The wood preservative potential of long-lasting Amazonian wood extracts

International Biodeterioration & Biodegradation 75 (2012) 146e149

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The wood preservative potential of long-lasting Amazonian wood extracts Alice M.S. Rodrigues a, b, e, *, Didier Stien c, d, Véronique Eparvier c, d, Laila S. Espindola e, Jacques Beauchêne f, Nadine Amusant f, g, Nicolas Leménager g, Christine Baudassé g, Lucien Raguin a,1 a

KLR SARL, PK8, RN2, 97351 Matoury, France Université des Antilles et de la Guyane, UMR EcoFoG, BP 709, 97387 Kourou, France c CNRS, UMR EcoFoG, Université des Antilles et de la Guyane, 97337 Cayenne, France d CNRS, Institut de Chimie des Substances Naturelles, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France e Laboratório de Farmacognosia, Universidade de Brasília, Brasília, Brazil f Cirad, UMR EcoFoG, BP 709, F-97387 Kourou, France g Laboratoire de Préservation, CIRAD, 73 rue JF Breton, Montpellier 34398, TA B 40-16, cedex 5, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 January 2012 Received in revised form 26 March 2012 Accepted 26 March 2012 Available online 24 October 2012

Investigations were carried out on the efficacy of extracts from seven Amazonian woods (Bagassa guianensis, Manilkara huberi, Sextonia rubra, Vouacapoua americana, Andira surinamensis, Handroanthus serratifolius, and Qualea rosea) with varying natural durability to reduce soft-rot degradation in a 6-wk soil-bed test. Six of the wood extracts had shown efficacy against soft-rot fungi. In particular, the preservation efficacies of B. guianensis, H. serratifolius, and S. rubra extracts were highly significant up to retention levels of 23, 25, and 12 kg m
3, respectively. Three extracts (A. surinamensis, H. serratifolius, and Q. rosea) were then tested against Gloeophyllum trabeum (brown rot) and Trametes versicolor (white rot), in an agar-block test. H. serratifolius wood extract was very efficient at protecting P. sylvestris samples at 5.1 kg m
3 against the brown rot. This extract could be used as a basis for new wood protectant formulations. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Rotting fungi Durable wood extracts Wood preservation Handroanthus serratifolius

1. Introduction Wood products are degraded primarily by fungi and insects (Tuor et al., 1995; Ohkuma, 2003; Schwarze, 2007). Consequently, lumber should be treated with preservatives when the wood is exposed to risk factors, such as high humidity or contact with the ground (Freeman et al., 2003; Schultz et al., 2007). In the Amazon, permanent pressure from xylophagous insects and fungi has prompted the appearance of highly resistant longliving ligneous species (Bultman and Southwell, 1976; Detienne et al., 1989; De Jesus et al., 1998; Carneiro et al., 2009). As a result, some woody species from the Amazonian forest of French Guiana are commercialized for residential construction and other outdoor applications because of their excellent resistance to decay. This durability is primarily caused by the presence of relatively highly levels of extractives with moderate to poor fungicidal efficacy in the

heartwood (Schultz et al., 1995; Schultz and Nicholas, 2000; Amusant et al., 2007; Rodrigues et al., 2010, 2011; Royer et al., 2010). The public’s negative perception of synthetic wood-protecting chemicals and the resulting regulations have encouraged us and others to investigate whether or not commercial waste from durable wood in French Guiana contains antifungal extractives potentially useful for wood preservation against fungal degradation (Onuorah, 2000; Kartal et al., 2006; Binbuga et al., 2008; Islam et al., 2009; Matan et al., 2009; Antwi-Boasiako and Damoah, 2010). Therefore, this study was undertaken to determine the antifungal efficiency of the extracts that were obtained from seven durable wood species, which were collected from logging waste in the Amazonian forest in French Guiana, and to evaluate the potential to transfer this natural durability to non-resistant heartwood and sapwood. 2. Materials and methods

* Corresponding author. Present address: CNRS, Institut de Chimie des Substances Naturelles, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France. Tel.: þ33 1 69 82 31 13; fax: þ33 1 69 07 72 47. E-mail address: [email protected] (A.M.S. Rodrigues). 1 In memoriam. 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2012.03.014

2.1. Wood extraction Mixed sapwood and heartwood from Sextonia rubra (Mez) van der Werff (Lauraceae), Qualea rosea Aublet (Vochysiaceae), Manilkara huberi (Ducke) Standl. (Sapotaceae), Vouacapoua

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americana Aubl., Andira surinamensis (Bondt) Split ex Pulle (Fabaceae), Handroanthus serratifolius (Vahl) S. Grose (Bignonaceae), and Bagassa guianensis Aubl. (Moraceae) was collected from logging waste in July 2007 in Régina, French Guiana. These woods are classified from 1 to 3 (“very durable” to “moderately durable”) on the durability scale (AFNOR, 1994) and represent some of the species most harvested by the wood industry in this country. Botanical identifications were performed at the French Guiana Herbarium (CAY), where a voucher specimen of each plant was deposited. The collected wood was dried at room temperature and ground into powder, and 3 kg from each one was extracted by cold maceration for 48 h with methanol or ethyl acetate, which was only used for S. rubra. After filtration, the extracted solutions were concentrated to dryness under reduced pressure below 30  C.

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obtained from SigmaeAldrich. For both the treated samples and positive controls, five identical samples were prepared for each retention level, and nine negative control samples were prepared. The treated samples were conditioned for 2 wk at 25  3  C and 70  5% relative humidity. Both the treated and untreated blocks were saturated with water while in contact with a humid filter paper at 8  C for 7 days, and their average initial hardness from four measurements was recorded with a durometer indenter (Hardmatic HH-300Ò, Mitotoyo, Japan). Subsequently, all of the samples were exposed to a mixture of sifted forest soil (4/5) and sand (1/5) at 28  5  C. During exposure water was added to maintain maximum soil water retention capacity. After 6 wk, the average hardness of each sample from four measurements was recorded again, which allowed for a determination of the hardness loss in the longitudinal direction as a measure of decay.

2.2. Wood samples 2.5. Agar-block decay test Schefflera morototoni (Aublet) Maguire, Steyermark and Frodin (average density 0.58 g cm
3) sapwood was chosen for the soil-bed test. The lumber was air-dried for 8 wk and cut into 2  2 cm (radial  tangential) sticks, which were further cut into blocks of 2  2  0.5 cm (radial  tangential  longitudinal). Scots pine (Pinus sylvestris L., average density 0.5 g cm
3), and European beech (Fagus sylvatica L., average density 0.7 g cm
3) sapwood were chosen for the agar-block decay test. The lumbers were stabilised under controlled conditions at 20  2  C and below 65  5% relative humidity for 8 wk. Samples were cut into wafers of 3 cm  1 cm  0.5 cm (R  T  L). 2.3. Fungal strains The fungal strains used in the agar-block decay test were Trametes versicolor (L.) Lloyd CTB 863 A (white-rot fungus) for beech sapwood and Gloeophyllum trabeum (Pers ex Fr.) Murrill for pine sapwood BAM Ebw 109 (brown-rot fungus). The strains are available at the CIRAD (UR40 Laboratory), Montpellier. Fungi were cultured on malt-agar solid medium, at 22  2  C and 70  5%HR. 2.4. S. morototoni block impregnation and soft-rot test The wood samples were treated to target four retention levels: 72, 36, 18, and 4.5 kg m
3. The actual retention levels were corrected based on the average weight gain of the blocks. The extracts were dissolved in a minimum amount of methanol or ethyl acetate (for the S. rubra extract). Treatments were performed by pipetting an adequate volume of each extract solution and depositing it directly on both 4 cm2 sides. The negative control samples were treated with methanol or ethyl acetate. The positive controls were treated to attain a retention level of 2.75 kg m
3 of chlorothalonil,

The wafers were treated using a full-cell process (10 min vacuum at 7 mBar, followed by 6.9 Bar of pressure for 2 h) in a minitreating receptacle. P. sylvestris blocks were treated at targeted retention levels of 40, 20, 10, and 5 kg m
3, while F. sylvatica blocks were treated at 52, 26, 13, and 6.5 kg m
3. The actual retention levels were corrected based on the weight gain after impregnation. Methanol was used as the solvent to prepare the Q. rosea (B), A. surinamensis (E), and H. serratifolius (F) extract solutions. The negative control samples were treated with methanol, and the positive controls were treated with a commercially available aqueous solution, a mixture of propiconazole (0.93%) and tebuconazole (0.3%) at a targeted retention level of 12.6 and 6.3 kg m
3 of the commercial product for P. sylvestris and F. sylvatica, respectively. These retention levels correspond to 0.15 kg m
3 propiconazole and 0.05 kg m
3 tebuconazole in Scots pine samples, and 0.06 kg m
3 propiconazole and 0.02 kg m
3 tebuconazole in European beech. A modification of the European standard EN113 (AFNOR, 1996) was used for the agar-block test (Archer et al., 1995). After impregnation, all of the wood blocks were sterilised under gamma ray irradiation. The initial mass of the wafers was recorded for four ovendried samples for each retention level and wood type (103  2  C, 18 h). These samples were used to calculate the initial mass of all the samples based on their relative humidity and were not used in the agar-bed assay because of the possible degradation of the treatments. Treated and untreated Scots pine sapwood blocks were exposed to the G. trabeum fungus, and the beech sapwood blocks were exposed to T. versicolor. Ten replicate blocks were prepared for the negative control, positive control, and each treatment (extract/ retention level). Jars containing malt-agar sterile solid medium were seeded with G. trabeum or T. versicolor as needed. Two treated

Table 1 Retention levels and corresponding hardness losses (as percentages) for S. morototoni wood samples treated with wood extracts.

S. rubra (2)a Q. rosea (3) M. huberi (1) V. americana (1) A. surinamensis (2) H. serratifolius (1) B. guianensis (1) Negative control Positive controle a

Extraction solvent

Hardness loss (Retention levels)

Ethyl acetate Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol

2.3%b ***c (56  1.9)d 7.5%*** (74  4.7) 17.8% (ns) (68  3.3) 7.8%*** (62  2.9) 2.8%*** (75  2.4) 3.6%*** (77  2.3) 4.4%*** (76  1.8) 23% 0.58%***

8.4%*** (29  2.4) 3.3%*** (31  2.6) 27.2% (ns) (34  1.6) 4.5%*** (35  1.9) 7.1%*** (38  1.7) 1.6%*** (42  2.1) 4.3%*** (45  1.5)

8.0%*** (12  2.1) 13.7%* (15  1.8) 21.4% (ns) (22  1.4) 12.9%* (21  1) 12.2%** (21  1.2) 10.8%*** (25  1.1) 1.5%*** (23  2.2)

25.5 (ns) (3.0  0.6) 15.5% (ns) (4.6  1.1) 14.1% (ns) (3.4  1.0) 18.7% (ns) (2.8  0.3) 11.2%* (2.3  1.0) 24.8% (ns) (2.9  1.4) 14.0% (ns) (2.7  0.4)

Natural resistance to soft-rot fungi-induced degradation (Tropix database: http://tropix.cirad.fr/): 1 ¼ very durable, 2 ¼ durable, 3 ¼ moderately durable. These results were obtained from an average of 20 measurements. c Significance levels relative to negative control: ***very significant, **significant, *weakly significant and (ns) not significant, as determined by ANOVA followed by the Tukey test. d These results are an average of 5 replicates,  standard deviations. e Chlorothalonil. b

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Table 2 Retention levels and corresponding mass losses (as percentages) for Scots pine and European beech wood samples treated with wood extracts. Mass loss (Retention levels) Scots pine Q. rosea A. surinamensis H. serratifolius Negative control Positive control

10.4%a (ns)b (41.2  0.9)c 1.4%*** (41.2  1.6) 1.9%*** (42.2  0.4) 17.9% 1.64%***

42.1% (ns) (18.9  0.3) 1.7%*** (20.4  0.6) 2.9%*** (20.6  0.4)

32.1% (ns) (10.3  0.1) 1.9%*** (10.2  0.1) 1.4%*** (10.3  0.1)

34.9% (ns) (5.1  0.1) 24.0% (ns) (4.8  0.2) 1.6%*** (5.1  0.1)

European beech Q. rosea A. surinamensis H. serratifolius Negative control Positive controld

5.3%*** (47.2  1.4) 1.7%*** (46.5  0.9) 4.5%*** (47.7  1.1) 45.0% 3.8%***

24.0% *** (22.3  1.0) 10.7%*** (22.7  0.7) 9.7%*** (23.9  0.5)

36.2% (ns) (12.1  0.4) 11.9%*** (11.4  0.4) 13.8%*** (12.4  0.2)

26.0% ** (6.1  0.4) 27.5%** (6.0  0.1) 17.8%*** (6.2  0.2)

a

These results were obtained from an average of 10 replicates. Significance levels relative to negative control: ***very significant, **significant, *weakly significant and (ns) not significant, as determined by ANOVA followed by the Tukey test. c These results are an average of 20 replicates,  standard deviations. d Chlorothalonil. b

blocks and two untreated wood wafers were placed in each jar as soon as the fungus had covered the solid medium. All test jars were incubated for 8 wk at 22  2  C and 70  5% relative humidity, then cleaned and dried at 103  2  C for 18 h. The preservative efficacy of the extracts was determined from the measurements of mass loss. 2.6. Data analyses The relative efficacy of each treatment was compared to the corresponding negative control and, in the case of agar-block tests, we also compared each treatment to a positive control, with a oneway ANOVA test. The Tukey post-hoc test was used to compare the treatments by pairs (multiple comparisons) at a significance level of P < 0.05. The XLSTAT software package was used for statistical analysis, and the GraphPad Prism 5 package was used for graphics. 3. Results 3.1. Soft-rot soil-bed assay The results of the soft-rot soil-bed assay are reported in Table 1. The M. huberi extract was inactive at all impregnation rates. All other extracts were significantly active. Q. rosea, A. surinamensis and H. serratifolius were chosen for further tests employing basidiomycete fungi because these woods have large amounts of logging waste. 3.2. Agar-block decay test Table 2 shows the percentage of mass lost by the Scots pine and beech blocks during the agar-block decay test in the presence of basidiomycete fungi. Extracts from H. serratifolius and A. surinamensis demonstrated very good efficacies for wood protection, and the former was as active as the positive control for the preservation of Scots pine wood. A comparison of the preservation efficiency of the positive control with that of the extracts was conducted with the Tukey’s multiple comparison test. The results show that several treatments possessed the same protection efficiency as the commercially available positive control. 4. Discussion The soft-rot ascomycete soil-bed assay demonstrated that treatment with all of the extracts, except that from M. huberi, significantly improved the durability of the wood. In particular, the

preservation efficacies of B. guianensis, H. serratifolius, and S. rubra extracts were highly significant up to retention levels of 23, 25, and 12 kg m
3, respectively. The ethyl acetate extract of S. rubra wood was reported to show moderate antifungal activity against human pathogens (Rodrigues et al., 2010). Two lactones, rubrenolide and rubrynolide with a lipophilic C10 side-chain, comprise a very high proportion of this extract, and rubrenolide exhibited antifungal properties. In addition, the S. rubra extract appears more promising for the preservation of termite-sensitive woods (Rodrigues et al., 2011), while the development of a lipophilic extract (ethyl acetate) appears to draw environmental issues (European Directive n 1999/13/CE, 1999). At the same time, the antifungal activity of other Handroanthus species (formerly named Tabebuia) has been reported previously, supporting the hypothesis that antifungal activity may be due to the presence of active quinones (Ali et al., 1998; Portillo et al., 2001). B. guianensis is a relatively rare species in the rainforest (Detienne et al., 1989; Schulze et al., 2008), and the extraction yield was low (2.7%). This extract contains mainly stilbenoids and moracins (Royer et al., 2011), which presumably account for its antifungal efficiency (Schultz et al., 1995; Jayasinghe et al., 2004). Therefore, we have only utilised B. guianensis extract as a model for the discovery of antifungal combinations, and we have decided to continue decay tests with the methanolic extracts from H. serratifolius, Q. rosea, and A. surinamensis wood. In the agar-block basidiomycete assay, H. serratifolius extract was most active and showed statistically significant preservation efficiency at all retention levels in Scots pine (Table 2). When compared to the positive control, statistical analysis demonstrated that the protection from this extract at all retention levels was not significantly different from the protection obtained with the commercial standard at the retention levels employed. Notably, H. serratifolius extract was efficient as the positive control at retention levels of 5.1 kg m
3 in Scots pine and 6.3 kg m
3 on beech. To a lesser extent, the A. surinamensis extract is also very efficient, while the Q. rosea extract did not show a protective effect, except at the highest retention level tested. 5. Conclusion Extracts obtained from very durable woods in the rainforest of French Guiana can be used as protectants for timber. Of the seven wood extracts we tested for the soft-rot soil-bed, the extract from H. serratifolius was the most effective. This extract was more active than the commercial standard used in our study at the retentions

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