Chemical characterization and allelopathic potential of volatile oil of Eucalyptus tereticornis against Amaranthus viridis

Abstract

The present study investigated the chemical composition and allelopathic potential of volatile oil from Eucalyptus tereticornis on growth and establishment of Amaranthus viridis, a wasteland weed. The volatile oil was rich in monoterpenoids and a total of 34 components were identified by gas chromatography/gas chromatography–mass spectroscopy analyses, constituting 98.52%. α-Pinene (32.5%) and 1,8-cineole (22.4%) were the two major constituents. The volatile oil and two major components were evaluated for their allelopathic potential against A. viridis. A significant reduction in early seedling growth and seedling vigor of A. viridis was observed in response to volatile oil or its major monoterpenes. Further, the contents of photosynthetic pigments, i.e. chlorophylls a and b, and carotenoids, and cellular respiration in oil-/monoterpene-treated seedlings were significantly reduced thereby indicating adverse effects of the oil on photosynthetic machinery and energy metabolism. Based on the study, it can be concluded volatile oil of E. tereticornis possess allelopathic potential and could be explored as bioherbicide for future weed management programs.

Keywords:

• Eucalyptus tereticornis
• volatile oil
• monoterpenes
• allelopathy
• bioherbicide

Introduction

Essential oils are complex mixture of volatile compounds in aromatic plants giving them a characteristic odor. These volatile oils are well used in pharmaceutical, agronomic, food and flavor industries, cosmetics and perfume industries due to their antiseptic properties (Batish et al. 2008). Further, they play a vital role in plant–plant, plant–animal, and plant–microbe interactions, act as pollinator attractants, provide defense strategy against herbivores (insects, pests, and pathogenic fungi), and also possess bactericidal, fungicidal, and medicinal properties (Bakkali et al. 2008; Batish et al. 2008). Allelopathic interactions, including the use of cover or smother crops or crop residues, have been regarded to hold a good promise for weed management (Singh et al. 2003). Of late, there has been a resurgence of interest in volatile oils and their constituent monoterpenes as potential candidates for weed and pest management (Batish et al. 2004, 2008; Singh et al. 2005; Dayan et al. 2009). It is thus pertinent to evaluate the phytotoxic/allelopathic effects of essential oils of aromatic plants.

Eucalyptus tereticornis (forest red gum; Family Myrtaceae) is an evergreen tree species planted extensively for paper industry under various afforestation/reforestation programs. The leaves are rich in essential oil that is used commercially in pharmaceutical, food, flavor, and perfumery industries (Singh et al. 2009). Further, these possess a range of biological activity including insecticidal, fungicidal, and antimicrobial (Batish et al. 2008). In spite of the studies demonstrating biological activities of essential oils from E. tereticornis (Singh et al. 2009), not much has been done to explore its allelopathic/phytotoxic activity. Therefore, the objective of present study was to investigate the effect of volatile essential oil from E. tereticornis on germination and growth of Amaranthus viridis, a wasteland weed, with a view to evaluate its weed-suppressing potential.

Material and methods

Extraction of oil

The volatile oil was extracted by hydro-distillation using Clevenger's apparatus (Singh et al. 2009) from freshly plucked leaves of E. tereticornis trees growing at Panjab University campus, Chandigarh, India. The plant was authenticated by the in-charge, Herbarium, Botany department, Panjab University. The leaves were boiled in distilled water for 3 h in round bottom flask fitted with condenser. The oil collected from nozzle of condenser was dried over anhydrous sodium sulphate and stored at 4°C until tested and analyzed. The leaves yielded 1.2% (v/w; on fresh weight basis) yellow colored oil.

Identification of oil

The chemical characterization of oil was done by gas chromatography (GC) and gas chromatography–mass spectroscopy (GC–MS) as per Singh et al. (2009). Briefly, gas chromatograph (Shimadzu GC-17A) equipped with flame ionization detector (FID) and a DB-5 column (60 m×0.25 mm; 0.25 µm film thickness) was used for analysis of oil. Helium was the carrier gas at flow rate of 1 ml min⁻¹. The injector and detector temperatures were set at 250 and 280°C while oven temperature was programmed from 50°C (held isothermally for 2 min) to 260°C (held for 3 min). Relative amounts of different components were determined by computerized peak area normalization without any correction factor and were based upon three injections of the oil.

Thereafter, analysis was done on Shimadzu QP 2010 mass spectrometer equipped with a fused silica (SGE BP 20) capillary wax column (30 m×0.25 mm thickness; 0.25 µm film thickness) using He as carrier gas at split ratio of 1:50 and linear velocity of 38.5 cm s⁻¹. The mass spectra were scanned in the range of m/z 40–600 amu. Different constituents were identified on the basis of co-elution, comparison of retention times with pure reference samples, retention indices (RI) relative to homologous series of n-alkanes, and computer matching of mass spectra with HP-5872 library (Hewlett–Packard) and compiled as per Adams (2007).

Allelopathic studies

Growth assays of E. tereticornis oil and its two major constituents were done against A. viridis in a laboratory bioassay. Seeds of A. viridis were purchased from Indian Agricultural Research Institute, New Delhi, India. Pre-imbibed (for 6 h) seeds of A. viridis were allowed to germinate in Petri dishes (15 cm diameter; volume = 350 cm³) lined with Whatman filter paper #1. The different concentrations (0.5, 1, and 2.5 µL) of E. tereticornis oil or its pure components were loaded on inner side of lid of Petri dishes. The Petri dishes were immediately sealed with adhesive tape and Parafilm. There were five independent (Petri dish) replicates for each treatment each with 30 seeds. A set of Petri dishes without treatment of oil served as control. All the Petri dishes were kept at 30±2°C temperature under 16 h/8 h light/dark conditions for 6 days in a growth chamber. On seventh day, the number of seeds germinated was counted, and the root and shoot length and seedling dry weight of emerged seedlings were measured. Further, the leaves were plucked for the estimation of chlorophyll content and cellular survivability.

Further, the seedling vigor index was calculated as percent germination × root length (Abdul-baki and Abderson 1973).

Estimation of chlorophyll content

Extraction of chlorophyll was done from cotyledonary leaves as per Hiscox and Israelstam (1979). Leaf discs (25 mg) were dipped in 4 ml of dimethyl sulphoxide and the test tubes were incubated at 60°C for 1 h. After 1 h, the absorbance was measured at 663 and 645 nm. The chlorophyll content was determined as per Arnon (1949) and expressed on dry weight basis as per Rani and Kohli (1991).

Determination of cell survivability

Cell survivability was determined indirectly using 2,3,5-triphenyl tetrazolium chloride (TTC) as per Batish et al. (2007). It provides an indirect method for measurement of cellular respiration. The absorbance was read at 530 nm and expressed as with respect to control. Respiring tissue reduces TTC to red colored triphenyl formazan by accepting electrons from mitochondrial electron transport chain. The absorbance of formazan is measured at 530 nm and values are expressed with respect to control.

Statistical analyses

The qualitative analysis of volatile of E. tereticornis determined by GC and GC–MS was based upon three injections of oil. All the experiments were repeated and data presented are mean values of two experiments. The allelopathic studies were carried out in a randomized block design with five replicates. The data presented here are mean±SE and analyzed by one-way ANOVA followed by comparison of mean values using post-hoc Tukey's test at P≤0.05.

Results and discussion

Chemical composition of Eucalyptus tereticornis leaf oil

Upon GC–MS analysis, a total of 34 components eluted between 2.6 and 33.3 min and constituting 98.52% were identified in volatile oil from leaves of E. tereticornis (Table 1). The oil, in general, was monoterpenoid in nature. α-Pinene was the major constituent (32.5%) followed by 1,8-cineole (22.4%), β-pinene (10.13%), β-eudesmol (5.89), α-eudesmol (3.45%), limonene (3.38%), α-terpineol (3.09%), and γ-terpinene (2.80%). All other components were found to be in low quantities (<2%; Table 1). Our findings are parallel to earlier findings depicting presence of α-pinene and 1,8-cineole as major constituents of essential oils from E. tereticornis (Pino et al. 2001; Cimanga et al. 2002; Ogunwande et al. 2003; Singh et al. 2009).

Table 1. Chemical characterization of volatile oil from leaves of Eucalyptus tereticornis.

Retention time	Compound^a	Percentage (%)^b	Retention index^c
2.60	α-Pinene	32.5	1038
2.88	α-Thujene	0.07	1077
2.97	Camphene	0.15	1083
3.42	β-Pinene	10.13	1114
3.63	Sabinene	0.05	1127
4.19	β-Myrcene	0.36	1163
4.25	α-Phyllandrene	1.31	1167
4.52	α-Terpinene	0.10	1172
4.87	Limonene	3.38	1204
5.08	1,8-Cineole	22.4	1212
5.88	γ-Terpinene	2.80	1245
6.50	Cymene	1.12	1272
6.78	Terpinolene	0.62	1283
12.68	β-Citronellal	1.40	1480
15.17	Isopulegol-I	0.42	1559
15.44	Isopulegol-II	0.32	1569
15.88	endo-Fenchol	0.34	1584
15.96	trans-β-Caryophyllene	0.42	1587
16.24	Aromadendrene	0.34	1595
16.41	Terpinen-4-ol	0.61	1601
17.99	trans-Pinocarveol	1.42	1653
18.32	Citronellyl acetate	0.16	1665
19.17	α-Terpineyl acetate	0.47	1693
19.33	α-Terpineol	3.09	1698
21.48	β-Citronellol (R)	0.52	1772
22.07	Myrtenol	0.41	1793
22.24	trans-p-mentha-1(7),8-dien-2-ol	0.09	1799
29.46	Globulol	0.41	2070
31.84	γ-Eudesmol	1.85	2166
32.03	Guaiol	1.09	2174
32.23	–(–)Aristolene	0.32	2182
32.30	Hinesol	0.42	2185
33.06	α-Eudesmol	3.45	2217
33.26	β-Eudesmol	5.89	2225
Notes: ^aCompounds presented in order of elution from the SGE-BP20 capillary column.
^bPercentage based on FID peak area normalization (n=3).
^cRetention index relative to n-alkanes (C 7–30) on the SGE-BP20 capillary column.

Eucalyptus tereticornis oil inhibits seedling growth of Amaranthus viridis

The root length of A. viridis decreased significantly in response to different concentrations of E. tereticornis oil, α-pinene or 1,8-cineole. With treatment of 0.5–2.5 µL oil, the root length was reduced by ∼7–72%, 3–64%, and 12–70% over control, respectively (Table 2). Likewise, the shoot length was also reduced in response to E. tereticornis volatile oil or its pure components though the reduction was lesser compared to roots. Upon exposure to 0.5 µL oil, the shoot length was reduced by ∼20%. At 2.5 µL E. tereticornis oil, α-pinene, and 1,8-cineole, the shoot length declined by 60%, 49%, and 67%, respectively, over control. The seedlings also exhibited reduction in dry weight in response to oil/monoterpenes. Upon exposure to 2.5 µL oil, α-pinene, and 1,8-cineole, dry weight reduced by 54%, 41%, and 49%, respectively. The maximum seedling vigor index was observed in control seedlings. It reduced drastically in oil-/monoterpenes-treated seedlings. The maximum reduction in seedling vigor index was noticed in E. tereticornis oil treatment followed by 1,8-cineole whereas it was the minimum in α-pinene (Table 2).

Table 2. Effect of Eucalyptus tereticornis oil/major monoterpenes on root and shoot length, seedling weight, and seedling vigor index of A. viridis measured after 7 days of exposure.

Concentrations (µL)	Root length (cm)	Shoot length (cm)	Seedling dry weight (mg)	Seedling vigor index (% germination×root length)
Control (0)	3.31±0.41a	2.76±0.16a	0.63±0.09a	330±0.0a
Eucalyptus tereticornis oil
0.5	3.07±0.23a	2.21±0.21b	0.51±0.06b	243±9.7b
1.0	2.13±0.17b	1.50±0.19c	0.32±0.07c	140±11.4c
2.5	0.91±0.18c	1.12±0.11d	0.29±0.08d	24±9.1d
α-Pinene
0.5	3.25±0.19a	2.38±0.15b	0.58±0.08a	270±11.2b
1.0	2.43±0.21b	1.91±0.14c	0.46±0.09b	184±10.09c
2.5	1.24±0.13c	1.41±0.13d	0.37±0.05c	93.5±8.91d
1,8-Cineole
0.5	2.91±0.14a	2.19±0.11b	0.51±0.06b	261b±8.58b
1.0	1.96±0.16b	1.65±0.13c	0.43±0.04c	192c±8.03c
2.5	1.08±0.09c	0.90±0.15d	0.32±0.07d	52d±7.98d
Note: Different alphabets within a column present significant difference at P≤0.05 according to Tukey's test.

To the best of our knowledge, no report is available regarding the allelopathic potential of volatile oils from E. tereticornis, though earlier studies have reported the allelopathic potential of its leaf and litter extracts (Puri and Khara 1991; Rizvi et al. 1999). In our study, the volatile oils from E. tereticornis leaves showed a strong allelopathic potential in terms of inhibition of germination (indicated by seedling vigor index), root and shoot lengths, and seedling weight. In general, the oil exhibited greater allelopathic effect compared to constitutive monoterpenes. This may be due to synergistic effect of monoterpenes present in volatile oil. In nature, allelochemicals released from plants show synergistic, antagonistic, or additive effects and bring about allelopathic expressions (Einhellig 1996).

Nevertheless, our findings are in agreement with earlier reports of allelopathic potential of volatile oils/constituent monoterpenes against other plants (Angelini et al. 2003; Azirak and Karaman 2008; Kordali et al. 2009; Young and Bush 2009; Kaur et al. 2010). Batish et al. (2004, 2006a, 2006b) reported allelopathic potential of monoterpene-rich oil from another species of Eucalyptus, i.e. E. citriodora, and observed a drastic decrease in germination of Triticum aestivum, Zea mays, Raphanus sativus, Cassia occidentalis, A. viridis and Echinochloa crus-galli. A 42 h exposure to volatile compounds from Allium ursinum revealed strong phytotoxic effects in terms of germination, and radicle elongation of Lactuca sativa, Amaranthus caudatus, and Triticum aestivum (Djurdjevic et al. 2004). Likewise, Mao et al. (2006) reported allelopathic effects of Vetiveria zizanioides against pea plants and citrus trees. However, the exact mechanism by which E. tereticornis volatile oil inhibits germination and growth is still unknown. Nevertheless, the volatile oils and their constituent monoterpenes have been demonstrated to negatively affect cell division, damage cell membranes, interfere with electron flow in respiration thus affect energy level of cell, or alter enzymatic activity (Abrahim et al. 2000; Gniazdowska and Bogatek 2005; Singh et al. 2006).

Besides the negative effect of volatile oil on germination and early seedling growth, a decline was noticed in chlorophyll a and b contents (Table 3). In general, a greater reduction was observed in chlorophyll a content compared to chlorophyll b. Upon exposure to volatile oil, the chlorophyll a and b contents were reduced in the range of 17–64% and 9–38%, respectively. On the other hand, chlorophyll a content declined in the range of 15–48% and 25–69% upon treatment with α-pinene and 1,8-cineole, respectively. However, chlorophyll b content decreased by 11–52% and 10–44% upon exposure to α-pinene and 1,8-cineole, respectively, over control. Further, the carotenoid content was also reduced upon exposure to volatile oil/monoterpenes. The maximum inhibition was observed when seeds were exposed to highest concentration (2.5 µL) of volatile oils of E. tereticornis (47%) followed by 1,8-cineole (up to 39%) and α-pinene (36%). Parallel to negative effects on photosynthetic pigments, the seedlings treated with oil/monoterpenes exhibited lesser cell survivability, and thus cellular respiration, compared to control. Upon exposure to 2.5 µL of E. tereticornis oil, α-pinene and 1,8-cineole, cell respiration declined to ∼60%, 36%, and 40%, respectively.

Table 3. Effect of Eucalyptus tereticornis oil or its pure components on chlorophylls a and b, carotenoids, and cellular survivability in A. viridis measured after 7 days of exposure.

Concentrations (µL)	Chlorophyll a (µg mg^−1)	Chlorophyll b (µg mg^−1)	Carotenoids (µg mg^−1)	Cell survivability (%)
Control (0)	3.67±0.18a	2.34±0.17a	1.90±0.09a	100±0.00a
E. tereticornis oil
0.5	3.05±0.12b	2.13±0.12b	1.78±0.08b	100±0.00a
1.0	2.76±0.15c	1.87±0.16c	1.56±0.07b	73.5±6.71b
2.5	1.34±0.09d	1.45±0.18d	1.01±0.07c	40.6±5.23c
α-Pinene
0.5	3.13±0.16b	2.09±0.14b	1.87±0.07a	90.2±5.46b
1.0	2.64±0.09c	1.65±0.09c	1.63±0.09b	75.4±5.21c
2.5	1.91±0.07d	1.12±0.08d	1.21±0.07c	64.3±3.56c
1,8-Cineole
0.5	2.77±0.12b	2.11±0.14b	1.69±0.08b	88.2±5.09b
1.0	2.52±0.14c	1.75±0.16c	1.43±0.09c	77.1±6.39b
2.5	1.15±0.12d	1.32±0.13d	1.16±0.06d	59.8±4.27c
Note: Different alphabets within a column present significant difference at P≤0.05 according to Tukey's test.

The present observations revealed that the photosynthetic pigments, i.e. chlorophylls a and b and carotenoids, and cellular respiration exhibited a significant decline in all E. tereticornis oil-/monoterpenes-treated seedlings. It is well established that volatile oils adversely affect photosynthesis by destroying chlorophyll (Zeng et al. 2001; Singh et al. 2005; Batish et al. 2006b). Zhou and Yu (2006) opined that allelochemicals inhibit chlorophyll accumulation in three ways, i.e. inhibition of chlorophyll synthesis, stimulation of chlorophyll degradation, and both. Volatile oil of Princeps utilis adversely affected photosynthesis by decreasing the stomatal conductance in Vicia faba (Rai et al. 2003).

A number of volatile allelochemicals have been reported to exert negative effects on respiration. Volatile oil from Mentha piperita and its constituent monoterpene pulegone strongly inhibited root respiration hence root development of cucumber (Mucciarelli et al. 2001). Monoterpenes being non-polar in nature act as hydrophobic uncouplers of oxidative phosphorylation (Terada 1981). In the present study, the interference of volatile allelochemicals with cellular respiration may be responsible for their inhibitory activity. Abrahim et al. (2000) demonstrated that α-pinene (1.0 and 5.0 mM) inhibited respiration in Zea mays roots. This may be due to monoterpenes being lipophilic in nature readily penetrate into mitochondrial membranes, alter dehydrogenases activity and impair respiratory metabolism (P[etilde]nuelas et al. 1996; Abrahim et al. 2000). It is clear from the results that volatile allelochemicals affect cellular energy metabolism thereby affecting other physiological processes associated with plant growth and development (Kramer 1983). However, other factors such as accessibility and uptake of volatile compounds by cell are still to be explored. Volatile compounds are important allelopathic agents. Because of their high vapor density, these after release from plants easily penetrate into the soil and create an environment around itself that is unfavorable to growth and development of other species (Scrivanti et al. 2003). This enables the plants to have more advantages over other species in the struggle for survival. Thus, based on the present study, we conclude that essential oils from E. tereticornis and its monoterpenes exhibit allelopathic properties against A. viridis and hold potential for use in weed management. However, in order to incorporate it as bioherbicide, there is a need to study long-term crop–weed allelopathic interactions under field conditions and to explore the physiological and biochemical mechanism of action.

Acknowledgements

Shalinder Kaur is thankful to University Grants Commission (UGC, New Delhi, India) for postdoctoral fellowship.