Bioactivity of Five Essential Oils Against Bruchidius incarnatus (Bohemann, 1833)

In the world, the faba bean beetle Bruchidius incarnatus (Coleoptera: Bruchidae) is an important insect-pest, especially on faba bean Vicia faba (Leguminosae) and it can infest field crops and cause severe damage in storage. Essential oils can be an alternative method to synthetic insecticides for pest management, due to their efficiency and environmental safety. The aim of the current study was to evaluate the toxicity and repellent activity of essential oils of camphor (Eucalyptus globules), castor (Ricinus communis), cinnamon (Cinnamomum zeylanicum), clove (Syzygium aromaticum) and mustard (Brassica rapa) against B. incarnatus adults. The treatments which contained essential oils at 0.5, 1, 2 and 4% and acetone (control) were applied. All essential oils with 4% concentration repelled the B. incarnatus adult except castor oil. The percentage of repellence was higher when used essential oil of cinnamon with 2 and 4% concentration compared with other essential oils and concentrations. In residual film experiment, the cinnamon oil had the highest toxicity rate on B. incarnatus adult fallowed by clove, camphor, mustard and the lowest effect was by castor oil. Based on our results, I can conclude that essential oils of camphor, cinnamon, clove and mustard have potential for use in the integrated management of B. incarnatus adult.


Introduction
The faba bean beetle, Bruchidius incarnatus (Coleoptera: Bruchidae) is associated with faba bean storage, where it can attack the whole faba bean grains in field and storeroom. Traditional organophosphates, such as malathion and pirimiphos-methyl are the most commonly used residual insecticides in stored grains (Arthur, 1996;Santos et al., 2009). Chemical insecticides can cause pest resistance, environmental and food contamination and toxicity to non-target organisms Tavares et al., 2010). Plants produce secondary metabolites many of which can have insecticidal properties, as an alternative to synthetic insecticides (Potenza et al., 2004). Plant extracts and essential oils have traditionally been used to kill or repel stored product insects (Arabi et al., 2008;Fouad et al., 2012;Tapondjou et al., 2005;Tinkeu et al., 2004). The insecticidal constituents of many essential oils against stored product insects are mainly monoterpenoids such as limonene, linalool, terpineol, carvacrol and myrcene (Ahn et al., 1998;Regnault-Roger and Hamraoui, 1995). Essential oils of several medical plant displayed considerable toxic, fumigant and repellent effects on adults of Bruchidae family (Mahfuz and Khalequzzaman, 2007;Mahmoudvand et al., 2011;Sabbour and Abd-El-Aziz, 2010).

Materials and methods
The insect Parent adults of faba bean beetle, Bruchidius incarnatus were obtained from laboratory stock cultures maintained at plant protection department, Faculty of Agriculture, Sohag University, Sohag, Egypt. They were reared in an environmentally controlled room at 25±2°C, 70±10% relative humidity (RH) and darkness. The food media used was whole faba bean grains.

The repellency test
The test was done in Petri dishes (9 cm diameter), containing filter papers inside (Whatman Nº 1, 9 cm diameter) in the dimension of the dishes. Solutions were prepared at concentrations of 0.5, 1, 2 and 4%. On one half of filter paper, uniformly, 0.5 mL of each concentration of the essential oils was applied, and on the other half only acetone adults of B. incarnates for each replicate. Data obtained were corrected using Abbott's formula (1925).

Results and discussion
Percentages of repellence (PR) values are shown in Fig.  1, Fig. 2 and Tab. 1. Four essential oils exhibited repellent activity against B. incarnates adult after 2 and 4 h. Data was applied. The treated and control half-discs were left at 10 minutes for the solvent to evaporate. On the center of each dish, 10 newly unsexed adults of B. incarnatus were placed. The treatments were repeated ten times. The repellency assay was placed in an environmentally controlled room at 25±2ºC, 70±10% RH and darkness. The number of beetles present in the control half (NC) and the treated half (NT) were recorded after 2 and 4 hours (h) (Olivero-Verbel et al., 2010).

Contact with a treated surface
Using a precision microsyringe, one mL of either each oil solution at 0.5, 1, 2 and 4% or acetone (control) was applied to the surface of a Petri dish (9 cm diameter, surface 63.6 cm 2 ) corresponding to dosages of 0.08, 0.16, 0.32 and 0.64 µL of oil/cm 2 . Each dish was left without direct sunlight for 10 min, after which 10 newly unsexed adults B. incarnates were placed in each one. The dishes were closed with a glass cover and kept in an environmentally controlled room (25±1°C, 70±10% RH and darkness). The mortality (%) was evaluated 24, 48, 72 and 96 h after starting the test (Tapondjou et al., 2005).
The design was entirely randomized with five oils with five concentrations and ten replications for each, with 10 in Tab. 1 showed that cinnamon oil had generally a more effective repellent (63%) after 2 h against adult B. incarnatus. However, castor oil had less PR values in all concentrations been used. The rest of essential oils had a moderate repellent action. A non significant difference showed between the essential oils with 0.5% concentration against B. incarnates after 2 and 4 h of treatment. In generally, the efficacy in respect of the repellency followed in the order: cinnamon > clove > camphor > mustard > castor.
The classes of repellency were higher with the cinnamon oil at 4% (classes IV) after 2 h of treatment compared with those from essential oils and other essential oils at 0.5, 1 and 2% (classes III, II and I) (Tab. 1).
The LD 50 for B. incarnatus beetles was recorded after 24, 48, 72 and 96 h from the beginning of treatment (Tab. 2). The essential oils from all five medical plants increased the mortality of the B. incarnatus adults. The LD 50 was decreased gradually in all the essential oils with increasing the days of exposure. Cinnamon oil revealed the highest residual toxicity effect followed by clove oil, camphor, mustard and the lowest effect was recorded in case of castor oil.
Our results showed that four of the tested essential oils (cinnamon, clove, camphor and Mustard) had a significant   (Ali et al., 1983) and S. zeamais (Costa et al., 2006). The presence of Allyl isothiocyanate (AITC), the main toxic compound formed from allyl glucosinolate hydrolysis (Mayton et al., 1996), was considered insecticidal substance biofumigation (Noble et al., 2002). The highest concentrations of AITC are found in some mustard, horseradish and wasabi species (Olivier et al., 1999;Yu et al., 2003). In our results, the LD 50 value was decreased gradually in all the essential oils with increasing the days of exposure. This results agree with the results obtained by Arannilewa et al. (2006) whose reported that an increase of mortality of S. zeamais adult associated with increasing the days of exposure in all concentrations of tested essential oils. Also, Arabi et al. (2008) and Ahmed (2006) reported that mortality of S. oryzae and O. surinamensis adults, respectively, was increased with the increase of the concentrations of camphor oil and increased the time of exposure.

Conclusions
Based on the present study, it could be concluded that essential oils of cinnamon, clove, camphor and mustard pose potential repellent and toxic activity against adults of B. incarnatus with higher effective was found by using cinnamon oil. The study demonstrates that these essential oils can play an important role in protection of faba bean grains from adults of B. incarnatus.
repellent and toxic effect on B. incarnates adults, suggests a wide spectrum of action from these essentials oils. Cinnamon oil had a highest possess repellency as well as toxicity effects against B. incarnates adult followed by clove > camphor > mustard and a lowest possess repellency as well as toxicity effects was castor oil. Cinnamon powder also was showed generally a more repellent effective on adults of Sitophilus granarius, Rhyzopertha dominica and T. castaneum (Shayesteh and Ashouri, 2010). The powders of Piper nigrum, Capsicum annuum and C. zeylanicum (Cinnamon plant) showed a repellent effect on S. zeamais (Salvadores et al., 2007). Cinnamaldehyde isolated from cinnamon oil was considered contact toxicity to both T. castaneum and S. zeamais (Huang and Ho, 1998).
The clove oil had also repellent activity on three important stored grain insect pests, R. dominica, S. oryzae and T. castaneum (Zeng et al., 2010). As well as, oil of clove is toxic to S. oryzae and R. dominica (Sighamony et al., 1986). However, extracts from clove plant had insecticidal effect to T. castaneum and S. zeamais (Ho et al., 1994), with the main chemical components of clove essential oil are phenylpropanoids such as carvacrol, thymol, eugenol, eugenol acetate, iso-eugenol and caryophyllene (Chaieb et al., 2007;Olivier et al., 1999).
The monoterpene camphor might have broad insecticidal activity against stored-product insects and act as a fumigant in Asplenium haussknechtii oil. Camphor from several Artemisia species reported that is toxic against stored-product beetles (Dunkel and Sears, 1998;Kordali et al., 2006;Negahban et al., 2007). Effect of mustard