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Article

Environment-Friendly Control Potential of Two Citrus Essential Oils against Aphis punicae and Aphis illinoisensis (Hemiptera: Aphididae)

by
Saqer S. Alotaibi
1,*,
Hadeer Darwish
1,
Ahmed K. Alzahrani
2,
Sarah Alharthi
3,
Akram S. Alghamdi
4,
Amal M. Al-Barty
4,
Mona Helal
5,
Amal Maghrabi
1,
Alaa Baazeem
4,
Hala A. Alamari
6 and
Ahmed Noureldeen
4
1
Department of Biotechnology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
2
College of Applied Medical Sciences, Northern Border University, P.O. Box 1321, Arar 91431, Saudi Arabia
3
Department of Chemistry, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
4
Department of Biology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
5
Pomology Department, Horticulture Institute, Agriculture Research Center, Giza 12619, Egypt
6
Institute of Biology and Environmental Research, National Center for Biotechnology, King Abdulaziz City for Science and Technology, P.O. Box 6086, Riyadh 11442, Saudi Arabia
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(9), 2040; https://doi.org/10.3390/agronomy12092040
Submission received: 4 July 2022 / Revised: 18 August 2022 / Accepted: 20 August 2022 / Published: 27 August 2022
(This article belongs to the Special Issue Biological Control as a Crucial Tool to Sustainable Food Production)
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Abstract

:
Aphids are serious pests of a wide range of agricultural crops, including pomegranates and grapevines. In addition, due to the negative environmental impacts of chemical insecticides, these pests are developing important resistance against aphicides. Therefore, one alternative method to control aphids is the use of essential oils (EO). The present study aimed to evaluate the insecticidal activity of Citrus aurantium and C. reticulata peel EO at different concentrations and with different exposure periods to pomegranate and grapevine aphids, Aphis punicae and A. illinoisensis via the topical application method under laboratory conditions. The results reveal that C. aurantium L. EO had greater toxicity against pomegranate and grapevine aphids, with LC50 of 0.37 and 0.82 μL/mL, respectively, at 48 h after application. The highest repellence effect was estimated for C. aurantium EO, at 2.5 μL/cm2, on A. punicae, with a value of 100% after an exposure time of 3 h, in contrast to the 88% repellence estimated for A. illinoisensis. The GC-MS investigation of both essential oils identified limonene, 3-carene, pinene, and p-cymene as active substances that could be attributed to the effects observed. Overall, our results offer a potential tool to control the two aphid species and could help in the development of integrated insect management in pomegranate and grapevine fields.

1. Introduction

One of the main pests in pomegranate orchards is the aphid, Aphis punicae Passerini (Hemiptera: Aphididae). Previously, it was considered a minor pest of pomegranate. However, in recent years, this pest has become serious and is appearing regularly all year round [1]. Nymphs and adults both feed on fruits, inflorescences, and leaves. Pomegranate aphid infestation causes pale and curled leaves, delayed growth, and dropped blossoms. In addition, the aphid spreads viral infections and secretes honey dew on which fungi grow, which affects crop quality and productivity [2]. A. illinoisensis Shimer, an invasive pest of grapevines, infests the undersides of young leaves, young terminal shoots [3], and fruit clusters, causing some grape berries to drop [4]. The pomegranate aphid is among the invasive pests recorded in the Mediterranean area, Asia, Africa, and the Indian subcontinent [5,6,7,8,9]. A. illinoisensis is a North American species [3]. Recently, this aphid has invaded Turkey [10], Greece [11], Iran [12], and has been discovered in Tunisia for the first time [13]. Even in Central Europe, it has been observed more recently in Slovenia [14] and France [15]. A. illinoisensis appears to produce anholocycles on grape plants in the continent of Eurasia, and no sexual morphs have been detected [16]. This species has been observed in Italy, namely on the island of Sicily and in the district of Catania [17], and for the first time in mainland Italy (Rome), apterous and winged viviparous females of this pest were found in 2021 on Vitis labrusca L. stems [18]. In Saudi Arabia, A. illinoisensis is also viewed as invasive due to the potential harm it could do to the production of grapes (the second most important economic fruit), particularly Taify cultivars produced in Taif region [19,20].
Aphids can reproduce quickly, so using insecticides frequently to control them has resulted in the emergence of resistance. Effective aphicides are very expensive, very poisonous, and have adverse environmental effects [21]. Although they have more negative to moderately harmful effects than malathion and pirimicarb on predators, neonicotinoid pesticides in Egypt can be viewed as potential tools for managing the pomegranate aphid, Aphis punicae, as outlined by [22]. According to research on the specialized pomegranate crops in Alicante, Spain, overuse of agrochemicals might potentially result in resistant insect populations [23]. Similarly, in Iran, it appears that using chemical pesticides to suppress this pest should be avoided because they have adverse effects on the environment, in addition to being harmful to the natural enemies and other beneficial insects that cause an outbreak [24]. Therefore, scientists are looking for substitute pesticides that are stronger against the two aphid species, don’t have many cytotoxic side effects on natural enemies, and are less hazardous to the environment [25].
Medicinal plants are the fundamental raw materials for various chemical drugs and food products. At present, more than one third of medical drugs are derived from botanic extracts [26]. A preliminary study on medicinal plant diversity in the flora of the Kingdom of Saudi Arabia found seven families with 254 species [27]. In particular, the Taif province of Saudi Arabia, has a wealth of such species, which are commercially and extensively cultivated in the production of essential oils (EOs). EOs from plant species are low-molecular-weight mixtures that are produced in large quantities by a variety of plant families, including the Asteraceae, Apiaceae, Lamiaceae, Rutaceae, Lauraceae, and Myrtaceae [28,29,30], whose monoterpenoids have received attention in recent years as potential pest control agents. These substances operate in a variety of ways, such as direct toxins, antifeedants or repellents, or by changing enzyme profiles [31]. Additionally, it was observed that a variety of parameters, including harvest year [32], cultivar [33], environmental conditions [34], and extraction system [35], had an impact on the essential oil composition of citrus fruits. The Rutaceae family includes plants of particular significance in potential applications in the medicinal [36,37], as well as the cosmetic [38,39], and industrial fields. These are the sour orange (Citrus aurantium L.) and mandarin orange (C. reticulata Blanco). It has long been speculated that the plant’s production of several unique EOs and antioxidants is efficiently produced in their skin or peel [40,41]. These EOs contain several compounds, such as monoterpenes [42], flavonoids, and other phytochemicals. Limonene is known to be abundant in the peel oils of the Citrus genus [43], and GC-MS analysis results [44] have shown that limonene is the primary constituent of bergamot (46.0%), lemon (75.7%), mandarin (71.9%), and sweet orange (83.8%) oils.
Recently, laboratory research into EOs and their ingredients for use in controlling pests in fruit trees has increased. A review of the literature reveals several papers have investigated the effects of EOs on aphids and stored-product pests [45,46,47,48,49], but few studies have focused on the aphicidal activity of citrus EOs. Gupta et al. [50] revealed, for the first time, the insecticidal effects of lemon (Citrus limon) peel aqueous extract against rose aphid (Macrosiphum roseiformis) and its influence on nontarget predators, Coccinella septempunctata, and Orius laevigatus on rose plants. The contact and residual toxicity of eleven EOs, including C. aurantium, C. sinensis, and C. limon, has been tested against the woolly beech aphid, Phyllaphis fagi (Hemiptera: Aphididae), with C. aurantium recording the highest residual toxicity (40%) of the Citrus species tested against the targeted insect [51]. Oregano (Origanum syriacum var. bevanii L.), cumin (Cuminum cyminum L.), anise (Pimpinella anisum L.), and eucalyptus (Eucalyptus camaldulensis Dehn) have all been successful as fumigants against cotton aphid (A. gossypii Glover) [52]. Additionally, the bioactivity of Tagetes minuta L. EO volatiles against the aphid species M. persicae, Acyrthosiphon pisum (Harris), and Aulacorthum solani (Kaltenbach) has been documented [53]. Moreover, [54] evaluated the efficacy of five plant materials on Aphis gossypii population in okra production and demonstrated that the aphid population was reduced more effectively by Tagetes minuta L. and Carica papaya L. Adverse effects of Thymus, Veronica, and Agrimonia Eos have also been demonstrated on cabbage. Although there have been numerous studies on the toxicity effects of Eos on aphids, little or no work has been carried out to find the effects of Eos on A. punicae and A. illinoisensis control. Therefore, this paper aims to investigate the biological activities and applications of C. aurantium and C. reticulata Eos as natural products on the mortality and repellency of pomegranate and grapevine aphids, A. punicae and A. illinoisensis, under laboratory conditions.

2. Materials and Methods

2.1. Plant Materials

Thirty (200 days old from blossom) sour (Citrus aurantium cv. Swingle) and mandarin (C. reticulata cv. Kinnow) full-mature orange fruits (6 fruits/plant) with orange color were picked from five plants (15 years old) each from local citrus farms (Taif, Saudi Arabia) that did not use any pesticides. The plants are grown in clay loam soil. The distance between the lines is 6 m and the distance between the trees is 5 m. The irrigation water that the plant receives (8–10 L/plant) in the drip system, and the fertilizers from soluble sources are injected with irrigation water into the drip network at the required rates in batches at a rate of once every one week. The botanical identification was conducted at the Departments of Biology and Biotechnology, Faculty of Sciences, Taif University, Saudi Arabia in January 2021.

2.2. Isolation of Essential Oils

Sour and mandarin orange fruits were completely rinsed with distilled water first, and then with absolute ethanol. Citrus fruit peels were removed. Fruit peels were allowed to air dry for two weeks at room temperature while being covered with a muslin cloth. Dried materials were pounded to powder in a mixer grinder (MRC LB20ES, Burnt Mill, Essex, CM20 2HU UK). For this experiment, 100 g of each material was added to 500 mL of distilled water and subjected to hydro-distillation for six hours using a Clevenger-type apparatus (Dolphin Labware, Mumbai, India). After distillation, 100 g of each plant’s dry material produced almost 4 mL of oil. To acquire the necessary amount of oil for further uses, the distillation was repeated. The essential oil distillates were filtered, dried over anhydrous sodium sulphate, and refrigerated at −4 °C until analysis [55].

2.3. GC–MS Analysis of Eos

An Agilent-Technologies (Little Falls, CA, USA) 6890N Network gas chromatographic (GC) system, equipped with an Agilent-Technologies 5975 inert XL Mass selective detector and an Agilent-Technologies 7683B series auto injector, was used for the gas chromatography/mass spectrometry (GC-MS) analysis of the phytochemical contents of essential oils. Compounds were separated on HP-5 MS capillary columns (30 m × 0.25 mm i.d., 0.25 µm film thickness; Little Falls, CA, USA). With a split ratio of 100:1, 1.0 µL of sample was injected in the split mode. An electron ionisation system, with an ionisation energy of 70 eV, was used for GC-MS detection. The temperature program for the column oven matched the one chosen for the GC analysis. At a flow rate of 1.5 mL/min, helium acted as a carrier gas. The injector and MS transfer line temperatures were set at 220 °C and 290 °C, respectively, while the mass scanning range ranged from 50 to 550 m/z.

2.4. Compound Identification

The documentation of the oil constituents would be based on the comparison of their retention indices relative to (C9–C24) n-alkanes with authentic compounds. Compounds were identified using their MS data compared to those from the NIST mass spectra library and published mass spectra [56].

2.5. Insects

Fresh leaves and buds of pomegranate and grapevine trees infested with the aphids, Aphis punicae and A. illinoisensis, respectively (about 50 not parasitised insects, homogenised in shape, age, and size per leaf or bud) were collected in plastic bags on the same experimental day from the farms of pomegranate and grapevine trees that had not used any pesticides at Taif, Saudi Arabia.

2.6. Bioassay

2.6.1. Toxicity Test

The toxicity of C. aurantium and C. reticulata oils was evaluated on A. punicae and A. illinoisensis via a topical application method described by Eidy et al. [57] with slight modification. Quantities of 0.5, 1, 2, 4, and 6 μL for each essential oil were diluted in 1 mL acetone [48]. Then, at each concentration, five replicates (each containing 20 apterae aphid individuals of the same size of the Petri dish) were employed, totalling 100 aphid individuals. Every Petri dish (9 cm) was lined with a thick layer (4–5 mm) of moistened cotton. Four Taify pomegranate or grapevine leaf discs measuring 1.5 cm in diameter were cut out and placed on the cotton layer in each dish. On the aphid body, 1 µL of each concentration was directly poured. A delicate camel hairbrush was used to softly move the adult aphid individuals on the leaf discs in the petri dishes. In the control treatments, insects were treated with the same volume of acetone for each aphid species. The Petri dishes were then sealed with Parafilm and kept under laboratory conditions of 25 ± 1 °C, 65 ± 3% relative humidity, and a 12 L:12 D light–dark cycle. Insects were counted as dead if their appendages did not move when a fine-point brush was used to touch them after 6, 12, 24, and 48 h. According to Abbott’s formula [58], mortality percentages were modified for mortality in control.

2.6.2. Repellence Test

Following Isman [59], the behavioral response (food preference) of aphids was examined by offering them two options in order to test whether they had any preference for or against a specific essential oil. In brief, pomegranate leaves were cleaned, dried, and cut into equal leaf discs measuring 1.5 cm in diameter. Then, leaf discs were impregnated for 30 sec in 2 mL of each essential oil diluted in acetone at doses of 0.125, 0.25, 0.50, 0.75, and 1 μL/cm2. The control leaf discs were treated with an equal volume of acetone. Leaf discs with and without EO treatments were air-dried for about 60 min to allow the solvent to evaporate. Two treated pomegranate leaf discs and two untreated ones were then kept in a Petri dish (9 × 1.5 cm) with moistened filter paper (Whatman No. 1, 8 cm diameter) and alternately arranged around the circumference of the Petri dish at the diagonal position. After that, 20 individuals of the A. punicae aphid were released at the center of the Petri dish. For A. illinoisensis, the previous procedures were followed. However, equal grapevine leaf discs were employed. The experiment was kept at 25 ± 1 °C and 65 ± 3% RH. At 30, 60, 90, 120, and 180 min following the release, observations on the number of aphids present on treated or untreated leaf discs were recorded. Equation used to determine repellency percentage: Repellence (%) = (Nc − Nt)/(Nc + Nt) × 100, where Nt = the number of insects on the treated discs after the exposure time And Nc = the number of insects on the untreated discs. Five replicates from each dose of the tested essential oil were taken.

2.7. Statistical Analysis

GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) was employed for all experiments’ statistical analyses. Two-way analysis of variance (ANOVA) with Duncan’s test was used to compare the means among the corrected mortalities. Using SPSS Version 23 (IBM Corp., Armonk, NY, USA), a t-test was performed on the LC50 and LC90 values, 95% confidence intervals of the lower and upper values, slope, intercept, and chi-square for both essential oils. A p-value of 0.05 or less indicates statistical significance. Results are expressed as the mean ± standard error (SE).

3. Results

3.1. Toxicity Assay

The data in Figure 1 show that the oil’s efficiency was directly related to concentration and exposure time, as the mortality of A. punicae adults ranged from 9 to 100% after topical application of C. aurantium oil at five concentrations for four exposure times. Median and high concentrations (2–6 µL EO/mL acetone) of the tested EO were the most efficient, causing 100% mortality after 48 h of exposure. When A. punicae was exposed to oil at concentrations between 2 and 6 µL/mL for a short time (8 h), the mortality percentages significantly increased, from 77 to 100%. For A. illinoisensis, the obtained results shown in Figure 2 illustrate that the highest significant values of mortality were 100% when a concentration of 6 µL/mL was used at 24 and 48 h exposure times followed by 98% at 6 µL/mL after 8 h exposure. On the other hand, only 2% of the mortality resulted from a 0.5 µL/mL treatment at 2 h after application.
For C. reticulata EO, the highest significant mortality of A. punicae and A. illinoisensis adult stages were represented in the highest concentration, 6 µL/mL, at all exposure times (Figure 3 and Figure 4). The highest percentages of A. punicae mortality (97% and 93%) were recorded at 6 µL/mL after 48 and 24 h (Figure 3), while those of A. illinoisensis mortality were 92.8% and 84.4% at 48 and 24 h, respectively (Figure 4). A statistical analysis of the A. punicae mortality results shows statistically significant differences (p < 0.05) between the mortality and the treatment of the tested EOs at all concentrations from 2 to 48 h (Table 1). The data in Table 1 show that at 48 h, C. aurantium EO was more effective against A. punicae adults than C. reticulata, as it recorded lower LC50 and LC90 values: 0.37 and 1.13 µL EO/mL acetone, respectively, for the former and 1.03 and 4.13 µL EO/mL acetone, respectively, for the latter. However, at 24 h, no significant difference was observed between the LC50 values of the two EOs. C. aurantium recorded 1.74 µL/mL, compared with 1.85 µL/mL for C. reticulata. As exposure time was extended in all treatments, the toxicity increased noticeably. As a result of the tested EOs, the individuals of A. punicae displayed varying degrees of homogeneity, with slope values ranging from 2.11 to 2.87 (Table 1). Similarly, Table 2 shows the toxicity of EOs, with C. aurantium more effective than C. reticulata after exposure periods of 24 h and 48 h against A. illinoisensis, with LC50 (1.09 and 0.82 µL/mL) and LC90 (3.47 and 2.15 µL/mL), respectively. The data in Table 2 also reveals that C. aurantium had the highest level of homogeneity for A. illinoisensis, with a slope value of 2.94.

3.2. Repellent Activity

The repellent percentage in aphid adults was increased by increasing both the doses of the two tested EOs and exposure times. Regarding the A. punicae results shown in Table 3, the highest significant repellency of the individuals was recorded as 100% and 98% for 2.5 µL C. aurantium oil/cm2, at 2 h and 3 h, respectively, followed by 92% for the same dose at 1.5 h, while 8% was the least significant repellency rating at 0.156 µL C. aurantium oil/cm2 after 30 min. For C. reticulata EO, the data presented in the same table indicate that the highest significant repellency of A. punicae adults after 3 h was 70% and 58% at 2.5 and 1.25 µL oil/cm2, respectively. For A. illinoisensis, the results presented in Table 4 show that the highest repellency percentage of adults increased when the dose of EOs was increased at all exposure times. C. aurantium showed its greatest substantial repellent activity at 2.5 µL oil/cm2 after 3 h, with a value of 88%, versus a value of 62% for C. reticulata after the same exposure time. However, the lowest significant repellency value was 2%, at 0.156 µL of C. aurantium oil/cm2. Furthermore, it was clear that C. reticulata EO at low doses does not perform well as an aphicide, since no repellency of A. illinoisensis was recorded at 0.156 and 0.312 µL/cm2 after a half-hour exposure.

3.3. Chemical Compositions of Essential Oils

The screened molecules found in the peels of both sour orange and mandarin samples containing EO compounds are limonene, 3-carene, α-pinene, and p-cymene (Table 5). Among all the identified compounds, limonene was the most prevalent, with a 96.98% area coverage and retention time of 4.738 and 91.86% and 4.768 for C. aurantium and C. reticulata oils, respectively. Some compounds were identified in only one plant, such as beta-guaiene and 2,7-bis(spirocyclopropane)bicyclo [2.2.1]heptan-5-one in C. aurantium, and carveol, cis-p-mentha-2,8-dien-1-ol, doconexent, methyl 6,8-octadeca diynoate, methyl 8,10-octadecadiynoate, (-) carvone, 3-cyclohexen-1-ol, 5-methylene-6-(1-methylethenyl)-, acetate, oxacyclotetradeca-4,11-diyne, and 5,8-dimethylene bicyclo [2.2.2]oct-2-ene in C. reticulata.

4. Discussion

The results of the present laboratory investigation shed light on the promising use of citrus Eos to suppress aphid insects attacking pomegranate and grapevine plants. Our results showed that both of the tested EOs at all concentrations and exposure times have been recorded as the toxicants against either A. punicae or A. illinoisensis. C. aurantium EO was more effective than C. reticulata. The results obtained were in accordance with [60,61], who evaluated the aphicidal effects of peel extracts of orange, C. sinensis, and C. sinensis, and C. paradise against wheat and cowpea aphids, respectively. They recorded that orange peel extract treatment consistently caused the highest level of wheat aphid mortality (65.69%) and the lowest LC50 value (62.3 µL/mL) for cowpea aphid. Furthermore, in the present study, A. punicae was more sensitive to both EOs than A. illinoisensis, which may be due to different insect morphology, behaviors, and inhibitory activity changes in biochemical biomarkers in A. punicae than in A. illinoisensis, as previously mentioned by [46,62]. In A. craccivora, it was found that the detoxification enzymes, Glutathione S-transferase and acetylcholinesterase, had significantly lower activities in insects treated with EOs than control.
The present investigation revealed for the first time that EOs of selected two citrus species possessed repellency towards A. punicae and A. illinoisensis. There were noticeable differences in the repellent actions of C. aurantium and C. reticulata essential oils, and all treatment doses efficiently repelled both aphids upon their instant application. C. aurantium EO also resulted in higher repellency than C. reticulata. In the same context, Fiaz et al. [63] found that lemon oil (at 5% concentration) had repellent and phago-deterrent effects against jassids and thrips. Numerous complex chemicals, including terpenoids and phenols, are responsible for the repellent properties of EOs. These compounds either prevent or disrupt insect feeding by rendering treated leaf surfaces unpleasant or unattractive [64]. As an alternative, these EOs might change the insects’ diet or interfere with their hormone balance, rendering their diet inedible [65].
Fourteen compounds were recorded in the screened molecules detected in the EOs of both sour orange and mandarin peels, of which limonene was the most prevalent in both C. aurantium and C. reticulata oils, with a higher percentage in C. aurantium than in C. reticulata. On the other hand, p-cymene appeared the least in the two tested oils. In the present study, results confirmed that the oil from C. aurantium and C. reticulata comprises mostly limonene, which is probably the reason for the insecticidal activities. Similarly, Sreepian et al. [66] identified 12 and 25 compounds in the EO of C. reticulata and C. aurantium fruit peels, respectively, revealing the potential of these citrus EOs as natural antibacterial agents, with the most prevalent component being limonene (62.9–72.5%). In China, monoterpene hydrocarbons were the main constituents of the EOs extracted from the fruit peels of physiologically dropped navel oranges (Citrus sinensis Osbeck cv. Newhall), Yangshuo kumquats (Citrus japonica Thunb), Nanfeng mandarins (Citrus reticulata Blanco cv. Kinokuni), Xunwu mandarins (Citrus reticulata), and limonene was the predominate compound for all citrus EOs [67]. Boughendjioua et al. [68] recorded the presence of 28 compounds in Algerian mandarin (Citrus reticulata) essential oil, constituting mainly D-limonene (85.10%), sabinene (2.49%), linalyl acetate (2.00%), copaene (1.80%), and α-pinene (1.75%). In Tunisia, [69] identified 37 compounds from C. aurantium EO, of which limonene was the most abundant (62.2%) and recorded the antimicrobial activity against a panel of pathogenic bacteria. However, analyzing the crude extract of peels of C. aurantium in Thailand by GC-MS revealed the presence of limonene as the major compound, accounting for 93.7% of the total and demonstrating the antiviral activity of L-limonene for the first time [70]. Furthermore, Sevindik et al. [71] in Turkey recorded (72.51%) limonene in the essential oil obtained from the C. aurantium central population, (77.27%) in samples originated from Germencik region, (79.77%) in samples from the Koçarlı population, and (95.70%) in samples from the Nazilli population.
Both limonene and linalool are poisonous for insects; they amplify the sensory nerve, causing considerable over-stimulation of the motor nerves, which may lead to convulsion and paralysis [50]. These findings were in conformity with the results of [72,73], who mentioned that the complexity of the chemical composition of the majority of unstable oils gives them low specificity because biological activity is not assigned to a single action mechanism since the wide variety of chemical groups allows for multiple targets in the cell. As recorded by [74] in Argentina, a mixture of ethanol extract of C. aurantium albedo and peel essential oil deterred Spodoptera frugiperda feeding by 46% and had the highest larval mortality (100%), attributing this action to the presence of D-limonene, which may be responsible for the feeding behaviour and toxic effects. C. aurantium has a higher potential for the production of terpenes than other plants in the citrus family, such as the sweet orange (C. sinensis) [75]. Many of these monoterpenes have been shown to have potential in medicinal applications to reduce inflammation [76] and as natural antioxidants [77] with numerous antimicrobial and antifungal properties [78,79,80,81,82,83,84,85]. Our GC-MS analysis also detected carene in the EO of both tested citrus species, whereas carvone was found in C. reticulata only. Carene and carvone are cyclic monoterpenes with an immense range of naturally occurring variations produced by many plants in the Rutaceae family as secondary metabolites [86]. They are used in the plant’s defence system and, as such, increase under oxidative stress. Many studies have also shown them to be experimentally inducible [87,88]. Methyl 8,10-octadecadienoate, oxacyclotetradeca-4,11-diyne, methyl 4,6-tetradecadiynoate, and methyl-4,7,10,13,16,19-docosahexaenoate are esters produced by the sour orange plant that exhibit antimicrobial/antifungal and biodiesel applications, while doconexent are omega-3 fatty acids prized for their medicinal properties and use as flavorants in several food products, such as cereals [89].
Our results were also in line with those recorded by [47], who mentioned that the EOs, especially those from Salvia sp., have been shown to be promising natural aphicides, repellents, and deterrents against A. punicae, and they are safe for important insect predators. In another study by [45], results indicated that arugula oil was the best treatment against two aphid species, Macrosiphum rosae and A. fabae. Abdelaal et al. [46] revealed that EO and nanoemulsion of Basilicum ocimum exhibited potential toxic activities against both laboratory and field strains of cowpea aphid, A. craccivora. Our techniques were used by Ezeonu et al. [90], Mansour et al. [91], and Akram et al. [92] to corroborate the mosquito-repelling and toxic effects of peel extracts of sweet orange (C. sinensis), lime (C. aurantifolia), rough lemon (C. jambhiri), and lemon (C. limon). As in this investigation, others have shown that EOs can be efficient insecticides for pest control in stored grains. Abad and Besheli [93] recorded the remarkable fumigant toxicity, repellent activity, and persistency effect of EO from the leaves of C. aurantium on three coleopteran stored-product pests. With topical treatment on the maize weevil, S. zeamais, Estrela et al. [94] evaluated the EOs of both Piper hispidinervum and P. aduncum and revealed 90–100% mortality. Restello et al. [95] evaluated the insecticidal influences of the EO from Tagetes patula on maize weevils and observed its effective influence at concentrations of 10 μL. Moreover, Changbunjong et al. [96] indicated that C. aurantium EO exhibits insecticidal activity against the stable fly Stomoxys calcitrans based on contact and fumigant toxicity, which can be achieved as an alternative to synthetic insecticides for controlling this pest, as well as chemical analysis of the essential oil showed the dominance of limonene (93.79%). The present results indicate the high toxicity of C. aurantium and C. reticulata towards aphids. Moreover, the presence of various monoterpenes (limonene, pinene, carene, p-cymene, and carvone), proven with mass spectrometry data are responsible for the possible insecticidal property.

5. Conclusions

In the current study, the two EOs extracted from peel displayed strong insecticidal activity against pomegranate and grapevine aphids at low concentrations and with shorter exposure times, showing higher toxicity of C. aurantium over C. reticulata and less sensitivity of A. punicae to both EOs than A. illinoisensis. Therefore, our results offer a reliable basis for promising and safe methods to develop insecticides based on these two EOs used in managing piercing–sucking insects, particularly pomegranate and grapevine aphids. More investigations should be completed on these two EOs or other plant products to evaluate their efficacy against other pests in the greenhouse and field.

Author Contributions

Conceptualisation: S.S.A. and A.N.; data curation: H.D., S.A. and A.N.; formal analysis: H.D., A.N. and M.H.; investigation: S.S.A., A.S.A., A.N. and H.A.A.; methodology: H.D., S.A., A.N., A.K.A., A.M.A.-B., A.M. and A.B.; project administration: S.S.A.; resources: H.D., S.A., A.S.A., A.N. and H.A.A.; validation: A.N., A.M.A.-B. and A.M.; visualization: A.N., H.D. and A.K.A.; writing—original draft: S.S.A. and A.N.; writing—review and editing: S.S.A., A.N., S.A. and A.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia through the project number 1-441-117.

Data Availability Statement

Not applicable.

Acknowledgments

The authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number 1-441-117.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 12 02040 g001 550
Figure 1. Mortality percentage (mean ± SE) of Aphis punicae exposed to different concentrations (0.5, 1, 2, 4, and 6 µL/mL) of C. aurantium essential oil at different exposure periods (2, 8, 24, and 48 h). The bars with the same letter annotation are not significantly different (according to the Duncan test, p < 0.05).
Figure 1. Mortality percentage (mean ± SE) of Aphis punicae exposed to different concentrations (0.5, 1, 2, 4, and 6 µL/mL) of C. aurantium essential oil at different exposure periods (2, 8, 24, and 48 h). The bars with the same letter annotation are not significantly different (according to the Duncan test, p < 0.05).
Agronomy 12 02040 g001
Agronomy 12 02040 g002 550
Figure 2. Mortality percentage (mean ± SE) of A. illinoisensis exposed to different concentrations (0.5, 1, 2, 4, and 6 µL/mL) of C. aurantium essential oil at different exposure times (2, 8, 24, and 48 h). Bars annotated with the same letter are not significantly different (p < 0.05, based on the Duncan test).
Figure 2. Mortality percentage (mean ± SE) of A. illinoisensis exposed to different concentrations (0.5, 1, 2, 4, and 6 µL/mL) of C. aurantium essential oil at different exposure times (2, 8, 24, and 48 h). Bars annotated with the same letter are not significantly different (p < 0.05, based on the Duncan test).
Agronomy 12 02040 g002
Agronomy 12 02040 g003 550
Figure 3. Mortality percentage (mean ± SE) of Aphis punicae exposed to different concentrations (0.5, 1, 2, 4, and 6 µL/mL) of C. reticulata essential oil at different exposure times (2, 8, 24, and 48 h). Bars annotated with the same letter are not significantly different (p < 0.05, based on the Duncan test).
Figure 3. Mortality percentage (mean ± SE) of Aphis punicae exposed to different concentrations (0.5, 1, 2, 4, and 6 µL/mL) of C. reticulata essential oil at different exposure times (2, 8, 24, and 48 h). Bars annotated with the same letter are not significantly different (p < 0.05, based on the Duncan test).
Agronomy 12 02040 g003
Agronomy 12 02040 g004 550
Figure 4. Mortality percentage (mean ± SE) of A. illinoisensis exposed to different concentrations (0.5, 1, 2, 4, and 6 µL/mL) of C. reticulata essential oil at different exposure times (2, 8, 24, and 48 h). Bars annotated with the same letter are not significantly different (p < 0.05, based on the Duncan test).
Figure 4. Mortality percentage (mean ± SE) of A. illinoisensis exposed to different concentrations (0.5, 1, 2, 4, and 6 µL/mL) of C. reticulata essential oil at different exposure times (2, 8, 24, and 48 h). Bars annotated with the same letter are not significantly different (p < 0.05, based on the Duncan test).
Agronomy 12 02040 g004
Table
Table 1. Toxicity values of C. aurantium and C. reticulata essential oils against Aphis punicae.
Table 1. Toxicity values of C. aurantium and C. reticulata essential oils against Aphis punicae.
Essential OilsExposure
Time (h)		LC50 µL/mL
(95% LCL–UCL)		LC90 µL/mL
(95% LCL–UCL)		Slope ± SEInterceptX2p-Value
Citrus aurantium23.95 (2.32–5.98)4.8 (2.98–6.69)2.42 ± 0.465.056.060.0131
82.31 (1.04–4.69)3.87 (1.6–5.1)2.87 ± 0.574.597.010.0154
241.74 (1.11–3.4)2.89 (1.36–4.47)2.26 ± 0.365.557.860.0082
480.37 (0.047–4.04)1.13 (0.89–2.33)2.25 ± 0.565.970.020.0276
Citrus reticulata24.28 (3.37–5.79)8.47 (6.63–12.97)2.11 ± 0.241.111.990.0030
82.92 (2.01–3.97)7.14 (5.58–10.91)2.20 ± 0.383.502.420.0110
241.85 (0.99–2.56)5.1 (4.07–7.28)2.44 ± 0.425.211.370.0100
481.03 (−0.09–1.73)4.13 (3.21–6.21)2.12 ± 0.498.031.280.0220
Lethal concentrations (LC50, LC90) are those that kill 50% of insects and 90% of insects, respectively. LCL is for lower confidence limit, UCL for higher confidence limit, X2 stands for chi-square value, SE for standard error, and p-value for probability.
Table
Table 2. Toxicity values of C. aurantium and C. reticulata essential oils against A. illinoisensis.
Table 2. Toxicity values of C. aurantium and C. reticulata essential oils against A. illinoisensis.
Essential OilsExposure
Time (h)		LC50 µL/mL
(95% LCL–UCL)		LC90 µL/mL
(95% LCL–UCL)		Slope ± SEInterceptX2p-Value
Citrus aurantium22.29 (1.91–3.69)5.71 (4.74–7.51)2.8 ± 0.284.04.030.0022
81.41 (1.23–3.92)4.69 (3.8–6.44)2.57 ± 0.424.622.740.0088
241.09 (1.88–3.68)3.47 (2.77–5.0)2.78 ± 0.284.91.830.0022
480.82 (2.0–3.87)2.15 (1.69–3.44)2.94 ± 0.295.252.120.0021
Citrus reticulata24.89 (4.06–6.31)8.33 (6.77–11.81)2.2 ± 0.12−0.752.550.0050
83.82 (3.06–4.89)7.35 (5.96–10.29)2.48 ± 0.190.671.620.0010
243.05 (2.34–3.89)6.31 (5.16–8.32)2.68 ± 0.301.721.820.0030
482.21 (1.49–2.91)5.24 (4.25–7.22)2.67 ± 0.383.731.320.0060
Lethal concentrations (LC50, LC90) are those that kill 50% of insects and 90% of insects, respectively. LCL is for lower confidence limit, UCL for higher confidence limit, X2 stands for chi-square value, SE for standard error, and p-value for probability.
Table
Table 3. Repellent activity of five doses of C. aurantium and C. reticulata essential oils against Aphis punicae.
Table 3. Repellent activity of five doses of C. aurantium and C. reticulata essential oils against Aphis punicae.
Essential OilsDose
(µL/cm2)		a Repellence %
30 min60 min90 min120 min180 min
Citrus aurantium0.156b 8 ± 3.720 ± 3.230 ± 3.236 ± 2.440 ± 0
0.31212 ± 3.726 ± 2.440 ± 3.250 ± 3.252 ± 3.7
0.62528 ± 244 ± 2.456 ± 2.460 ± 064 ± 2.4
1.2550 ± 3.266 ± 2.472 ± 3.776 ± 2.486 ± 2.4
2.570 ± 3.282 ± 292 ± 3.798 ± 2100 ± 0
Citrus reticulata0.1560 ± 04 ± 2.48 ± 222 ± 4.936 ± 2.4
0.3122 ± 28 ± 216 ± 2.434 ± 2.446 ± 2.4
0.6256 ± 2.424 ± 2.438 ± 246 ± 2.450 ± 0
1.2516 ± 2.438 ± 246 ± 2.452 ± 258 ± 3.7
2.522 ± 248 ± 252 ± 256 ± 2.470 ± 3.2
a In this experiment, each treatment was represented by five replicates, each containing twenty adult insects. b Each column’s numbers showed the repellence ± standard error.
Table
Table 4. Repellent activity of five doses of C. aurantium and C. reticulata essential oils against A. illinoisensis.
Table 4. Repellent activity of five doses of C. aurantium and C. reticulata essential oils against A. illinoisensis.
Essential OilsDose
(µL/cm2)		a Repellence %
30 min60 min90 min120 min180 min
Citrus aurantium0.156b 2 ± 24 ± 2.414 ± 426 ± 2.432 ± 2
0.3126 ± 2.410 ± 3.218 ± 230 ± 3.240 ± 0
0.6258 ± 218 ± 228 ± 236 ± 2.446 ± 2.4
1.2518 ± 228 ± 244 ± 2.444 ± 2.456 ± 4
2.532 ± 242 ± 250 ± 3.268 ± 3.788 ± 3.7
Citrus reticulata0.1560 ± 00 ± 02 ± 26 ± 2.414 ± 2.4
0.3120 ± 02 ± 26 ± 2.46 ± 2.418 ± 2
0.6254 ± 2.48 ± 3.712 ± 3.718 ± 232 ± 2
1.258 ± 214 ± 2.414 ± 224 ± 2.442 ± 2
2.522 ± 226 ± 2.434 ± 2.444 ± 2.462 ± 3.7
a Each treatment in this experiment was represented by five replicates, each containing twenty adult insects. b The numbers in each column are indicated to repellence ± standard error.
Table
Table 5. The identified chemical compounds contained in the peel of C. aurantium and C. reticulata.
Table 5. The identified chemical compounds contained in the peel of C. aurantium and C. reticulata.
No.CompoundsC. aurantiumC. reticulata
DetectR.T (Min.)Area %DetectR.T (Min.)Area %
1Limonene+4.73896.98+4.76891.86
2α-Pinene+3.020
3.930		0.42
0.88		+3.026
3.946		0.33
0.40
33-Carene+3.629
4.285
6.371		0.31
0.27
0.63		+3.640
13.846		0.04
0.17
4p-Cymene+4.6050.06+4.6200.04
5beta-Guaiene+16.5700.23---
62,7-Bis
(spirocyclopropane)bicyclo [2.2.1]heptan-5-one		+8.7850.22---
7cis-p-Mentha-2,8-dien-1-ol---+7.1501.08
8Carveol---+7.264
9.454
9.808		0.94
0.86
0.41
9Doconexent---+8.801
8.914
13.294
13.732		0.19
0.34
0.53
0.72
10Methyl 6,8-octadeca
diynoate and Methyl 8,10-octadecadiynoate		---+6.887
11.851
12.193		0.26
0.56
0.28
11(-) Carvone---+10.0890.29
123-Cyclohexen-1-ol, 5-methylene-6 (1methylethenyl)-, acetate---+12.4660.19
13Oxacyclotetradeca-4,11-diyne---+13.0800.13
145,8-Dimethylene
bicyclo
[2.2.2]oct-2-ene		---+8.3590.10
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Alotaibi, S.S.; Darwish, H.; Alzahrani, A.K.; Alharthi, S.; Alghamdi, A.S.; Al-Barty, A.M.; Helal, M.; Maghrabi, A.; Baazeem, A.; Alamari, H.A.; et al. Environment-Friendly Control Potential of Two Citrus Essential Oils against Aphis punicae and Aphis illinoisensis (Hemiptera: Aphididae). Agronomy 2022, 12, 2040. https://doi.org/10.3390/agronomy12092040

AMA Style

Alotaibi SS, Darwish H, Alzahrani AK, Alharthi S, Alghamdi AS, Al-Barty AM, Helal M, Maghrabi A, Baazeem A, Alamari HA, et al. Environment-Friendly Control Potential of Two Citrus Essential Oils against Aphis punicae and Aphis illinoisensis (Hemiptera: Aphididae). Agronomy. 2022; 12(9):2040. https://doi.org/10.3390/agronomy12092040

Chicago/Turabian Style

Alotaibi, Saqer S., Hadeer Darwish, Ahmed K. Alzahrani, Sarah Alharthi, Akram S. Alghamdi, Amal M. Al-Barty, Mona Helal, Amal Maghrabi, Alaa Baazeem, Hala A. Alamari, and et al. 2022. "Environment-Friendly Control Potential of Two Citrus Essential Oils against Aphis punicae and Aphis illinoisensis (Hemiptera: Aphididae)" Agronomy 12, no. 9: 2040. https://doi.org/10.3390/agronomy12092040

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