and M. huberi the highest content of lignin; both are known as resistant to xylophagous organisms and were also most resistant to the tested fungus. C. ferrea has the lowest content of extractives and D. regia of lignin; both species did not inhibit the fungus Phanerochaete chrysosporium. H. stigonocarpa occupies an intermediate position in content of extractives and lignin as well in resistance to P. chrysosporium. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Lignin content Extractives Hymenaea stigonocarpa Anadenanthera colubrina Caesalpinia ferrea Manilkara huberi Delonix regia Phanerochaete chrysosporium
1. Introduction The chemical composition of wood is complex. The two major chemical components in wood are the macromolecular cell wall components, carbohydrate (65e75%) and lignin (18e35%), and an array of low-molecular-mass compounds as extractives (4e10%) (Pettersen, 1984). Hydrophilic extractives comprise a great diversity of compounds, such as flavonoids (anthocyanins, flavanols, flavonols and flavones) and several classes of non-flavonoids (phenolic acids, tannins, stilbenes) (Harborne, 1989). Wood extractives are also lipophilic substances consisting mainly of triglycerides, fatty acids, diterpenoid resin acids, sterols, waxes and steryl esters (Fengel and Wegener, 1989). Natural durability or decay resistance is the ability of wood to prevent biological degradation (Eaton and Hale, 1993). After cellulose, lignin is the second most abundant type of biopolymers on the earth and provides plant resistance to microbial
* Corresponding author. Tel.: þ55 8121268347; fax: þ55 8121268346. E-mail address: [email protected] (M.S. Nascimento). 0964-8305/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2010.08.001
degradation, markedly influencing the natural durability of wood (Syafh et al., 1988; Tuomela et al., 2000). Although extractives contribute merely a few percent to the entire wood composition, they are very important to trees as defense mechanisms against microbial attack (Silva et al., 2007). According to Amusant et al. (2007) there is no doubt that extractives are the most significant factor influencing the durability of wood due to their fungicidal and antioxidant activity. Among the extractives, phenolic compounds are important to the plants for normal growth development and defense against infection and injury (Jerez et al., 2007). They play a key role as antioxidants due to the presence of aromatic hydroxyl groups, which enable them to scavenge free radicals. Different organisms can deteriorate wood, but the greatest damage is caused by fungi. White-rot basidiomycete fungi are the only known microorganisms in nature that are capable of degrading lignin completely. Phanerochaete chrysosporium has been well studied as a model strain because of its specialized ability to degrade lignin, while leaving the white cellulose nearly untouched (Kersten and Cullen, 2007; Hu et al., 2009). The aim of this work was to investigate the natural resistance of five different tropical woods to P. chrysosporium and to observe
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whether resistance correlates with the extractive- or lignin content of the wood.
These samples were autoclaved for 1 h at 121 3 C. The acidsoluble lignin was determined spectrophotometrically at 280 nm wavelength.
2. Materials and methods 2.1. Wood samples The wood from five different species, namely Hymenaea stigonocarpa, Anadenanthera colubrina, Caesalpinia ferrea, Delonix regia and Manilkara huberi were investigated. The information about the trees and their habitats was provided in Table 1. 2.2. Chemical analysis of wood 2.2.1. Extractives content The rotor-milled wood samples (1 g) were extracted with cyclohexane and then with ethanol (500 ml) in a soxhlet apparatus for 4 h to remove extractives. The lower polarity solvent, cyclohexane, was used for the first step to remove lipophilic compounds, and the more polar ethanol was used for the second extraction step to remove hydrophilic compounds. The extract solutions were concentrated in a rotary evaporator. The wood samples free of extractives were dried at 37 C, weighed and used for the determination of lignin. 2.2.2. Phytochemical tests of the plants The wood samples were air dried at room temperature, powdered and subsequently subjected to phytochemical screening, using procedures described by Costa (1982), to identify the main classes of secondary metabolites (Table 3). 2.2.3. Lignin content The lignin content was evaluated following the TAPPI method (Tappi, 1996), which is based on the isolation of Klason lignin after hydrolysis of the polysaccharides (cellulose and hemicellulose) by concentrated sulfuric acid (72%). After filtering of the insoluble lignin the hydrolyzed samples (triplicate) were put in a water bath for 2 h at 30 1 C and later were transferred to an Erlenmeyer flask and diluted to 4% acid concentration with distilled water.
2.2.4. Total phenol contents The total phenolic content was estimated by the FolineCiocalteu colorimetric method, based on the procedure of Waterman and Mole (1994), and the results are expressed as gallic acid equivalents (GAE). Standard concentrations of gallic acid between 0.78 and 50 mg ml1 were used to prepare calibration curves. 0.5 ml of wood extract methanol/water (8/2) or gallic acid (standard phenolic compound) was mixed with FolineCiocalteu reagent (2.5 ml, 10% diluted with distilled water) and aqueous solution sodium carbonate (2.0 ml of 7.5%). The mixture was kept for 5 min at 50 C; the absorbance at 760 nm was measured. The analyses were done in triplicate and the mean value was calculated. 2.2.5. DPPH radical scavenging assay DPPH (1,1-Diphenyl-2-picryl-hydrazyl) scavenging activity of wood extracts was determined according to the method described by Brand-Willians et al. (1995) with slight modifications. 0.2 ml of the ethanolic extract (100 mg ml1 in methanol) was added to 4 ml of DPPH methanolic solution (43 mg ml1). This method is based on the reduction of a methanol solution of DPPH in the presence of a hydrogen donating antioxidant due to the formation of the nonradical form DPPH-H. The antioxidanteradical reactions were conducted for 30 min in the dark at ambient temperature. This transformation results in a change of color from purple to yellow, which is measured spectrophotometrically by the disappearance of the purple color at 515 nm. Ascorbic acid was used as standard. 2.3. Fungi The white-rot fungus P. chrysosporium from Collection of Tropical Culture (CCT 1999) Fundação André Tosello, was used in this study. Cultures were maintained on malt extract agar at 30 C for 10 days. The fungus was then incubated on solid culture medium containing 15 g of agar, 15 g of yeast extract and 1000 ml distilled water.
Table 1 Key characteristics of tested wood species. Wood
Characteristic and use
References
Hymenaea stigonocarpa Occurrence: Bolívia, Brazil Family: Caesalpinioideae Common name: jatobá-do-cerrado Anadenanthera colubrina Occurrence: Bolivia, Brazil, Peru Family: Mimosoideae Common name: Angico-branco, angico-de-caroço Caesalpinia ferrea Occurrence: Brazil Family: Caesalpinioideae Common name: jucá, pau-ferro Manilkara huberi Occurrence: Brazil, Venezuela, French Guiana Family: Sapotaceae Common name: maçaranduba, beefwood, bulletwood, sapotilla Delonix regia Occurrence: native to Madagascar but introduced to tropical areas. Family: Caesalpinioideae Common name: Flamboyant, flame of the forest, poinciana
Natural resistance soft-rot fungi and termites, furniture, used in building and naval industries.
Lorenzi, 1992 Santana et al., 2010
Natural resistance soft-rot fungi and termites, used in furniture, leather tanning, building and naval industries.
Lorenzi, 1998 Santana et al., 2010
Durable wood is used in building industry and manufacture of furniture, guitars, fiddles and pipes.
Lorenzi and Matos, 2002
High durability natural, indicated for use on fences, posts and floors. Exported to U.S. market, Japan and some European countries
Lorenzi, 1998
Soft and weak wood limiting its use in carpentry, subject to termite attack.
Schütt and Lang, 2004 Lorenzi et al., 2003
L.S. Oliveira et al. / International Biodeterioration & Biodegradation 64 (2010) 711e715
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Table 2 Percent extractives in the studied wood species. Extraction procedure (%)
Hymenaea stigonocarpa
Anadenanthera colubrina
Caesalpinia ferrea
Manilkara huberi
Delonix regia
Cyclohexane Ethanol Total
1.5 0,6 52 72
0.33 0.08 82 92
1.0 0.5 3.2 0,3 4.3 0.4
0.6 0.2 7.1 0,5 7.7 0,4
0.8 0.4 41 51
2.4. Laboratory test on the susceptibility of woods to the white-rot fungus Phenerochaete chrysosporium The laboratory tests to evaluate the natural durability of wood to degradation by P. chrysosporium were carried out based on the procedure of Kamida et al. (2005) with slight modifications. 5 g of finely powdered wood samples were weighed in Erlenmeyer flasks (125 ml) and humidified with 1 ml of tap water. Three replicates were used for extracted and unextracted woods. Non-inoculated, sterilized wood was used as a biotic control. Three disks (6 mm in diameter) from solid culture medium inoculated with the fungus were transferred to each Erlenmeyer flask. The growth of P. chrysosporium mycelium was evaluated after 4 weeks at 30 C. 3. Results and discussion Table 2 displays the extractives of woods. The amount of cyclohexane-extractives was always lower than ethanol-extractives. The total content of extractives (extracted gradually with cyclohexane and ethanol) was the greatest in A. colubrina (9% of the total dry weight of wood), followed by M. huberi (7.7%) and H. stigonocarpa (7%), D. regia (5%) and C. ferrea (4.3%). These values agree rather well with other woods reported in the literature. A study carried out by Santana and Okino (2007) with 36 Brazilian tropical timbers found extractive content between 17.3% and 0.9%, but only eight of the thirty six species showed extractive content higher than 10%. In that study M. huberi had an extractives content of 8% so is comparable with our results. Species with high contents of extractives are of interest for future studies because chemical compounds from the crude extracts can be used for pharmaceuticals, dyes, cosmetics, perfumes and natural antioxidants (Santana and Okino, 2007). High extractive content is known to result in good natural durability (Windeisen et al., 2002). The result of the preliminary phytochemical analysis of the extract of wood species is shown in Table 3. The phytochemical studies revealed the presence of flavonoids, terpenes and steroids in all analyzed woods. Tannin presence was very strong in A. colubrina. Saponins were found only in M. huberi indicating that, among the studied woods, there were significant differences in the amount and in the composition of extractives. Phenolic compounds, such as tannins and flavonoids, have antimicrobial and antioxidative properties and are involved in the defense against fungi and other microorganisms (Boudet, 2007). Saponins have been reported to prevent antimicrobial activity (Sparg et al., 2004). Secondary metabolites (extractives) are present in all plants, generally as mixtures that can be highly diverse. A large number of studies have demonstrated the importance of these metabolites as
plant defense compounds. A high diversity of extractives in high concentration provides a more effective protection against herbivores than single compounds or low diversity in both low and high concentrations (Castellanos and Espinosa-Garcia, 1997). The diversity (Table 2) as well as the concentration of phenolic compounds (Table 3) was more pronounced in A. colubrina and M. huberi in sharp contrast to D. regia and C. ferrea. Table 4 provides the quantification of lignin. According to Syafh et al. (1988) lignin is the most important non-toxic factor that limits the growth of the microorganisms in wood biodegradation. M. huberi presents the highest content of total lignin (30%). In this study, D. regia had the lowest concentration of lignin (22.3%), thus suggesting that lignin may be the main factor for the low resistance of this wood. Among 36 woods analyzed, Santana and Okino (2007) found an acid-soluble lignin (ASL) content of 0.7e1.8% and an acidinsoluble lignin (AIL) of 26.7e37%. The values for M. huberi were 34% AIL and and 0.9% ASL. The lignin content for all woods was similar and typical for tropical hardwoods, except for D. regia. These results suggest that differences in the content and structure of lignin may influence the decay resistance caused by wooddecaying fungi (Syafh et al., 1988). The content of phenolics (mg g1) in ethanol extracts was determined from a regression equation of the calibration curve (y ¼ 0.004663x þ 0.0565, R2 ¼ 0.99) and expressed in Gallic Acid Equivalents (GAE). As shown in Table 5, there is a close correlation between the phenolic content and the % DPPH radical quenched. The ethanol extract from H. stigonocarpa contained the highest amount of total phenolics and, consequently, the highest antioxidant activity, followed by A. colubrina, C. ferrea, D. regia, and M. huberi. Although D. regia has higher phenolic contents than M. huberi, it displays the lowest antioxidant activity. These results indicate that phenolic composition among both woods differ widely in terms of chemical composition. The influence of extractives on the growth of P. chrysosporium in the tested woods is shown in Table 6. The extracted and unextracted wood of M. huberi inhibited fungal growth completely, confirming that lignin plays an important role in natural resistance of this wood (Syafh et al., 1988; Tuomela et al., 2000). This observation suggests that the content of saponins is not responsible for the natural resistance of M. huberi against fungal attack since decay resistance was not affected by their extraction (Table 3). Among all the studied woods, A. colubrina was the only species where the influence of extractives was very well observed. Fungal growth was entirely inhibited by unextracted wood but intense growth formed in the presence of extracted wood (Fig. 1). Rowell et al. (2005) states that wood resistance to fungal attack is attributed mainly to the presence of extractives that are toxic to
Table 3 Preliminary phytochemical analysis of extractives detected in the woods. Classes of extractives
Extraction procedure
Hymenaea stigonocarpa
Anadenanthera colubrina
Caesalpinia ferrea
Manilkara huberi
Delonix. regia
Saponins Tannins Alkaloids Flavonoids Steroids and terpenoids
Frothing Ferric chloride Dragendorff, Mayer Shinoda Oxalo-boric acid LiebermanneBuchard
þ þ þþ þþ
þþþ þþþ þþ
þ þ þ
þþþ þ þþ þþ
þ þþ
() not detected; (þ) detected. The number of positive signs indicates the intensity of the reactions (Costa, 1982).
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Table 4 Lignin content of the studied woods species. Lignin (%)
Hymenaea stigonocarpa
Anadenanthera colubrina
Caesalpinia ferrea
Manilkara huberi
Delonix regia
Acid-soluble (ASL) Acid-insoluble (AIL) Total
0.6 0.4 27 1 28 1
1.81 0.03 25.9 0.9 27.6 0.9
1.7 0.3 26 4 28 4
1.40 0.06 28.6 0.4 30.0 0.4
0.3 0.1 22 1 22.3 1
Table 5 Total phenolic content and DPPH scavenging activity of phenolic extractives from analyzed woods. Methanol/water extract
Hymenaea stigonocarpa
Anadenanthera colubrina
Caesalpinia ferrea
Manilkara huberi
Delonix regia
mg GAE g1 of extracts % DPPH radical quenched
0.248 0.009 91.00 0.050
0.231 0.003 71.90 0.099
0.151 0.005 21.51 0.358
0.069 0.004 5.99 0.050
0.092 0.007 3.07 0.14
Table 6 Phanerochaete chrysosporium growth in the studied woods species. Wood samples
Hymenaea stigonocarpa
Unextracted þ wood Extracted þþ wood
Anadenanthera Caesalpinia Manilkara Delonix colubrina ferrea huberi regia
þþþ
þþþ
þþ
þþþ
þþþ
() no growth; (þ) weak, (þþ) medium and (þþþ) abundant growth of Phanerochaete chrysosporium.
xylophagous organisms, thus providing the wood with natural durability. In addition, A. colubrina contains a high content of phenolic compounds and high antioxidant activity (Table 5) and also has good natural resistance to termites (Silva et al., 2007; Santana et al., 2010). According to Schultz and Nicholas (2000), the radical
scavenging activity of phenolic compounds may further accelerate fungal death by scavenging the radicals produced by fungus and reducing fungal nutrition necessary for repairing cell wall injuries, thus resulting in the strong synergy of antifungal activity. As can be seen in Fig. 1, unextracted wood of H. stigonocarpa stimulates only weak growth of P. chrysosporium, whereas fungal growth in the presence of extracted wood is far more pronounced, confirming that the extractives could not completely inhibit the growth of the fungus but acted as a primary barrier to prevent colonization. Although H. stigonocarpa extractives did not inhibit completely fungus growth, this wood is known for its natural resistance (Table 1). According to Rudman (1965), heartwood extractives from durable species are not always toxic against a broad spectrum of wood-destroying fungi but are often specific to a limited number of species. Apparently, the extractives of H. stigonocarpa do not exhibit strong antifungal activity against P. chrysosporium used in this study.
Fig. 1. The growth of Phanerochaete chrysosporium showing on extracted and unextracted woods from Hymenaea stigonocarpa (left) and Anadenanthera colubrina (right).
Fig. 2. The growth of Phanerochaete chrysosporium showing on extracted and unextracted woods from Delonix regia (left) and Caesalpinia ferrea (right).
L.S. Oliveira et al. / International Biodeterioration & Biodegradation 64 (2010) 711e715
Fig. 2 shows similar behavior for C. ferrea and D. regia in respect to fungal growth. The extractives from both woods showed weak antifungal activities. Usually flavonoids and tannins are responsible for antioxidant activity, which sequester the free radicals produced by fungi during the decay process (Schultz and Nicholas, 2000). C. ferrea and D. regia have a lower content of extractives (Table 2) and phenolic compounds (Table 5) than other woods. These factors may explain the low resistance of both woods against P. chrysosporium. 4. Conclusion The durability of A. colubrina can be attributed mainly to the presence of phenolic compounds, particularly tannins and flavonoids that inhibit fungal growth. Although the wood of M. huberi contains large quantities of extractives, especially saponins, they are not the only compounds responsible for antifungal activity. The high lignin content and the chemical composition of the lignin seem to be the major reasons for the high decay resistance of this wood. The naturally low durability of C. ferrea and D. regia wood against P. chrysosporium may be the result of various factors, including the low quantity of extractives, the low antioxidant activity of the phenolic extractives, and the structural composition of the lignin. Acknowledgements The authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for grants and fellowships. References Amusant, N., Moretti, C., Richard, B., Prost, E., Nuzillard, J.M., Thevenon, M.F., 2007. Chemical compounds from Eperua falcata and Eperua grandiflora heartwood and their biological activities against wood destroying fungus (Coriolus versicolor). Holz als Roh- und Werkstof 65, 23e28. Boudet, A.M., 2007. Evolution and current status of research in phenolic compounds. Phytochemistry 68, 22e24. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Food Science and Technology 28, 25e30. Castellanos, I., Espinosa-Garcia, F.J., 1997. Plant secondary metabolite diversity as a resistance trait against Insects: a test with Sitophilus granarius (Coleoptera: Curculionidae) and Seed secondary metabolites. Biochemical Systematics and Ecology 25, 591e602. Costa, A.F., 1982. Farmacognosia, 2nd ed., Vol. 3. Fundação Calouste Gulbenkian, Lisboa. Eaton, R.A., Hale, M.D.C., 1993. Wood: decay, pests and protection. Chapman & Hall, London.
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Fengel, D., Wegner, G., 1989. Wood chemistry, ultra structure, reactions. Walter de Gruyter, Berlin. Harborne, J.B., 1989. Methods in plant biochemistry, plant phenolics, Vol. 1. Academic Press, London. Hu, M., Zhang, W., Wu, Y., Gao, P., Lu, X., 2009. Characteristics and function of a lowmolecular-weight compound with reductive activity from Phanerochaete chrysosporium in lignin biodegradation. Bioresource Technology 100, 2077e2081. Jerez, M., Selga, A., Sineiro, J., Torres, J.L., Núñez, M.J., 2007. A comparison between bark extracts from Pinus pinaster and Pinus radiata: antioxidant activity and procyanidin composition. Food Chemistry 100, 439e444. Kamida, H.M., Durran, T.L.R., Monteiro, R.T.R., Armas, E.D., 2005. Biodegradação de efluente têxtil por Pleurotus sajor-caju. Química Nova 28, 629e632. Kersten, P., Cullen, D., 2007. Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium. Fungal Genetics and Biology 44, 77e87. Lorenzi, H., 1992. Árvores brasileiras: manual de identificação e cultivo de plantas arbóreas nativas do Brasil. Plantarum, São Paulo. Lorenzi, H., 1998. Árvores brasileiras: manual de identificação e cultivo de plantas arbóreas do Brasil. Plantarum, São Paulo. Lorenzi, H., Matos, F.J.A., 2002. Plantas medicinais do Brasil: nativas e exóticas. Plantarum, Nova Odessa. Lorenzi, H., Souza, H.M., Torres, M.A.V., Bacher, L.B., 2003. Árvores Exóticas no Brasil: Madereiras, ornamentais e aromáticas. Plantarum, Nova Odessa. Pettersen, R.C., 1984. The chemical composition of wood. In: Rowell, R.M. (Ed.), The chemistry of solid wood. Advances in chemistry series 207. American Chemical Society, Washington, pp. 57e126. Rowell, R.M., Pettersen, R., Han, J.S., Rowell, J.S., Tshabalala, M.A., 2005. Cell wall chemistry. In: Rowell, R.M. (Ed.), Handbook of wood chemistry and wood composites. Boca Raton, CRC Press, Washington, pp. 35e74. Rudman, P., 1965. Fine structure of wood. Nature 208, 55e56. Santana, A.L.B.D., Maranhão, C.A., Santos, J.C., Cunha, F.M., Conceição, G.M., Bieber, L.W., Nascimento, M.S., 2010. Antitermitic activity of extractives from three Brazilian hardwoods against Nasutitermes corniger. International Biodeterioration and Biodegradation 64, 7e12. Santana, M.A.E., Okino, E.Y.A., 2007. Chemical composition of 36 Brazilian Amazon forest wood species. Holzforschung 61, 469e477. Schultz, T.P., Nicholas, D.D., 2000. Naturally durable heartwood: evidence for a proposed dual defensive function of the extractives. Phytochemistry 54, 47e50. Schütt, P., Lang, U.M., 2004. Bäume der tropen. Nikol Verlagsgesellschaft, Hamburg. Silva, C.A., Monteiro, M.B.B., Brazolin, S., Lopez, G.A.C., Richter, A., Braga, M.R., 2007. Biodeterioration of brazilwood Caesalpinia echinata Lam. (Leguminosae e Caesalpinioideae) by rot fungi and termites. International Biodeterioration and Biodegradation 60, 285e292. Sparg, S.G., Light, M.E., van Staden, J., 2004. Review e biological activities and distribution of plant saponins. Journal of Ethnopharmacology 94, 219e243. Syafh, W., Yoshimoto, T., Samejima, M., 1988. Effect of lignin structure on decay resistance of some tropical wood. Bulletin of Tokyo University Forest 80, 69e77. Tappi Test Method T 222 om-88, 1996. Acid-insoluble lignin in wood and pulp. In: Tappi test methods. Tappi Press, Atlanta. Tuomela, M., Vikman, M., Hatakka, A., Itavaara, M., 2000. Biodegradation of lignin in a compost environment: a review. Bioresource Technology 72, 169e183. Waterman, P.G., Mole, S., 1994. Analysis of phenolic plant metabolites. Blackwell Scientific Publications, London. Windeisen, E., Wegener, G., Lesnino, G., Schumacher, P., 2002. Investigation of the correlation between extractives content and natural durability in 20 cultivated larch trees. Holz als Roh- und Werkstoff 60, 373e374.
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Natural resistance of five woods to Phanerochaete chrysosporium degradation Luciana S. Oliveira a, Andréa L.B.D. Santana a, Cláudia A. Maranhão a, Rita de Cássia M. de Miranda b, Vera Lúcia A. Galvão de Lima c, Suzene I. da Silva d, Márcia S. Nascimento b, *, Lothar Bieber a a
Departamento de Química Fundamental e CCEN, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, Pernambuco, Brazil Departamento de Antibióticos e CCB, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, Pernambuco, Brazil c Departamento de Economia Doméstica, Universidade Federal Rural de Pernambuco, Dois Irmãos, 52171-030 Recife, Pernambuco, Brazil d Departamento de Botânica, Universidade Federal Rural de Pernambuco, Dois Irmãos, 52171-030 Recife, Pernambuco, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 May 2010 Received in revised form 5 August 2010 Accepted 6 August 2010 Available online 28 September 2010
This research evaluated the natural resistance of five woods to the white-rot wood-destroying fungus Phanerochaete chrysosporium under laboratory conditions and in nature. The studied species were Hymenaea stigonocarpa, Anadenanthera colubrina, Caesalpinia ferrea, Manilkara huberi and Delonix regia. The natural resistance to decay is one of the most important properties of wood, mainly assigned to lignin and extractives of wood. A. colubrina has the highest content of extractives and M. huberi the highest content of lignin; both are known as resistant to xylophagous organisms and were also most resistant to the tested fungus. C. ferrea has the lowest content of extractives and D. regia of lignin; both species did not inhibit the fungus Phanerochaete chrysosporium. H. stigonocarpa occupies an intermediate position in content of extractives and lignin as well in resistance to P. chrysosporium. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Lignin content Extractives Hymenaea stigonocarpa Anadenanthera colubrina Caesalpinia ferrea Manilkara huberi Delonix regia Phanerochaete chrysosporium
1. Introduction The chemical composition of wood is complex. The two major chemical components in wood are the macromolecular cell wall components, carbohydrate (65e75%) and lignin (18e35%), and an array of low-molecular-mass compounds as extractives (4e10%) (Pettersen, 1984). Hydrophilic extractives comprise a great diversity of compounds, such as flavonoids (anthocyanins, flavanols, flavonols and flavones) and several classes of non-flavonoids (phenolic acids, tannins, stilbenes) (Harborne, 1989). Wood extractives are also lipophilic substances consisting mainly of triglycerides, fatty acids, diterpenoid resin acids, sterols, waxes and steryl esters (Fengel and Wegener, 1989). Natural durability or decay resistance is the ability of wood to prevent biological degradation (Eaton and Hale, 1993). After cellulose, lignin is the second most abundant type of biopolymers on the earth and provides plant resistance to microbial
* Corresponding author. Tel.: þ55 8121268347; fax: þ55 8121268346. E-mail address: [email protected] (M.S. Nascimento). 0964-8305/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2010.08.001
degradation, markedly influencing the natural durability of wood (Syafh et al., 1988; Tuomela et al., 2000). Although extractives contribute merely a few percent to the entire wood composition, they are very important to trees as defense mechanisms against microbial attack (Silva et al., 2007). According to Amusant et al. (2007) there is no doubt that extractives are the most significant factor influencing the durability of wood due to their fungicidal and antioxidant activity. Among the extractives, phenolic compounds are important to the plants for normal growth development and defense against infection and injury (Jerez et al., 2007). They play a key role as antioxidants due to the presence of aromatic hydroxyl groups, which enable them to scavenge free radicals. Different organisms can deteriorate wood, but the greatest damage is caused by fungi. White-rot basidiomycete fungi are the only known microorganisms in nature that are capable of degrading lignin completely. Phanerochaete chrysosporium has been well studied as a model strain because of its specialized ability to degrade lignin, while leaving the white cellulose nearly untouched (Kersten and Cullen, 2007; Hu et al., 2009). The aim of this work was to investigate the natural resistance of five different tropical woods to P. chrysosporium and to observe
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whether resistance correlates with the extractive- or lignin content of the wood.
These samples were autoclaved for 1 h at 121 3 C. The acidsoluble lignin was determined spectrophotometrically at 280 nm wavelength.
2. Materials and methods 2.1. Wood samples The wood from five different species, namely Hymenaea stigonocarpa, Anadenanthera colubrina, Caesalpinia ferrea, Delonix regia and Manilkara huberi were investigated. The information about the trees and their habitats was provided in Table 1. 2.2. Chemical analysis of wood 2.2.1. Extractives content The rotor-milled wood samples (1 g) were extracted with cyclohexane and then with ethanol (500 ml) in a soxhlet apparatus for 4 h to remove extractives. The lower polarity solvent, cyclohexane, was used for the first step to remove lipophilic compounds, and the more polar ethanol was used for the second extraction step to remove hydrophilic compounds. The extract solutions were concentrated in a rotary evaporator. The wood samples free of extractives were dried at 37 C, weighed and used for the determination of lignin. 2.2.2. Phytochemical tests of the plants The wood samples were air dried at room temperature, powdered and subsequently subjected to phytochemical screening, using procedures described by Costa (1982), to identify the main classes of secondary metabolites (Table 3). 2.2.3. Lignin content The lignin content was evaluated following the TAPPI method (Tappi, 1996), which is based on the isolation of Klason lignin after hydrolysis of the polysaccharides (cellulose and hemicellulose) by concentrated sulfuric acid (72%). After filtering of the insoluble lignin the hydrolyzed samples (triplicate) were put in a water bath for 2 h at 30 1 C and later were transferred to an Erlenmeyer flask and diluted to 4% acid concentration with distilled water.
2.2.4. Total phenol contents The total phenolic content was estimated by the FolineCiocalteu colorimetric method, based on the procedure of Waterman and Mole (1994), and the results are expressed as gallic acid equivalents (GAE). Standard concentrations of gallic acid between 0.78 and 50 mg ml1 were used to prepare calibration curves. 0.5 ml of wood extract methanol/water (8/2) or gallic acid (standard phenolic compound) was mixed with FolineCiocalteu reagent (2.5 ml, 10% diluted with distilled water) and aqueous solution sodium carbonate (2.0 ml of 7.5%). The mixture was kept for 5 min at 50 C; the absorbance at 760 nm was measured. The analyses were done in triplicate and the mean value was calculated. 2.2.5. DPPH radical scavenging assay DPPH (1,1-Diphenyl-2-picryl-hydrazyl) scavenging activity of wood extracts was determined according to the method described by Brand-Willians et al. (1995) with slight modifications. 0.2 ml of the ethanolic extract (100 mg ml1 in methanol) was added to 4 ml of DPPH methanolic solution (43 mg ml1). This method is based on the reduction of a methanol solution of DPPH in the presence of a hydrogen donating antioxidant due to the formation of the nonradical form DPPH-H. The antioxidanteradical reactions were conducted for 30 min in the dark at ambient temperature. This transformation results in a change of color from purple to yellow, which is measured spectrophotometrically by the disappearance of the purple color at 515 nm. Ascorbic acid was used as standard. 2.3. Fungi The white-rot fungus P. chrysosporium from Collection of Tropical Culture (CCT 1999) Fundação André Tosello, was used in this study. Cultures were maintained on malt extract agar at 30 C for 10 days. The fungus was then incubated on solid culture medium containing 15 g of agar, 15 g of yeast extract and 1000 ml distilled water.
Table 1 Key characteristics of tested wood species. Wood
Characteristic and use
References
Hymenaea stigonocarpa Occurrence: Bolívia, Brazil Family: Caesalpinioideae Common name: jatobá-do-cerrado Anadenanthera colubrina Occurrence: Bolivia, Brazil, Peru Family: Mimosoideae Common name: Angico-branco, angico-de-caroço Caesalpinia ferrea Occurrence: Brazil Family: Caesalpinioideae Common name: jucá, pau-ferro Manilkara huberi Occurrence: Brazil, Venezuela, French Guiana Family: Sapotaceae Common name: maçaranduba, beefwood, bulletwood, sapotilla Delonix regia Occurrence: native to Madagascar but introduced to tropical areas. Family: Caesalpinioideae Common name: Flamboyant, flame of the forest, poinciana
Natural resistance soft-rot fungi and termites, furniture, used in building and naval industries.
Lorenzi, 1992 Santana et al., 2010
Natural resistance soft-rot fungi and termites, used in furniture, leather tanning, building and naval industries.
Lorenzi, 1998 Santana et al., 2010
Durable wood is used in building industry and manufacture of furniture, guitars, fiddles and pipes.
Lorenzi and Matos, 2002
High durability natural, indicated for use on fences, posts and floors. Exported to U.S. market, Japan and some European countries
Lorenzi, 1998
Soft and weak wood limiting its use in carpentry, subject to termite attack.
Schütt and Lang, 2004 Lorenzi et al., 2003
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Table 2 Percent extractives in the studied wood species. Extraction procedure (%)
Hymenaea stigonocarpa
Anadenanthera colubrina
Caesalpinia ferrea
Manilkara huberi
Delonix regia
Cyclohexane Ethanol Total
1.5 0,6 52 72
0.33 0.08 82 92
1.0 0.5 3.2 0,3 4.3 0.4
0.6 0.2 7.1 0,5 7.7 0,4
0.8 0.4 41 51
2.4. Laboratory test on the susceptibility of woods to the white-rot fungus Phenerochaete chrysosporium The laboratory tests to evaluate the natural durability of wood to degradation by P. chrysosporium were carried out based on the procedure of Kamida et al. (2005) with slight modifications. 5 g of finely powdered wood samples were weighed in Erlenmeyer flasks (125 ml) and humidified with 1 ml of tap water. Three replicates were used for extracted and unextracted woods. Non-inoculated, sterilized wood was used as a biotic control. Three disks (6 mm in diameter) from solid culture medium inoculated with the fungus were transferred to each Erlenmeyer flask. The growth of P. chrysosporium mycelium was evaluated after 4 weeks at 30 C. 3. Results and discussion Table 2 displays the extractives of woods. The amount of cyclohexane-extractives was always lower than ethanol-extractives. The total content of extractives (extracted gradually with cyclohexane and ethanol) was the greatest in A. colubrina (9% of the total dry weight of wood), followed by M. huberi (7.7%) and H. stigonocarpa (7%), D. regia (5%) and C. ferrea (4.3%). These values agree rather well with other woods reported in the literature. A study carried out by Santana and Okino (2007) with 36 Brazilian tropical timbers found extractive content between 17.3% and 0.9%, but only eight of the thirty six species showed extractive content higher than 10%. In that study M. huberi had an extractives content of 8% so is comparable with our results. Species with high contents of extractives are of interest for future studies because chemical compounds from the crude extracts can be used for pharmaceuticals, dyes, cosmetics, perfumes and natural antioxidants (Santana and Okino, 2007). High extractive content is known to result in good natural durability (Windeisen et al., 2002). The result of the preliminary phytochemical analysis of the extract of wood species is shown in Table 3. The phytochemical studies revealed the presence of flavonoids, terpenes and steroids in all analyzed woods. Tannin presence was very strong in A. colubrina. Saponins were found only in M. huberi indicating that, among the studied woods, there were significant differences in the amount and in the composition of extractives. Phenolic compounds, such as tannins and flavonoids, have antimicrobial and antioxidative properties and are involved in the defense against fungi and other microorganisms (Boudet, 2007). Saponins have been reported to prevent antimicrobial activity (Sparg et al., 2004). Secondary metabolites (extractives) are present in all plants, generally as mixtures that can be highly diverse. A large number of studies have demonstrated the importance of these metabolites as
plant defense compounds. A high diversity of extractives in high concentration provides a more effective protection against herbivores than single compounds or low diversity in both low and high concentrations (Castellanos and Espinosa-Garcia, 1997). The diversity (Table 2) as well as the concentration of phenolic compounds (Table 3) was more pronounced in A. colubrina and M. huberi in sharp contrast to D. regia and C. ferrea. Table 4 provides the quantification of lignin. According to Syafh et al. (1988) lignin is the most important non-toxic factor that limits the growth of the microorganisms in wood biodegradation. M. huberi presents the highest content of total lignin (30%). In this study, D. regia had the lowest concentration of lignin (22.3%), thus suggesting that lignin may be the main factor for the low resistance of this wood. Among 36 woods analyzed, Santana and Okino (2007) found an acid-soluble lignin (ASL) content of 0.7e1.8% and an acidinsoluble lignin (AIL) of 26.7e37%. The values for M. huberi were 34% AIL and and 0.9% ASL. The lignin content for all woods was similar and typical for tropical hardwoods, except for D. regia. These results suggest that differences in the content and structure of lignin may influence the decay resistance caused by wooddecaying fungi (Syafh et al., 1988). The content of phenolics (mg g1) in ethanol extracts was determined from a regression equation of the calibration curve (y ¼ 0.004663x þ 0.0565, R2 ¼ 0.99) and expressed in Gallic Acid Equivalents (GAE). As shown in Table 5, there is a close correlation between the phenolic content and the % DPPH radical quenched. The ethanol extract from H. stigonocarpa contained the highest amount of total phenolics and, consequently, the highest antioxidant activity, followed by A. colubrina, C. ferrea, D. regia, and M. huberi. Although D. regia has higher phenolic contents than M. huberi, it displays the lowest antioxidant activity. These results indicate that phenolic composition among both woods differ widely in terms of chemical composition. The influence of extractives on the growth of P. chrysosporium in the tested woods is shown in Table 6. The extracted and unextracted wood of M. huberi inhibited fungal growth completely, confirming that lignin plays an important role in natural resistance of this wood (Syafh et al., 1988; Tuomela et al., 2000). This observation suggests that the content of saponins is not responsible for the natural resistance of M. huberi against fungal attack since decay resistance was not affected by their extraction (Table 3). Among all the studied woods, A. colubrina was the only species where the influence of extractives was very well observed. Fungal growth was entirely inhibited by unextracted wood but intense growth formed in the presence of extracted wood (Fig. 1). Rowell et al. (2005) states that wood resistance to fungal attack is attributed mainly to the presence of extractives that are toxic to
Table 3 Preliminary phytochemical analysis of extractives detected in the woods. Classes of extractives
Extraction procedure
Hymenaea stigonocarpa
Anadenanthera colubrina
Caesalpinia ferrea
Manilkara huberi
Delonix. regia
Saponins Tannins Alkaloids Flavonoids Steroids and terpenoids
Frothing Ferric chloride Dragendorff, Mayer Shinoda Oxalo-boric acid LiebermanneBuchard
þ þ þþ þþ
þþþ þþþ þþ
þ þ þ
þþþ þ þþ þþ
þ þþ
() not detected; (þ) detected. The number of positive signs indicates the intensity of the reactions (Costa, 1982).
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Table 4 Lignin content of the studied woods species. Lignin (%)
Hymenaea stigonocarpa
Anadenanthera colubrina
Caesalpinia ferrea
Manilkara huberi
Delonix regia
Acid-soluble (ASL) Acid-insoluble (AIL) Total
0.6 0.4 27 1 28 1
1.81 0.03 25.9 0.9 27.6 0.9
1.7 0.3 26 4 28 4
1.40 0.06 28.6 0.4 30.0 0.4
0.3 0.1 22 1 22.3 1
Table 5 Total phenolic content and DPPH scavenging activity of phenolic extractives from analyzed woods. Methanol/water extract
Hymenaea stigonocarpa
Anadenanthera colubrina
Caesalpinia ferrea
Manilkara huberi
Delonix regia
mg GAE g1 of extracts % DPPH radical quenched
0.248 0.009 91.00 0.050
0.231 0.003 71.90 0.099
0.151 0.005 21.51 0.358
0.069 0.004 5.99 0.050
0.092 0.007 3.07 0.14
Table 6 Phanerochaete chrysosporium growth in the studied woods species. Wood samples
Hymenaea stigonocarpa
Unextracted þ wood Extracted þþ wood
Anadenanthera Caesalpinia Manilkara Delonix colubrina ferrea huberi regia
þþþ
þþþ
þþ
þþþ
þþþ
() no growth; (þ) weak, (þþ) medium and (þþþ) abundant growth of Phanerochaete chrysosporium.
xylophagous organisms, thus providing the wood with natural durability. In addition, A. colubrina contains a high content of phenolic compounds and high antioxidant activity (Table 5) and also has good natural resistance to termites (Silva et al., 2007; Santana et al., 2010). According to Schultz and Nicholas (2000), the radical
scavenging activity of phenolic compounds may further accelerate fungal death by scavenging the radicals produced by fungus and reducing fungal nutrition necessary for repairing cell wall injuries, thus resulting in the strong synergy of antifungal activity. As can be seen in Fig. 1, unextracted wood of H. stigonocarpa stimulates only weak growth of P. chrysosporium, whereas fungal growth in the presence of extracted wood is far more pronounced, confirming that the extractives could not completely inhibit the growth of the fungus but acted as a primary barrier to prevent colonization. Although H. stigonocarpa extractives did not inhibit completely fungus growth, this wood is known for its natural resistance (Table 1). According to Rudman (1965), heartwood extractives from durable species are not always toxic against a broad spectrum of wood-destroying fungi but are often specific to a limited number of species. Apparently, the extractives of H. stigonocarpa do not exhibit strong antifungal activity against P. chrysosporium used in this study.
Fig. 1. The growth of Phanerochaete chrysosporium showing on extracted and unextracted woods from Hymenaea stigonocarpa (left) and Anadenanthera colubrina (right).
Fig. 2. The growth of Phanerochaete chrysosporium showing on extracted and unextracted woods from Delonix regia (left) and Caesalpinia ferrea (right).
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Fig. 2 shows similar behavior for C. ferrea and D. regia in respect to fungal growth. The extractives from both woods showed weak antifungal activities. Usually flavonoids and tannins are responsible for antioxidant activity, which sequester the free radicals produced by fungi during the decay process (Schultz and Nicholas, 2000). C. ferrea and D. regia have a lower content of extractives (Table 2) and phenolic compounds (Table 5) than other woods. These factors may explain the low resistance of both woods against P. chrysosporium. 4. Conclusion The durability of A. colubrina can be attributed mainly to the presence of phenolic compounds, particularly tannins and flavonoids that inhibit fungal growth. Although the wood of M. huberi contains large quantities of extractives, especially saponins, they are not the only compounds responsible for antifungal activity. The high lignin content and the chemical composition of the lignin seem to be the major reasons for the high decay resistance of this wood. The naturally low durability of C. ferrea and D. regia wood against P. chrysosporium may be the result of various factors, including the low quantity of extractives, the low antioxidant activity of the phenolic extractives, and the structural composition of the lignin. Acknowledgements The authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for grants and fellowships. References Amusant, N., Moretti, C., Richard, B., Prost, E., Nuzillard, J.M., Thevenon, M.F., 2007. Chemical compounds from Eperua falcata and Eperua grandiflora heartwood and their biological activities against wood destroying fungus (Coriolus versicolor). Holz als Roh- und Werkstof 65, 23e28. Boudet, A.M., 2007. Evolution and current status of research in phenolic compounds. Phytochemistry 68, 22e24. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Food Science and Technology 28, 25e30. Castellanos, I., Espinosa-Garcia, F.J., 1997. Plant secondary metabolite diversity as a resistance trait against Insects: a test with Sitophilus granarius (Coleoptera: Curculionidae) and Seed secondary metabolites. Biochemical Systematics and Ecology 25, 591e602. Costa, A.F., 1982. Farmacognosia, 2nd ed., Vol. 3. Fundação Calouste Gulbenkian, Lisboa. Eaton, R.A., Hale, M.D.C., 1993. Wood: decay, pests and protection. Chapman & Hall, London.
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