Simple Summary: Research about innovative sustainable ecofriendly pesticides is a key topic of global interest, aiming to reduce synthetic inputs in agriculture, to protect biodiversity, and to ensure food safety to consumers. Botanical substances, and in particular essential oils, are among the most promising natural pesticides, as can be seen from the incredibly large number of published studies in the last two decades. Nevertheless, most research is limited to laboratory studies, leaving a gap between scientific studies and field applications. In this scenario, the aim of this paper was to evaluate the feasibility of an innovative nano-insecticide containing sweet orange essential oil as the active ingredient against a key aphid pest in real conditions. Due to its high polyphagy, Aphis gossypii is considered a key pest of many crops, and it can feed on hundreds of plant species belonging to the families Cucurbitaceae, Malvaceae, Solanaceae, Rutaceae, and Asteraceae. The control of this pest mainly relies on synthetic insecticides whose adverse effects on the environment and human health are encouraging researchers to explore innovative, alternative solutions. In this scenario, essential oils (EOs) could play a key role in the development of ecofriendly pesticides. In this study, the development of a citrus peel EO-based nano-formulation and its biological activity against A. gossypii both in the laboratory and field were described and evaluated. The phytotoxicity towards citrus plants was also assessed. The developed nano-insecticide highlighted good aphicidal activity both in the laboratory and field trials, even at moderate EO concentrations. However, the highest tested concentrations (4 and 6% of active ingredient) revealed phytotoxic effects on the photosynthetic apparatus; the side effects need to be carefully accounted for to successfully apply this control tool in field conditions.
Keywords: Aphis gossypii; botanical; nano-emulsion; integrated pest management; side effect
The widespread indiscriminate and often over-use of conventional insecticides poses several risks for the environment and human health. In addition, the ongoing emergence of insecticide resistance of several pests is undermining the range of control tools available to farmers [[
Among the insect pests that cause the greatest agricultural production losses worldwide, Aphis gossypii Glover (Homoptera: Aphididae) is considered a key pest of a variety of crops, causing direct damage through both feeding on young leaves and twigs and by transmitting a series of plant viruses, including the Citrus tristeza virus (CTV) and the cucumber mosaic virus (CMV) [[
The aim of this experimental activity was the development of a Citrus peel EO-based nano-insecticide for the control of A. gossypii, testing its efficacy both in laboratory condition and in the field. In addition, the phytotoxic effects of the developed formulations were tested on citrus plants. The choice of using Citrus sinensis EO was made taking into consideration its widespread availability at a reasonable cost. Furthermore, being considered a byproduct of the orange industry, the choice of blood–sweet orange EO to develop an alternative biopesticide to control Citrus pests strongly relies on the general idea of a circular economy and on the specific concept of circular agriculture. On the basis of the 4R approach (reduce, reuse, recycle, and recover), circular agriculture aims to reduce external inputs by using in-house resources to improve the whole sustainability of the agroindustry and agroecosystems [[
Blood–sweet orange essential oil (EO) was obtained from fruit harvested in the same orchard in which the field trials were subsequently carried out. Fresh citrus fruits were hand harvested, washed, peeled, and grinded to reduce the peels to small pieces. The EO was extracted by a traditional steam distillation apparatus (Albrigi Luigi s.r.l., Verona, Italy) and recovered in a separation funnel placed after the condenser. The recovered EO was dried over anhydrous sodium sulphate and stored at 5 °C in dark vials for further analyses and for the nano-emulsion's formulation.
GC/MS analyses were performed with a Thermo Fisher TRACE 1300 gas chromatograph equipped with a MEGA-5 capillary column (30 m × 0.25 mm; coating thickness, 0.25 μm) and a Thermo Fisher ISQ LT ion trap mass detector (emission current: 10 microamps; count threshold: 1 count; multiplier offset: 0 volts; scan time: 1.00 s; prescan ionization time: 100 microseconds; scan mass range: 30–300 m/z; ionization mode: EI). The following analytical conditions were employed: injector and transfer line temperature at 250 and 240 °C, respectively; oven temperature programmed from 60 to 240 °C at 3 °C min
The Citrus sinensis EO nano-emulsion (EO-NE) was prepared using the self-emulsifying process followed by sonication [[
The average droplet size and size distribution (polydispersity index—PDI) were measured by using a dynamic light scattering (DLS) particle size analyzer (Z-sizer Nano, Malvern Instruments) at 25 °C. In addition, the particle charge was quantified as zeta potential using a Z-Sizer nano (Malvern Instruments) at 25 °C. To attain the correct measurements from the instrument, 0.5 mL of EO-NE was diluted in 100 mL of double-distilled water, and the aliquots (1 mL for droplet dimension and 0.75 mL for droplet surface charge) of the diluted emulsion were analyzed. The physical characteristics of EO-NE were tested after 24 h from the preparation and monthly for one year. Three replicates of fourteen cycles were provided for the tested sample. Three samples were analyzed as replicates for every tested measure.
The original cotton aphid colony came from an organic citrus crop (cv. Tarocco) placed in Calabria (southern Italy). The aphid specimens used in laboratory experiments were reared on young zucchini plants grown in a 36 m
To evaluate the efficacy of the developed formulation, the zucchini seedlings (each provided with 4 well-expanded leaves) were infested with 30 specimens, and then, after they were fixed to the leaf surface (1 h approx.), the infested plants were sprayed until runoff using a 2 L power-pack aerosol hand sprayer (Dea
Mortality was assessed 24, 36, and 48 h after the beginning of the experiments. Specimens were considered dead when they remained immobile after being stimulated with a fine paintbrush. Each treatment was replicated 5 times.
The field trials were carried out during spring 2021 in a 4-hectare citrus orchard under integrated pest management located at Locri (province of Reggio Calabria, Italy) (38°14′50.2″ N 16°16′10.2″ E) at 20 m above the sea level, where no chemical treatments were applied one year before the trials. The trees were 30-year-old orange trees (cv. Tarocco nucellare) planted in a 5 × 6 m grid. The experiments were carried out in spring when the young shoots were highly infested by aphids. Preliminary investigations were carried out a few days before the beginning of the experiments to determine the aphid population density and specificity (>95% belonging to the target specie A. gossypii). The different treatments were applied to citrus trees using a 5 L power-pack aerosol hand sprayer (Eurospin
For each EO-NE dilution and treatment, four randomly selected trees were sprayed in a completely randomized model. Aphid mortality was assessed for each tree on four previously labeled infested shoots after the same time intervals carried out in the laboratory experiment (i.e., 24, 36, and 48 h from the treatments).
The field treatment efficacy was calculated as follows:
(
where R represents the reduction rate due to the treatments calculated as follows:
(
where P represents the population (number of alive specimens) registered in each sample.
Phytotoxicity analyses were carried out by photosynthetic rate measurements as a sensible physiological trait for different reasons: a non-destructive and quick method, the best ecotoxicological method [[
The photosynthetic rate measurements were carried out on at least three healthy leaves of five orange trees treated with EO-NE solutions at the same application rates used in the laboratory trials (i.e., 1, 2, 4, and 6 g × 100 mL
A calibrated portable photosynthesis system (LI-6400; LI-COR, Inc.; Lincoln, NE) was used to measure the photosynthetic rate (μmol (CO
Dependent variables were tested for homogeneity and normality of variance (Levene and Shapiro–Wilk tests, respectively), and because they met the ANOVA assumptions (p < 0.05), no data transformation was required.
The laboratory efficacy of the tested formulations was corrected for negative control mortality using Abbott's formula [[
Probit analysis was performed in order to estimate the median lethal concentrations (LC
The photosynthetic rate results were analyzed by two-way ANOVA with the treatment and time as the main factors with different levels (CTR, 1, 2, 4, and 6% for the concentration; 0, 1, and, 6 days for the time) and concentration–time interaction. Then, Tukey's HSD post hoc test was used to compare the means of the photosynthetic rate data of each treatment within each time.
All statistical analyses were performed using the software SPSS v. 22 (IBM, Armonk, NY, USA).
A total of 29 different compounds were detected in the blood–sweet orange EO. D-limonene was the most abundant compound (93.35%) detected, followed by β-myrcene (3.38%), and α-pinene (1.14%), whereas the abundance of the other 26 compounds was lower than 1%. Only three compounds (α-cubebene, γ-muurolene, and caryophyllene oxide) were only in traces (an abundance less than 0.01%) (Table 1).
Monoterpene hydrocarbons (98.59%) represented the great majority of the detected chemicals, while oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and non-terpene aldehyde ranged from 0.73 to 0.01%. The developed nano-emulsion had particle dimensions of 131.36 ± 0.50 nm with a polydispersity index (PDI) of 0.11 and a surface charge (ξ potential) of −23.76 ± 0.75 mV.
In the laboratory trials, the aphid mortality registered for the negative control after 24, 36, and 48 h from the treatment was 0, 1.6, and 4%, respectively, whereas the positive control (deltamethrin) caused the complete mortality of all the exposed insects immediately after 24 h from the treatment. In all the time intervals (
Both the EO-NE concentration as well as the time after the treatment significantly affected aphid mortality (concentration: F = 464.05; df = 3; p < 0.01; time: F = 10.43; df = 2; p < 0.01), whereas their interaction did not affect the efficacy (F = 1.328; df = 6; p > 0.05). The lowest tested concentration (i.e., 1% of EO in the nano-emulsion) was able to kill less than 15% of the exposed insects. Conversely, the mortality caused by nano-emulsions containing 2, 4, and 6% of EO ranged from 38.67 to 100%, with the two highest doses (i.e., 4 and 6%) provoking the death of almost all tested insects just after 24 h (mortality = 89.33 ± 3.86 and 96 ± 2.67%, respectively). In detail, the insecticidal activities recorded for 4 and 6% EO-NE were similar without accountable statistical differences between the two dilutions (p > 0.05) (Figure 1).
In the field, the efficacy (reported as field treatment efficacy) of the developed nano-insecticide showed a similar trend compared to that described in the laboratory trials (Figure 2). The concentration used, as well as the time between the treatment and the sampling, had a significant effect on aphid mortality (concentration: F = 505.793; df = 3; p < 0.01; time: F = 22.008; df = 2; p < 0.01); however, similar to laboratory trials, their interaction (concentration × time) did not determine statistical differences (p = 0.05). Indeed, the mortality induced by the two highest tested concentrations was not significantly different, regardless of time.
The recorded mortality, 24 h after the treatments, ranged from 4.88 ± 6.16 to 89.39 ± 6.34% depending on the EO-NE concentration. In the sampling carried out 36 h after the treatments, the aphicidal activity increased, and at the two highest application rates (i.e., 4 and 6%), the efficacy reached 85.68 ± 4.66 and 91.85 ± 5.16%, respectively. Lastly, 48 h after the treatments, aphid mortality on the plants treated with the EO-NE containing 4 and 6% of EO was in both cases higher than 90%. The positive control (deltamethrin) caused higher mortality rates than those recorded for the EO-treated plants; indeed, after 24 h no aphids were recorded alive in the samples treated with deltamethrin.
The highest EO-NE concentrations (i.e., 4 and 6%) caused toxic effect on the photosynthetic apparatus of citrus plants. One day after treatment, the photosynthetic rate was lower in the plants treated with EO-NE applied at 4 and 6% of a.i. than in the other tested concentrations and the water control (concentration–time interaction: F = 2.8274; df = 8, p < 0.01). The photosynthetic rate was reduced by 52% and 51% in plants treated with 4 and 6% of a.i., respectively, compared to the water control. The phytotoxic effects induced by the two highest concentrations was observed up to six days after treatment, while in the plants treated with EO-NE at 1 and 2% of a.i., no phytotoxic effects were highlighted throughout the trial (Figure 3).
The compounds detected in C. sinensis EO were almost totally represented by monoterpene hydrocarbons, and among them, D-limonene accounted for nearly the entire blend of the EO. The chemical composition of C. sinensis EO can be influenced by different seasonal and climatic parameters, as well as the ripening stage, cultivar, and growing localities [[
Eos presenting insecticidal activity against target pests usually affect different biological pathways, mainly related to the nervous system of insects (i.e., octopamine receptor, GABA channel, and ACHE activity) [[
To the best of our knowledge, so far, the evaluation of EO-based insecticides against the cotton aphid has been conducted exclusively under laboratory conditions; thus, their evaluation under field operating conditions was lacking. In this scenario, the present study firstly demonstrated the efficacy of these insecticide formulations also in the field. The field trials highlighted that EO-NE caused a field treatment efficacy comparable to the positive control, deltamethrin, after 48 h from the application. Most of the previous studies about aphid species performed fumigation trials to determine the insecticidal activity of the EOs toward the target pests (e.g., [[
Concerning crops, the ecotoxicological tests of pesticides are based on growth inhibition and/or visualization symptoms on representative non-target plant species [[
During the development and evaluation of new control tools against pests, a series of variables must be considered to achieve potentially transferable results in real operative conditions. As an example, despite the fact that many studies have evaluated the pesticidal activity of EOs, only a few papers also took into account the undesirable effects toward non-target species, including crop plants. In this scenario, deeper knowledge about the ecotoxicological impact and environmental fate of botanical-based pesticides is still needed.
In this paper, the aphicidal potential of the developed Citrus EO nano-formulation was demonstrated, although further improvements are auspicial to reduce the phytotoxic effects at the highest application rates towards the crops. Nevertheless, lower EO concentrations were safe for the citrus trees and demonstrated good insecticidal activity against A. gossypii, suggesting that a compromise between the release rate and the concentration of the a.i. can be the key strategy to overcome the negative effects on the crop granting effective pest control.
Graph: Figure 1 Mean percent mortality ± SE of A. gossypii adults exposed to different concentrations of Citrus sinensis EO nano-emulsion after 24, 36, and 48 h in laboratory trials. Different letters indicate statistical differences among the different treatments at the same exposure time (ANOVA, p < 0.05).
Graph: Figure 2 Mean percent mortality ± SE of A. gossypii adults exposed to different concentrations of Citrus sinensis EO nano-emulsion after 24, 36 and 48 h in field trials. Different letters indicate statistical differences among the different treatments at the same exposure time (ANOVA, p < 0.05).
Graph: Figure 3 Photosynthetic rate (µmol CO2 cm−2 s−1) ± SE registered in the plants at 0, 1, and 6 days after treatments with different concentrations of Citrus sinensis EO nano-emulsion. Different letters indicate statistical differences among the different treatments at the same exposure time (ANOVA, p < 0.05).
Table 1 Chemical composition of the essential oil extracted from sweet orange fruit (cv. Tarocco nucellare).
Compound a LRI Exp b LRI Literature c Retention Time Relative Peak Area α-thujene 928 931 6.52 0.01 α-pinene 935 939 6.70 1.14 Camphene 950 953 7.11 0.01 Sabinene 975 976 7.77 0.50 β-pinene 979 980 7.89 0.03 β-myrcene 993 991 8.24 3.38 Octanal 1007 1001 8.68 0.33 δ-3-Carene 1013 1011 8.90 0.18 D-limonene 1033 1031 9.64 93.35 Terpinolene 1090 1088 11.68 0.02 Linalool 1102 1098 12.11 0.54 Trans-p-mentha-2,8-dienol 1123 1118 13.05 0.02 Cis-limonene oxide 1133 1134 13.46 0.04 Trans-limonene oxide 1138 1139 13.66 0.04 Citronellal 1155 1153 14.42 0.03 α-terpineol 1192 1189 16.01 0.07 Decanal 1212 1208 16.85 0.10 α-cubebene 1356 1351 23.11 tr d α-copaene 1367 1376 23.57 0.04 β-cubebene 1381 1390 24.17 0.04 Z-β-caryophyllene 1410 1406 25.38 0.02 E-β-caryophyllene 1420 1418 25.78 0.04 α-caryophyllene 1444 1454 26.78 0.01 γ-muurolene 1468 1477 27.73 tr Germacrene D 1473 1481 27.92 0.02 Valencene 1484 1491 28.40 0.02 α-muurolene 1492 1499 28.71 0.01 δ-cadinene 1515 1524 29.61 0.04 Caryophyllene oxide 1578 1581 32.02 tr Class compound Relative abundance Monoterpene hydrocarbons 98.59 Oxygenated monoterpenes 0.73 Aldehydes 0.43 Sesquiterpene hydrocarbons 0.24 Oxygenated sesquiterpenes 0.01
Table 2 Toxicity of sweet orange essential oil nano-emulsion against Aphis gossypii.
Time (h after Treatment) LC50a (g hg−1–95%FL b) LC90a (g hg−1–95%FL) Χ2 c Significance 24 2.27 (1.92–2.65) 4.35 (3.61–5.76) 0.364 ns d 36 1.88 (1.59–2.21) 3.56 (2.94–4.79) 1.478 ns 48 1.48 (1.22–1.74) 2.86 (2.34–4.03) 1.607 ns Significance * *
Conceptualization, F.L., O.C., V.P., A.S. and G.G.; methodology, F.L., O.C., A.S. and G.G.; validation, R.C., P.Z., I.L. and A.M.; formal analysis, O.C, R.C., A.S. and G.G.; investigation, F.L., R.C., P.Z., I.L. and A.M.; resources, V.P.; data curation, O.C, A.S. and G.G.; writing—original draft preparation, F.L., O.C. and G.G.; writing—review and editing, all the authors; supervision, O.C. and G.G.; project administration, V.P.; funding acquisition, V.P. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
The data will be available from the corresponding author upon reasonable request.
The authors declare no conflict of interest.
By Francesca Laudani; Orlando Campolo; Roberta Caridi; Ilaria Latella; Antonino Modafferi; Vincenzo Palmeri; Agostino Sorgonà; Paolo Zoccali and Giulia Giunti
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