Cinnamomum camphora var. linaloolifera essential oil on pest control: Its effect on Trialeurodes vaporariorum (Hemiptera: Aleyrodidae)

This study evaluated the insecticidal effect of Cinnamomum camphora var. linaloolifera essential oil and its major compound, linalool, on Trialeurodes vaporariorum. Essential oil treatments were applied at 0.5, 1.0, 1.5, and 2.0% v/v on eggs, nymphs, and adult individuals and linalool equivalent concentrations on nymphs of T. vaporariorum. The negative controls water and Tween-80 (0.5% v/v) and a positive control (spiromesifen 0.05% v/v for eggs and nymphs; pymetrozine 0.04% m/v for adults) were also used. The essential oil of C. camphora at 2.0% v/v caused egg mortality of 49.7%; same concentration caused the highest nymph mortality (88.5%). For adults, the essential oil at 2.0% v/v caused the highest mortality (40.0%) after 48 h, not changing from 48 to 72 h. The essential oil and linalool performed similarly relative to the nymphs, whose mortalities have not differed statistically, with exception of the concentrations of 0.5 and 1.0% v/v. Regarding the chemical control, the concentration of 2.0% v/v has had similar mortality for nymphs; for eggs and adults, the essential oil caused lower mortality (49.4 and 40.0%) than the synthetic pesticide (65.0 and 72.0%). The essential oil of C. camphora may be a potential control alternative for T. vaporariorum, especially regarding the Integrated Pest Management (IPM) practices.


Introduction
Trialeurodes vaporariorum (Westwood, 1856) is considered the main pest in ornamental and vegetables in protected cultivations (Lourenção et al., 2008). More than 250 species of plants are susceptible to this insect (Zapata et al., 2016), whose attacks cause economic losses to the agricultural sector (CABI, 2017).
T. vaporariorum feeds on sap (phloem) by introducing its buccal apparatus in plant leaves, causing mechanical damage to the plant tissues. The damage is characterized by localized spots and yellowing and fall of leaves; if the infestation reaches critical levels, wilting and reduction of the growth of the host plants may also be seen (Capinera, 2008).
Most of the phloem ingested is not metabolized by T. vaporariorum, being excreted as honeydew, a sugary substance rich in carbohydrates (Henneberry et al., 1999). The honeydew covers fruits and leaves and serves as food to a saprophyte fungus, rendering the plant surface blackish and sticky (Perring et al. 2018). This indirect damage fosters the development of a fungus (Capnodium sp.), which harms the leaves' physiological processes, causing them to wilt and fall, hastening the development cycle of the cultivation (Barbosa et al., 2002). T. vaporariorum may also act as a vector in transmitting viral diseases, which causes severe damage and production loss in several kinds of vegetables (Capinera, 2008). This insect's management is carried out mainly by using synthetic pesticides (Michereff Filho & Inoue-Nagata, 2015).
In general, large-spectrum pesticides are used without considering the negative impacts that come from the excessive use of chemical control because, in many cases, several applications are necessary, which results in excessive use of synthetic molecules. Hence, pesticide-resistant individuals of T. vaporariorum are continuously selected (Erdogan et al., 2012;Ovčarenko et al., 2014).
The integration of several control techniques may be useful in the reduction of T. vaporariorum infestations. Natural products, such as plant extracts and essential oils, may be used as tools to help control this pest. Essential oils could be considered as large-spectrum pesticides (have several modes of action) and low risk (due to the quick volatilization and limited field permanence) (Koul et al., 2008). Unlike synthetic pesticides, essential oils may act by several mechanisms, reducing the probability of the individuals becoming resistant due to the diverse composition of active compounds in essential oils (Pavela & Benelli, 2016).
Essential oils are complex mixtures of naturally occurring compounds, which are secondary metabolites of plant metabolism and may contain more than 300 compounds at different concentrations (Sell, 2006). Several factors determine the chemical composition of essential oils, both biotic and abiotic (luminosity, water stress, attack by pests and pathogens), which may be correlated (Morais, 2009;Pandey et al., 2014;Campos et al., 2019;Silvestre et al., 2019).
Many essential oils obtained from plants of several botanical families show some insecticidal activity or repellent on arthropods. In general, essential oils may be absorbed by the cuticle, inhaled, or ingested by the insects (Regnault-Roger & Hamraoui, 1995). Inside the several families, the genus Cinnamomum highlights itself, which is already cited in the literature as presenting biological activity (Nerio et al., 2010).
The chemical composition of C. camphora essential oil is already presented and discussed in the literature; its major compound, linalool, is a monoterpene whose repellent and insecticidal properties were addressed by other researchers, suggesting the potential use to control T. vaporariorum (Cansian et al., 2010(Cansian et al., , 2015Tambwe et al., 2014).
This work aimed to evaluate the chemical composition of the essential oil of C. camphora var. linaloolifera and investigate the effect of the essential oils on eggs, nymphs, and adults of T. vaporariorum, also evaluating the activity of linalool on nymphs of this pest, aiming to get an alternative form of control for this insect.

Methodology
The experiments and the evaluation of the insecticidal activity of the C. camphora essential oil on T. vaporariorum were carried out in the Laboratory of Pest Management of the University of Caxias do Sul, Caxias do Sul, RS, Brazil, from August 2017 to November 2019.

Plant material for essential oil extraction
The plant material of Cinnamomum camphora var. linaloolifera was collected in the Institute of Biotechnology of UCS (geographical coordinates: 29°09'47.8"S 51°08'37.4"W). After the collection, the material underwent a visual inspection with the remotion of inorganics and decaying plant material. The leaves were separated and dried at room temperature (22-25 °C) for four days in a kiln with forced air circulation.

Essential oil extraction and synthetic major compound
The essential oil was extracted by steam distillation for two hours. The volume of obtained essential oils was measured using a 25 mL glass graduated cylinder. The essential oil was kept in amber flasks, and 0.5 mL was sent to chromatographic analysis. The material was stored under refrigeration at 4±2 °C until use in the bioassays.
The synthetic compound linalool (CAS 78-70-6, purity of 99.2%) was purchased from Merck (Germany) and was kept under refrigeration at 4±2 °C until use in the bioassays.

Chromatographic analysis
The analyses were carried out in the Analytical Central of the Institute of Biotechnology of the UCS, based on the procedures described by Silvestre et al. (2019) and Vicenço et al. (2020). The GC-FID analysis was carried out using a Hewlett Packard 6890 Series gas chromatograph, equipped with an HP-Chemstation data processing unit. An HP-Innowax column (30 m x 320 µm i.d.) with 0.50 µm film thickness (Hewlett Packard, Palo Alto, USA) was used. Temperature programming was: 40 °C for 8 min, from 40 °C to 180 °C at 3 °C·min -1 , from 180 to 230 °C at 20 °C·min -1 , 230 °C for 10 min; injector temperature of 250 °C; split ratio 1:50, FID detector at 250 °C; hydrogen as carrier gas at 34 kPa, injected volume of 1 µL, diluted in hexane (1:10).
The GC/MS analysis was carried out using a gas chromatograph, coupled with an HP 6890/MSD5973 mass spectrometer, equipped with an HP-Chemstation and the Wiley 275 spectra library. An HP-Innowax fused silica capillary column (30 m x 320 µm i.d.) with 0.50 µm film thickness was used. The temperature program used was the same as GC-FID; interface at 280 °C; split ratio 1:100; helium as carrier gas at 56 kPa; flow ratio of 1.0 mL·min -1 ; ionization energy of 70 eV; injected volume of 1 µL diluted in hexane (1:10).
The essential oils components were identified by comparison between the obtained spectra and the ones from the Wiley library (GC/MS) and by comparison of the calculated linear retention indexes (LRI's) and their literature references. The LRI values were calculated using the Van den Dool and Kratz equation and a C8-C30 alkane series solution as standard.

Bioassays
Tests were performed with eggs, nymphs, and adult individuals of T. vaporariorum had an experimental design completely randomized. All tests were carried out in an air-conditioned room at 25±2 °C, 50±5% RH, and a photoperiod of 14:10 h (light:dark).
For the three stages of T. vaporariorum, the treatments were the concentration of essential oil and negative and positive controls. Five concentrations of C. camphora essential oil were tested: 0.1, 0.5, 1.0, 1.5, and 2.0% v/v. Also, treatments with the major compound were carried out with nymphs. The same concentrations were used but adjusted to the major compound's content in the essential oil, according to Equation 1.
Being 'C' the adjusted concentration, 'D' the theoretical dose, and 'T' the major compound's content in the essential oil.
All treatments (essential oil and major compound) were emulsified using Tween-80 ® (0.5% v/v). The recommended pesticides for the control of the T. vaporariorum were used as positive controls. For the adult phase, it was used pymetrozine in the recommended dose of 0.04 wt.% (Chess ® , Syngenta), and for the juvenile phase (eggs and nymphs) it was used spiromesifen in the recommended dose of 0.05% v/v. (Oberon ® , Bayer S.A.). Two treatments were used as negative controls: water and Tween-80 ® 0.5% v/v.
For the tests with the eggs of T. vaporariorum, tomato plants (S. lycopersicum cv. Micro-Tom) with 30 days old underwent visual inspection to exclude plants with any sign of disease or infestation. The selected plants had their leaves removed, except for one leaf. After, 20 adult individuals, not sexed, were transferred from the colony using a manual aspirator.
The plants were covered using a voile fabric to avoid insect escape, and they were kept ovipositing for 48 h. After this period, 1 the adults were removed, and the leaves with the eggs were treated in the abaxial direction with the essential oil/major compound solutions using a manual sprayer. The treated plants were kept in an air-conditioned room as previously described and the mortality/toxicity evaluations were carried out after eight days. It was counted the number of individuals that reached the first nymphal stage. It was used five replicates for each treatment, being each replicate composed of a plant.
For the tests with the nymphs of T. vaporariorum, 20 adult individuals were transferred using a manual aspirator to tomato plants (S. lycopersicum cv. Micro-Tom) with 30 days old. The plants were covered using a voile fabric to avoid insect escape, and they were kept ovipositing for 48 h. After this period, the adults were removed, and the plants were kept in an airconditioned room as previously described. When the nymphs reached the second nymphal stage (which was observed using a magnifier), the treatments were sprayed in the abaxial direction of the leaves using a hand sprayer. The plants were kept in an air-conditioned room, and the evaluation of the mortality/toxicity was carried out after eight days by counting the number of nymphs that reached the fourth nymphal stage and the dead ones. It was used five replicates for each treatment, being each replicate composed of a plant.
For the test with the adult individuals of T. vaporariorum, it was used the methodology described by Müller et al. (2018).
Leaves of tomato plant (S. lycopersicum cv. Micro-Tom) with 30 days old were cut and washed with tap water. After, both leaves sides were sprayed with 1 mL of treatments and kept 30 min away from sunlight to dry on an absorbent paper. Each leaf was put in glass flasks (7 cm of diameter and 9 cm of height) with moistened floral foam. Each flask was infested with five adult individuals of T. vaporariorum with the age of 5-7 days by using a manual aspirator. The flasks were closed using black TNT fabric and kept in an air-conditioned room. The mortality percentage of the individuals was evaluated in 24, 48, and 72 h after infestation. It was used five replicates for each treatment and five individuals for each replicate, totaling 25 individuals for each treatment.

Statistical analysis
Data normality was evaluated using the Kolmogorov-Smirnov test. The parametric data were analyzed by analysis of variance (ANOVA), followed by least significant difference (LSD) test at a 5% significance level (p <0.05). The Spearman correlation coefficient was also evaluated at a 5% significance level, aiming to verify possible relationships between mortality data and the concentrations of essential oil/major compound. The data that presented significant correlations were used to generate polynomial regressions to observe the data behavior. The statistical analyses were carried out using the Statistical Package for the Social Sciences software (SPSS for Windows 17.0) (Green & Salkind, 2005).

Bioassays
It was possible to observe a decrease in the number of nymphs that emerged with an increase in essential oil concentration. There was a difference in the mortality of eggs from the concentration of 0.1% v/v of C. camphora essential oil, differing from the negative controls (water and Tween-80 ® ). However, essential oil treatments presented a lower mortality rate of eggs than the positive control (spiromesifen 0.05% v/v). The mortality data of eggs and nymphs of T. vaporariorum are presented in Table 2. Means followed by the same letter in column do not differ statistically by least significant difference (LSD) test at 5% probability. Source: Authors (2021).
The number of eggs that have not hatched has not differed statistically in the essential oil concentrations of 1.0, 1.5, and 2.0% v/v, whose mortality varied between 30.9 and 49.4%. The essential oil concentration of 2.0% v/v had a poorer performance than the positive control (spiromesifen 0.05% v/v).
For the nymphs (Table 2), it was possible to observe that the concentrations of 1.5 and 2.0% v/v have not differed statistically; there was no difference of the concentration of 2.0% v/v relative to the chemical control (88.5 and 98.3% of individuals mortality, respectively). Also, the concentration of 1.5% v/v has not differed from the concentration of 1.0% v/v, causing nymph mortalities of 78.0 and 83.4%, respectively. The essential oil concentration of 0.1% v/v has not differed from the two negative controls (water and Tween-80 ® ).
The Spearman correlation coefficient between the essential oil concentration of the eggs and nymphs' mortality were 0.7854 and 0.8991, respectively; both were significant at a 5% probability. Figure 2 presents the regression graphs between the essential oil concentration and the egg and nymph mortality of de T. vaporariorum. It was observed that the mortality of eggs and nymphs followed a quadratic pattern relative to the dose of essential oil, with determination coefficients (R 2 ) of 0.6918 for eggs and 0.9729 for nymphs. It is important to note the difference between the egg and nymph mortalities (60 and 90% for essential oil concentration of 2.0% v/v), although the regression's overall aspect was similar for both bioassays.
The evaluation of the adult individuals of T. vaporariorum (Table 3)   Means followed by the same letter in column do not differ statistically by least significant difference (LSD) test at 5% probability. Source: Authors (2021).
The determination of the Spearman correlation coefficient indicated the existence of three significant correlations between the mortalities of adult individuals at 24 (0.4167), 48, and 72 h (0.7553 in both). Figure 3 presents the regression data of the adult mortality relative to the evaluation times and the concentration of C. camphora essential oil. The bioassay that compared the essential oil of C. camphora and linalool, its major compound, (Table 4), showed that linalool caused higher mortality percentages of nymphs at the concentrations of 0.5 and 1.0% v/v when compared to the essential oil. Means followed by the same letter, uppercase in row (OE kind) and lowercase in column (concentration) do not differ statistically by the least significant difference (LSD) test at 5%. * -The concentration of the major compound was adjusted according to its content in the essential oil. Source: Authors (2021).
However, at higher concentrations (from 1.5% v/v on), no statistical difference was observed between the two treatments. Moreover, essential oil from 1.5% v/v on, linalool and chemical control (spiromesifen) have had a similar performance, in which the mortality percentages of T. vaporariorum nymphs were above 90% for both treatments.
The test also indicated that linalool was the primary insecticidal agent in the essential oil of C. camphora var.
linaloolifera. As the concentration of essential oil increased, the mortality percentage increased considerably; from 1.5% v/v on, the essential oil's toxic effect has not differed from the one of linalool. These results may indicate that the major compound (linalool) concentration in the essential oil was sufficient to suppress possible minor compounds' antagonistic effects.
The Spearman correlation coefficient indicated a significant correlation between nymph mortality and linalool concentration (0.8443), beyond the already established correlation with C. camphora essential oil (0.8891). Figure 4 presents the regression graphs of T. vaporariorum nymph mortality relative to both linalool and C. camphora essential oil concentrations. Evaluating the graph shows that T. vaporariorum nymphs' mortality data exposed to both the essential oil and linalool fitted similarly, with a quadratic fit. It indicates both treatments presented the same toxic effect on nymphs of this pest, with mortalities ranging from 85 to 90% for a concentration of 2.0% v/v.

Essential oil yield and composition
Relative to C. camphora essential oil, other authors reported similar results, which also characterized linalool as the major compound of this essential oil in contents ranging from 84 to 95 wt.% (Cansian et al., 2010(Cansian et al., , 2015Nenaah & Ibrahim, 2011;Tomazoni et al., 2017).
The compounds found in the essential oil are classified into two main chemical groups, varying according to the metabolic pathway from which they originated: terpenes (mono-, sesqui-, and diterpenes) and, in lower contents, phenylpropanoids (Regnault-Roger et al., 2012). In plants, monoterpenes act as defense agents against pathogens due to allelochemical and repellent activities, among other functions (Chand et al., 2017).
The physiological expression of secondary metabolism of plants may differ due to the stage of development (Hansted et al., 1994;Raguso & Pichersky, 1999). The proportion of monoterpenes, for example, depends on circadian rhythm and varies according to the plant stage (Clark & Menary, 1981). Some plant species also have different chemotypes, i.e., they are botanically and genetically identical. However, they differ in the level of gene expression, which is linked to the activation of several metabolic pathways due to specific alleles' presence or activation (Regnault-Roger et al., 2012).
Essential oil composition also may be changed during the extraction process. Water kind and quality, acidity, and extraction temperature may cause oxidation and hydrolysis of terpenes. The storage conditions are also quite crucial since the degradation possibilities are many. These essential oil changes occur mostly by polymerization and oxidation reactions (Simões et al., 2017).

Insecticidal activity of C. camphora essential oil on T. vaporariorum
Literature is scarce about the effect of the essential oil of C. camphora in the juvenile phase (eggs and nymphs) of T.
vaporariorum, neither other species, having only studies relative to the adult phase. The essential oil of C. camphora has had toxicity on Sitophilus zeamais, causing 100% mortality at the concentration of 0.114% v/v (Cansian et al., 2015).
The behavior observed in the present work may indicate that, at higher concentrations (1.5 and 2.0% v/v), the effect of the essential oil on egg and nymph mortality becomes constant, not increasing in higher concentrations. Pereira et al. (2018), using essential oil from the fruits of Zanthoxylum riedelianum to control second-instar nymphs of Bemisia tabaci, reported a loglogistic fit of the data in function of the concentration, with mortalities in the range of 50-80%.
Essential oils contain several compounds that can exert repellent and insecticidal mechanisms. Many studies have demonstrated that these compounds have biological activity and can cause adverse effects on several pests (Dhifi et al., 2016).
The toxicity of essential oil on insects, nematodes, fungi, bacteria, and other organisms may be attributed to the terpenic compounds present in these oils (Regnault-Roger et al., 2012;Pavela & Benelli, 2016). The quick action of these natural products against insects may be indicative of neurotoxic action. There is evidence of interference of terpenes with the neuromodulator octopamine or GABA-modulated calcium channels (Priestley et al., 2003;Isman, 2006). They can also affect acetylcholinesterase, octopamine, sodium channels, among other mechanisms (Huignard et al., 2008;Regnault-Roger et al., 2012;Campos et al., 2018). However, the essential oils' action capacity is poorly understood, since the toxic effect may differ according to the quality and quantity of the compounds present in the essential oil (Campos et al., 2018).
Considering the crescent necessity of the development and use of more environmentally friendly molecules, the C.
camphora essential oil presents itself as a feasible alternative due to the relatively high oil yield (about 2.0% v/w) and the high content of linalool (the linaloolifera variety). The use of natural molecules over synthetic ones is also interesting due to the biodegradability, low presence of residues, and a slower resistance development by the target pests (Cansian et al., 2015;Pavella & Benelli, 2016;Campos et al., 2018).
According to the EPA (Environmental Protection Agency, 2019), to register a pesticide, the substance must cause at least 90% of the target individuals' mortality. In the present study, the chemical pesticides caused a T. vaporariorum egg mortality of 65% and an adult mortality of 72% after 72 h of evaluation, indicating that this pest may have developed some resistance mechanism against these pesticides (spiromesifen and pymetrozine). Zhou et al. (2019) reported a linear increase in the mortality of Aphis pomi with time when exposed to small concentrations of Dracocephalum integrifolium essential oil; however, as the essential oil concentration increased, the effect was more pronounced, reaching higher mortalities at the same time.
Taking into account the IPM method, a comprehensive control using several mechanisms (natural predators, natural and synthetic chemicals, management procedures) also helps in hindering resistance development and increasing the overall control efficiency without excessive use of any of the control methods (Nerio et al., 2012;Regnault-Roger et al., 2012). In this sense, the leaf essential oil of C. camphora as a control tool in the integrated management of T. vaporariorum may be a valuable and important tool in promoting more eco-friendly and efficient management of the cultures affected by this insect.

Conclusion
The chemical analysis of the essential oil of C. camphora showed linalool as the major compound, with 92.34 wt.%.
According to the results, the C. camphora essential oil presented a moderate activity on eggs and adult individuals of T.
vaporariorum and was successful in controlling the nymphs of this pest. Thus, this essential oil may be an effective alternative and an option in the management of this insect, especially in light of the MIP procedures.