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Allelopathic potential of essential oils isolated from peels of three citrus species
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Allelopathic potential of essential oils isolated from peels of three citrus species ⁎
Salma A. El Sawia, Mohamed E. Ibrahimb, Kowthar Gad El-Rokiekc, , Samia Amin Saad El-Dinc a
Pharmacognosy Department, National Research Centre, 12622 Dokki, Egypt Medicinal and Aromatic Plants Research Department, National Research Centre, 12622 Dokki, Egypt c Botany Department, National Research Centre, 12622 Dokki, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Essential oils Heliantus annus Portulaca oleracea Lupinus albus
In this study the essential oils were extracted from the peels of Citrus sinensis (sweet orange), Citrus aurantium (bitter orange) and Citrus reticulata (mandarins) by hydrodistillation using a Clevenger- type apparatus for 4 h. The essential oils of all samples were subjected to Gas chromatography–mass spectrometry. The results indicated that the major compounds were D-limonene, terpinene and myrcene. Essential oils from these species were tested against seed germination and seedling growth of Heliantus annus (sunflower), Portulaca oleracea (purslane), Lupinus albus (field lupine) and Malva parviflora (Egyptian mallow) in laboratory experiments at different concentrations. The assay results showed that the essential oil extracted from C. reticulata peels was the most inhibitory followed by the essential oil extracted from C. aurantium and C. sinensis peels. Essential oils of C. reticulata and C. aurantium peels inhibited completely seed germination and seedling growth of H. annus at all concentrations. Portulaca oleracea seed germination and seedling growth were inhibited completely by essential oil of C. aurantium (2%). The essential oil of C. sinensis (3%) inhibited 100% of germination and growth of Lupinus albus. And. M. parviflora germination was 100% inhibited by C. aurantium (3%) and C. reticulata (1%) peel oils. The results obtained suggested that citrus peel essential oils present significant growth inhibitory property of the listed species and has potential to be used in allelopathic processes.
1. Introduction Weeds caused serious problems in agriculture because they compete with the main crops for water and nutrient uptake causing great loss in the yield (Oerke, 2006; Murphy et al., 2008). Thus, lack of adequate nutrition may result in poor crop yield, regardless of crop quality. The use of synthetic herbicides is the main method for controlling weeds and increasing crop yield (Kraehmer et al., 2014). However, continuous use of the synthetic herbicides produces resistant species of weeds in addition to reducing crop quality and environmental contamination. Therefore, replacing the synthetic herbicides is the strategy for safe environment and producing crops in good qualities. Allelopathy is defined as the effect(s) of one plant on other plant (s) through the release of chemical compounds (allelochemicals) in the environment by leaching, exudation, volatilization (release of essential oils), or decomposition (El-Rokiek and El-Nagdi, 2011). Some of these compounds, at certain concentrations, are phytotoxic to receiving organisms, but at other concentrations they are also stimulating (Rice, 1984). Plants that produce these allelochemicals called allelopathic
⁎
plants. Essential oils are one of the most important natural plant products derived from aromatic plants and can be widely applied to plant pests as an environmentally friendly. These products are characterized by low toxicity as well as low cost that can be exploited as a chemical and biological environmental approach to resist the effects of different pests. Since essential oils contain a range of natural chemical compounds, the probability of their resistant strains being less than traditional insecticides based on one active ingredient, which can lead to resistance pests (Dayan et al., 2009). Previous studies have shown that essential oils from a number of higher plants are known to possess greater toxicity and are responsible for allelopathic activity that reduce the growth of neighbouring crops (Kaur et al., 2011; Mehmet et al., 2016). The orange peel after juice extraction is rich in essential oil. Essential oil of several species is able to inhibit the germination and growth of other species (Duke et al., 2002). The orange peel essential oil is highly bioactive posses' strong inhibitory effects on the germination and initial growth of several species (Sharma and Tripathi, 2006;
Corresponding author. E-mail address: [email protected] (K.G. El-Rokiek).
https://doi.org/10.1016/j.aoas.2019.04.003 Received 13 October 2018; Received in revised form 11 April 2019; Accepted 24 April 2019 0570-1783/ 2019 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Salma A. El Sawi, et al., Annals of Agricultural Sciences, https://doi.org/10.1016/j.aoas.2019.04.003
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2.2.3. Identification of essential oil components The chemical constituents of orange peel essential oils were identified based on the data base of mass spectra from the MS library (software Wiely138 and NBS75 database) i. e. The oil constituents were identified by comparing the spectra of these compounds with known chemical compounds that were previously analyzed and their spectra were stored in library software (Wiely138 and NBS75 database). Also, the obtained data were confirmed by injecting authentic samples of different components found in the oil under the same conditions. The obtained data were confirmed by injecting authentic samples of different components in GC–MS under the same conditions and in comparison, with the data obtained from the literature (Wehrli and Kováts, 1959; Adams, 1995).
Ali and Çelik, 2007). Moreover, even the aqueous methanol extracts of Citrus junos peel, inhibited the growth of the roots and shoots of alfalfa (Medicago sativa L.), cress (Lepidium sativum L.), crabgrass (Digitaria sanguinalis L.), lettuce (Lactuca sativa L.), timothy (Pheleum pratense L.), and ryegrass (Lolium multiflorum Lam.) as has been reported by Katonoguchi (2004). The genus Citrus, belonging to family Rutaceae, includes about 140 genera and 1300 species. Citrus sinensis (Orange), Citrus reticulata (tangerine) and Citrus aurantium (sour orange), are some important fruits of the genus Citrus (Anwar et al., 2008). Citrus are defined as one of the world's largest fruit crops produced in many tropical or subtropical countries. The countries of the Mediterranean region are the main producers of citrus (Schimmenti et al., 2013). Citrus and their by-products have high economic value, because of their multiple uses in many industries such as cosmetics, food and medical industries (Silalahi, 2002). Citrus processing industry annually generates tons of waste, including orange peel, which is produced from the extraction of citrus juice in food industry (Rivas et al., 2008). It has been suggested that using citrus waste for various applications such as fibber pectin and flavonoids (Inoue et al., 2010). However, they still get rid of large amount of waste every year (Pourbafrani et al., 2010). So, this causes significant economic and environmental problems as a result of the lack of site waste disposal, and the accumulation of organic materials (Tripodo et al., 2004). Waste from the citrus processing industry, after extraction of juice, corresponds to about 50% of the raw fruit processing, can be used as a potential source of precious derivatives (El-Adawy et al., 1999). The citrus peels, commonly treated as agro-industrial waste, are a potential source of secondary plant metabolites and essential oils (Andrea et al., 2003). In the present work essential oils from peels of three citrus species were tested in vitro against Heliantus annus, Portulaca oleracea, Lupinus albus and Malva parviflora.
2.3. Seed germination and seedling growth inhibition 2.3.1. Preparation of the essential oil concentrations Before assaying the oils on seedling growth, simple test was carried out to find the more active concentrations. Series of essential oil concentrations starting from 0.25 to 3% (v/v) was tested against the species under study (El-Rokiek and El-Nagdi, 2011). The simple assay test revealed complete death of all species at concentrations 2 and 3% (v/v) of C. reticulata. So, the proper concentrations assayed by C. reticulata peel essential oil was 0.25, 0.5 and 1% (v/v). The concentrations of the essential oil 1, 2, 3% (v/v) for C. sinensis, C. aurantium are appropriate. The essential oils isolated from the three citrus species were dissolved in distilled water with the help of ethanol. 2.3.2. Testing allelopathic activity Petri dishes bio test were carried out for screening the different concentrations of the essential oil isolated from the three citrus species peels 1, 2, 3% (v/v) for C. sinensis, C. aurantium and 0.25, 0.5 and 1% (v/v) for C. reticulata, on seedling growth of Helianthus annus (sunflower), Portulaca oleracea (purslane), Lupinus albus (field lupine) and Malva parviflora. Seeds of H. annus, P. oleracea, L. albus and M. parviflora (Egyptian mallow) were germinated in sterilized Petri dishes (9 cm diameter) containing 1-layer filter paper Whatman No. 3. Ten milliliters of the isolated essential oils of citrus species peel were added to each Petri dish. Distilled water was added to five Petri dishes serving as control. The germination of seeds was carried out in laboratory with temperature controlled at 32 ± 1 °C and 17.5 ± 1 °C for H. annus, P. oleracea. However, the Petri dishes containing the seeds of L. albus and M. parviflora were incubated at 24 °C. Each treatment was represented by five replicates; each Petri dish contained 10 seeds (for H. annus and L. albus) and 20 seeds (for P. oleracea and M. parviflora) represented one replicate. Five days later 5 mL of the isolated essential oils of citrus species peel were added to each Petri dish. Records on the percentage of germination were carried out by counting the germinating seeds. Shoot and root lengths as well as fresh mass of seedlings/Petri dish were also determined 8 days after germination. The experiment was repeated twice and the presented results are the mean of the two experiments. The treatments were arranged at complete randomized design.
2. Material and methods 2.1. Plant material and isolation of essential oils Fruits of citrus of three species: Citrus sinensis L- cultivar balady orange, Citrus aurantium and Citrus reticulata were obtained from the farms of the Egyptian Ministry of Agriculture in 30 December 2017. C. sinensis was checked for defects, insect damage, disease, surface colour change and other defects, to ensure the quality of the final product. For preparation and processing of citrus peels, the fruits were peeled and dried in a shaded place for 15 days. The essential oils of all citrus species extracted by hydro-distillation using a Clevenger-type apparatus for 4 h. The essential oil for all samples were subjected to GC/MS. 2.2. Chemical analysis 2.2.1. Gas chromatography GC analysis was performed using a Shimadzu GC- 9A gas chromatograph equipped with a DB5 fused silica column (30 m × 0.25 mm i.d., film thickness 0.25 μm). Oven temperature was held at 40 °C for 5 min and then programmed until 250 °C at a rate of 4 °C/min. Injector and detector (FID) temperature were 260 °C; helium was used as a carrier gas with a linear velocity of 32 cm/s.
2.4. Statistical analysis The generated data were subjected to analysis of variance (ANOVA) by using completely randomized design and means compared by Fisher's Least Significant Difference (LSD) 5% probability level (Snedecor and Cochran, 1980).
2.2.2. Gas chromatography/mass spectrometry Gas-chromatograph apparatus was used. A capillary DB5 (methyl‑silicone containing 5% phenyl groups) column (30 m × 0.25 mm i.d.) was used. Temperature program: 2 min at 60 °C, 60–100 °C (2 °C/ min) and 100–250 °C (5 °C/min). Helium was used as the carrier gas at a flow rate of 1.0 mL/min. Injection volume: 1.0 μL at a 1:50 split. A mass spectrometer (EI-MS 70 eV) was used with using a spectral range of m/z 40–350.
3. Results 3.1. Essential oil percentage The percentage of essential oils of orange peel recorded 0.73% (v/ w) based on dry weight for C. sinensis peel, while, these values reduced 2
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Table 1 Components of citrus peel essential oils extracted from three citrus species. Compounds
α‑Pinene Sabinene β‑Pinene Myrcene α‑Phellandrene 3‑Carene D‑Limonene Trans-ocimene γ‑Terpinene α‑Terpinolene Linalool Terpinen‑4‑ol α‑Terpineol Decanal Cis-Carveol Linalyl acetate Perilla aldehyde Thymol e-Citral Neryl acetate Geranyl acetate Dimethyl anthranilate caryophyllene Germacrene‑D Valencene Total Group MH OM SH VC
Compound class
MH MH MH 991 1005 1011 1031 1050 1062 1088 1098 1177 1189 1204 1229 1257 1271 1290 1341 1365 1383 1402 1418 1480 1491
Kovat index
939 976 980 MH MH MH MH MH MH MH OM OM OM VC OM VC OM OM OM VC VC VC SH SH SH
Percentage in the essential oil of the citrus species Citrus sinensis (Sweet orange)
Citrus aurantium (Bitter orange)
Citrus reticulata (Mediterranean mandarin)
1.70 0.44 nd 4.42 nd 0.55 86.02 nd 0.95 0.16 0.23 2.24 0.82 1.55 nd 0.23 nd nd nd nd nd nd nd nd 0.46 99.77
0.52 nd 0.19 1.44 nd nd 91.59 0.20 0.15 0.37 nd nd nd 0.08 nd 0.55 nd nd nd 0.22 0.33 0 0.34 0.17 nd 96.15
0.80 0.26 0.57 0.22 0.22 0.15 76.44 nd 10.64 nd nd 0.22 1.73 0.12 nd 0.14 0.32 0.29 nd nd nd 0.29 nd nd 0.14 92.55
94.24 3.29 0.46 1.78
94.46 nd 0.51 1.18
89.30 2.56 0.14 0.55
nd = not detected, MH = Monoterpene hydrocarbons, OM = Oxygenated monoterpenes, OS = oxygenated sesquiterpenes, SH=Sesquiterpene hydrocarbons, VC=Various compounds, CG-MS = Gas chromatography–mass spectrometry.
3.2.3. Citrus aurantium Thirteen compounds identified in C. aurantium peel essential oil are similar to most other citrus essential oils and differ only in their concentrations. Analysis of the essential oil of C. aurantium indicated that it is made essentially from MH which constitute the main class (Table 1). OM group was found as the second class followed by VC and SH group. Limonene compound was also found as the major constituent of C. aurantium essential oil, while the other compounds were found at very low concentrations. Neryl acetate, geranyl acetate, β-caryophyllene and germacrene‑D were found only in C. aurantium.
to 1.88% and 0.98% (v/w) for C. aurantium and mandarin peel, respectively (Table 1). 3.2. Essential oil constituents 3.2.1. C. sinensis The volatile constituents of C. sinensis peel from Egypt were identified by GC/MS. Thirteen components have been identified that represented nearly 99.77% for C. sinensis cultivar orange peel (Table 1). Essential oil of balady orange peel was characterized by high amounts of D-limonene, terpinene and myrcene. Some other minor terpenoids components such as, pinene, sabinene, 3-carene, terpinolene, terpinen4 ol, terpineol, decanal, cis carveol, linalyl acetate, e-citral, valencene and linalyl acetate were detected in balady orange peel essential oils. Essential oil analysis pointed out that monoterpenes hydrocarbons group was the main class of C. sinensis peel essential oil.
3.3. Seed germination and seedling growth inhibition 3.3.1. Seed germinaton percent evaluaton Peel essential oils solutions of citrus species at different concentrations showed significant inhibitory effects on seed germination of tested species (Table 2). The degree of inhibition depends on essential oil type, species response as well as concentrations. The inhibition increased with increasing essential oil concentration. Germination percentage in H. annus seeds was reduced to 70 and 60% by using essential oil of C. sinensis at concentrations 1 and 2% as compared to the control, while complete inhibition was obtained by using concentration 3%, as well as all concentrations of C. aurantium and C. reticulata. Germination of P. oleracea seeds was inhibited completely by the highest concentrations of C. sinensis and C. aurantium at 2 and 3% as well as C. reticulata at 1%. The germination of L. albus inhibited completely by peel essential oils of C. aurantium at 3%. Germination of seeds of M. parviflora was the least effective by essential oils of citrus species. Seed germination of M. parviflora was completely inhibited by essential oils of C. aurantium at 3% and C. reticulata at 1%.
3.2.2. Citrus reticulata Analysis of peel essential oil composition of C. reticulata recorded that, sixteen components have been identified that represented nearly 92.55%. The essential oil of mandarin was characterized by high amounts of D-limonene and γ-terpinene. Significant amounts of some other terpenoids compositions such as α-phellandrene and α-terpineol were detected (Table 1). Also, monoterpenes hydrocarbons group was found in a high concentration compared with other terpenoids group such as OM, SH and VC. The chemical content of Mandarin essential oil is similar to C. sinensis essential oil with difference in the concentration of compounds in both essential oils. Mandarin essential oil also contains some additional compounds, at low concentrations such as α-phellandrene, perilla aldehyde, thymol and dimethyl anthranilate. 3
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Table 2 Effect of essential oils of Citrus sinensis, Citrus aurantium and Citrus reticulata on the percentage of germination of Helianthus annus, Portulaca oleracea, Lupinus albus and Malva parviflora. Source of essential oil
Essential oil (%)
H. annus
P. oleraceae
L. albus
M. parviflora
Control C. sinensis
0 1 2 3 1 2 3 0.25 0.50 1
100a 70.0b 60.0c 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0
100.0a 85.0b 35.0c 0.0 16.5e 0.0 0.0 100.0a 21.5d 0.0 7.6
100.0a 50.0e 35.5g 33.3g 43.5f 13.5h 0.0 90.0b 80.0c 57.2d 3.5
100.0a 96.5b 93.5b 83.5c 60.0d 20.0e 0.0 93.5b 86.5c 0.0 5.0
C. aurantium
C. reticulata
LSD
Table 4 Effect of essential oil of Citrus sinensis, Citrus aurantium and Citrus reticulata on root lengths of Helianthus annus, Portulaca oleracea, Lupinus albus and Malva parviflora. Source of essential oil
Control C. sinensis
C. aurantium
C. reticulata
Essential oil (%)
0 1 2 3 1 2 3 0.25 0.50 1
LSD
Mean values in the same column for each trait followed by the same lower-case letter are not significantly according to Fisher's Least Significant Difference (LSD) test at p ≤ 0.05.
Control C. sinensis
C. aurantium
C. reticulata
LSD
0 1 2 3 1 2 3 0.25 0.50 1
Source of essential oil
L. albus
M. parviflora
108.33a 43.61b 43.33b 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.26
23.10a 8.86d 3.98f 0.00 2.1.00b 2.1.00b 0.00 16.00c 6.66e 0.00 1.96
62.50a 6.65c 3.88c 3.88c 9.45b 6.65c 0.00 12.90b 8.90bc 4.45c 4.85
43.00a 38.50a 38.35ab 33.70b 6.70cd 3.00d 3.00d 11.70c 5.35d 0.00 5.90
M. parviflora
141.66a 26.83b 20.00c 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.54
24.10a 5.98c 2.00d 0.00 1.33d 1.33d 0.00 20.50b 3.33d 0.00 2.30
63.75a 24.55b 11.25d 3.35e 13.45 cd 7.35d 0.00 17.80c 12.33c 5.0e 5.87
55.00a 41.7b 41.70b 18.35c 8.90de 3.40f 0.00 10.00d 6.70e 0.00 3.26
Table 5 Effect of essential oil of Citrus sinensis, Citrus aurantium and Citrus reticulata on the fresh biomass of seedlings of Helianthus annus, Portulaca oleracea, Lupinus albus and Malva parviflora.
Shoot length of seedling (mm) P. oleraceae
L. albus
3.3.4. Seedling fresh mass/Petri dish Different species of citrus peel essential oils caused significant reduction in fresh mass (mg) of seedlings of all tested species (Table 5). Complete seedling death was realized in H. annus at all concentrations of peel essential oils of C. aurantium and C. reticulata in addition to the highest concentration (3%) of C. sinensis. Portulaca. oleracea seedlings mass reached to zero at the highest concentrations of the essential oils of all tested citrus species (3%, 3% and 1%). Seedling mass of L. albus reached zero at the highest concentration (3%) of C. aurantium. The corresponding results in M. parviflora were obtained by peel essential oils of C. aurantium at concentrations 2 and 3% as well as C. reticulata at 1% (the highest concentration).
Table 3 Effect of essential oil of Citrus sinensis, Citrus aurantium and Citrus reticulata on shoot lengths of Helianthus annus, Portulaca oleracea, Lupinus albus and Malva parviflora.
H. annus
P. oleraceae
reduction in P. oleracea seedling root length ranged from14.9% in 0.25% of C. reticulata treated seedlings to 94.5% in concentrations 1 and 2% of C. aurantium treated seedlings. There are no growing seedlings with using the highest concentrations of essential oil solutions of the three citrus species. Seedling root length in both L. albus and M. parviflora was less sensitivethan their corresponding shoot lengths in some concentrations. It worthy to mention that using 1% of C. sinensis essential oils solution was stimulator to seedling root length of M. parviflora as compared to untreated seedlings (Table 4).
3.3.3. Seedling root length Seedlings root length in both H. annus and P. oleracea were more sensitive to essential oils of citrus species peels than seedlings shoot length. The reduction in seedling root length in H. annus recorded 85 and 88.9% by using C. sinensis peel essential oils at concentrations 1 and 2% reaching to no growing seedlings by the other treatments. The
Essential oil (%)
H.annus
Mean values in the same column for each trait followed by the same lower-case letter are not significantly according to Fisher's Least Significant Difference (LSD) test at p ≤ 0.05.
3.3.2. Seedling shoot length A significant reduction in H. annus shoot length of seedlings induced by C. sinensis at concentrations 1 and 2% was observed (Table 3). This inhibition reduced to 60% of the untreated seedlings. There are no growing seedlings at concentration 3% and at all concentrations of the other two species. Seedling shoot length of P. oleracea was reduced significantly under untreated seedlings by using concentrations 1 and 2% of C. sinensis and C. aurantium as well as 0.25 and 0.5 of C. reticulata. There are no seedlings growing at the highest concentrations of peel essential oils of all species. The reduction in seedling shoot length of L. albus ranged from 79.36 to 93.8% of the untreated seedlings reaching to complete death by using concentrations 3% of essential oils of C. aurantium. The inhibition in seedling shoot length was higher with C. sinensis peel essential oil. The reduction in seedling shoot length of M. parviflora ranged from 10.46 in C. sinensis treated seedlings to 93% in C. aurantium treaded seedling reaching to complete death by using concentrations 1% of essential oils of C. reticulata.
Source of essential oil
Root length of seedling (mm)
Control C. sinensis
C. aurantium
C. reticulata
LSD
Mean values in the same column for each trait followed by the same lower-case letter are not significantly according to Fisher's Least Significant Difference (LSD) test at p ≤ 0.05.
Essential oil (%)
0 1 2 3 1 2 3 0.25 0.50 1
Fresh biomass (mg) H. annus
P. oleraceae
L. albus
M. parviflora
2031.33a 929.56b 715.66c 0.00 0.00 0.00 0.00 0.00 0.00 000 49.00
140.00a 66.00b 12.00c 0.00 5.00c 3.00c 0.00 24.50c 21.50c 0.00 32.00
2800.00a 2100.00c 1650.00f 860.00 1980.00d 1500.00g 0.00 2200.00c 1840.00e 2380.00b 118
3535.00a 1480.00b 740.00c 470.00d 320.00e 0.00 0.00 150.00f 47.66f 0.00 109.00
Mean values in the same column for each trait followed by the same lower-case letter are not significantly according to Fisher's Least Significant Difference (LSD) test at p ≤ 0.05. 4
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4. Discussion
well as seedling fresh mass with the increase in the extracted essential oil concentrations. This suggestion was coincided with that obtained by De Oliveira et al. (2015) on lettuce, the authors reported that the degree of inhibition in seedling growth was proportional with the concentration of the essential oil. Through the results was concluded that the effect of citrus peel essential oil on seedling growth was different between citrus species. C. aurantium and C. reticulata peel essential oil caused reduction in seedling growth more than C. sinensis (Table 1). The present results are in accordance to those published by Saad and Abdelgaleil (2014) which reported a correlation between the chemical composition of essential oils and their effects on germination and seedling growth.
Essential oils and their constituents are being explored for weed and pest management (Mehmet et al., 2016). Bioactive terpenoids constitute an important part of the defensive mechanisms of a large number of organisms and represent a source of active compounds of potential use in the agricultural field (Macías et al., 2006). Citrus sinensis (Orange), Citrus reticulata (tangerine) and Citrus aurantium (sour orange) are some important fruits of genus citrus belonging to the Rutaceae or Rue family. Citrus peel essential oils are reported to be one of the richest sources of bioactive compounds (secondary metabolites), namely coumarins, flavonoids, carotenes, terpenes and linalool etc. (Mondello et al., 2005) that have natural antimicrobial properties (Viuda-Martos et al., 2008). Orange peel essential oils seem to be highly bioactive (Sharma and Tripathi, 2006; Ali and Çelik, 2007). By means of the results of Tables 2, 3, 4 and 5 it was possible to observe significant reductions in germination percentage, shoot seedling length, root seedling length and fresh mass of total seedlings of H. annus, P. oleracea, L. albus as well as M. parviflora. A complete inactivation of seedling growth was obtained especially at the highest concentrations of the essential oils tested. In this context, Ribeiro and Lima (2012) reported severe reduction in shoot and root length of Euphorbia heterophylla and Ipomoea grandifolia seedlings by peel essential oils of Citrus sinensis L. In addition, the authors mentioned that a number of dead plants of E. heterophylla were found (7.16 from 10 seedling were dead). Orange peel extracts inhibited the growth of Lemna minor (Erukainure et al., 2016) indicating a phytotoxic potential at the highest concentration (1000 μg/mL). The essential oils extracted from different plants have allelopathic effects on different species (Kaur et al., 2011; De Oliveira et al., 2015). Rosemary (Rosmarinus officinalis), artemisia (Artemisia comprises) and lavender (Lavandula angustifolia) essential oils as for example have strong allelopathic effects and prevents weed germination and growth of P. oleracea, (Dudai et al., 1999). Many plant species release phytotoxic monoterpenes, such as pinene, limonene, p‑cymene and 1,8‑cineole, that inhibit the development of weeds (Angelini et al., 2003). The inhibition of germination and seedling growth can be explained by the presence of monoterpenes in the essential oils which have phytotoxic properties that may cause anatomical and physiological changes in plant seedlings leading to accumulation of lipid globules in the cytoplasm, reduction in some organelles such as mitochondria, due to inhibition of DNA synthesis or disruption of membranes surrounding mitochondria and nuclei (Tabana et al., 2013; Bouajaj et al., 2014). In the present study the results of analysis of C. sinensis peel essential oils indicated that essential oil was characterized by high amounts of D‑limonene, terpinene and myrcene (Table 1). The essential oil of C. reticulata peels was characterized by high amounts of D‑limonene and γ‑terpinene in addition to significant amounts of some other terpenoids compositions such as α‑phellandrene and α‑terpineol (Table 1). Compounds of the monoterpene class were also found in a high concentration. Thirteen compounds identified in C. aurantium peel essential oil are similar to most other citrus essential oils and differ only in their concentrations (Table 1). Limonene was found as being the principal constituent of C. aurantium oil, while the other compounds were found at very low concentrations. Neryl acetate, geranyl acetate, β‑caryophyllene and germacrene‑D were detected only in C. aurantium. The present study confirms the allelopathic property of C. sinensis, C. aurantium and C. reticulata peel essential oil on H. annus, P. oleracea, L. albus and M. parviflora (Tables 1, 2, 3 and 4). The current results are also in confirmation with the finding of many workers that suggested the potent phytotoxic activity of plants essential oils is correlated to a high amount of oxygenated monoterpenes (Astani et al., 2009; De Almeida et al., 2010; Ersilia et al., 2018). The results of the current investigation referred to increasing in inhibition of germination percentage, seedling shoot and root length as
5. Conclusion The peel essential oil of C. sinensis, C. aurantium and of C. reticulata induced significant inhibition of the seed germination and seedling growth of Heliantus annus, Portulaca oleracea, Lupinus albus and Malva parviflora. The detected allelopahtic property of the essential oils studied represent a promising source for the developing of biodegradable and non-toxic natural herbicides. Researches involving the effect of these essential oils on cultures realized on conditions of the greenhouse and the fields are suggested. Acknowledgements The authors are thankful to the National Research Centre, Egypt, for the provision of the laboratories and facilitating this work. References Adams, R.P., 1995. Allured, Carol Stream, Illinois, and U.S.A. ISBN:(0-931710-42-1). Ali, Ö., Çelik, A.T., 2007. Cytotoxic effects of peel extracts from Citrus limon and Citrus sinensis. Caryologia. 60, 48–51. Andrea, V., Nadia, N., Teresa, R.M., Andrea, A., 2003. Analysis of some Italian lemon liquors (Limoncello). J. Agric. Food Chem. 51, 4978–4983. Angelini, L.G., Carpanese, G., Cioni, P.L., Morell, I., Macchia, M., Flamini, G., 2003. Essentialoils from Mediterranean Lamiaceae as weed germination inhibitors. J. Agric. Food Chem. 51, 6158–6164. Anwar, F., Naseer, R., Bhanger, M.I., Ashraf, S., Talpur, F.N., Aladedunye, F.A., 2008. Physico-chemical characteristics of citrus seeds and seed oils from Pakistan. J. American essential oil Chemist Soc. 85, 321–330. Astani, A., Reichling, J., Schnitzler, P., 2009. Comparative study on the antiviral activity of selected monoterpenes derived from essential oils. Phytother. Res. 24, 673–679. Bouajaj, S., Romane, A., Benyamna, A., Amri, I., Hanana, M., Hamrouni, L., Romdhane, M., 2014. Essential oil composition, phytotoxic and antifungal activities of Ruta chalepensis L. leaves from high Atlas Mountains (Morocco). Natl. Prod. Res. 28, 1910–1914. Dayan, F.E., Cantrell, C.L., Duke, S.O., 2009. Natural products in crop protection. Bioorg. Med. Chem. 17, 4022–4034. De Almeida, R., Fernando, L., Fernando, F., Mancini, E., de Feo, V., 2010. Phytotoxic activities of Mediterranean essential oils. Molecules. 15, 4309–4323. De Oliveira, C.M., das Graças Cardoso, M., Ionta, M., Gomes Soares, M., de Andrade Santiago, J., et al., 2015. Chemical characterization and in vitro antitumor activity of the essential oils from the leaves and flowers of Callistemon viminalis. Am. J. Plant Sci. 6, 2664–2671. Dudai, N., Poljakoff-Mayber, A., Mayer, A.M., Putievsky, E., Lerne, H.R., 1999. Essential oils as allelochemicals and their potential use as bioherbicides. J. Chem. Ecol. 25, 1079–1089. Duke, S.O., Dayan, F.E., Rimando, A.M., Schralder, K.K., Aliotta, G., Oliva, A., Romagni, J.G., 2002. Chemicals from nature for weed management. Weed Sci. 50, 138–151. El-Adawy, T.A., Rehman, E.H., El-Bedawy, A.A., Gafar, A.M., 1999. Properties of some Citrus seeds. Part 3. Evaluation as a new source of potential essential oil. Nahrung. 43, 385–391. El-Rokiek, K.G., El-Nagdi, W.M., 2011. Dual effects of leaf extracts of Eucalyptus citriodora on controlling purslane and root-knot nematode in sunflower. J. Plant Protec. Res. 51, 121–129. Ersilia, A., Sumalan, R.M., Danciu, C., Obistioiu, D., Negrea, M., Poiana, M., Rus, C., Radulov, I., Pop, G., Dehelean, C., 2018. Synergistic antifungal, allelopathic and antiproliferative potential of Salvia officinalis L., Thymus vulgaris L. essential oils. Molocules. 23, 1–15. Erukainure, O., Ebuehi, O.A.T., Chaudhary, M.I., Mesaik, M.A., Elemo, G.N., 2016. Orange peel extracts: chemical characterization, antioxidant, antioxidative burst, and phytotoxic activities. J. Dietary Supplements. 13, 1–10. Inoue, T., Tsubaki, S., Ogawa, K., Onishi, K., Azuma, J.-I., 2010. Isolation of hesperidin from peels of thinned Citrus unshiu fruits by microwave-assisted. Food Chem. 123,
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acid fermentation on orange peel autohydrolysate. Agric. Food Chem. 56, 2380–2387. Saad, M.M.G., Abdelgaleil, S.A.M., 2014. Allelopathic potential of essential oils isolated from aromatic plants on Silybum marianum L. Glob. Adv. Res. J. Agric. Sci. 3, 289–297. Schimmenti, E., Borsellino, V., Galati, A., 2013. Growth of citrus production among the Euro-Mediterranean countries: political implications and empirical findings. Spanish J. Agric. Res. 11, 561–577. Sharma, N., Tripathi, A., 2006. Fungitoxicity of the essential oil of Citrus sinensis on postharvest pathogens. World J. Microbiol. Biotech. 22, 587–593. Silalahi, J., 2002. Anticancer and health protective properties of Citrus fruit components. Asia Pacific J. Clinic. Nutr. 11, 79–84. Snedecor, G.W., Cochran, W.G., 1980. Statistical Methods, 7th ed. The Iowa State University Press, Ames, Iowa, IA, USA, pp. 507. Tabana, A., Saharkhiza, M.J., Hadian, J., 2013. Allelopathic potential of essential oils from four Satureja spp. Biol. Agric. Hort. 29, 244–255. Tripodo, M.M., Lanuzza, F., Micali, G., Coppolino, R., Nucita, F., 2004. Citrus waste recovery: a new environmentally friendly procedure to obtain animal feed. Bioresour. Technol. 91, 111–115. Viuda-Martos, M., Ruiz-Navajas, Y., Ferna'ndez-Lo'pez, J., Pe'rez-A'lvarez, J., 2008. Antifungal activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils. Food Control 19, 1130–1138. Wehrli, A., Kováts, E., 1959. Gas-chromatographische Charakterisierung organischer Verbindungen. Teil 3: Be rechnung der Retentionsindices aliphatischer, alicyclischer und aromatischer Verbindungen. Helv. Chim. Acta 42, 2709–2736.
542–547. Katonoguchi, H., 2004. Allelopathic potential of Citrus junos fruit waste from food processing industry. Bioresource Techn. 94, 211–214. Kaur, S., Singh, H.P., Batish, D.R., Kohli, R.K., 2011. Chemical characterization and allelopathic potential of volatile oil of Eucalyptus tereticornis against Amaranthus viridis. J. Plant Interactions 6, 297–302. Kraehmer, H., Laber, B., Rosinger, C., Schulz, A., 2014. Herbicides as weed control agents: state of the art: I. Weed control research and safener technology: the path to modern agriculture. Plant Physiol. 166, 1119–1131. Macías, F.A., Chinchilla, N., Varela, R.M., Molinillo, J.M., 2006. Bioactive steroids from Oryza sativa L. Steroids. 71, 603–608. Mehmet, A., Mavi, K., Uremis, I., 2016. Bio-herbicidal effects of oregano and rosemary essential oils on germination and seedling growth of bread wheat cultivars and weed. Romanian Biotechn. Letters. 21, 11149–11159. Mondello, L., Casill, A., Tranchida, P.Q., Dugo Dugo, P.G., 2005. Comprehensive twodimensional G.C. for the analysis of Citrus essential oils. Flavour Fragrance J. 20, 136–140. Murphy, K.M., Dawson, J.C., Jones, S.S., 2008. Weed suppression in spring wheat landraces and modern cultivars. Field Crops Res. 105, 107–115. Oerke, E.C., 2006. Crop losses to pests. J. Agric. Sci. 144, 31–43. Pourbafrani, M., Forgács, G., Horváth, I., Niklasson, S.C., Taherzadeh, M.J., 2010. Production of biofuels, limonene and pectin from citrus wastes. Bioresour. Technol. (11), 4246–4250. Ribeiro, J.P.N., Lima, M.I.S., 2012. Allelopathic effects of orange (Citrus sinensis L.) peel essential oil. Acta Botanica Brasilica. 26, 256–259. Rice, E.L., 1984. Allelopathy, 2nd Ed. Academic press. New Yourk, pp. 424. Rivas, B., Torrado, A., Torre, P., Converti, A., Domínguez, J.M., 2008. Submerged citric
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Allelopathic potential of essential oils isolated from peels of three citrus species ⁎
Salma A. El Sawia, Mohamed E. Ibrahimb, Kowthar Gad El-Rokiekc, , Samia Amin Saad El-Dinc a
Pharmacognosy Department, National Research Centre, 12622 Dokki, Egypt Medicinal and Aromatic Plants Research Department, National Research Centre, 12622 Dokki, Egypt c Botany Department, National Research Centre, 12622 Dokki, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Essential oils Heliantus annus Portulaca oleracea Lupinus albus
In this study the essential oils were extracted from the peels of Citrus sinensis (sweet orange), Citrus aurantium (bitter orange) and Citrus reticulata (mandarins) by hydrodistillation using a Clevenger- type apparatus for 4 h. The essential oils of all samples were subjected to Gas chromatography–mass spectrometry. The results indicated that the major compounds were D-limonene, terpinene and myrcene. Essential oils from these species were tested against seed germination and seedling growth of Heliantus annus (sunflower), Portulaca oleracea (purslane), Lupinus albus (field lupine) and Malva parviflora (Egyptian mallow) in laboratory experiments at different concentrations. The assay results showed that the essential oil extracted from C. reticulata peels was the most inhibitory followed by the essential oil extracted from C. aurantium and C. sinensis peels. Essential oils of C. reticulata and C. aurantium peels inhibited completely seed germination and seedling growth of H. annus at all concentrations. Portulaca oleracea seed germination and seedling growth were inhibited completely by essential oil of C. aurantium (2%). The essential oil of C. sinensis (3%) inhibited 100% of germination and growth of Lupinus albus. And. M. parviflora germination was 100% inhibited by C. aurantium (3%) and C. reticulata (1%) peel oils. The results obtained suggested that citrus peel essential oils present significant growth inhibitory property of the listed species and has potential to be used in allelopathic processes.
1. Introduction Weeds caused serious problems in agriculture because they compete with the main crops for water and nutrient uptake causing great loss in the yield (Oerke, 2006; Murphy et al., 2008). Thus, lack of adequate nutrition may result in poor crop yield, regardless of crop quality. The use of synthetic herbicides is the main method for controlling weeds and increasing crop yield (Kraehmer et al., 2014). However, continuous use of the synthetic herbicides produces resistant species of weeds in addition to reducing crop quality and environmental contamination. Therefore, replacing the synthetic herbicides is the strategy for safe environment and producing crops in good qualities. Allelopathy is defined as the effect(s) of one plant on other plant (s) through the release of chemical compounds (allelochemicals) in the environment by leaching, exudation, volatilization (release of essential oils), or decomposition (El-Rokiek and El-Nagdi, 2011). Some of these compounds, at certain concentrations, are phytotoxic to receiving organisms, but at other concentrations they are also stimulating (Rice, 1984). Plants that produce these allelochemicals called allelopathic
⁎
plants. Essential oils are one of the most important natural plant products derived from aromatic plants and can be widely applied to plant pests as an environmentally friendly. These products are characterized by low toxicity as well as low cost that can be exploited as a chemical and biological environmental approach to resist the effects of different pests. Since essential oils contain a range of natural chemical compounds, the probability of their resistant strains being less than traditional insecticides based on one active ingredient, which can lead to resistance pests (Dayan et al., 2009). Previous studies have shown that essential oils from a number of higher plants are known to possess greater toxicity and are responsible for allelopathic activity that reduce the growth of neighbouring crops (Kaur et al., 2011; Mehmet et al., 2016). The orange peel after juice extraction is rich in essential oil. Essential oil of several species is able to inhibit the germination and growth of other species (Duke et al., 2002). The orange peel essential oil is highly bioactive posses' strong inhibitory effects on the germination and initial growth of several species (Sharma and Tripathi, 2006;
Corresponding author. E-mail address: [email protected] (K.G. El-Rokiek).
https://doi.org/10.1016/j.aoas.2019.04.003 Received 13 October 2018; Received in revised form 11 April 2019; Accepted 24 April 2019 0570-1783/ 2019 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Salma A. El Sawi, et al., Annals of Agricultural Sciences, https://doi.org/10.1016/j.aoas.2019.04.003
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2.2.3. Identification of essential oil components The chemical constituents of orange peel essential oils were identified based on the data base of mass spectra from the MS library (software Wiely138 and NBS75 database) i. e. The oil constituents were identified by comparing the spectra of these compounds with known chemical compounds that were previously analyzed and their spectra were stored in library software (Wiely138 and NBS75 database). Also, the obtained data were confirmed by injecting authentic samples of different components found in the oil under the same conditions. The obtained data were confirmed by injecting authentic samples of different components in GC–MS under the same conditions and in comparison, with the data obtained from the literature (Wehrli and Kováts, 1959; Adams, 1995).
Ali and Çelik, 2007). Moreover, even the aqueous methanol extracts of Citrus junos peel, inhibited the growth of the roots and shoots of alfalfa (Medicago sativa L.), cress (Lepidium sativum L.), crabgrass (Digitaria sanguinalis L.), lettuce (Lactuca sativa L.), timothy (Pheleum pratense L.), and ryegrass (Lolium multiflorum Lam.) as has been reported by Katonoguchi (2004). The genus Citrus, belonging to family Rutaceae, includes about 140 genera and 1300 species. Citrus sinensis (Orange), Citrus reticulata (tangerine) and Citrus aurantium (sour orange), are some important fruits of the genus Citrus (Anwar et al., 2008). Citrus are defined as one of the world's largest fruit crops produced in many tropical or subtropical countries. The countries of the Mediterranean region are the main producers of citrus (Schimmenti et al., 2013). Citrus and their by-products have high economic value, because of their multiple uses in many industries such as cosmetics, food and medical industries (Silalahi, 2002). Citrus processing industry annually generates tons of waste, including orange peel, which is produced from the extraction of citrus juice in food industry (Rivas et al., 2008). It has been suggested that using citrus waste for various applications such as fibber pectin and flavonoids (Inoue et al., 2010). However, they still get rid of large amount of waste every year (Pourbafrani et al., 2010). So, this causes significant economic and environmental problems as a result of the lack of site waste disposal, and the accumulation of organic materials (Tripodo et al., 2004). Waste from the citrus processing industry, after extraction of juice, corresponds to about 50% of the raw fruit processing, can be used as a potential source of precious derivatives (El-Adawy et al., 1999). The citrus peels, commonly treated as agro-industrial waste, are a potential source of secondary plant metabolites and essential oils (Andrea et al., 2003). In the present work essential oils from peels of three citrus species were tested in vitro against Heliantus annus, Portulaca oleracea, Lupinus albus and Malva parviflora.
2.3. Seed germination and seedling growth inhibition 2.3.1. Preparation of the essential oil concentrations Before assaying the oils on seedling growth, simple test was carried out to find the more active concentrations. Series of essential oil concentrations starting from 0.25 to 3% (v/v) was tested against the species under study (El-Rokiek and El-Nagdi, 2011). The simple assay test revealed complete death of all species at concentrations 2 and 3% (v/v) of C. reticulata. So, the proper concentrations assayed by C. reticulata peel essential oil was 0.25, 0.5 and 1% (v/v). The concentrations of the essential oil 1, 2, 3% (v/v) for C. sinensis, C. aurantium are appropriate. The essential oils isolated from the three citrus species were dissolved in distilled water with the help of ethanol. 2.3.2. Testing allelopathic activity Petri dishes bio test were carried out for screening the different concentrations of the essential oil isolated from the three citrus species peels 1, 2, 3% (v/v) for C. sinensis, C. aurantium and 0.25, 0.5 and 1% (v/v) for C. reticulata, on seedling growth of Helianthus annus (sunflower), Portulaca oleracea (purslane), Lupinus albus (field lupine) and Malva parviflora. Seeds of H. annus, P. oleracea, L. albus and M. parviflora (Egyptian mallow) were germinated in sterilized Petri dishes (9 cm diameter) containing 1-layer filter paper Whatman No. 3. Ten milliliters of the isolated essential oils of citrus species peel were added to each Petri dish. Distilled water was added to five Petri dishes serving as control. The germination of seeds was carried out in laboratory with temperature controlled at 32 ± 1 °C and 17.5 ± 1 °C for H. annus, P. oleracea. However, the Petri dishes containing the seeds of L. albus and M. parviflora were incubated at 24 °C. Each treatment was represented by five replicates; each Petri dish contained 10 seeds (for H. annus and L. albus) and 20 seeds (for P. oleracea and M. parviflora) represented one replicate. Five days later 5 mL of the isolated essential oils of citrus species peel were added to each Petri dish. Records on the percentage of germination were carried out by counting the germinating seeds. Shoot and root lengths as well as fresh mass of seedlings/Petri dish were also determined 8 days after germination. The experiment was repeated twice and the presented results are the mean of the two experiments. The treatments were arranged at complete randomized design.
2. Material and methods 2.1. Plant material and isolation of essential oils Fruits of citrus of three species: Citrus sinensis L- cultivar balady orange, Citrus aurantium and Citrus reticulata were obtained from the farms of the Egyptian Ministry of Agriculture in 30 December 2017. C. sinensis was checked for defects, insect damage, disease, surface colour change and other defects, to ensure the quality of the final product. For preparation and processing of citrus peels, the fruits were peeled and dried in a shaded place for 15 days. The essential oils of all citrus species extracted by hydro-distillation using a Clevenger-type apparatus for 4 h. The essential oil for all samples were subjected to GC/MS. 2.2. Chemical analysis 2.2.1. Gas chromatography GC analysis was performed using a Shimadzu GC- 9A gas chromatograph equipped with a DB5 fused silica column (30 m × 0.25 mm i.d., film thickness 0.25 μm). Oven temperature was held at 40 °C for 5 min and then programmed until 250 °C at a rate of 4 °C/min. Injector and detector (FID) temperature were 260 °C; helium was used as a carrier gas with a linear velocity of 32 cm/s.
2.4. Statistical analysis The generated data were subjected to analysis of variance (ANOVA) by using completely randomized design and means compared by Fisher's Least Significant Difference (LSD) 5% probability level (Snedecor and Cochran, 1980).
2.2.2. Gas chromatography/mass spectrometry Gas-chromatograph apparatus was used. A capillary DB5 (methyl‑silicone containing 5% phenyl groups) column (30 m × 0.25 mm i.d.) was used. Temperature program: 2 min at 60 °C, 60–100 °C (2 °C/ min) and 100–250 °C (5 °C/min). Helium was used as the carrier gas at a flow rate of 1.0 mL/min. Injection volume: 1.0 μL at a 1:50 split. A mass spectrometer (EI-MS 70 eV) was used with using a spectral range of m/z 40–350.
3. Results 3.1. Essential oil percentage The percentage of essential oils of orange peel recorded 0.73% (v/ w) based on dry weight for C. sinensis peel, while, these values reduced 2
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Table 1 Components of citrus peel essential oils extracted from three citrus species. Compounds
α‑Pinene Sabinene β‑Pinene Myrcene α‑Phellandrene 3‑Carene D‑Limonene Trans-ocimene γ‑Terpinene α‑Terpinolene Linalool Terpinen‑4‑ol α‑Terpineol Decanal Cis-Carveol Linalyl acetate Perilla aldehyde Thymol e-Citral Neryl acetate Geranyl acetate Dimethyl anthranilate caryophyllene Germacrene‑D Valencene Total Group MH OM SH VC
Compound class
MH MH MH 991 1005 1011 1031 1050 1062 1088 1098 1177 1189 1204 1229 1257 1271 1290 1341 1365 1383 1402 1418 1480 1491
Kovat index
939 976 980 MH MH MH MH MH MH MH OM OM OM VC OM VC OM OM OM VC VC VC SH SH SH
Percentage in the essential oil of the citrus species Citrus sinensis (Sweet orange)
Citrus aurantium (Bitter orange)
Citrus reticulata (Mediterranean mandarin)
1.70 0.44 nd 4.42 nd 0.55 86.02 nd 0.95 0.16 0.23 2.24 0.82 1.55 nd 0.23 nd nd nd nd nd nd nd nd 0.46 99.77
0.52 nd 0.19 1.44 nd nd 91.59 0.20 0.15 0.37 nd nd nd 0.08 nd 0.55 nd nd nd 0.22 0.33 0 0.34 0.17 nd 96.15
0.80 0.26 0.57 0.22 0.22 0.15 76.44 nd 10.64 nd nd 0.22 1.73 0.12 nd 0.14 0.32 0.29 nd nd nd 0.29 nd nd 0.14 92.55
94.24 3.29 0.46 1.78
94.46 nd 0.51 1.18
89.30 2.56 0.14 0.55
nd = not detected, MH = Monoterpene hydrocarbons, OM = Oxygenated monoterpenes, OS = oxygenated sesquiterpenes, SH=Sesquiterpene hydrocarbons, VC=Various compounds, CG-MS = Gas chromatography–mass spectrometry.
3.2.3. Citrus aurantium Thirteen compounds identified in C. aurantium peel essential oil are similar to most other citrus essential oils and differ only in their concentrations. Analysis of the essential oil of C. aurantium indicated that it is made essentially from MH which constitute the main class (Table 1). OM group was found as the second class followed by VC and SH group. Limonene compound was also found as the major constituent of C. aurantium essential oil, while the other compounds were found at very low concentrations. Neryl acetate, geranyl acetate, β-caryophyllene and germacrene‑D were found only in C. aurantium.
to 1.88% and 0.98% (v/w) for C. aurantium and mandarin peel, respectively (Table 1). 3.2. Essential oil constituents 3.2.1. C. sinensis The volatile constituents of C. sinensis peel from Egypt were identified by GC/MS. Thirteen components have been identified that represented nearly 99.77% for C. sinensis cultivar orange peel (Table 1). Essential oil of balady orange peel was characterized by high amounts of D-limonene, terpinene and myrcene. Some other minor terpenoids components such as, pinene, sabinene, 3-carene, terpinolene, terpinen4 ol, terpineol, decanal, cis carveol, linalyl acetate, e-citral, valencene and linalyl acetate were detected in balady orange peel essential oils. Essential oil analysis pointed out that monoterpenes hydrocarbons group was the main class of C. sinensis peel essential oil.
3.3. Seed germination and seedling growth inhibition 3.3.1. Seed germinaton percent evaluaton Peel essential oils solutions of citrus species at different concentrations showed significant inhibitory effects on seed germination of tested species (Table 2). The degree of inhibition depends on essential oil type, species response as well as concentrations. The inhibition increased with increasing essential oil concentration. Germination percentage in H. annus seeds was reduced to 70 and 60% by using essential oil of C. sinensis at concentrations 1 and 2% as compared to the control, while complete inhibition was obtained by using concentration 3%, as well as all concentrations of C. aurantium and C. reticulata. Germination of P. oleracea seeds was inhibited completely by the highest concentrations of C. sinensis and C. aurantium at 2 and 3% as well as C. reticulata at 1%. The germination of L. albus inhibited completely by peel essential oils of C. aurantium at 3%. Germination of seeds of M. parviflora was the least effective by essential oils of citrus species. Seed germination of M. parviflora was completely inhibited by essential oils of C. aurantium at 3% and C. reticulata at 1%.
3.2.2. Citrus reticulata Analysis of peel essential oil composition of C. reticulata recorded that, sixteen components have been identified that represented nearly 92.55%. The essential oil of mandarin was characterized by high amounts of D-limonene and γ-terpinene. Significant amounts of some other terpenoids compositions such as α-phellandrene and α-terpineol were detected (Table 1). Also, monoterpenes hydrocarbons group was found in a high concentration compared with other terpenoids group such as OM, SH and VC. The chemical content of Mandarin essential oil is similar to C. sinensis essential oil with difference in the concentration of compounds in both essential oils. Mandarin essential oil also contains some additional compounds, at low concentrations such as α-phellandrene, perilla aldehyde, thymol and dimethyl anthranilate. 3
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Table 2 Effect of essential oils of Citrus sinensis, Citrus aurantium and Citrus reticulata on the percentage of germination of Helianthus annus, Portulaca oleracea, Lupinus albus and Malva parviflora. Source of essential oil
Essential oil (%)
H. annus
P. oleraceae
L. albus
M. parviflora
Control C. sinensis
0 1 2 3 1 2 3 0.25 0.50 1
100a 70.0b 60.0c 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0
100.0a 85.0b 35.0c 0.0 16.5e 0.0 0.0 100.0a 21.5d 0.0 7.6
100.0a 50.0e 35.5g 33.3g 43.5f 13.5h 0.0 90.0b 80.0c 57.2d 3.5
100.0a 96.5b 93.5b 83.5c 60.0d 20.0e 0.0 93.5b 86.5c 0.0 5.0
C. aurantium
C. reticulata
LSD
Table 4 Effect of essential oil of Citrus sinensis, Citrus aurantium and Citrus reticulata on root lengths of Helianthus annus, Portulaca oleracea, Lupinus albus and Malva parviflora. Source of essential oil
Control C. sinensis
C. aurantium
C. reticulata
Essential oil (%)
0 1 2 3 1 2 3 0.25 0.50 1
LSD
Mean values in the same column for each trait followed by the same lower-case letter are not significantly according to Fisher's Least Significant Difference (LSD) test at p ≤ 0.05.
Control C. sinensis
C. aurantium
C. reticulata
LSD
0 1 2 3 1 2 3 0.25 0.50 1
Source of essential oil
L. albus
M. parviflora
108.33a 43.61b 43.33b 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.26
23.10a 8.86d 3.98f 0.00 2.1.00b 2.1.00b 0.00 16.00c 6.66e 0.00 1.96
62.50a 6.65c 3.88c 3.88c 9.45b 6.65c 0.00 12.90b 8.90bc 4.45c 4.85
43.00a 38.50a 38.35ab 33.70b 6.70cd 3.00d 3.00d 11.70c 5.35d 0.00 5.90
M. parviflora
141.66a 26.83b 20.00c 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.54
24.10a 5.98c 2.00d 0.00 1.33d 1.33d 0.00 20.50b 3.33d 0.00 2.30
63.75a 24.55b 11.25d 3.35e 13.45 cd 7.35d 0.00 17.80c 12.33c 5.0e 5.87
55.00a 41.7b 41.70b 18.35c 8.90de 3.40f 0.00 10.00d 6.70e 0.00 3.26
Table 5 Effect of essential oil of Citrus sinensis, Citrus aurantium and Citrus reticulata on the fresh biomass of seedlings of Helianthus annus, Portulaca oleracea, Lupinus albus and Malva parviflora.
Shoot length of seedling (mm) P. oleraceae
L. albus
3.3.4. Seedling fresh mass/Petri dish Different species of citrus peel essential oils caused significant reduction in fresh mass (mg) of seedlings of all tested species (Table 5). Complete seedling death was realized in H. annus at all concentrations of peel essential oils of C. aurantium and C. reticulata in addition to the highest concentration (3%) of C. sinensis. Portulaca. oleracea seedlings mass reached to zero at the highest concentrations of the essential oils of all tested citrus species (3%, 3% and 1%). Seedling mass of L. albus reached zero at the highest concentration (3%) of C. aurantium. The corresponding results in M. parviflora were obtained by peel essential oils of C. aurantium at concentrations 2 and 3% as well as C. reticulata at 1% (the highest concentration).
Table 3 Effect of essential oil of Citrus sinensis, Citrus aurantium and Citrus reticulata on shoot lengths of Helianthus annus, Portulaca oleracea, Lupinus albus and Malva parviflora.
H. annus
P. oleraceae
reduction in P. oleracea seedling root length ranged from14.9% in 0.25% of C. reticulata treated seedlings to 94.5% in concentrations 1 and 2% of C. aurantium treated seedlings. There are no growing seedlings with using the highest concentrations of essential oil solutions of the three citrus species. Seedling root length in both L. albus and M. parviflora was less sensitivethan their corresponding shoot lengths in some concentrations. It worthy to mention that using 1% of C. sinensis essential oils solution was stimulator to seedling root length of M. parviflora as compared to untreated seedlings (Table 4).
3.3.3. Seedling root length Seedlings root length in both H. annus and P. oleracea were more sensitive to essential oils of citrus species peels than seedlings shoot length. The reduction in seedling root length in H. annus recorded 85 and 88.9% by using C. sinensis peel essential oils at concentrations 1 and 2% reaching to no growing seedlings by the other treatments. The
Essential oil (%)
H.annus
Mean values in the same column for each trait followed by the same lower-case letter are not significantly according to Fisher's Least Significant Difference (LSD) test at p ≤ 0.05.
3.3.2. Seedling shoot length A significant reduction in H. annus shoot length of seedlings induced by C. sinensis at concentrations 1 and 2% was observed (Table 3). This inhibition reduced to 60% of the untreated seedlings. There are no growing seedlings at concentration 3% and at all concentrations of the other two species. Seedling shoot length of P. oleracea was reduced significantly under untreated seedlings by using concentrations 1 and 2% of C. sinensis and C. aurantium as well as 0.25 and 0.5 of C. reticulata. There are no seedlings growing at the highest concentrations of peel essential oils of all species. The reduction in seedling shoot length of L. albus ranged from 79.36 to 93.8% of the untreated seedlings reaching to complete death by using concentrations 3% of essential oils of C. aurantium. The inhibition in seedling shoot length was higher with C. sinensis peel essential oil. The reduction in seedling shoot length of M. parviflora ranged from 10.46 in C. sinensis treated seedlings to 93% in C. aurantium treaded seedling reaching to complete death by using concentrations 1% of essential oils of C. reticulata.
Source of essential oil
Root length of seedling (mm)
Control C. sinensis
C. aurantium
C. reticulata
LSD
Mean values in the same column for each trait followed by the same lower-case letter are not significantly according to Fisher's Least Significant Difference (LSD) test at p ≤ 0.05.
Essential oil (%)
0 1 2 3 1 2 3 0.25 0.50 1
Fresh biomass (mg) H. annus
P. oleraceae
L. albus
M. parviflora
2031.33a 929.56b 715.66c 0.00 0.00 0.00 0.00 0.00 0.00 000 49.00
140.00a 66.00b 12.00c 0.00 5.00c 3.00c 0.00 24.50c 21.50c 0.00 32.00
2800.00a 2100.00c 1650.00f 860.00 1980.00d 1500.00g 0.00 2200.00c 1840.00e 2380.00b 118
3535.00a 1480.00b 740.00c 470.00d 320.00e 0.00 0.00 150.00f 47.66f 0.00 109.00
Mean values in the same column for each trait followed by the same lower-case letter are not significantly according to Fisher's Least Significant Difference (LSD) test at p ≤ 0.05. 4
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S.A. El Sawi, et al.
4. Discussion
well as seedling fresh mass with the increase in the extracted essential oil concentrations. This suggestion was coincided with that obtained by De Oliveira et al. (2015) on lettuce, the authors reported that the degree of inhibition in seedling growth was proportional with the concentration of the essential oil. Through the results was concluded that the effect of citrus peel essential oil on seedling growth was different between citrus species. C. aurantium and C. reticulata peel essential oil caused reduction in seedling growth more than C. sinensis (Table 1). The present results are in accordance to those published by Saad and Abdelgaleil (2014) which reported a correlation between the chemical composition of essential oils and their effects on germination and seedling growth.
Essential oils and their constituents are being explored for weed and pest management (Mehmet et al., 2016). Bioactive terpenoids constitute an important part of the defensive mechanisms of a large number of organisms and represent a source of active compounds of potential use in the agricultural field (Macías et al., 2006). Citrus sinensis (Orange), Citrus reticulata (tangerine) and Citrus aurantium (sour orange) are some important fruits of genus citrus belonging to the Rutaceae or Rue family. Citrus peel essential oils are reported to be one of the richest sources of bioactive compounds (secondary metabolites), namely coumarins, flavonoids, carotenes, terpenes and linalool etc. (Mondello et al., 2005) that have natural antimicrobial properties (Viuda-Martos et al., 2008). Orange peel essential oils seem to be highly bioactive (Sharma and Tripathi, 2006; Ali and Çelik, 2007). By means of the results of Tables 2, 3, 4 and 5 it was possible to observe significant reductions in germination percentage, shoot seedling length, root seedling length and fresh mass of total seedlings of H. annus, P. oleracea, L. albus as well as M. parviflora. A complete inactivation of seedling growth was obtained especially at the highest concentrations of the essential oils tested. In this context, Ribeiro and Lima (2012) reported severe reduction in shoot and root length of Euphorbia heterophylla and Ipomoea grandifolia seedlings by peel essential oils of Citrus sinensis L. In addition, the authors mentioned that a number of dead plants of E. heterophylla were found (7.16 from 10 seedling were dead). Orange peel extracts inhibited the growth of Lemna minor (Erukainure et al., 2016) indicating a phytotoxic potential at the highest concentration (1000 μg/mL). The essential oils extracted from different plants have allelopathic effects on different species (Kaur et al., 2011; De Oliveira et al., 2015). Rosemary (Rosmarinus officinalis), artemisia (Artemisia comprises) and lavender (Lavandula angustifolia) essential oils as for example have strong allelopathic effects and prevents weed germination and growth of P. oleracea, (Dudai et al., 1999). Many plant species release phytotoxic monoterpenes, such as pinene, limonene, p‑cymene and 1,8‑cineole, that inhibit the development of weeds (Angelini et al., 2003). The inhibition of germination and seedling growth can be explained by the presence of monoterpenes in the essential oils which have phytotoxic properties that may cause anatomical and physiological changes in plant seedlings leading to accumulation of lipid globules in the cytoplasm, reduction in some organelles such as mitochondria, due to inhibition of DNA synthesis or disruption of membranes surrounding mitochondria and nuclei (Tabana et al., 2013; Bouajaj et al., 2014). In the present study the results of analysis of C. sinensis peel essential oils indicated that essential oil was characterized by high amounts of D‑limonene, terpinene and myrcene (Table 1). The essential oil of C. reticulata peels was characterized by high amounts of D‑limonene and γ‑terpinene in addition to significant amounts of some other terpenoids compositions such as α‑phellandrene and α‑terpineol (Table 1). Compounds of the monoterpene class were also found in a high concentration. Thirteen compounds identified in C. aurantium peel essential oil are similar to most other citrus essential oils and differ only in their concentrations (Table 1). Limonene was found as being the principal constituent of C. aurantium oil, while the other compounds were found at very low concentrations. Neryl acetate, geranyl acetate, β‑caryophyllene and germacrene‑D were detected only in C. aurantium. The present study confirms the allelopathic property of C. sinensis, C. aurantium and C. reticulata peel essential oil on H. annus, P. oleracea, L. albus and M. parviflora (Tables 1, 2, 3 and 4). The current results are also in confirmation with the finding of many workers that suggested the potent phytotoxic activity of plants essential oils is correlated to a high amount of oxygenated monoterpenes (Astani et al., 2009; De Almeida et al., 2010; Ersilia et al., 2018). The results of the current investigation referred to increasing in inhibition of germination percentage, seedling shoot and root length as
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