Nanoparticles loading essential oils as alternative solution for the postharvest control of the date moth Ectomyelois ceratoniae (Pyralidae) †

: Ectomyelois ceratoniae (Pyralidae) is the main insect pest attacking dates in field and storage in Tunisia. Postharvest protection is relied on fumigants. This work investigates fumigant toxicity of 2-hydroxypropyl-beta-cyclodextrin (HP-β -CD) loading Rosmarinus officinalis essential oil and its major constituents (1,8-cineole, α -pinene, camphor, borneol) against E. ceratoniae larvae and adults. Results revealed that mortalities reached 56.86; 60.71; 64.28; 50 and 75% respectively for the crude essential oil, α -pinene, camphor, borneol and 1,8-cineole after 30 storage days at the concentration 90 µl/l air. Also, results indicated that 1,8-cineole, camphor and α -pinene showed the strongest inhibitory effects of adults emergence.


Introduction
Essential oils and their compounds exhibited a variety of biological functions including insecticidal activities [1,2].Indeed, various studies reported the potential of many plant derived essential oils against insect pests mainly those infesting stored products [3,4].
Despite the promising potential of EO, their applications are limited due to their degradation and volatilizations because of their volatile components [5].So, the usage of controlled-release formulations could be an interesting option for the development of bioinsecticide [6].In this respect, in last years, cyclodextrin were successfully used as enclusion matrix to encapsulate bioactive substances including essential oils' compounds [7].Encapsulation prolongs the active compounds viability [8]; increase the compounds solubility in water [9]; and prevent from light and environmental degradation factors [10].Rosemary (Rosmarinus officinalis L.) is native to Mediterranean region [11].Rosemary essential oil is kwnown by its biological activities like antifungal [12], antibacterial [13], antioxidant properties [14] and insecticidal potential [15].
The present research was designed to explore the fumigant toxicity of the 2-hydroxypropyl-beta-cyclodextrin (HP-β-CD) loading R. officinalis essential oil or its major constituents (1,8-cineole, α-pinene, camphor,borneol) nanoparticles against larvae and adults of the date moth Ectomyelois ceratoniae for different storage periods.

Plant materials and essential oils extraction
Arial parts of spontaneously grown R. officinalis were collected from Korbous arboretum (36° 48' 59" N 10° 34' 07" E 401) in March 2020.Samples were air-dried at room temperature for two weeks.Essential oils were obtained by hydrodistillation using a modified Clevenger-type device for 4 h.Essential oils were dried using anhydrous sodium sulphate.Yields were calculated based on plant materials dry weight.Chromatographic analyzes were performed using an Agilent-Technologies 6890 N Network GC system equipped with a flame ionization detector and HP-5MS capillary column (30 mx0.25mm, film thickness 0.25 µ m; Agilent-Technologies, Little Falls, CA, USA).

Encapsulation in HP-β-CD
Excess amounts from standards (1,8-cineole, pinene, camphor, borneol ) were added to 5 ml HP-β-CD solutions (10,25,50,75,100 mM).Mixtures were shaken at 25°C for 24h then filtered using a 0.45 µ m membrane filter.HPLC method was used to determine the oil and standards concentrations in the filtrates.Encapsulation efficiency was calculated according to the equation: EE (%) = (mexp/mi)*100 where mexp is the mass of the oil or standards experimentally determined by HPLC in the inclusion complex solution and mi is the mass of the oil or standards initially used to prepare the inclusion complex.

Larval mortality and adult emergence
Larval mortality was assessed for three essential oil concentrations, respectively 30, 60 and 90 µ l/l air.For the standards, the respective used concentrations were 30; 60 and 90 µ g/l air.For each trial, three replications were performed.2 cm diameter Whatman filter papers were impregnated with the different oil concentrations.Impregnated filter paper was attached to the covers of 1L glass bottle.Each bottle contained 20 unsexed larvae (placed in dates).Mortality was recorded after 7, 15, 30 and 37 days.Abbott formula [17] was used to assess the mortality.
Moths' emergence was registered daily for each trial.Percentage of emerged adults was determined by formula of Arivoli [18].

Lethal concentration and Lethal time bioassays
Probit analysis [19]) was used to determine the respective lethal concentration (LC50) and lethal Time (LT50) values for the various treatments after 15 days of exposure.

Formulation insecticidal efficacy
Same procedure was employed to evaluate the larvicidal and adulticidal effect of the formulations.For that, the solid HP-β-CD/R.officinalis and HP-β-CD /standards inclusion complex were placed in 1L glass bottles containing 20 unsexed larvae and 10 adults.Mortality data was assessed by Abbott's formula [17], adult emergence percentages were determined by Arivoli formula [18].

Statistical analysis
Data were subjected to analysis of variance ANOVA using SPSS software version 20 (IBM Corporation NY, USA).Duncan's test was employed to notice significant differences at the level 0.05%.Values presented the means of three replications and expressed as the mean  SD.

Essential oil yield and chemical composition
The chemical composition of R. officinalis essential oil is reported in Table 1 as percentages of chemical classes.Results showed that R. officinalis essential is dominated by Monoterpenes with respective percentages of 61.88% for oxygenated Monoterpenes and 34.47% for Monoterpenes hydrocarbons.On the other hand, extraction yield was 0.6%.

Encapsulation efficiency
The Encapsulation Efficiency Encapsulation of the various compounds (α-pinene, 1,8-cineole, Camphor)r and R. officinalis essential oil in HP-β-CD inclusion complex are presented in Table 2. Results indicated that the encapsulation efficiency varied among compounds and the essential oil.The highest EE value was obtained for 1,8-cineole with 63%.However, α-pinene, Camphor and the essential oil presented close respective EE values of 20, 23 and 22%.

Insect mortality
The insecticidal activities of free and encapsulated compounds and R. officinalis essential oil were illustrated in figure 1.
Results (Figure 1) revealed that fumigant toxicity varied between free and encapsulated compounds.For all compounds and R. officinalis essential oil, toxicity was greater in the case of free compared to encapsulation.Moreover, results indicated that toxicity increased for encapsulated compounds over exposure periods.

Figure 1.
Mortalities (%) of fifth instar larvae of Ectomyelois ceratoniae exposed to free and encapsulated (HP-β-CD inclusion complex) R. officinalis essential oil and the compounds (Letters denotes significant differences at P0.05 in percent mortality for each exposure period (uppercase) and each concentration (lowercase); Duncan's test).a Units LC50 = µ m/l air b 95% lower and upper confidence limits are shown in parentheses.

Lethal concentrations
Results pointed out that 1,8-cineole and α-pinene were more toxic than R. officinalis and Camphor.Results revealed that lethal time values varied among compounds and essential oil.1,8-cineole and Camphor were more toxic compared to α-pinene and R. officinalis essential oil.

Discussion
The postharvest control of E. ceratoniae is mainly based on the use of synthetic fumigants [20].Recently, the use of essential oils has gained attention as pest management agents, due to their insecticidal proprieties [21].
Copyright: © 2021 by the authors.Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licen ses/by/4.0/).

Table 1 .
Chemical classes of R. officinalis essential oil collected from arboretum of Korbous and extraction yield

Table 3
cited the lethal concentrations values of R. officinalis essential oil and the three standards determined by Probit method.

Table 4
reported the lethal time values of R. officinalis essential oil and the three standards determined by Probit method.