Durable Woods and Antifungal Activity of Their Essential Oils: Case of Tetraclinis articulata (Vahl) Masters and Cedrus atlantica Manetti

2.1 Material used for EOs extraction and chemical analysis

Trunk wood and root burl samples of T. articulata were collected from sweepings of craft processing workshops in Khemisset Region (central plate of Morocco), while samples of C. atlantica sawdust were collected from wood sawmill in the region of Azrou (Middle Atlas Mountains of Morocco). Sawdust was then sieved into particles of 1 mm size and triplicate samples of 250 g were subjected to hydrodistillation for 4 hours to obtain pure essential oils (EOs).

The chemical analysis and component identification of EOs were performed by gas chromatography (GC-FID) and by gas chromatography coupled with a mass spectroscopy (GC-MS). The identification of EO components was achieved by comparison of their retention indices (RI) relative to (C8-C22) n-alkanes with those of known compounds, and by comparison of similar mass spectra using Wiley/NBS mass spectral library of the GC-MS data system and other published mass spectra [19]. The percentage compositions of samples were calculated according to the area of the chromatographic peaks using the total ion current.

2.2 Material used for bioassay

Bioactivity of EOs extracted from T. articulata and C. atlantica woods was assessed in bioassay against the following wood-decaying basidiomycetes fungi: Gloeophyllum trabeum (BAM Ebw.109 strain), Oligoporus placenta (FPRL. 280 strain), Coniophora puteana (BAM Ebw. 15 strain), and Trametes versicolor (CTB 863 A strain). Fungi strains originated from the mycological collection of the Laboratory of Botany, Mycology and Environment, Faculty of Sciences in Rabat, Morocco.

2.3 Experimental

Bioassays of EOs were performed by direct contact of fungi strains on agar medium according to the method reported by Remmal et al. [20]. Oils were first diluted in a sterile solution of tap water-agar at 0.2% in order to obtain a homogeneous mixture, then distributed in test tubes containing 13.5 ml of sterilized malt-agar medium (20 g/l malt extract and 15 g/l agar), and kept at 45°C in a water bath. To obtain the final oil concentrations in the culture medium ranging from 1/250 to 1/5000 v/v, aseptic volumes of 1.5 ml of different dilutions were then added to those tubes before pouring EO-medium mixtures into Petri dishes. Additional control dishes containing only 13.5 ml of culture medium and agar solution at 0.2% (SA) alone were also prepared. Inoculation of Petri dishes was made by two 0.5 cm² fragments of 10 days old fungal culture in malt-agar. For each treatment, three repetitions were prepared and incubated in the dark for 7 days at 22°C. At the end of each bioassay, minimal inhibitory concentration (MIC) [21] was determined for each fungus.

2.4 Results and discussion

According to the bioassay conducted on oils extracted from T. articulata and C. atlantica woods, a significant inhibitory effect on the four tested wood-decaying fungi was observed (Table 1) with different levels of inhibition. T. articulata root burl wood EOs showed, however, a strong inhibitory action against those fungi strains with oil dilutions over 1/4000 v/v. G. trabeum fungus was the most sensitive to the inhibitory effect of this essential oil since it was inhibited by concentrations between 1/5000 for T. articulata root burl oil and 1/1000 v/v for Atlas cedar oil. O. placenta was the most resistant strain since its growth inhibition was not reached until 1/400 concentrations for C. atlantica oil and 1/800 for T. articulata trunk wood oil (Table 1).

Previous studies by our team [5, 8] showed that T. articulata and C. atlantica woods were classified as very durable to durable (DC 1 and 2) and means of mass loss of test specimens was below 5.20% compared to those of Scot pine wood (control) (40.70%). According to their durability indexes (X) determined by NF EN 350-1 and CEN/TS 15083-1 standards [22, 23] and the biological risks defined by EN 335-2 standard [24], natural durability levels of those woods against wood-decaying fungi allow them to access high-risk classes of biological attacks 4 and 5 for an end-use without preservative treatment regarding decay fungi [25, 26]. Compared to similar studies on Moroccan coniferous woods (Figure 1), the natural durability of native Atlas cedar wood is similar to that of C. atlantica heartwood (DC 1 and 2) originated from a south Italian plantation [7], whereas Pinus halepensis and P. pinaster woods were considered as less durable (DC 4) [6, 27]. Generally, pine woods contain less active extractives than those of Cupressaceae. Adamopoulos et al. [28] reported that the weakness of natural durability of both heartwood and sapwood of Pinus leucodermis is related to low presence of bioactive extractives that can inhibit the brown-rot fungus, Coniophora puteana.

In addition, other works by our team [8, 29] revealed that EOs of C. atlantica wood is dominated by ketones (52.05%) and alcohols (26.58%), while those of thuya are dominated by alcohols (about 55–78%) and sesquiterpenes (13–22%) (Table 2). Major components of C. atlantica oil are, respectively, E-γ-atlantone, E-α-atlantone, 5-isocedranol, 9-iso-thujopsanone, cedranone, Z-α-atlantone, cedroxyde, and 14-hydroxy-δ-cadinene [8] (Table 3).

EOs of T. articulata woods are rich in thymol, 3-tert-butyl-4-methoxyphenol, cedrol, and α-cedrene (Table 3). The oil of T. articulata trunk wood contains phenols such as α- and β-acorenol, cedrol, and totarol, along with terpenes as α-cedrene that can protect this wood from fungi decay. The strong inhibitory effect of T. articulata root burl oil is probably due to its oxygenated fraction rich in phenols as thymol, 3-tert-butyl-4-methoxyphenol, and cedrol. A previous work already highlighted this significant antibacterial activity at low concentrations [30]. Abundance of tropolones and phenols in Cupressaceae woods may explain their high natural durability grades against wood-decaying fungi as reported by Haluk and Roussel [16] and by more recent works [13, 30, 31]. Early investigations found that thujaplicines (tropolones) of Thuja plicata possesses strong inhibitory effect against the wood blue stain fungi and many wood-decaying fungi [32].

Natural durability of C. atlantica wood can be correlated to bioactivity of its oils essentially rich in sesquiterpene ketones as atlantones [9, 14]. In the literature, African padauk (Pterocarpus soyauxii) medium-heavy heartwood manifests a remarkable decay resistance attributed to its specific extractive compounds [33]. Moreover, extractive compounds obtained from black locust heartwood were able to increase the native durability of European beech against wood-decaying fungi from class 5 (not durable) to class 3 (moderately durable) [34].

The strong antifungal activity of T. articulata root burl wood EOs is probably related to their alcohol fraction, rich in thymol and 3-tert-butyl-4-methoxyphenol, which confer them this significant bioactivity. The combined action of two phenolic compounds, such as thymol and carvacrol, was previously reported [17, 35]. Action of phenolic compounds on fungi is primarily based on the inhibition of fungal enzymes containing SH group in their active site [36, 37]. The antifungal activity of the EOs of C. atlantica wood can be correlated to its sesquiterpene ketones, which are mainly atlantones (about 40.61%). Bioassays conducted with pure α-atlantones extracted from Decalepis hamiltonii revealed great inhibitory activity against pests and molds [38]. In our study, alcohols present in C. atlantica oil in significant amount (more than 26%), such as isocedranol, tumerol, himachalol, and cedrol, may also be involved in the inhibitory effect of this oil. Other compounds such as cadinenes (monoterpene hydrocarbon) could also have a great antimicrobial property [39]. However, synergistic action of two or more components of essential oils extracted from thuya and Atlas cedar woods can also be involved in the observed bioactivity reported in our study.

Antifungal activity of several thyme species oils was recently tested successfully against wood-decaying fungi especially those of Thymus bleicherianus [17]. Furthermore, EOs of T. articulata root burl showed an antibacterial activity two to six times greater compared to that of reference antibiotic and were more effective on Staphylococcus aureus (Gram⁺) and Escherichia coli (Gram⁻) with significant bacteriostatic and bactericidal effects [40]. Regarding inhibition mechanisms of active compounds of EOs, it was reported that volatile alcohols can act on both lytic and synthetic enzyme pathways and growth inhibition consequently occurred after breaking off natural extension hyphae of fungi [41, 42].

Active compounds contained in oils of T. articulata and C. atlantica woods, which were successfully tested against wood-decaying fungi, may protect and ensure good natural durability levels for these woods ranging from very durable (DC1) to durable (DC2) classes.

Findings of this study could allow us to consider recovering wastes from T. articulata and C. atlantica wood processing for the extraction of bioactive oils and use them as biocide especially in preservative treatment of less-durable woods such as those of pines. Also, these results are in favor of more protection for these threatened species and for the rehabilitation in their natural environment. Natural compounds of T. articulata and C. atlantica oils can then replace the use of current petrochemical compounds that are becoming more and more criticized for their harmful effect on human health and the environment.