Figures
Abstract
The essential oils from four Eucalyptus species (E. spathulata, E. salubris, E. brockwayii and E. dundasii) have been previously confirmed to have stronger inhibitory effects on germination and seedling growth of silverleaf nightshade (Solanum elaeagnifolium Cav.). The aqueous volatile fractions (AVFs) were the water soluble volatile fractions produced together with the essential oils (water insoluble fractions) during the steam distillation process. The aim of this study was to further assess the phytotoxicity of AVFs from the four Eucalyptus species and their chemical composition. The fresh leaves of the four Eucalyptus species were used for the extraction of AVFs. The AVFs were tested for their phytotoxic effects on the perennial weed, silverleaf nightshade under laboratory conditions. The chemical compositions of the AVFs were determined by gas chromatograph–mass spectrometry (GC-MS). Our results showed that the AVFs had strong inhibition on the germination and seedling growth of silverleaf nightshade. The inhibition index increased with the increasing concentrations of AVFs. The inhibitory effects of the AVFs varied between different Eucalyptus species. The AVF from E. salubris demonstrated the highest inhibitory activity on the weed tested, with complete inhibition on germination and seedling growth at a concentration of 75%. The GC-MS analysis revealed that 1,8-cineole, isopentyl isovalerate, isomenthol, pinocarvone, trans-pinocarveol, alpha-terpineol and globulol were the main compounds in the AVFs. These results indicated that all AVFs tested had differential inhibition on the germination and seedling growth of silverleaf nightshade, which could be due to the joint effects of compounds present in the AVFs as these compounds were present in different quantities and ratio between Eucalyptus species.
Citation: Zhang J, An M, Wu H, Liu DL, Stanton R (2014) Phytotoxic Activity and Chemical Composition of Aqueous Volatile Fractions from Eucalyptus Species. PLoS ONE 9(3): e93189. https://doi.org/10.1371/journal.pone.0093189
Editor: Jamshidkhan Chamani, Islamic Azad University-Mashhad Branch, Iran (Islamic Republic of)
Received: December 16, 2013; Accepted: March 1, 2014; Published: March 28, 2014
Copyright: © 2014 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Meat and Livestock of Australia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Eucalyptus has been reported to have a range of bioactivity, including antimicrobial, antiviral, fungicidal, insecticidal, anti-inflammatory, anti-nociceptive and anti-oxidant activities [1]–[3]. It was also reported that essential oil from eucalyptus has strong phytotoxic effects against many weeds, such as Parthenium hysterophorus, Cassia occidentalis, Echinochloa crus-galli, Bidens pilosa, Amaranthus viridis, Rumex nepalensis, Leucaena leucocephala, Casuarina pusilla and Leptospermum myrsinoides [4]–[7].
Similarly, aqueous extracts or leachates of eucalyptus have also been documented to possess bioactivities [8]–[11]. Aqueous leachates from fresh leaves of Eucalyptus globulus Labill. were suppressive to the establishment of vegetative propagules and early seedling growth of purple nutsdge (Cyperus rotundus L.) and bermuda grass (Cynodon dactylon L.) [8]. Khan et al. [9] reported that aqueous extracts from E. camaldulensis L. significantly inhibited weed germination and seedling growth. The water soluble fractions of Eucalyptus dundasii obtained during steam distillation were reported to be phytotoxic to the germination and growth of annual ryegrass (Lolium rigidum Gaudin) and barley grass (Hordeum glaucum Steud.) [11]. The phytotoxic activity of eucalyptus extracts obtained by different methods suggests that they may have potential herbicidal activities.
Field observations have identified that there was limited vegetation within the dripline of four Eucalyptus species: E. spathulata, E. salubris, E. brockwayii and E. dundasii. The presence of these Eucalyptus species also suppressed the understorey growth of silverleaf nightshade (Solanum elaeagnifolium Cav.).
Silverleaf nightshade is a deep-rooted, summer-growing perennial weed of the Solanaceae family that is a declared noxious weed in several countries [12]–[13]. It has been recently listed as one of the Weeds of National Significance in Australia [14]. The management of this weed includes cultural, mechanical, chemical and biological controls [12]. In the absence of reliable and effective control options, alternative control options are needed for the effective management of this weed.
Our previous study has confirmed that the phytotoxicity of essential oils (water insoluble fractions) from leaves of these four Eucalyptus species on silverleaf nightshade [15]. The essential oils were extracted by steam distillation from the leaves of the four Eucalyptuses. Meanwhile, an aqueous volatile fraction (AVF) was also obtained during the steam distillation. The aim of this study was to further assess the phytotoxicity of AVFs from the four Eucalyptus species and their chemical composition.
Materials and Methods
Plant Materials and Chemicals
Approximately two kilograms of fresh leaves of E. spathulata, E. salubris, E. brockwayii and E. dundasii were randomly collected from 6-year old trees grown in the field at Ungarie (Long. 146°55′41.33″, Lat. 33°35′53.06″), New South Wales (NSW), Australia. E. melliodora was included as a control species as it had no suppression on the understory vegetation as compared with above four Eucalyptus species underneath which silverleaf nightshade and other vegetation were suppressed. The fresh leaves of E. melliodora were collected from Wagga Wagga campus, Charles Sturt University and used as a control species. The leaves were then stored in a cool room (10°C) before steam distillation. Seeds of silverleaf nightshade were collected from a field site at Culcairn (Long. 147°10′7.75″, Lat. 35°35′38.11″), NSW in 2008. No specific permissions were required for these locations/activities because this was done on public area. The field studies did not involve endangered or protected species.
Steam Distillation
AVFs were extracted by steam-distillation according to Wu et al. [11]. Three hundred grams of fresh leaves of eucalyptus leaves were cut into 5 mm strips and subjected to steam-distillation for 2.5 h using a Pyrex oil distillation apparatus with a flat bottom flask (2 L) containing 1,200 ml distilled water to generate steam. The volatile components from the leaves were condensed through a cooling tube. Two volatile fractions, which included condensed water and the fractions (defined as “essential oil”) afloat on it, were obtained. The former was collected through a separation funnel and designated as the AVF (full strength, 100%), which was stored in a sealed bottle at 4°C before use.
Bioassays of AVFs on Weed Germination and Growth
The bioassay protocol developed by Wu et al. [16] was adopted. A concentration series [0 (water control), 25, 50, 75, 100%] was made up from the full strength (100%) solutions of AVF. Seeds of silverleaf nightshade were pre-treated by soaking in water for 24 h to remove the mucous coating for improved germination [17], then air-dried and stored prior to use. Fifty pre-treated seeds were put in each Petri dish (9 cm diameter) lined with one layer of Whatman No. 1 filter paper. An aliquot (5 ml) of each concentration of AVF was added to the Petri dish. The Petri dishes were then sealed with parafilm and maintained in a growth incubator with a diurnal temperature cycle of 25°C in light and 15°C in dark and a 12 h photoperiod. A randomized complete block design with three replicates was used. Seeds with >1 mm radical growth were considered as germinated. The germination rate, root and shoot lengths were measured after 20 days of incubation.
Solid Phase Microextraction (SPME) Procedure
SPME is a rapid, solvent-free, low-cost technique for sample preparation and has been successfully used for the extraction of analytes from aqueous and gaseous samples [18]–[19]. In this work, the analytes in AVFs were extracted by using a manual SPME holder (Sigma–Aldrich/Bellefonte, PA, USA) before gas chromatograph–mass spectrometry (GC-MS) analysis. The fiber was conditioned prior to use by heating it at 250°C for 30 min in the GC injecting unit during a blank run. The fiber was then exposed to the AVF in 10-ml glass vials for 1 min with magnetic stirring at room temperature. After extraction, analytes on the fiber were immediately desorbed in the injector port of the gas chromatograph, separated and detected as described in the following section.
Chemical Analysis of AVFs
The AVFs were analyzed by GC-MS with the use of J & W DB-5 fused silica capillary column (30 m×0.25 mm×0.25 µm) in a Varian 3800 gas chromatograph directly coupled to a Varian Saturn 2000 Ion Trap (ITD) mass spectrometer controlled by a Saturn GC/MS workstation (v5.2). Gas chromatography operating conditions followed those described by Adams [20]: 240°C injector and transfer line temperature; 60 to 250°C at 3°C/min oven temperature, with a final hold time of 8.67 min at 250°C (total run time 72.0 min); Helium carrier gas; Splitless. Mass spectrometry acquisition parameters were: full scan with scan range 41–415 amu; 1.0 s scan time; 1 count threshold; AGC mode on; 5 microscans; 1.8 min filament delay. Column head pressure was adjusted to 13.0 psi.
Compounds were identified by comparing their Kovats retention indices (KI), retention times and their mass fragmentation pattern with data in Adams [20], aided with NIST mass spectra library in the spectrometer database. The retention times of a homologous series of n-alkanes (C8–C20) were determined under the same operating conditions and used for the calculation of KI. Quantification of volatile components in AVFs was carried out by peak area normalisation measurements. The relative percentage of each component was calculated by dividing its GC-MS response (expressed as peak area) by the total peak area of chromatogram (set as 100%) of all components.
Statistical Analysis
The dose-response data were subjected to the analysis of whole-range assessment proposed by An et al. [21]. The whole-range assessment considers overall effect/response across the whole range of application rates, instead of assessing the effect of each individual rate on test species. The program WESIA (Whole-range Evaluation of the Strength of Inhibition in Allelopathic-bioassay) developed by Liu et al. [22] was used to calculate the inhibition index based on the following equation:Where the 0, D1, D2, … Dn are the dose-concentrations tested and the R(0), R(D1), R(D2), … R(Dn) are the corresponding responses, respectively. The Dc is the threshold dose at which response equals the value of control and above which the responses are inhibitory. The f(D) represents the response function. The inhibition index is a summary of the overall biological response of an organism to a tested allelochemical or equivalent and provides a relative strength indicator of biological response. Large values indicate that the species is sensitive or that the allelochemical possesses strong phytotoxic potential/biological activity, whilst small values indicate tolerance or weak potential/biological activity.
Results
AVFs on Germination of Silverleaf Nightshade
All AVFs tested inhibited the germination of silverleaf nightshade, depending on the concentration and the species (Table 1, Figure 1). The inhibition increased with the increasing concentrations. The AVFs from E. spathulata, E. salubris, E. brockwayii and E. dundasii reduced the germination rate of silverleaf nightshade by 39.8%, 92.0%, 42.0% and 35.2% respectively at a concentration of 50% and by 85.2%, 100.0%, 84.1% and 67.1% respectively at a concentration of 100%. However, the inhibition differed between species. The AVFs of the four Eucalyptus species selected had a higher inhibition than that of E. melliodora (the control species). E. salubris showed the inhibitoriest activity, with no germination observed at a concentration of 75% whereas the germination was reduced by only 36.4% at the same concentration of the AVF from the control species, E. melliodora. The inhibition potential was ranked in a decreasing order as E. salubris, E. brockwayii, E. spathulata, E. dundasii and E. melliodora based on the whole range assessment (Table 1).
The Eucalyptus species tested were E. spathulata, E. salubris, E. dundasii, E. brockwayii and E. melliodora. Germination of the bioassay without AVFs (water control) was taken as 100%. The germination of the bioassays with different concentrations of AVFs was then calculated relative to the water control. Error bars show standard error of the means (n = 3).
AVFs on Seedling Growth of Silverleaf Nightshade
It was also observed that the root length of silverleaf nightshade seedlings was decreased when exposed to the AVFs, depending on the concentration and the species (Table 1, Figure 2). The higher concentrations of AVFs used resulted in higher inhibitory effects on silverleaf nightshade. The root growth was inhibited by 85.6%, 88.2%, 78.0% and 71.9% respectively at a concentration of 50% and by 90.2%, 100.0%, 90.1% and 91.1% respectively at a concentration of 100% for AVFs from E. spathulata, E. salubris, E. brockwayii and E. dundasii. The AVF of E. salubris demonstrated the highest inhibition on root growth, whereas the AVF of E. melliodora was the least inhibitory. The inhibition potential was ranked in a decreasing order similar to the germination inhibition reported above.
The Eucalyptus species tested were E. spathulata, E. salubris, E. dundasii, E. brockwayii and E. melliodora. Root length of the bioassay without AVFs (water control) was taken as 100%. The root lengths of the bioassays with different concentrations of AVFs were then calculated relative to the water control. Error bars show standard error of the means (n = 3).
The AVFs also significantly suppressed the shoot growth of silverleaf nightshade seedlings (Table 1, Figure 3). This inhibition became more severe with increased dose used. The application of the AVF of E. salubris resulted in 73.1% inhibition at a concentration of 25% and 100% inhibition when the concentration was 75%. Different degrees of inhibition were again observed between species. The inhibition potential was ranked in a decreasing order as E. salubris, E. spathulata, E. brockwayii, E. dundasii and E. melliodora. However, the inhibition index was lower than that in root growth for all species (Table 1), indicating that the root growth of silverleaf nightshade was more sensitive to AVFs than shoot growth.
The Eucalyptus species tested were E. spathulata, E. salubris, E. dundasii, E. brockwayii and E. melliodora. Shoot length of the bioassay without AVFs (water control) was taken as 100%. The shoot lengths of the bioassays with different concentrations of AVFs were then calculated relative to the water control. Error bars show standard error of the means (n = 3).
Chemical Analysis of AVFs by GC-MS
The AVF composition from the four eucalyptus leaves was analyzed by GC-MS. The results were presented in Table 2 and Figure 4. There were 32 compounds identified in the AVF from E. spathulata leaves. It was dominated by 1,8-cineole (74.0%), pinocarvone (3.8%), trans-pinocarveol (7.2%), alpha-terpineol (2.5%) and globulol (1.4%). A total of 29 compounds were identified in the AVF of E. salubris. The main components were 1,8-cineole (47.5%), isomenthol (15.9%), pinocarvone (1.5%), trans-pinocarveol (4.7%) and alpha-terpineol (1.9%). Thirty-five compounds in the AVF of E. brockwayii were identified, with the predominant compounds being 1,8-cineole (37.1%), isopentyl isovalerate (5.9%), pinocarvone (4.2%), trans-pinocarveol (7.0%), alpha-terpineol (5.8%) and globulol (6.9%). GC–MS analyses also led to the identification of 34 different compounds in AVF of E. dundasii, with 1,8-cineole (80.1%), pinocarvone (2.2%), trans-pinocarveol (4.3%), alpha-terpineol (2.1%) and globulol (1.0%) as the main components.
Discussion
The bioassay showed that all AVFs tested had differential inhibition on the germination and seedling growth of silverleaf nightshade. The AVFs from E. spathulata, E. salubris, E. brockwayii and E. dundasii had stronger phytotoxic effect on silverleaf nightshade when compared with the control species E. melliodora. Among these four selected species, E. salubris had the highest inhibition index for germination, root and shoot growth of silverleaf nightshade. These results confirmed that the AVFs from Eucalyptus species had phytotoxicity on weed plant.
The identification and quantification of AVFs revealed that 1,8-cineole was the most abundant components in the AVFs of all Eucalyptus species tested. 1,8-Cineole has been confirmed to have many bioactivities [1], [23]. The herbicidal activity of 1,8-cineole has been tested on a wide range of weed species, including E. crus-galli, Cassia obtusifolia [24], Ageratum conyzoides L. [25] and Amaranthus viridis [26]. It has been successfully used as a lead compound in the development of an active grass herbicide for use in broadleaf crops such as soybeans [Glycine max (L.) Merr.] [27]. Therefore, 1.8-cineole, as the most dominant component in AVFs, may account for the phytotoxicity observed. However, the % 1,8-cineole in the AVFs did not fully explain the differences in phytotoxicities between the species. As showed in Tables 1–2 and Figure 4, the relative percentage (37.1%) and GC-MS response in terms of the peak area for 1,8-cineole in AVF of E. brockwayii was the lowest among the four Eucalyptus species selected, whereas the inhibition potential on silverleaf nightshade was moderate and even higher than that of E. dundasii with the highest relative percentage (80.1%) of 1,8-cineole. These results indicated that 1,8-cineole was not the most potent compound for the observed biological activities of AVFs. Other compounds including minor components could also contribute to the overall suppression of AVFs. In fact, many other individual compounds identified in eucalyptus oils, such as alpha-terpineol, citronellal, citronellol and alpha-pinene, have been confirmed to have phytotoxic activity [1], [28]. The chemical composition of AVFs varied between species. The combined effects of these compounds and the difference in chemical composition between Eucalyptus species may be used to explain the difference in their phytotoxicities against silverleaf nightshade. Further research on the phytotoxicities of AVFs and individual compounds from these Eucalyptus species on more weed plants will improve our understanding the relation between their bioactivity and chemical composition.
It was further found from Table 2 that the main compounds in AVFs were also present in the corresponding essential oils identified previously [15]. These results indicated that both extracts from eucalyptus leaves contained aqueous and phytotoxic compounds, which could be easily leached onto the ground. However, further investigation under field conditions is needed to determine the concentration of these bioactive compounds in the soil.
Conclusions
The results obtained in this study indicated that the AVFs from the selected Eucalyptus species had strong phytotoxic effects on the germination and seedling growth of silverleaf nightshade. The chemical analysis showed that the main components of the AVFs were 1,8-cineole, isopentyl isovalerate, isomenthol, pinocarvone, trans-pinocarveol, alpha-terpineol and globulol, depending on species. The AVF inhibition between species was different on the weed, which could be due to the joint effects of compounds present in the AVFs as these compounds were present in different quantities and ratio between Eucalyptus species.
Acknowledgments
We thank Mr. Robert Thompson of New South Wales Department of Primary Industries for his assistance in sample collection of eucalyptus leaves.
Author Contributions
Conceived and designed the experiments: JZ MA HW. Performed the experiments: JZ MA HW. Analyzed the data: JZ DLL MA HW. Contributed reagents/materials/analysis tools: MA RS. Wrote the paper: JZ MA HW.
References
- 1. Zhang JB, An M, Wu H, Stanton R, Lemerle D (2010) Chemistry and bioactivity of Eucalyptus essential oils. Allelopathy J 25: 313–330.
- 2. Li XF, Xue F, Wang JA, Huang D, Wang LX, et al. (2012) Allelopathic potential of Artemisia frigida and successional changes of plant communities in the northern China steppe. Plant Soil 341: 383–398.
- 3. Martins C, Natal-da-Luz T, Sousa JP, Gonçalves MJ, Salgueiro L, et al. (2013) Effects of essential oils from Eucalyptus globulus leaves on soil organisms involved in leaf degradation. PLoS ONE 8 (4) e61233
- 4. Del Moral R, Willis RJ, Ashton DH (1978) Suppression of coastal heath vegetation by Eucalyptus baxteri. Aust J Bot 26: 203–219.
- 5. Singh HP, Batish DR, Setia N, Kohli RK (2005) Herbicidal activity of volatile oils from Eucalyptus citriodora against Parthenium hysterophorus. Ann Appl Biol 146: 189–194.
- 6. Batish DR, Setia N, Singh HP, Nidhi S, Shalinder K, et al. (2006) Chemical composition and inhibitory activity of essential oil from decaying leaves of Eucalyptus citriodora. Z Naturforsch C 61: 52–56.
- 7. Setia N, Batish DR, Singh HP, Kohli RK (2007) Phytotoxicity of volatile oil from Eucalyptus citriodora against some weedy species. J Environ Biol 28: 63–66.
- 8. Chandra Babu R, Kandasamy OS (1997) Allelopathic effect of Eucalyptus globulus Lahill. On Cyperus rotundus L. and Cynodon dactylon L. Pers. J Agro Crop Sci 179: 123–126.
- 9. Khan MA, Hussain I, Khan EA (2008) Suppressing effects of Eucalyptus camaldulensis L. on germination and seedling growth of six weeds. J Weed Sci Res 14: 201–207.
- 10.
Stanton R, Wu H, An M, Lemerle D (2008) Home among the gum trees – not necessarily so for silverleaf nightshade. In: Proceedings of the 16th Australian Weeds Conference. Cairns, p 330–332.
- 11. Wu H, Zhang JB, Stanton R, An M, Liu DL, et al. (2011) Allelopathic effects of Eucalyptus dundasii on germination and growth of ryegrass and barley grass. Allelopathy J 28: 87–94.
- 12. OEPP/EPPO (2007) Solanum elaeagnifolium. Bull OEPP/EPPO Bull 37: 236–245.
- 13.
USDA-NRCS (2005) The PLANTS Database, Version 35. National Plant Data Center, Baton Rouge (US). http://plant.susda.gov. Accessed 15 November 2013.
- 14.
AWC (Australian Weeds Committee) (2012) Weeds of national significance. http://www.weeds.org.au/WoNS/. Accessed 10 April 2013.
- 15. Zhang JB, An M, Wu H, Liu DL, Stanton R (2012) Chemical composition of essential oils of four Eucalyptus species and their phytotoxicity on silverleaf nightshade (Solanum elaeagnifolium Cav.) in Australia. Plant Growth Regul 68: 231–237.
- 16. Wu H, Pratley J, Lemerle D, Haig T (1999) Crop cultivars with allelopathic capability. Weed Res 39: 171–180.
- 17. Stanton R, Wu H, Lemerle D (2012) Factors affecting seed persistence and germination of silverleaf nightshade (Solanum elaeagnifolium) in southern Australia. Weed Science 60: 42–47.
- 18. Deng C, Li N, Zhang X (2004) Rapid determination of essential oil in Acorus tatarinowii Schott. by pressurized hot water extraction followed by solid-phase microextraction and gas chromatography–mass spectrometry. J Chromatogr A 1059: 149–155.
- 19. Cuevas-Glory LF, Pino JA, Santiago LS, Sauri-Duch E (2007) A review of volatile analytical methods for determining the botanical origin of honey. Food Chem 103: 1032–1043.
- 20.
Adams RP (1995) Identification of essential oil components by gas chromatography mass spectroscopy (2nd ed.). Carol Stream: Allured Publishing Corporation.
- 21. An M, Pratley JE, Haig T, Liu DL (2005) Whole-Range Assessment: A simple method for analysing allelopathic dose-response data. Nonlinearity Biol Toxicol Med 3: 245–60.
- 22. Liu DL, An M, Wu H (2007) Implementation of WESIA: Whole-range evaluation of the strength of inhibition in allelopathic-bioassay. Allelopathy J 19: 203–14.
- 23. Van Vuuren SF, Viljoen AM (2007) Antimicrobial activity of limonene enantiomers and 1,8-cineole alone and in combination. Flavour and Frag J 22: 540–544.
- 24. Romagni JG, Allen SN, Dayan FE (2000) Allelopathic effects of volatile cineoles on two weedy plant species. J Chem Ecol 26: 303–313.
- 25. Singh HP, Batish DR, Kohli RK (2002) Allelopathic effect of two volatile monoterpenes against billy goat weed (Ageratum conyzoides L). Crop Prot 21: 347–350.
- 26. Kaur S, Singh HP, Batish DR, Kohli RK (2011) Chemical characterization and allelopathic potential of volatile oil of Eucalyptus tereticornis against Amaranthus viridis. J Plant Interact 6: 297–302.
- 27. Baum SF, Karanastasis L, Rost TL (1998) Morphogenetic effect of the herbicide Cinch on Arabidopsis thaliana root development. J Plant Growth Regul 17: 107–114.
- 28. Vaughn SF, Spencer GF (1993) Volatile monoterpenes as potential parent structures for new herbicides. Weed Sci 41: 114–119.