Efficiency of extracts from Amazonian species in protecting the wood of Hevea brasiliensis Muell. Arg. against xylophagous fungi

2.1 Obtaining the rubber tree and preparing the samples

The rubber trees (H. brasiliensis), clone IAN 873 (Table 1), came from a 27-year-old plantation located at Fazenda Santa Maria, Pacotuba district, municipality of Cachoeiro de Itapemirim, Espírito Santo, Brazil (latitude 20°72′44.4″S, longitude 41°32′43.6″W). The climate is tropical highland (Cwa), with rainfall poorly distributed throughout the year, with rainy summers and dry winters, according to the Köpen classification. The average temperature of the coldest month is below 20 °C and the hottest is above 27 °C (Oliveira 2007).

Central planks approximately 10 cm thick were removed from each tree, and the sides containing the sapwood were separated to obtain cubic samples with 1.9 cm edges, which were used for impregnation, biological testing, and physical characterization (basic density and the coefficients of linear contraction [radial and tangential] and anisotropy, determined according to the Brazilian Standard – NBR 7190-1, Brazilian Association of Technical Standards – ABNT (2022)) (Figure 1).

Figure 1:

Steps to obtain the sample for the test. Stage 1: log splitting, stage 2: central plank and sides containing sapwood, stage 3: sample dimensions for the biological test (a) and physical tests (b).

2.2 Obtaining and preparing solutions for the impregnation of the wood

The extracts impregnated in the rubber tree wood were obtained from industrial processing residues (sawdust) of Amazonian woods with high natural durability, such as leopardwood (R. montana), angelim vermelho (D. excelsa), and angelim pedra (H. flavum). The sawdust was collected from the company Mil Madeiras Preciosas Ltda. (Precious Woods Amazon – PWA), located on Highway AM 363, in the rural area of the municipality of Itacoatiara, Amazonas, Brazil (latitude 03°00′S, longitude 58°30′W).

The sawdust was dried in a drying oven (50–60 °C) for 24 h for subsequent solubilization in ethyl alcohol, according to the methodology described by (Nogueira et al. 2002). The impregnation solutions were obtained by solubilizing the sawdust in 96 °GL ethanol, as described by (Brocco et al. 2024). For this purpose, the sawdust:solvent mixture was used in a 1:5 ratio. The material obtained was filtered in a Büchner funnel with filter paper with a porosity of 14 µm, adjusted to a Büchner funnel. Suction in a vacuum pump was carried out to eliminate fine sawdust particles. The solvent was removed in a rotary evaporator at 60 ± 2 °C and the solid crude extract was dried in an oven at 50 ± 2 °C. The treatment solutions were obtained by dissolving the crude extracts in ethyl alcohol at concentrations of 2, 4, and 8 %.

The pH of the total extracts was obtained using a digital pH meter (Alfakit, AT-355, Santa Catarina, Brazil), which was calibrated with standardized buffer solutions (4, 7, and 10). The measurement was carried out at a temperature of 25 ± 2 °C and the result was obtained by contacting the electrode with the solution, as mentioned by (Silva 2021).

The cold bath method was used to impregnate the rubber tree wood with the extract solutions and their respective concentrations, with a contact time of 5 h between the solution and the samples. After impregnation, the samples were dried in a drying oven at 50–60 °C and the retention of the extracts, for the respective concentrations, was calculated.

2.3 Chemical identification of the main compounds isolated from the extracts

The analyses to identify the main organic compounds present in the alcoholic extracts obtained from the residues (sawdust) from the mechanical processing of R. montana, D. excelsa, and H. flavum wood, used for the impregnation of rubber wood, were carried out at the Central Analytical Laboratory of the Federal University of Espírito Santo (UFES), Alegre Campus, Alegre, Espírito Santo, Brazil. The extracts were subjected to analysis by gas chromatography coupled with mass spectrometry (GC/MS) in a Shimadzu GC-2010 and GCMS-QP2010 PLUS spectrophotometer (Kyoto, Japan). The column used was a Restek Rtx-5MS (30 m × 0.25 mm × 0.25 µm). 1 µL of each sample was inserted into the injector in split mode, which was at a temperature of 270 °C.

The analysis began at 75 °C, followed by a heating ramp of 5 °C min⁻¹ to 230 °C, remaining at this temperature for an additional 39 min (70 min of analysis). The ionization source and interface temperatures were both 230 °C. All mass spectra were recorded using electron impact ionization at 70 eV. Helium was used as the carrier gas at a constant flow rate of 1.0 mL min⁻¹. Similar methods have been reported in the literature (Adams 2017; Barbosa et al. 2006; Brocco et al. 2020).

Monitoring was performed in SCAN mode to scan between 30 and 700 mz⁻¹. The main compounds were identified based on the mass spectrum compared with the Wiley library, 7th edition, and by comparing the mass spectra obtained with those existing in the National Institute of Standards and Technology – NIST library, using the relative retention index (Adams 2017). The results were expressed in terms of sample area (%), retention time (min), and molecular weight of the compounds.

2.4 Efficiency of extracts against xylophagous fungi

To test the efficiency of plant extracts obtained from Amazonian forest species in the biological protection of H. brasiliensis wood, the teak extract (Tectona grandis L. f.) used in the research carried out by (Brocco et al. 2024) was used as a comparison standard. The impregnated sample and control (non-impregnated) sample were exposed to the action of brown-rot fungi, Neolentinus lepideus (Fr.) Redhead & Ginns (Mad 534) and Rhodonia placenta (formerly Postia placenta) (Fr.) Niemelä, K. H. Larss. & Schigel (Mad 698R) and white-rot fungi Polyporus fumosus (Pers ex Fries) (FCC 496 A) and Trametes versicolor (L.) Lloyd. (Mad 697), according to the American Wood Protection Association – AWPA E 10-22 (2024a).

The test was prepared in 600 mL bottles, filled with 300 g of soil, water retention capacity (24.05 %) and pH (6.28) according to AWPA E 10-22 (2024a). The soil in each bottle was moistened to 130 % of its retention capacity by adding 63 mL of distilled water and two Pinus elliottii Engelm. sapwood wooden feeders were added to each flask and sterilized in an autoclave at 103 kPa and 121 °C for 30 min. Pine feeders were used for both brown and white rot fungi, since the fungi used grow well in this type of wood.

After cooling the bottle, fragments (≈5 × 5 mm) obtained from pure cultures of the fungi were inoculated into the feeders. After the fungus had developed in the feeders and colonized the soil, the samples were added, totaling six repetitions for each situation (fungus × extract × concentration) and controls (rubber and pine wood). The test was kept in an air-conditioned room (25 ± 2 °C and 65 ± 5 % relative humidity – RH) for 12 weeks. After this period, the flasks were opened, the wood samples removed and cleaned (removal of the fungal mycelium) (Figure 2), dried in a drying oven (same initial conditions of the experiment) and the mass loss evaluated according to the resistance class (Table 2).

Figure 2:

Bottles containing pine wood feeders and xylophagous fungi (a), samples exposed to fungi (b), and samples after 12 weeks (c).

2.5 Statistical analysis of data

In the test, the effects of wood extract (three levels) and solution concentration (three levels) were evaluated, 3 × 3 factorial, totaling nine treatments. Each treatment had six, for each extract and concentration, totaling 288 samples. Four types of fungi were used in the test. They were not compared with each other since they cause different damage to the wood. Before carrying out the analysis of variance (ANOVA), the normality of the data (Lilliefors test, p < 0.05) and the homogeneity of the variances (Cochran test, p < 0.05) were checked. When necessary, the mass loss values were transformed.

The mass loss values (%) were transformed into arcsen [square root (mass loss/100)]. These transformations, suggested by (Steel et al. 1997), were necessary to ensure normality and homogeneity of the variances (respectively), and allow the data to be analyzed using ANOVA. The Tukey test (p < 0.05) was used to analyze and evaluate the tests for the interaction and factors found to be significant by the F test (p < 0.05).

3.1 Physical properties of rubber tree wood, pH, and retention of extracts

Basic density directly influences the physical (dimensional stability) and mechanical properties of wood. The average value of this characteristic found for rubber tree wood was 530 kg m⁻³, which is considered average, according to the Comisión Panamericana de Normas Técnicas – COPANT (1974) and Silveira et al. (2013). The tangential and radial shrinkage values were 5.6 % and 2.4 % respectively, and the anisotropy coefficient was 2.36. It is considered a low quality wood (≥2.0), as mentioned by (Durlo and Marchiori 1992).

The average basic density value was similar to that found by (Portal-Cahuana et al. 2022). However, the anisotropy coefficient obtained was 1.74, lower than that found in this study. This value may have been influenced by age and edaphoclimatic characteristics. Santana et al. (2001) obtained a basic density of 490 kg m⁻³ for the same rubber tree clone used in this study, which was lower than that obtained.

Regarding shrinkage (tangential and radial) and the anisotropy coefficient, Santana et al. (2001) found values of 5.1 %, 2.3 %, and 2.2 respectively, corroborating this study. For the rubber tree samples obtained along the stem, Raia et al. (2018) found a medium basic density of 580 kg m⁻³ and an anisotropy of contraction greater than 2.0, which are in accordance with the values obtained in this study.

For applications involving dimensional stability of the wood, such as furniture, flooring, and paneling, it is recommended that the wood present an anisotropy coefficient close to 1, thus producing better quality products in terms of this characteristic (Oliveira and Silva 2003).

The average pH of the extracts was acidic, with values of 6.1, 5.5, and 3.5 for angelim pedra, angelim vermelho, and leopardwood, respectively. Figure 3 presents the average retention of these extracts in rubber tree wood at the different tested concentrations. Retention values ranged from 2.5 to 25.4 kg m⁻³, according to the concentrations of the solutions used (2, 4 and 8 %).

Figure 3:

Retention averages and standard deviations of the extracts and concentrations of the species impregnated in rubber tree wood.

Concerning the pH values, the leopardwood extract had the lowest value. Acidic extracts tend to provide greater resistance to xylophagous fungi. This value is outside the optimum limit for the development of wood-rotting fungi (Schmidt 2006).

Regarding the impregnation of marupá wood (Simarouba amara Aubl.) with extracts obtained from the wood and bark of Amazonian species to protect against subterranean termites (Nasutitermes sp.), Barbosa et al. (2007) found retention variations between 4.9 and 6.7 kg m⁻³. Brocco et al. (2017) obtained retention for teak extract ranging from 21 to 23 kg m⁻³ for pine wood and 10.5–15.6 kg m⁻³ for teak sapwood. For the extract of residue (sawdust) from the industrial processing of teak wood, Brocco et al. (2024) obtained a value of 36–40 kg m⁻³ for Southern pine – SYP (Pinus taeda L.) sapwood. In summary, coniferous woods tend to absorb more product than hardwoods.

Factors such as the anatomy of the wood influence the amount of product absorbed by the wood. The anatomical structure of coniferous wood is relatively simple compared to hardwoods. This contributes to greater penetration and impregnation of preservative products in the wood (Gonzaga 2006; Sales-Campos et al. 2003).

3.2 Analysis and characterization of the main extracted compounds

Table 3 and Figures 4–6 show the main compounds identified in the GC-MS analysis, along with sample area, retention time, and molecular weight for the identification of alcoholic extracts of residues from the Amazonian species (R. montana, D. excelsa, and H. flavum), used to protect rubber tree wood (H. brasiliensis) against wood-decaying fungi. The column relating to the sample area (%), Table 3, represents the relative peak area of each compound, calculated as a percentage of the total area of all detected compounds in the extract. This indicates the relative composition of the extract, rather than the absolute concentrations in the wood or the total extractives content. These results revealed distinct chemical profiles among the species, with a predominance of flavonoids, phenols, and phenylacetic acid as the main classes of bioactive compounds against white and brown rot fungi.

Figure 4:

Bioactive compounds identified in extracts of Roupala montana.

Figure 5:

Bioactive compounds identified in extracts of Dinizia excelsa.

Figure 6:

Bioactive compounds identified in extracts of Hymenolobium flavum.

In R. montana extract, phenols and flavonoids, such as 5-pentadecylresorcinol (44.9 %) and 4H-1-Benzopyran-4-one (39.4 %) were the most abundant compounds, calculated based on these relative peak areas. These compounds are known for their alteration in fungal cell membranes, in addition to acting as inhibitors of spore synthesis, which are essential for fungal growth and reproduction (Barbosa et al. 2006; Doloking et al. 2022; Hammami et al. 2015; Kumar et al. 2014). The compound ethyl oleate (8.04 %) was also present, being an unsaturated fatty acid ester that, according to Zhang et al. (2013), may present antimicrobial activity by interfering with the permeability of the fungal plasma membrane. The results indicate that R. montana extracts were effective, especially at concentrations of 4 % and 8 %, promoting significant inhibition of the growth of fungi such as Gloeophyllum trabeum (Pers.) Murrill. and T. versicolor.

D. excelsa extracts had a majority composition of flavonoids such as 2H-1-Benzopyran-7-ol (39.5 %) and flavan (9.78 %), in addition to phenols such as isoeugenol (4.03 %) and 2-methoxy-4-propylphenol (2.13 %). The antifungal action of these compounds occurs by inhibiting the enzymatic activity of wood-decaying fungi, as reported by (Barbosa et al. 2006; Brocco et al. 2024; Doloking et al. 2022; Hassan et al. 2019; Kumar et al. 2014; Zhang et al. 2022), who demonstrated the efficacy of flavonoids in destabilizing the fungal cell wall. Furthermore, the presence of the styrenic derivative 3,4-dimethoxy styrene (32.57 %) may have enhanced this action through oxidative mechanisms (Oelschlägel et al. 2018; Zhang et al. 2022). The extract’s performance was more expressive at concentrations of 8 %, resulting in high protection of the robberwood.

In the H. flavum extract, the major compound was benzeneacetic acid (58.07 %), followed by levoglucosan (18.74 %) and 1,3-benzenediol (15.82 %). Benzeneacetic acid, classified as a phenylacetic acid, acts as a natural antimicrobial, promoting acidification of the fungal intracellular environment, which compromises its viability, as reported by (Hwang et al. 2001). Furthermore, levoglucosan, a byproduct of carbohydrate pyrolysis, may have synergistic antifungal properties (Abdul Karim and Mazlan 2024; Brocco et al. 2024; Ekhuemelo et al. 2024; Hwang et al. 2001; Juntarachat et al. 2013; Sablík et al. 2016). Concentrations of 4 % and 8 % of the extract provided good stability of rubber wood against xylophagous fungi, with observable inhibition in the darkening and loss of wood mass.

3.3 Biological resistance to brown-rot fungi

The analysis of variance of the mass loss data indicated significant results by the F test for brown-rot fungi in all the situations tested (Table 4). It was noted that the angelim pedra (AP), angelim vermelho (AV), leopardwood (LF), and teak (TC) extracts, in the concentrations (2, 4, and 8 %), were efficient in controlling the growth of the N. lepideus fungus, being classified as resistant (AWPA E 30-22, 2024b) (Figure 7).

Figure 7:

Average mass loss, standard deviation, and resistance classes of impregnated wood, for extracts and concentrations, to attack by brown-rot fungi.

This can be attributed to the phenolic compounds present in these extracts identified by GC/MS (Table 3 and Figures 4–6), which may be possible inhibitors to this fungus. The pine control had a mass loss of 34.56 %, considered of moderate resistance according to the aforementioned standard.

For the R. placenta fungus, the wood impregnated with the extracts of leopardwood, at concentrations of 2 and 4 %, and teak, at 4 and 8 %, obtained lower mass loss, classified as resistant, AWPA E 30-22 (2024b), Figure 7. Those made from angelim pedra and angelim vermelho acted less efficiently, providing moderate resistance to the impregnated samples. It is worth noting that rubber tree wood did not have good retention for the angelim pedra and angelim vermelho extracts (Figure 3).

Regarding the controls (rubber and pine), for both fungi, the pine lost more mass. According to Schmidt (2006), this wood has a low density, simple anatomical characteristics, and a low proportion of phenolic extractives, which allows it to be easily deteriorated by these microorganisms.

In order to evaluate the biological resistance of rubber tree wood, treated and untreated with ethylene, subjected to brown-rot fungi (Gloeophyllum sepiarium (Wulfen) P. Karst. and Gloeophyllum striantum (Fr.) Murrill), Cherdchim and Satansat (2016) observed that they promoted greater mass loss in the treated wood. However, Yingprasert et al. (2023) found that extracts of Acacia mangium Willd. bark improved the resistance of rubber tree wood against attack by the brown rot fungus (Gloeophyllum striatum), corroborating the data from this research.

To evaluate the preservative potential of the ethanol extract of teak heartwood (T. grandis), impregnated in the sapwood, and Pinus sp. wood, Brocco et al. (2017) and Brocco et al. (2024) observed an improvement in the resistance of the woods to the fungi P. placenta and N. lepideus, indicating the influence of natural compounds on the resistance of low-durability woods.

According to Schmidt (2006) and Goodell et al. (2020), brown-rot fungi mainly use carbohydrate polymers in the wood’s cell wall, and although they provide a more heterogeneous attack, they cause greater mass losses than white-rot fungi.

3.4 Biological resistance to white-rot fungi

For accelerated rotting to white-rot fungi, results show that the biological resistance of the wood differed according to the fungi tested. The resistance of rubberwood impregnated with the extracts tested was higher than the controls when compared to the P. fumosus fungus. In this case, no significant difference was observed by the F test among the concentrations tested (Table 5).

The extracts of angelim pedra (AP) and leopardwood (LF) provided greater resistance to the wood, which was classified as resistant. However, those obtained from angelim vermelho (AV) and teak (TC) provided improvements in wood resistance (Table 5), classified as moderate resistance (Figure 8). The pine wood had a loss of mass that classified it as non-resistant. This shows that the fungus used was vigorous enough to consume the wood.

Figure 8:

Average mass loss, standard deviation, and resistance classes of impregnated wood, for extracts and concentrations, to attack by white rot fungi.

Regarding T. versicolor, it was observed that the factors extract, concentration, and interaction were significant according to the F test (Table 5). However, its attack on the wood of rubber trees (SG) and pine trees (PN) was similar to that caused by P. fumosus (Table 5). The extracts provided similar resistance gains for all the concentrations tested since the 2 % concentration already provided sufficient retention to control the fungi (Figure 3).

For the 2 % concentration, the leopardwood extract provided better resistance than the others. Nevertheless, for the 4 % concentration, the protection provided was similar to the teak and angelim pedra extracts. The same behavior was observed for the 8 % concentration. The results were not influenced by the retention of the extracts (Figure 3). The samples impregnated with angelim pedra extract at concentrations of 4 and 8 %, leopardwood (2 and 4 %), and teak (4 and 8 %) were classified as resistant and the others as moderately resistant (Figure 8). In general, leopardwood extract was more efficient in protecting rubber tree wood, at concentrations of 2 and 4 %, against the white-rot fungi tested.

The difference between the fungi P. fumosus and T. versicolor may be associated with the form of attack. For Corymbia maculata (Hook.) K. D. Hill & L. A. S. Johnson wood subjected to attack by the fungi Postia (now Rhodonia) placenta, N. lepideus, and P. fumosus, Paes (2002) observed that P. fumosus caused less deterioration than the others.

For extracts obtained from the bark of A. mangium, Yingprasert et al. (2023) observed that the higher concentrations among those studied (10, 15, and 20 %) resulted in lower mass loss of rubber tree wood. The inverse result to the one obtained in this study, in which the leopardwood extract at concentrations of 2 and 4 % provided the best results. This may be related to the leaching of the excess of the extract during the period of exposure to the fungus. Or the size of the particles in the more concentrated solutions was larger than the natural cavities present in the wood, making it difficult for them to penetrate.

For the teak extract, the results obtained by Brocco et al. (2017), at a concentration of 4 % in ethanol, contributed to the protection of the wood (teak and Pinus sp. sapwood). To evaluate the natural durability of 28 Amazonian species subjected to attack by different rotting fungi, Carneiro et al. (2009) found that angelim pedra wood showed natural resistance to the T. versicolor fungus. Regarding the intensity of attack by xylophagous agents on wood stored in yards and warehouses of sawmills in the state of Amapá, Costa and Cabral (2020) found that jatobá (Hymenaea spp.), angelim pedra, and angelim vermelho are hardly attacked.

The results found in the literature review and those obtained in this study indicate that the most important factors in wood biodegradability indices are the levels of retention and the quality of the extractives. These factors also provided the rubber tree wood with better resistance to the white-rot and brown-rot fungi tested.