Essential oils of Pinus nigra J.F. Arnold subsp. laricio Maire: Chemical composition and study of their herbicidal potential

2.1 Plant material

The needles of 50 year old P. nigra J.F. Arnold subsp. laricio Maire were collected during October 2009 (autumn season) from the Souinet arboreta of the National Institute of Researches on Rural Engineering, Water and Forests. Five samples collected from more than 5 different trees were harvested and mixed for homogenization. The experimental site is located in Ain Draham, in the north of Tunisia at an altitude of 492 m, where humid climate prevails. The voucher specimen of the plant (No. Pn.950) was prepared and deposited at the herbarium division of the Institute.

2.2 Isolation of the essential oils

Three replications of 100 g of air-dried and finely grounded raw materials were submitted to hydrodistillation for 5 h with 500 mL distilled water using a Clevenger type apparatus according to the European Pharmacopoeia (2004). The oil obtained was collected and dried over anhydrous sodium sulfate and stored in sealed glass vials in a refrigerator at 4 °C prior to analysis. Yield based on dry weight of the sample was calculated (w/w%).

2.3 Gas chromatography analysis/mass spectrometry analysis conditions

2.4 Statistical analysis

Data obtained from essential oil analysis, seed germination and seedling growth assays were expressed as mean values and were subjected to one-way analysis of variance (ANOVA), using the SPSS 13.0 software package. Differences between means were tested through Student–Newman–Keuls (SNK) and values of p ⩽ 0.05 were considered significantly different.

3.1 Chemical composition

The hydro-distillation of dried P. nigra ssp. laricio needles gave a yellowish essential oil (yields 0.6%, w/w). The qualitative and quantitative analytical results of identified compounds by GC and GC–MS are shown in Table 1. Twenty-seven constituents accounting for 97.9% of total oil composition were identified. The oil was dominated by oxygenated diterpenes (38%) and sesquiterpenes hydrocarbons (41.4%) but monoterpenes were represented by a small quantity (10.5%). The major components were manool oxide (38%), germacrene D (16.7%), δ-cadinene (9%), (E)-caryophyllene (8.9%), α-cadinol (4.3%), limonene (2.6%), α-pinene (2.1%) and other components which are present in appreciable amounts. To the best of our knowledge, there are many reports on the chemical composition of P. nigra essential oils. On the other hand, our results were different from the literature by the low amounts of α and β-pinene and the high levels of 13-epi-manool oxide. The chemical composition of P. nigra ssp. dalmatica essential oil from needles was previously investigated by Chalchat and Gorunovic in Italy (Chalchat and Gorunovic, 1995). They found α-pinene, borneol and (E)-caryophyllene as the major compounds. In Turkey, α-pinene (22.2–43.18%), β-pinene (22.4–34.1%), germacrene D (6.45–14.9%) and (E)-caryophyllene (5.65–9.21%) were considered to be the major components of essential oils of P. nigra Arnold (Sezik et al., 2010). Essential oil of P. nigra Arnold from central Italy was studied by Macchioni et al. (2003), with α-pinene, germacrene D, (E)-caryophyllene and β-pinene as the major compounds. But, the chemical composition from our essential oil shows similarity with P. nigra ssp. laricio of Corsica with α-pinene, manool oxide, germacrene D, β-myrcene, (E)-caryophyllene and limonene were found to be the main constituents (Rezzi et al. 2001). This is the first report which indicates that manool oxide is the major component in the essential oil from P. nigra ssp. laricio cultivated in Tunisia. The above results suggest that the great variations in the chemical composition of the essential oil from P. nigra grown in Tunisia and those from other countries may be due to many factors such as subspecies (laricio and dalmatica), geographic factors, genetic background of tree, harvest time and extraction method.

3.2 Herbicidal effects of the oil on weed germination and seedling growth

The phytotoxic effects of P. nigra oil were tested on seed germination and seedling growth of S. arvensis, T. campestre and P. canariensis, common weeds in Tunisia. Table 2 shows that essential oil strongly inhibited the germination and seedling growth of tested weeds in a dose dependent manner with the effect being significantly more effective on S. arvensis than T. campestre and P. canariensis. Indeed, at lower concentrations from 1 to 3 μL/mL⁻¹ for S. arvensis and from 1 to 4 μL/mL⁻¹ for T. campestre and P. canariensis, the germination and seedling growth of weeds were partially reduced. However, at high concentrations (4 μL/mL⁻¹ for S. arvensis and 5 μL/mL⁻¹ for T. campestre and P. canariensis), the germination and seedling growth of all tested weeds were totally inhibited. These results are in agreement with literature (Vokou et al., 2003; De Martino et al., 2010; Amri et al., 2011, 2012). Indeed, in recent reports, we have shown the herbicidal effects of some species essential oils belonging different families that Pinaceae, (Amri et al., 2011, 2012, 2013). According to these studies, Pine species were shown to possess a potent herbicidal activity, recently, it has been demonstrated that Pinus taeda, Pinus pinea, Pinus halepensis and Pinus patula displayed inhibitory effects against the germination and seedling growth of weeds and cultivated crops (Amri et al., 2011, 2012, 2013; Kennedy et al., 2011), also. Our data agree with the literature on the inhibitory activity exerted by essential oils against weed germination and seedling growth of weeds and cultivated crops and their phytotoxicity was generally attributed to the allelopathic potential of some terpenes (Vokou et al., 2003; De Martino et al., 2010). It has been shown that the herbicidal effects of essential oils resulted from the combined reactions of several compounds including addition, synergetic and antagonistic (Vokou et al., 2003). Looking at the chemical composition of the oil (Table 1) of P. nigra, it is shown that more than 11 compounds are known to have herbicidal activity; α-pinene, β-pinene, β-myrcene, limonene, δ-3-carene and γ-terpinene are six hydrocarbonated monoterpenes that are present in our oil, indeed, these compounds have been reported to have herbicidal activities (Vokou et al., 2003; De Martino et al., 2010). Linalool and α-terpineol are two oxygenated monoterpene alcohols, linalyl acetate and isobornyl acetate are two esters; these compounds are present in the oil of P. nigra in different percentages and they are known for their potential herbicidal activity (Vokou et al., 2003).

In addition, in our study, the oil was rich in sesquiterpenes (E)-caryophyllene and manool oxide (oxygenated diterpene) which are known for their phytotoxic effects (De Feo et al., 2002; Singh et al., 2006). Numerous studies showed the phytotoxic potential of essential oil isolated from different plants. Singh et al. (2006) have demonstrated that exposure of seedling to α-pinene inhibited seedling growth causing oxidative damage in the root tissue. Kil et al. (2000) reported that (E)-caryophyllene was an important sesquiterpene of essential oil of Artemisia lavandulaefolia, which suppressed the seedling growth of Achyranthes japonica; Wang et al. (2009) showed that (E)-caryophyllene at the dose of 3 mg/L significantly inhibited the germination rates and seedling growth of Brassica campestris and Raphanus sativus. Generally, both monoterpenoids and sesquiterpenoids appear to be involved in these allelopathic interactions, for this reason the herbicidal activity of our oil was attributed to the presence of both sesquiterpenes and monoterpenes and the synergism between components does play an important role. Although the exact mechanisms of essential oil on germination and seedling growth inhibition remain unclear however, such inhibitory effects could be caused by allelochemicals interfering with physiological and biochemical processes in target species (Weir et al., 2004; Blanco, 2007).