Mediterranean Wild Herbs As Grain Protectants †

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Introduction
Numerous agricultural products are damaged by pests and diseases during their development and maturation in the field [1−3].These products are stored in facilities, where gradual manipulations are taking place, such as processing, industrialization, packaging and transportation [4].Infestations by insects, mites, fungi and rodents are particularly common in these places [3].Stored products are infested by insect pests that cause qualitative and quantitative losses up to 10% of the world production in developed countries and more than 20% in developing countries [5].
The essential oils (EOs) of several plants are known to serve as efficient insecticides [6][7][8].In Greece, sufficient quantities of various wild herbs are available for extraction.A review of the literature showed that little is known on the application of EOs as grain protectants [6−8].In this context, it is challenging to investigate the insecticidal properties as grain protectants of EOs derived from wild herbs of the Mediterranean Basin.It is worth noting that in recent years the bioactivity of EOs from plants originated in the Mediterranean Basin has been reported, indicating their insecticidal potential [9,10] Therefore, the aim of this study was to investigate the efficacy of Citrus limon, Echinophora tenuifollia ssp.sibthropiana, Juniperus phoenicea, Laurus nobilis, Origanum majorana and Origanum vulgare ssp.hirtum, commonly found in Mediterranean, as grain protectants, against two key stored-product insect pests, i.e. the khapra beetle, Trogoderma granarium  Everts (Coleoptera: Dermestidae) and the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae).

Materials and Methods
Trogoderma granarium and T. castaneum were cultured on wheat at 30 °C, 65% relative humidity and continuous darkness.The EOs of C. limon, E. tenuifollia ssp.sibthropiana, J. phoenicea, L. nobilis, O. majorana and O. vulgare ssp.hirtum were tested as wheat protectants.All the EOs were formulated to micro emulsions, with food grade emulsifier [11] and were applied as wheat coating.A stock solution of EO and Tween 20 (1:1) was prepared from each plant.The EOs stock solutions were tested at the concentrations of 250, 500 and 1000 ppm, where water with 0.05% Tween 20 served as control.Sprays were performed on plates.Quantities of 0.20 kg of wheat were sprayed each with 1 mL of the test solution as thin layers.The sprayed wheat quantities were inserted separately in plastic canisters and were shaken for 10 min to realize the balanced distribution of the EOs on the entire wheat masses.Three subsamples of 10 g were obtained and placed in Petri dishes (9 cm diameter, 1.5 cm height).The covers of the dishes bore a circular hole (1.50 cm diameter).The holes were covered by muslin cloth that allowed the ventilation of the inside space of the dishes.Larvae of T. castaneum (2−4 mm length) and adults of T. castaneum (< 1 week old), were used in the experiments.The mortality of T. castaneum larvae was determined after 14 d of exposure, while the mortality of T. granarium adults was determined after 7 d of exposure due to short adult longevity of this species.
Mortality data obtained from the dose-response trials were subjected to probit analysis and LD50, values, i.e., the minimal inhibition dose of an EO to cause 50% mortality on the studied population, were estimated.The statistical analysis was conducted using Polo Plus [12].

Results
Mortality in the control treatments was low (<5%) for both species, therefore no correction was considered necessary for the mortality values.The results of the laboratory toxicity bioassays are shown in Table 1.
Table 1.Mean LD50 values and 95% confidence intervals (ppm) for Trogoderma granarium adults and Tribolium castaneum larvae 7 and 14 days after application with several essential oils, respectively.Concerning T. castaneum larvae, the mean LD50 values of C. limon, E. tenuifollia ssp.sibthropiana and O. majorana, i.e., 416.1, 424.1 and 454.5 ppm, respectively, were significantly lower compared to the mean LD50 value of J. phoenicea (649.4 ppm).In addition, the mean LD50 values of J. phoenicea, L. nobilis and O. vulgare ssp.hirtum, i.e., 649.4,539.4 and 490.5 ppm, respectively, did not significantly differ compared to the mean LD50 values of all the other EOs.

Discussion
T. granarium is considered one of the most important quarantine insects in various countries of the world [13].Quarantine insects have potential economic importance in an area where they are not yet present or where they are present but are not widespread and are not controlled by phytosanitary authorities [14,15].Quarantine insects disrupt the marketing of agricultural stored products between countries and geographical areas within countries (14).In addition, T. castaneum is widely spread in agricultural and food storage worldwide [16].
The preservation of nutritional and commercial value of stored agricultural products is highly based on their protection from insect pests [3].Our study clearly shows the effectiveness of the EOs of six Mediterranean wild herbs against two notorious storedproduct insect species.The insecticide activity of EOs as grain protectants has only recently been studied.For example, the EOs obtained from Mentha longifolia, Dysphania ambrosioides, Carlina acaulis and Pimpinella anisum were efficient against the larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrychidae) and T. granarium [6].The potential of Hazomalania voyronii essential oil-to protect wheat infestations by T. castaneum, T. confusum and Tenebrio molitor has also been highlighted [7].Trogoderma granarium suffered high mortality after the application of EOs obtained from Ferula assa-foetida and F. gummosa [8].
We revealed that EOs can exhibit a significant range of pesticidal activities.Treatments with synthetic contact insecticides is a common practice against storedproduct insect pests [17−19].However, food safety is linked with integrated pest management, aiming at the reduced use of synthetic pesticides [20,21].Our results are towards this direction, as we showed that the EOs of several Mediterranean wild herbs have the potential to serve as efficient tools against the tested major stored-product insect pests.
We expect our results to have bearing on the management of these notorious storedproducts insect species.Further research on the insecticidal activity of several Mediterranean wild herbs as grain protectants will gather more information towards an efficient, eco-friendly management of several storage pests.
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/licenses /by/4.0/).
Means in the same column followed by different letters are significantly different.For T. granarium adults, the mean LD50 values of J. phoenicea, L. nobilis and O. majorana, i.e., 501.3, 506.9, and 440.6, respectively, were significantly lower compared to C. limon (745.3 ppm).The mean LD50 values of E. tenuifollia ssp.sibthropiana (619.1 ppm) and O. vulgare ssp.hirtum (530.7 ppm) did not significantly differ in comparison to the mean LD50 values of all other EOs.